TESIS DE DOCTORADO
SEARCH FOR BIOMARKERS RELATED TO
RHINITIS AND DIFFERENT ASTHMA
PHENOTYPES BY SERUM PROTEOMICS
AND IMMUNOASSAYS
Juan José Nie o Fon a igo
ESCUELA DE DOCTORADO INTERNACIONAL
PROGRAMA DE DOCTORADO EN MEDICINA MOLECULAR
SANTIAGO DE COMPOSTELA
2019
DECLARACIÓN
DO AUTOR/A DA
TESE
SEARCH FOR BIOMARKERS RELATED TO RHINITIS AND DIFFERENT
ASTHMA PHENOTYPES BY SERUM PROTEOMICS AND
IMMUNOASSAYS
D./Dna. Juan José Nie o Fon a igo
P esen o a miña ese, seguindo o p ocedemen o axei ado ao Regulamen o,
e decla o que:
1) A ese aba ca os esul ados da elabo ación do meu aballo.
2) De selo caso, na ese aise e e encia ás colabo acións que i o es e
aballo.
3) A ese é a e sión de ini i a p esen ada pa a a súa de ensa e
coincide coa e sión en iada en o ma o elec ónico.
4) Con i mo que a ese non inco e en ningún ipo de plaxio dou os
au o es nin de aballos p esen ados po min pa a a ob ención
dou os í ulos.
En San iago de Compos ela, 26 de Ma zo de 2019.
Asdo. Juan José Nie o Fon a igo
AUTORIZACIÓN
DO
DIRECTOR
/
TITOR DA TESE
SEARCH FOR BIOMARKERS RELATED TO RHINITIS AND DIFFERENT
ASTHMA PHENOTYPES BY SERUM PROTEOMICS AND
IMMUNOASSAYS
D./Dna. F ancisco Ja ie Salgado Cas o
D./Dna. Mon se a Noguei a Ál a ez
INFORMA/N:
Que a p esen e ese, co espóndese co aballo ealizado po D/Dna. Juan
José Nie o Fon a igo, baixo a nosa di ección, e a
u o izamos
a súa
p esen ación
, conside ando
que eúne os
equisi os
esixidos no
R
egulamen o
de Es udos de
Dou o amen o da USC,
e
que
como
di ec o es des a
non inco e nas causas de
abs ención es ablecidas
na Lei
40/2015.
En San iago de Compos ela, 26 de Ma zo de 2019.
Asdo. Asdo.
F ancisco Ja ie Salgado Cas o Mon se a Noguei a Ál a ez
CONFLICTO DE INTERÉS
SEARCH FOR BIOMARKERS RELATED TO RHINITIS AND
DIFFERENT ASTHMA PHENOTYPES BY SERUM PROTEOMICS AND
IMMUNOASSAYS
D. Juan José Nie o Fon a igo
Decla o no ene ningún ipo de con lic o de in e eses, ni ninguna
elación económica, pe sonal, polí ica, in e és inancie o ni
académico que pueda in lui en es e abajo.
En San iago de Compos ela, 26 de Ma zo de 2019.
Fdo. Juan José Nie o Fon a igo
Es a esis y los abajos de i ados se han ealizado en el Cen o de
In es igación en Biología de la Uni e sidad de San iago de
Compos ela (CIBUS) – Facul ad de Biología, Campus ida,
Uni e sidade de San iago de Compos ela g acias al inanciamien o
de los p oyec os (121/2012) po la Sociedad Española de
Neumología y Ci ugía To ácica, (SEPAR) y el (PI13/02046) del
Ins i u o de Salud Ca los III, Minis e io de Economía y
Compe i i idad (Fondo de In es igación Sani a ia, FIS; co-
inanciado po ondos FEDER de la Unión Eu opea)
El au o de es a esis, D. Juan José Nie o Fon a igo, ha sido
ben icia io de la beca p edoc o al de la Xun a de Galicia (DOG
Núm. 122, Páx. 27453, Mé co es, 29 de xuño de 2016), Modalidad B
(Co inanciadas po Fondo Social Eu opeo, FSE).
Todos los abajos ealizados en es a esis cuen an con la ap obación
del Comi é de É ica de In es igación Clínica de Galicia
(CEIC)(2011/001), y odos los pa icipan es en los mismos han
i mado un consen imien o in o mado.
Juan José Nie o Fon a igo
AIMS OF THE THESIS ............................................................. 85–87
CHAPTER I ........................................................................................ 89–119
In oduc ion ............................................................................................................... 91–92
Ma e ial and Me hods ............................................................................................... 92–96
Resul s ..................................................................................................................... 96–108
Discussion ............................................................................................................. 108–112
Re e ences ............................................................................................................ 112–117
Supplemen a y In o ma ion ................................................................................. 118–119
CHAPTER II .............................................................................. 121–155
In oduc ion .......................................................................................................... 123–125
Ma e ial and Me hods .......................................................................................... 125–129
Resul s .................................................................................................................. 129–139
Discussion ............................................................................................................. 139–146
Re e ences ............................................................................................................ 146–153
Supplemen a y In o ma ion ................................................................................. 154–155
CHAPTER III............................................................................. 157–204
In oduc ion .......................................................................................................... 159–161
Ma e ial and Me hods .......................................................................................... 161–165
Resul s .................................................................................................................. 165–180
Discussion ............................................................................................................. 181–185
Re e ences ............................................................................................................ 186–194
Supplemen a y In o ma ion ................................................................................. 195–204
CHAPTER IV ............................................................................. 205–258
In oduc ion .......................................................................................................... 207–210
Ma e ial and Me hods .......................................................................................... 210–213
Resul s .................................................................................................................. 213–233
Discussion ............................................................................................................. 233–238
Re e ences ............................................................................................................ 239–248
Supplemen a y In o ma ion ................................................................................. 249–258
GENERAL DISCUSSION ...................................................... 259–278
1. INNATE IMMUNE SYSTEM BIOMARKERS OF ALLERGIC ASTHMA (AA)
(Chap e I) .................................................................................................. 259–265
2. CD26, ADAPTIVE IMMUNE CELLS, AND ASTHMA PHENOTYPES
INDEX
(Chap e s II and III) ...................................................................................... 265–273
3. SEARCHING FOR BIOMARKERS IN SERUM SAMPLES FROM PATIENTS WITH
RHINITIS OR DIFFERENT ASTHMA PHENOTYPES/SEVERITIES (Chap e IV) ..... 273–278
CONCLUSIONS ...................................................................... 279–283
REFERENCES (In oduc ion and Gene al Discussion) ...... 285–342
APPENDIX I .................................................................................... 343
ABBREVIATIONS
I
ABBREVIATIONS
2-DE:
Two dimension elec opho esis
2D-DIGE:
2D Fluo escence Di e ence Gel Elec opho esis
AA:
Alle gic as hma
ADA:
Adenosine deaminase
AECs:
Ai way epi helial cells
AHSG:
α-2-HS-glycop o ein
AHR:
Ai way hype esponsi eness
APCs:
An igen p esen ing cells
BALF:
B onchoal eola la age luid
BB:
B onchial biopsies
BCR:
B-cell ecep o
BIM:
Bcl-2 in e ac ing media o
CFH:
Complemen ac o H
CFI:
Complemen ac o I
CPLLs:
Combina o ial pep ide ligand lib a ies
CSF:
Ce eb ospinal luid
DAMPs:
Damage associa ed molecula pa e ns
DPP:
Dipep idyl pep idase p o ein
EBC:
Exhaled b ea h condensa e
ELISAs:
Enzyme-linked immunoso ben assays
ESI:
Elec osp ay ionisa ion
EU:
Eu opean Union
FeNO:
F ac ional exhaled ni ic oxid
GWAS:
Genome-wide associa ion s udies
HDM:
House dus mi e
HSPG2:
Hepa an sulpha e p o eoglycan 2
ICS:
Inhaled cos icos e oids
IGFs:
Insuline-like g ow h ac o s
IGFALS:
Insulin-like g ow h ac o binding p o ein, acid
labile subuni
IGFBPs:
Insulin-like g ow h ac o binding p o eins
Juan José Nie o Fon a igo
II
ILCs:
Inna e lymphoid cells
iNKT:
In a ian na u al kille T cells
iTRAQ:
Isoba ic ag o ela i e and absolu e quan i a ion
iT egs:
Induced egula o y T cells
LAP:
Low abundan p o eins
LBP:
Lipopolysaccha ide binding p o ein
LC:
Liquid ch oma og aphy
LC-MS/MS:
Liquid ch oma og aphy coupled o mass
spec ome y
LMW:
Low molecula weigh p o eins
LPS:
Lipopolysaccha ide
LTQ:
Linea ap quad upole
MALDI-TOF:
Ma ix-Assis ed Lase Deso p ion/Ioniza ion-
ime-o - ligh
mCD14:
Memb ane CD14
MHC:
Majo his ocompa ibili y complex
MS:
Mass spec ome y
NAA:
Non-alle gic as hma
NB:
Nasal b ushing
NK:
Na u al kille
NLF:
Nasal la age luid
NSE:
Neu on-speci ic enolase
nT egs:
Na u al egula o y T cells
ORM1/AGP:
O osomucoid/α-1-acid glycop o ein 1
OVA:
O albumin
PAMPs:
Pa hogen associa ed molecula pa e ns.
P o ein AMBP:
α-1-mic oglobulin/bikunin p ecu so
PRRs:
Pa hogen ecogni ion ecep o s
PCs:
P incipal componen s
RP:
Re e se phase
sCD14:
Soluble CD14
ABBREVIATIONS
III
sCD25:
Soluble CD25
sCD26:
Soluble CD26
SCX:
S ong ca ion exchange
SNPs:
Single nucleo ide polymo phisms
TCM:
Cen al-memo y T cells
TCR:
T-cell ecep o
TEM:
E ec o -memo y T cells
TEMRA:
Te minally-di e en ia ed e ec o T cells
Te :
E ec o T cells
T H:
Follicula helpe T cells
TH:
Helpe T cells
TLRs:
Toll-like ecep o s
TN:
Naï e T cells
T eg:
Regula o y T cells
TSCM:
S em cell memo y T cells
UPLC:
Ul a pe o mance liquid ch oma og aphy
US:
Uni ed S a es
WT:
Wild ype
ABSTRACT
3
s hma is a he e ogeneous disease wi h se e al clinical pheno ypes and
molecula endo ypes. Howe e , he speci ic connec ion be ween
as hma pheno ypes and he unde lying pa hological ea u es is di icul o
explain. Thus, he o e all aim o he p esen hesis was o sea ch o
bioma ke s associa ed wi h hini is and di e en pheno ypes (alle gic and
non-alle gic) and se e i ies (in e mi en -mild and mode a e-se e e) o
as hma, which could ha e an applica ion in he diagnosis, p ognosis o
ea men o his disease. The mechanisms unde lying as hma a e mul iple
and complex, bu he immune sys em plays a key ole in he
pa hophysiology o his disease. The e o e, we decided o u he s udy he
ole o he immune sys em in di e en pheno ypes o se e i ies o as hma
h ough he analysis o ce ain bioma ke s p e iously ela ed o his
disease: CD14 (inna e immune sys em) and CD26/CD126 (adap i e immune
sys em).
CD14 is a ecep o mainly exp essed on monocy es, which
pa icipa es in he lipopolysaccha ide (LPS) signalling. Ou esul s show ha
bo h he exp ession o CD14 on monocy es and he no malized le els o
soluble CD14 (sCD14) in se um a e educed in alle gic as hma s. heal hy
con ols. This educ ion can be explained by he expansion o CD14low cells,
p obably non-classical monocy es wi h a high capaci y o di e en ia e in o
M2 mac ophages. In addi ion, sCD14 le els a e associa ed wi h he
p omo e SNP o CD14 (-159 C/T). Thus, subjec s wi h he C allele and CC
geno ype ha e lowe le els o sCD14, as well as inc eased isk o alle gic
as hma. On he o he hand, CD26 is a pep idase mainly exp essed by helpe
T (TH) lymphocy es. Ou esul s e idence a high co ela ion be ween he
exp ession o CD26 on hose cells and DPP4 ac i i y (~ soluble CD26/sCD26)
in i o. Mo eo e , he exp ession o his molecule di e en ia es sub ypes
o T cells (TH17>>TH1>TH2>T eg), as well as disc imina es be ween cells
wi h di e en s ages o di e en ia ion: cen al-memo y T cells (TCM,
CD26high, CD45RA-CCR7+CD28+), naï e T cells (TN, CD26in ,
CD45RA+CCR7+CD28+), and e minally di e en ia ed o e ec o -memo y T
cells (TEM o TEMRA, CD26low, CD45RA+/-CCR7-CD28-). In addi ion, he e is an
A
Juan José Nie o Fon a igo
10
bioma cado es sé icos en asma (aplicable a o as pa ologías ambién),
así como la ealización de un es udio p ospec i o, no di igido, pa a
iden i ica nue os ma cado es biológicos asociados con dis in os
eno ipos y/o se e idades de es a en e medad. Pa a ello, as p oba
di e en es me odologías, se ha desa ollado un p o ocolo que consis e
en una eliminación de lipop o eínas del sue o, el en iquecimien o en
p o eínas de media-baja abundancia median e lib e ías de
hexapép idos combina o ios (CPLLs) (comp esión del ango dinámico
de abundancias), la gene ación de pép idos íp icos, su ma caje
median e eac i os iTRAQ, y el accionamien o, iden i icación y
cuan i icación ela i a median e c oma og a ía líquida en ase e e sa
acoplada a espec ome ía de masas en ándem (LC-MS/MS).
Capí ulo I
El es udio que se expone en el capí ulo I u o como obje i o el es udio
de la exp esión de CD14 en la supe icie de monoci os, así como el
análisis de los ni eles de CD14 soluble, su elación con el SNP del
p omo o de CD14 (-159 C/T) y la suscep ibilidad de su i asma
alé gica. Todo es o en una población bien de inida (adul os,
caucásicos, bajos ni eles de endo oxina) y con un amaño mues al
ela i amen e ele ado (277 pacien es con asma alé gica y 277
con oles sanos). Los esul ados de es e es udio demos a on un
aumen o del núme o de monoci os en pacien es con asma alé gica
pocas eces desc i o p e iamen e. Sin emba go, la exp esión de CD14
en la supe icie de es as células, así como la concen ación de CD14
soluble (no malizado po el núme o de monoci os) en sue o es aban
educidos en los pacien es con asma. Dicha educción pod ía debe se a
la expansión de monoci os con eno ipo CD14-, p obablemen e de ipo
“no clásico” (CD14-CD16+) y con al a capacidad de di e encia se a
mac ó agos isula es M2. Pe o además, a ello se pod ía suma el
e ec o del SNP CD14 (-159 C/T) sob e los ni eles de sCD14. De
SUMMARY IN SPANISH
11
hecho, nues os esul ados apun an a mayo es ni eles de sCD14 en
indi iduos po ado es del alelo T y del geno ipo TT. Además, an o el
alelo T como el geno ipo TT de dicho SNP es án asociados a un
meno iesgo de su i asma alé gica. O dicho de o o modo, nues os
esul ados sugie en un papel pa a el alelo C o el geno ipo CC del SNP
(-159 C/T) a la ho a de gene a bajos ni eles de CD14 soluble y
aumen a el iesgo de desa olla asma alé gica de mayo g a edad.
Los meno es ni eles de CD14/CD14 soluble en indi iduos con asma
alé gica ienen sen ido biológico, ya que es a molécula cons i uye
jun o con “Toll-like Recep o 4” (TLR4) un complejo esponsable de
la señalización del lipopolisacá ido bac e iano (LPS) en
monoci os/mac ó agos. Dicha ía desencadena la p oducción de
ci oquinas como IL-12, p omo iendo la di e enciación TH1 en
de imen o de la TH2.
Finalmen e, debido al aumen o de monoci os ci culan es
de ec ado en pacien es con asma alé gica, du an e la ealización de
es e es udio ambién se analiza on los ni eles de la enolasa neu onal
especí ica (NSE), una molécula que se ha is o aumen ada as
ac i ación de eosinó ilos y mac ó agos en algunas en e medades.
Nues os esul ados mues an una co elación posi i a en a las
concen aciones sé icas de es a molécula e IgE o al, ambas
aumen adas en pacien es con asma alé gica. De hecho, la e iciencia
diagnós ica e aluada a a és de los alo es de “a ea unde he cu e”
AUC de las cu as “Recei e Ope a ing Cha ac e is ic” (ROC)
demues an unos alo es muy simila es de e ec i idad diagnós ica
pa a NSE e IgE, sub ayando un posible uso como bioma cado de es e
eno ipo asmá ico.
Juan José Nie o Fon a igo
12
Capí ulos II y III
Del mismo modo que sucede con CD14 en monoci os, CD26 es una
pep idasa con ac i idad DPP4 que mues a una especial abundancia en
lin oci os T colabo ado es (TCD4+; sis ema inmune adap a i o), que
p esen a una o ma soluble, y que ambién ha sido asociada al asma
po di e sos au o es. Los es udios lle ados a cabo en los capí ulos II y
III ienen como obje i o p o undiza en es a úl ima asociación en
elación con los dis in os eno ipos asmá icos y se e idades.
CD26/DPP4 es una p o eína “plu iempleada” (“moonligh ing”), lo que
hace que su papel en asma sea complejo de es udia . Además, la
p ác ica ausencia de es udios de e isión bibliog á ica sob e las
unciones de es a molécula y sus posibles consecuencias en asma
impulsó el abajo que se mues a en el anexo I de la p esen e esis
(Anexo I). Es e p opo ciona una isión gene al y es uc u ada de las
dis in as unciones p o- y an i-in lama o ias de CD26, así como de las
posibles implicaciones en la isiopa ología asmá ica. Es udios p e ios
en nues o g upo habían demos ado una mayo abundancia de CD26
en las células T e ec o as que en células T egulado as. Además, o os
au o es habían demos ado que los ni eles de CD26 den o de las
células e ec o as e an bas an e a iables: TH17>> TH1> TH2> T eg.
Con esa idea en men e, nues o abajo (Capí ulo II) demos ó po
p ime a ez en cul i o in i o un aumen o de exp esión de CD26 en
lin oci os TH e ec o es humanos as ac i ación, especialmen e en
condiciones que p omo ían una di e enciación TH17, pe o ambién
una co elación posi i a con la ac i idad DPP4 soluble (una medida
indi ec a de CD26 soluble). Po an o, nues os esul ados suge ían
que los ni eles de CD26/DPP4 soluble in i o es aban in luenciados
po el núme o de lin oci os T CD4+ así como po su g ado de
ac i ación y eno ipo (TH1, TH2, o TH17), de modo que los ni eles
de CD26 soluble en sue o pod ían es a e lejando, como una “huella
dac ila ”, la con acción o expansión de unas subpoblaciones TH sob e
SUMMARY IN SPANISH
13
o as; dicho de o o modo, pod ía habe di e encias en la exp esión de
CD26 en los lin oci os TH y en los ni eles de su iso o ma soluble
dependiendo del eno ipo y se e idad asmá icas. Po an o, es a
hipó esis inicial ue examinada en es udios con pacien es.
En consonancia con dicha hipó esis de pa ida y ambién con
los esul ados de un es udio p e io de 2007 publicado po Saman ha
Wei-Man Lun y colabo ado es, la exp esión de CD26 en lin oci os TH
ue mayo en pacien es con asma alé gica en compa ación con los
con oles sanos. Sin emba go, los ni eles de CD26 es aban,
con a iamen e, educidos en el sue o de es os mismos pacien es; lo
mismo ocu ía con CD25 soluble, o o ma cado de ac i ación. Dado
que no se obse a on cambios en los po cen ajes de células T
egulado as (una población CD26low), es os meno es ni eles de
CD26/CD25 solubles ue on elacionados con la expansión de una
subpoblación TH e ec o a con eno ipo CD25low/CD26low/CD127low
(células “ iple low” o Tlow) en asma alé gica. Además, dicha
expansión ue con i mada en el Capí ulo III, donde además se
p o undizó en la ca ac e ización eno ípica de es a población. Así, se
io que las células Tlow e an células TH en un a anzado es ado de
di e enciación, con eno ipo “e ec o de memo ia” (TEM) o
“ e minalmen e di e enciado” (TEMRA), ca ac e izadas po la pé dida
de ma cado es de supe icie como CD27, CD28, CCR7 o CD127. Po
an o, la exp esión de CD26 en células TH y sus ni eles en sue o son
indica i os sob e odo de las dis in as e apas de di e enciación naï e-
memo ia: células de memo ia cen al (TCM, CD26high), células naï e
(TN, CD26in e media e) y células e ec o as de memo ia o e minalmen e
di e enciadas (TEM o TEMRA, CD26-/low).
De igual o ma que en pacien es con asma alé gica, ambién
hemos is o un descenso de CD26 soluble en pacien es con asma no
alé gica. Sin emba go, no se obse ó una expansión signi ica i a en
es os pacien es de poblaciones TH con eno ipo TEM/TEMRA (Tlow).
Juan José Nie o Fon a igo
14
Po an o, decidimos en oca el análisis sob e los lin oci os T CD4-,
que incluyen células B, NK, NKT y lin oci os T-γδ. Además, se
analiza on no sólo los ni eles de CD26, sino ambién los de
CD126/IL-6Rα, ya que IL-6 iene un papel impo an e en la
gene ación de lin oci os TH17, ambos con ele ancia en asma no
alé gica. En el conjun o de lin oci os T CD4-, se obse ó una ele ada
co elación en e la exp esión de CD26 y CD126. También se
documen ó en pacien es con asma no alé gica un aumen o del
po cen aje de lin oci os T CD4- en a anzado es ado de di e enciación
(TEM/TEMRA) y con eno ipo CD26- o CD126-. Además, a pesa de no
obse a cambios en los po cen ajes de los dis in os ipos celula es
den o de los lin oci os T CD4-, nues os esul ados sí e idencia on un
aumen o de la p opo ción de células T-γδ con bajos ni eles de CD26
en es os pacien es. Po an o, ambos eno ipos asmá icos (asma
alé gica y asma no alé gica) p esen an un pa alelismo en elación con
la expansión de células CD26-/low al amen e di e enciadas
(CD27lowCD28lowCCR7lowCD127low) pe enecien es a di e en es
linajes: TH en asma alé gica y CD4- T-γδ en asma no alé gica; la
educción de los ni eles de CD26 solubles en ambos eno ipos
asmá icos se ía una “imp on a” de dicha expansión en el sue o de los
pacien es.
La educción de CD26 de memb ana o soluble en asma,
especialmen e el eno ipo no alé gico, debe ene se en cuen a a la is a
del papel modulado de CD26 sob e ci oquinas (p.ej., IL-3, GM-CSF)
o quimioquinas (p.ej., RANTES o eo axina); especialmen e eniendo
en cuen a el papel inhibido que dicha unción iene sob e la a acción
quimio ác ica de células e ec o as impo an es en asma, como los
lin oci os TH o T-γδ. Po ejemplo, eo axina es una quimioquina muy
po en e en la a acción de eosinó ilos y células TH2 a luga es de
in lamación, cuya unción se e uncada po el co e p o eolí ico
p oducido po CD26. Lo mismo pasa con SDF-1α/CXCL12 o las
SUMMARY IN SPANISH
15
quimioquinas inducidas po IFN-γ (CXCL9-11), que son
quimioa ayen es de células TH1 a luga es de in lamación. Es as
unciones inhibido as de CD26 deben sob e odo ene se en cuen a en
pacien es con diabe es ipo-II que es án ecibiendo o an a ecibi
glip inas (inhibido es de la ac i idad DPP4 de CD26) y que además
p esen an asma (p.ej., asma no alé gica asociada a obesidad), ya que
se pod ía ag a a el es ado del pacien e.
El es udio expues o en el capí ulo III ambién mos ó una
educción de los ni eles de CD126/IL-6Rα en monoci os, neu ó ilos
y lin oci os TH en pacien es con asma mode ado-g a e en
compa ación con los pacien es con asma in e mi en e-le e. Es e
esul ado es indica i o de un papel de CD126/IL-6Rα en la se e idad
del asma, posiblemen e a a és de un mecanismo denominado ans-
señalización en células que son CD126-gp130+. En dicho mecanismo
in e iene una e sión soluble de CD126, que es gene ada po
p ocesos de p ocesamien o al e na i o del mRNA o median e
p o eólisis de la o ma anclada a memb ana. Finalmen e, es e es udio
ambién sugie e un de ec o uncional (no numé ico) en las células T
egulado as de pacien es con o me aumen a la se e idad de la
en e medad. Es e de ec o depende ía de una meno p oducción ne a de
adenosina, un nucleósido inmunosup eso , como consecuencia de la
p esencia de mayo es ni eles de CD26 y meno es de CD39 en las
células T egulado as de pacien es con asma mode ado-g a e
compa ado con las co espondien es a pacien es con asma
in e mi en e-le e.
Capí ulo IV
En la úl ima pa e de es a esis se plan eó como obje i o la
iden i icación de p o eínas de baja abundancia de sue o que si iesen
como bioma cado es po enciales de eno ipos o se e idad asmá ica.
Pa a ello se desa olló un mé odo basado la eliminación de
Juan José Nie o Fon a igo
16
lipop o eínas del sue o y el en iquecimien o de p o eínas de baja
abundancia median e el uso de lib e ías pep ídicas alea o ias (CPLLs).
Pos e io men e, es as mues as en iquecidas en p o eínas poco
abundan es ue on ipsinizadas, los pép idos ue on e ique ados
median e ma cajes isobá icos (iTRAQ) y las p o eínas de las dis in as
mues as (asma alé gica mode ado-g a e, asma alé gica in e mi en e-
le e, ini is, asma no alé gica mode ado-g a e, asma no alé gica
in e mi en e-le e, sanos) ue on iden i icadas y cuan i icadas median e
LC-MS/MS (LTQ-O bi ap). Pa a pode ene en cuen a la
a iabilidad biológica, los sue os de cada uno de los seis g upos
mues ales ue on alea o iamen e sepa ados en dos subg upos y
combinados (“pooles biológicos”). A su ez, cada uno de esos “pooles
biológicos” ue analizado po iplicado median e LC-MS/MS.
También ue empleado un con ol in e no con el obje i o de
no maliza las señales.
En conjun o, el es udio p o eómico cuan i a i o de ec ó 217
p o eínas sé icas, den o de las cuales 26 mos aban una abundancia
di e encial en e g upos. Los es udios uncionales (“gene on ology”,
GO) han demos ado que es os bioma cado es po enciales
desempeñan p ocesos cla e en la pa ogénesis del asma, ales como
ac i ación del complemen o, espues a inmune, espues a a es ímulos
bió icos, o espues a inmune mediada po células B. Los es udios de
en iquecimien o (Reac ome), po su pa e, mues an un
en iquecimien o en la ía “Regulación del anspo e de ac o es de
c ecimien o simila es a insulina (IGFs) y cap ación po p o eínas de
unión a ac o es de c ecimien o simila es a insulina (IGFBPs)” en
asma alé gica. A es a ía pe enece IGFALS, un posible bioma cado
de asma alé gica (especialmen e la mode ado-g a e) que hemos
con i mado ya median e ELISA. O as p o eínas, en cambio, es án
pendien es de alidación, como po ejemplo HSPG2 o la p o eína
AMBP, cuya concen ación sé ica ha sido encon ada aumen ada en
SUMMARY IN SPANISH
17
pacien es con asma alé gica. Po o o lado, ambién se han is o
cambios en di e sas p o eínas elacionadas con la ac i ación y
egulación de odas las ías del complemen o en asma no alé gica.
En e ellas cabe des aca los ac o es de complemen o I (CFI) o H
(CFH), así como la p o eína MASP1; odas ellas es án ele adas en el
eno ipo no alé gico, y ambién pendien es de una pos e io
alidación. Jun o con lo an e io men e comen ado sob e las células T-
γδ, el papel de los ac o es del complemen o habla nue amen e de las
implicaciones del sis ema inmune inna o en el asma no alé gica. Ello
asemeja a es a en e medad a una pa ología au oinmune y explica, a su
ez, su edad más a día de apa ición y la mayo incidencia en muje es.
Po an o, es os esul ados p elimina es mues an cambios en un g upo
de p o eínas sé icas que pod ían se ú iles pa a di e encia di e en es
eno ipos asmá icos, se i pa a p onos ica la se e idad de la
en e medad, o se dianas de u u as e apias di igidas.
Juan José Nie o Fon a igo
26
be ound in samples om he le uppe lobe o he lung [33].
In e es ingly, as hma suscep ibili y has also been associa ed wi h
changes in he coloniza ion o lowe ai ways by mic obes. Indeed,
some wo ks ha e shown ha as hma ics displayed a highe
p edominance o P o eobac e ia (a phylum o g am-nega i e bac e ia)
han subjec s wi hou as hma, whe e Bac e iode es (ano he phylum o
g am-nega i e bac e ia) a e mo e equen [1, 33, 34].
The e is a disag eemen abou he na u e o he unde lying
mechanism ha explains he highe ela i e abundance o phylum
P o eobac e ia in as hma ics, bu i is b oadly accep ed ha he
immune sys em is implica ed. Mic obes p oduce subs ances such as
LPS, lagellin, o lipo eichoic acid. These exogenous and highly
conse ed molecules a e collec i ely called pa hogen-associa ed
molecula pa e ns (PAMPs). PAMPs a e ecognized by pa e n
ecogni ion ecep o s (PRRs), which a e memb ane-bound (e.g., Toll-
like ecep o s/TLRs) o cy oplasmic (e.g., NOD-like ecep o s, RIG-I-
like ecep o s) ecep o s mainly exp essed by inna e immune cells
(e.g., monocy es, mac ophages, dend i ic cells, neu ophils,
eosinophils, epi helial cells) [35]. PRRs also iden i y damage-
associa ed molecula pa e ns (DAMPs) o ala mins, ano he subse o
molecules eleased his ime by hos cells du ing in lamma ion o
when hey a e damaged o dead. Ac i a ion o PRRs leads o inna e
cells o elease soluble ac o s (e.g., cy okines, chemokines) and
exp ess co-s imula o y molecules, which in u n unchain he adap i e
immune esponse.
CD14 is a co- ecep o ha allows he de ec ion o bac e ial LPS
along wi h LPS binding p o ein (LBP), TLR4 and MD2. T igge ing o
CD14/TLR4 leads o he elease o IL-12/IL-18 by
monocy e/mac ophages, wo cy okines ha a ou he TH1
di e en ia ion, in de imen o he TH2-media ed adap i e esponses
[36-38]. This shi in he TH1-TH2 axis was he classical explana ion
INTRODUCTION
27
o he hygiene hypo hesis. Howe e , ce ain helmin hic in ec ions
(e.g., Schis osomiasis) igge TH2- ype immune esponses and
p e en as hma [39]. Mo eo e , TH1 cells could e en inc ease he
pa hogenici y o TH2 cells, which makes i di icul o explain he
hygiene e ec h ough a simple TH1-TH2 cell coun e balance.
Nowadays, i is belie ed ha he e is a loss o T eg cells in subjec s
li ing in “hygiene en i onmen s”, which u ns in o inc eased TH2 cell
immuni y and highe suscep ibili y o de elop as hma/alle gy [39, 40].
CD14/TLR4 is a he c oss oads whe e naï e lymphocy es ha e o
make he decision be ween TH1 and TH2 e ec o esponses so ha
polymo phisms in hese genes could impac se iously o as hma
de elopmen o se e i y [41].
4. INNATE IMMUNITY IN ASTHMA
The inna e immuni y is he i s line o de ence agains pa hogens.
This is an immedia e and ela i ely non-speci ic esponse in which
di e en cellula popula ions and soluble ac o s a e implica ed.
Be ween hem, we a e going o pu special emphasis on he ole o
ai way epi helial cells (AECs), inna e lymphoid cells (ILCs), γδ-T
lymphocy es, and monocy es in as hma.
4.1. Ai way epi helial cells (AECs) and inna e lymphoid cells
(ILCs) in as hma.
The i s cellula componen implica ed in he pa hogenesis o as hma
is he ai way epi helial ba ie . AECs a e con inuously exposed o
di e en pollu an s, pa icula e ma e , alle gens and pa hogens.
PAMPs om mic oo ganisms (e.g., LPS) and enzymes de i ed om
pa hogens o alle gens (e.g., dus mi e alle gen De p1) in e ac wi h
Juan José Nie o Fon a igo
28
AECs h ough PRRs (e.g., TLRs) and p o ease-ac i a ed ecep o s,
espec i ely. This in e ac ion unleashes he po en ial o epi helial cells
o p oduce a se o cy okines and chemokines implica ed in as hma
pa hogenesis [42, 43], whose bes examples a e TSLP ( hymic s omal
lymphopoie in), IL-25 and IL-33. PRRs ac i a ion by DAMPs o
p o ease-ac i a ed ecep o s clea age by endogenous p o einases (e.g.,
h ombin, plasmin, kallik eins, ma ix me allop o einases, elas ases)
play a complemen a y ole in AECs ac i a ion.
As we s a e abo e, he genes encoding bo h IL-33 and i s
ecep o (ST2/IL-1R1L) a e as hma suscep ibili y genes [17, 44, 45].
IL-33 is a membe o he IL-1 supe amily o cy okines, bu i is also
conside ed an ala min o DAMP; i.e. an in acellula molecule
eleased in esponse o cell inju y which is discha ged o ex acellula
space a e cell damage (nec osis o auma) [46]. IL-33 is
cons i u i ely exp essed by al eola ype-II pneumocy es, and i s
exp ession is enhanced upon ai way in lamma ion [47, 48],
pa icula ly in alle gic as hma (AA) [49, 50]. Addi ionally,
mac ophages a e bo h he sou ce and a ge o his cy okine [51]. IL-
33 is in ol ed in he esponse o bo h helmin hs and i al in ec ion
[52, 53], h ough i s binding o i s ecep o (ST2/IL-1R1L). ST2 is
exp essed by a my iad o immune cells, including mas cells,
basophils, eosinophils, dend i ic cells, and ILCs [51]. Binding o IL-
33 o ansmemb ane ST2 igge s di e en signalling ou es. Fo
example, IL-33 induces he ac i a ion o he NF-κB pa hway in
human mas cells s imula ing he elease o di e en cy okines (e.g.,
GM-CSF, TNF-α, IL-1β, IL-6, IL-8, o IL-13) and chemokines (e.g.,
CXCL8, CCL1) [54].
Simila ly, TSLP is a cy okine encoded by a gene associa ed
wi h a highe isk o AA [17]. TSLP (IL-2 amily o cy okines), as
well as IL-25 (IL-17 amily o cy okines; IL-17E), a e eleased by
AECs in esponse o alle gens (e.g., HDM) ha possess p o ease
INTRODUCTION
29
ac i i y and a e capable o ac i a ing p o ease-ac i a ed ecep o s like
PAR2 [55]. Bo h cy okines display inc eased le els in as hma ic
pa ien s [56, 57]. TSLP is c i ical o he main enance o alle gic
ai way in lamma ion h ough inducing TH2- ype cy okines
p oduc ion, boos ing TH9 cells di e en ia ion, inhibi ing T eg
unc ion, and ac i a ing basophils/mas cells [43, 58-60]. Mo eo e ,
hino i us in ec ion boos le els o TSLP in a mouse model o AA,
unde lying he po en ial ole o his cy okine in as hma exace ba ion
[61]. IL-25, o i s pa , is mainly associa ed wi h eosinophils
ac i a ion. Howe e , i is wo h highligh ing ha he he e odime ic
ecep o o his cy okine (IL-17RA/IL-17RB) is exp essed in many
immune cell ypes such as an igen p esen ing cells (APCs), in a ian
na u al kille T cells (iNKT), AECs, and ILC2/nuoci es [43].
Al oge he , IL-25, TSLP, and pa icula ly IL-33 a e esponsible o
he ea ly alle gic esponse in as hma, d i ing he ac i a ion o ILC2
cells [43, 52, 55].
Upon ac i a ion by IL-25, TSLP and IL-33, hese ILC2 cells
become one o he majo p oduce s o IL-5 and IL-13 [62], wo
cy okines wi h a pi o al ole in he induc ion and main enance o AA
[63]. Recen ly, se e al wo ks in bo h mice and he human sys em ga e
suppo o an impo an ole o his subse in as hma. These s udies
ha e shown ha ac i a ed ILC2 cells no only p oduce IL-5 and IL-13,
bu also IL-3, IL-4, IL-6, IL-8, o GM-CSF [ e iewed in 62].
Mo eo e , his unc ion o ILC2 cells is enabled by human mas cells
ia p os aglandin D2 [64-66]. The e o e, ILC2 lymphocy es migh be
a b idge be ween he inna e and he adap i e immune sys em.
Apa om ILC2 cells, o he inna e immune cells a e
becoming mo e ele an o as hma pa hogenesis, especially in he
ligh o as hma he e ogenei y and he p esence o di e en disease
pheno ypes, such as non-alle gic as hma (NAA). Fo example,
al e na i e ILCs subse s such as na u al kille (NK) cells, ILC1, o
Juan José Nie o Fon a igo
30
ILC3 cells could be ele an o as hma. Bo h NK and ILC1 cells ha e
been linked o he pa hogenesis o his diso de [67, 68], bu mo e
in e es ing is he ole o IL-17-p oducing ILC3 cells in obesi y- ela ed
as hma [69]. Howe e , mo e s udies a e necessa y o asce ain he
possible oles o ILC1 and ILC3 in his disease.
4.2. Monocy es in as hma pa hogenesis
Monocy es ep esen abou 10% o ci cula ing leukocy es and hei
hal -li e is a ound 3 days in human blood [70]. These cells a e
“ci cula ing mac ophages” ha can u n in o issula in lamma o y
cells also capable o gene a ing IL-33. This is illus a ed in mouse
models, whe e ci cula ing monocy es a e ec ui ed o he lung upon
HDM-induc ion o become ac i e p oduce s o IL-33 [71].
Human blood monocy es ha e been subdi ided in o h ee
disc e e subpopula ions based on he su ace exp ession o CD14 and
CD16: “classical” CD14++CD16- cells, ep esen ing ~ 90% o
ci cula ing monocy es; “non-classical” CD14+CD16++ monocy es; and
“in e media e” CD14++CD16+ monocy es [72-74]. Howe e , he las
subse is conside ed a ansi ional subpopula ion mo e han a ue
sub ype [75]. The e is an inc ease in ma u i y om classical
monocy es, which display an an i-apop o ic and p oli e a i e s a e, o
non-classical subse s, ha ing an an i-p oli e a i e and apop o ic s a e
[75].
CD14++CD16- classical monocy es espond o LPS ia TLR2
and TLR4, p oducing eac i e oxygen species and a se o cy okines
and chemokines (e.g., IL-6, IL-8 o CCL-3) [76]. Mukhe jee e al.
ha e shown ha hese cells display high le els o CD36 and CD163
(sca enge ecep o s), which e idence he phagocy ic na u e o his
subse [76]. Addi ionally, in e media e monocy es and non-classical
INTRODUCTION
31
monocy es a e cha ac e ized by a high co-s imula o y abili y, as hey
exp ess CD80 and CD86 [77]. Non-classical monocy es espond o
nucleic acids (damaged cells) and i uses ia TLR7 and TLR8,
sec e ing TNF-α and IL-1β [76] and hey a e also inc eased du ing
sepsis [77].
All ypes o monocy es a e able o di e en ia e in o monocy e-
de i ed mac ophages o dend i ic cells. Indeed, non-classical
monocy es display high le els o CD11a and CTSL1, which allow
hem o in il a e and di e en ia e in M2- ype mac ophages [78].
Mac ophage-de i ed monocy es ha e bo h p o-in lamma o y and an i-
in lamma o y oles. M1 mac ophages o classically-ac i a ed
mac ophages a e mainly p o-in lamma o y cells which elease
cy okines and chemokines such as IL-1β, IFN-γ, IL-8, IP-10, IL-23,
TNF-α, o RANTES a e pa hogen in ec ion. These cy okines a e
po en induce s o TH1 and TH17-d i en in lamma ion [79], which is
impo an in non-alle gic lung in lamma ion. Mo eo e , highe le els
o M1- ype mac ophages ha e been desc ibed in less se e e AA
compa ed o se e e alle gic disease, highligh ing hei possible an i-
alle gic ole [80]. On he o he hand, M2 mac ophages consis o ou
majo g oups o cells: M2a cells, which sec e e IL-4 and IL-13; M2b
cells, which sec e e p o-in lamma o y (IL-6, IL-1β, and TNF) and
an i-in lamma o y (IL-10 and TGF-β) cy okines; M2c cells, mainly
IL-10 p oduce s and T eg cell-induce s; and M2d cells (Figu e 3)
[80]. M2a- ype mac ophages ha e been ela ed o AA in se e al
wo ks. They a e IL-13 p oduce s, a cy okine implica ed in mucus
p oduc ion, issue emodelling, and ib osis ha d i es he
cha ac e is ic TH2- ype in lamma ion in AA [81]. These cells a e also
IL-33 p oduce s (see abo e) [82], and hey exp ess a wide a ie y o
chemokines (CCL-17, CCL-18, CCL-22, and CCL-24), which a ac
eosinophils and TH2 cells o he in lamed issue [80, 83].
Juan José Nie o Fon a igo
32
Figu e 3. Schema ic ep esen a ion o sub ypes o monocy e-de i ed
mac ophages du ing in lamma ion. In he p esence o a di e en s imulus,
monocy es can be di e en ia ed in M2a, M2b, M2c, and M2d cells, which ha e
ei he p oin lamma o y o an i-in lamma o y oles in as hma h ough he sec e ion o
di e en cy okines and chemokines.
4.2.1. CD14 and as hma
As commen ed abo e, some pa hogens display se e al ypes o
PAMPs, such as LPS in g am-nega i e bac e ia o lipo eichoic acid in
g am-posi i e. These molecules a e ecognized by PRRs, which a e
localized on inna e immune cells such as monocy es o mac ophages.
[35]. The e a e di e en ypes o PRRs bu he mos s udied a e he
TLRs. The e a e 10 ypes o TLRs (TLR1 o 10) in humans, each o
hem capable o ecognizing di e en pa hogen componen s. Fo
INTRODUCTION
33
example, TLR2 ecognizes mic obial lipopep ides, whe eas TLR3
ecognizes dsRNA om he i al genome [84].
TLR4 o ms pa o he ecep o o bac e ial LPS and is
mainly exp essed by monocy es, mac ophages and dend i ic cells [84,
85]. LBP cap u es LPS monome s and ans e s hem o CD14, a co-
ecep o and a suscep ibili y gene o as hma and alle gy [86, 87]. LPS
is hen deli e ed o he complex MD2/TLR4, which o m a hexame
complex ( wo LPS/TLR4/MD2). Al hough he e a e MyD88-
independen TLR-4 signalling pa hways, he main ou e begins wi h
he sequen ial ec ui men o wo adap o p o eins: TIRAP ( oll-
in e leukin 1 ecep o domain con aining adap o p o ein) and MyD88
(Myeloid di e en ia ion p ima y esponse 88) [85, 88, 89]. Then,
IRAK kinases (IRAK4 and IRAK1 o 2) a e ec ui ed and
phospho yla ed, allowing he binding o he ubiqui in p o ein ligase
TRAF6 and he subsequen ec ui men o TAK1 and IKK complex.
Finally, bo h MAP kinases and ea ly NF-κB pa hways a e ac i a ed
(Figu e 4) [90-92].
The hexame complex (TLR4-MD2-LPS)2 can be in e nalized
ia endosomes, which induces a signal ansduc ion pa hway
dependen on he ec ui men o TRIF/TRAM. This pa hway ends up
wi h he ac i a ion o NF-κB and IRF3 (Figu e 4) [91]. Finally, all
hese signal ansduc ion pa hways p omo e he exp ession o genes
encoding TH1 and TH17- ype cy okines and chemokines, such as
ype-I In e e on, IL-12, IL-1, IL-6, TNF-α, IL-8, o RANTES [90-
92]. This se o cy okines d i es he induc ion o a TH1 and TH17-
ype in lamma ion, in de imen o a TH2- ype pola iza ion. Indeed,
MyD88-de icien mice had a comp omised ac i a ion o an igen-
speci ic TH1 cells, bu no TH2 [93].
Juan José Nie o Fon a igo
34
Figu e 4. TLR4-CD14 signalling pa hway. The o ma ion o he he e odime
complex (TLR4/MD2/LPS)2 igge s wo sequen ial signalling pa hways. The is o
hem s a s wi h he ec ui men o MyD88 and TIRAP and inish wi h he ea ly-
phase ac i a ion o NFκB, i s ansloca ion o he nucleus and he induc ion o
in lamma o y cy okines such as IL-12 o IL18. Secondly, he TLR4/MD2/LPS-
hexame complex can be in e nalized in o endosomes, whe e h ough he
ec ui men o TRAM and TRIF, leads o he ac i a ion o la e-phase NFκB, and
hus, he induc ion o ype I IFN.
In addi ion, memb ane-ancho ed CD14 (mCD14) acili a es
he esponse o TLRs (TLR2, TLR3, TLR4) o o he PAMPs (e.g.,
i al nucleic acids and componen s om G am-posi i e and nega i e
INTRODUCTION
35
bac e ia) o DAMPs (e.g., endogenous molecules like ce amide, u a e
c ys als, o amyloid pep ides) [94]. Hence, changes in he abundance
o de ec s in he unc ion o CD14 (e.g., gene ic ac o s like SNPs)
could be ele an o he de elopmen o alle gen-speci ic TH2 cells
and AA pa hogenesis.
4.2.1.1. Soluble CD14 (sCD14) and as hma
mCD14 is a ached o he cell su ace by means o a
glycophospha idylinosi ol ancho age, bu his p o ein is also
de ec able as a soluble o m (sCD14) in plasma. sCD14 is eleased by
a con olled mechanism (e.g., LPS, CpG, TNF-α) in ol ing ei he
enzyma ic clea age (e.g., elas ase, phospha idylinosi ol phospholipase
C) o di ec sec e ion by exocy osis [94-96]. sCD14 can display
opposi e unc ions. On he one hand, sCD14 can mimic he unc ion o
he mCD14 by ans e ing LPS om LBP o TLR4; he e o e sCD14
po en ia es he induc ion o ype-1 o ype-17 p oin lamma o y
cy okines by endo oxin [96]. In addi ion, sCD14 quali ies CD14-
nega i e cells (e.g., endo helial o epi helial cells) o espond o LPS
[97]. Re e sely, sCD14 can ac as a sca enge ecep o ans e ing
LPS o plasma lipop o eins and p e en ing he ac i a ion o TLR4-
NF-κB pa hway [98].
sCD14 is eleased om monocy es upon s imula ion wi h
TLR ligands (e.g., LPS, CpG, lagellin) o p o-in lamma o y
cy okines (e.g., IL-6 o IL-1β) [38]. Mo eo e , plasma sCD14 also
beha es as a posi i e acu e phase p o ein (APP+). This means ha
sCD14 le els inc ease du ing in ec ion, in lamma ion o upon
exposu e o hepa ocy es o IL-6 [94]. Hence, sCD14 can be
conside ed a non-speci ic monocy e ac i a ion ma ke [38]. sCD14
ea u es wo se um iso o ms, wi h 49 and 55 kDa, and is also clea ed
in plasma by p o eases like ca hepsin D o yield 13 kDa N- e minal
Juan José Nie o Fon a igo
42
ai ways [150, 151] and show a s ong associa ion wi h as hma disease
se e i y [151]. Fu he mo e, i has also been de ec ed in pe iphe al
blood o as hma ics he expansion o a subse o ci cula ing e ec o
memo y IL-6Rα/CD126high CD8+ T cells ha p oduce TH2 cy okines
[152]. Howe e , mos o he s udies in as hma ha e been ocused on
CD4+ T cells, especially TH2 cells and AA.
5.1. The ole o TH2 cells in as hma
TH2 cells ha e been conside ed since a long ime ago majo playe s in
AA. GATA3 and STAT6 a e key ansc ip ions ac o s o he
de elopmen o TH2 cells, and hei e ec o ype-2 cy okines (IL-4,
IL-5, IL-9, IL-13, and IL-25) a e impo an o humo al immune
esponses, de ence agains ex acellula pa asi es (e.g., helmin hs), and
AA pa hogenesis.
IL-4 is cy okine p oduced, amongs o he s, by basophils and
ILC2 cells, essen ial o p oli e a ion, su i al, and di e en ia ion o
TN cells o TH2 lymphocy es [153]. This in e leukin induces B-cell
p oli e a ion, B-cell class swi ching o IgE syn hesis, and plasma cell
di e en ia ion [154]. The e o e, IL-4 plays an impo an ole in he
ea ly alle gic phase. This cy okine ac s ia he IL-4Rα, which can be
in complex wi h he common cy okine ecep o γ-chain (IL-2Rγc;
ype-I IL-4 ecep o ) o wi h he IL-13Rα1 ( ype-II IL-4 ecep o [154,
155]. The las one is also a ecep o o IL-13, which sha es some
unc ions wi h IL-4.
Howe e , IL-13 binds o ano he ecep o wi h highe a ini y,
IL-13Rα2 [155]. Compa ed o IL-4, IL-13 is a la e-ac ing (e ec o
phase) p o-in lamma o y cy okine ha induces mucus p oduc ion,
AHR, and lung ib osis. Indeed, blockade o IL-13 p e en s and
e e ses mucus sec e ion [156]. IL-13 is esponsible o he ansi ion
INTRODUCTION
43
o Cla a cells (also known as b onchiola exoc ine cells) o globe
cells (me aplasia), whose main ole is o sec e e a g oup o
glycop o eins called mucins capable o a ac ing wa e molecules
[157, 158]. IL-13 also leads o goble cell hype plasia (cell
p oli e a ion) and induces he exp ession o se e al genes ela ed o
mucus hype sec e ion: MUC5AC, MAPK13, GABAA-R, o
TMEM16A [159-162]. When he amoun and speed in ol ed in
mucus p oduc ion a e excessi e, his gene a es a hick sec e ion in he
ai ways ha canno be easily emo ed by cilia o cough, d i ing o an
ai low obs uc ion ( oge he wi h AHR) cha ac e is ic o as hma.
Di e en s udies in mice ha e epo ed ha IL-13 could a ec di ec ly
o AECs o /and smoo h muscle cells o p oduce AHR [163, 164].
Mo eo e , he induc ion o MUC5AC o ce ain con ac ile media o s
such as ni ic oxide by IL-13 appea s o con ibu e o he de elopmen
o AHR in as hma ics [165, 166]. Ano he unc ion o IL-13 is he
s imula ion o myo ib oblas s p oli e a ion and hei ec ui men o
ai way issues, leading sub-epi helial ib osis [167].
IL-5, o i s pa , p omo es he ma u a ion, di e en ia ion, and
su i al o eosinophils. Mo eo e , his cy okine ac as a chemokine
o his leukocy e subse and enhances i s e ec o unc ion [168].
Indeed, he o e exp ession o IL-5 in mice induces he expansion o
eosinophils bo h in bone ma ow and pe iphe al blood [168, 169],
whe eas he absence o IL-5Rα abolishes he eosinophilia and AHR
compa ed o wild- ype (WT) mice [168, 170]. In addi ion, some
s udies ha e no iced educed eosinophils in lux o he lung epi helium
and less AHR a e he use o an i-IL-5 an ibodies [168]. Rec ui men
o eosinophils and TH2 cells o lung epi helium d i es he la e-phase
alle gic esponse in as hma due o he elease o e ec o media o s
such as he majo basic p o ein, eosinophil-de i ed neu o oxin o
pe oxidase [171, 172].
Juan José Nie o Fon a igo
44
5.2. Pa icipa ion o o he e ec o TH subse s in as hma: TH1
and TH17 lymphocy es
TH2 cells ha e been he mos s udied CD4+ T subse in as hma o e
ime, pa icula ly in he alle gic pheno ype. Howe e , o he TH
subpopula ions display impo an oles in he pa hology o his
complex disease, such as TH1 o TH17 cells. As we ha e s a ed abo e
(see sec ion 3.2), bo h TH2 and TH1 cells we e belie ed o ha e
opposi e oles p omo ing o inhibi ing as hma, espec i ely.
TH1 cells di e en ia e om TN lymphocy es in esponse o IL-
12 (an APC-de i ed cy okine). This subse is cha ac e ized by he
exp ession o he ansc ip ion ac o s T-be and STAT4, and he
sec e ion o IFN-γ and o he e ec o cy okines such as IL-2, TNF, o
LT-α [146]. TH1 cells a e esponsible o he p o ec ion agains
i uses and o he in acellula pa hogens [146]. The i s s udies made
on he ole o TH1 lymphocy es in as hma desc ibed his subse as
p o ec i e. Fo example, child en li ing on a ms and exposed o high
endo oxin (LPS) le els we e ound o be less p edisposed o as hma o
alle gies. Indeed, LPS exposu e by means o CD14-TLR4 d i es he
di e en ia ion owa ds TH1- ins ead o TH2- ype esponses, as s a ed
abo e. Fu he mo e, IL-12 p e en s he p oduc ion o IL-4 and
inhibi s he de elopmen o TH2 cells, downmodula ing AA
in lamma ion [37]. Howe e , a po en ial p oin lamma o y ole o
TH1 cells in as hma is now eme ging. Indeed, some i uses like
human hino i uses induce he sec e ion o IL-33 and TSLP, which
also igge TH1 esponses and a e esponsible o acu e as hma
a acks [173]. Addi ionally, adul s wi h ea men - e ac o y se e e
as hma ha e inc eased le els o TH1 cells in hei lungs, highligh ing
he possible ole o his subse on he se e i y o his diso de [174].
INTRODUCTION
45
TH17 keep mucosal ba ie s i and acili a e pa hogen
clea ance a mucosal su aces [175]. In he p esence o IL-1β, IL-6,
and IL-23, TN cells di e en ia e in o TH17 lymphocy es; TGF-β also
appea s o play a ole in TH17 di e en ia ion [146]. Th ough i s
in e ac ion wi h IL-6R (gp130 + IL-6Rα/CD126), IL-6 induces he up-
egula ion o e inoic acid ecep o - ela ed o phan ecep o gamma
(ROR-γ ). ROR-γ and ROR-α a e wo ansc ip ion ac o s ha d i e
he de elopmen o TH17 cells [176-178]. Mo eo e , IL-6 can also
in e ac wi h soluble IL-6Rα, ac i a ing CD126-gp130+ cells [179,
180]. Indeed, his p ocess, also known as ans-signalling, is impo an
o he main enance o se e al in lamma o y diseases such as as hma
[181-184]. Apa om ROR-γ , TH17 lymphocy es can be
dis inguished by he exp ession o ano he ansc ip ion ac o
(STAT3) and he su ace ecep o s CCR6 and IL23R. TH17 cells
ca y ou many o hei ac i i ies by eleasing se e al e ec o
cy okines om he IL-17- (IL-17A, IL-17F), he IL-10- (IL-22), and
he IL-12- amily (IL-23) [185, 186].
Some IL-17 polymo phisms ha e been associa ed wi h highe
as hma isk, like o ins ance IL-17A s4711998(A/G), IL-17F
s1889570(C/T), and IL-17A s3819024(A/G) [187]. IL-17 has been
ela ed o ai way diseases due o i is di ec ly connec ed wi h he
p oduc ion o p o-in lamma o y (e.g., IL-8) and p o- ib o ic cy okines
(e.g., IL-6, IL-11), as well as ma ix me allop o eases om
in lamma o y cells (e.g., eosinophils), AECs, and ib oblas s [188-
190]. These p o-in lamma o y cy okines p omo e lung in lamma ion
by means o he ec ui men o mac ophages and neu ophils [191,
192]. Addi ionally, Chang e al. desc ibed ha IL17-A, IL-17F, and
IL-22 can induce ai way smoo h muscle cells p oli e a ion and
he e o e p omo e ai way emodelling [193].
Juan José Nie o Fon a igo
46
Se e al wo ks ha e been sugges ed a possible ole o TH17
cells in as hma pa hogenesis [194]. Fo example, he absence o ei he
IL-17A o IL-17F is enough o supp ess AA induced by HDM in mice
[195]. Bo h IL-17 and neu ophils le els a e augmen ed in as hma ic
subjec s wi h highe body mass index [196], highligh ing he ole o
IL-17 in wo se con ol in obese adul s wi h as hma [69]. On he o he
hand, Zao e al. epo ed an inc ease in bo h he pe cen age o TH17
cells and he p oduc ion o TH17-de i ed cy okines (IL-17 and IL-25)
in pe iphe al blood om pa ien s wi h AA and in ac i a ed pe iphe al
blood mononuclea cells (PBMCs) om alle gic as hma ics compa ed
o heal hy con ols. Mo eo e , hey also e idenced highe le els o
bo h IL-17 and IL-22 cy okines as disease se e i y inc eases (i.e.
Con ols < Mild as hma < Mode a e as hma < Se e e as hma),
highligh ing he possible implica ion o his subse in as hma se e i y
[197, 198]. Fu he mo e, TH17 cells ha e also been implica ed in
s e oid- esis an as hma [199]. Glucoco icoids ha e been able o
induce BIM (Bcl-2 in e ac ing media o ), a p o-apop o ic p o ein
which an agonizes Bcl-2. The p edominance o BIM o e Bcl-2
induces he ac i a ion o BAX, and he subsequen ac i a ion o
caspases, leading o apop osis [200, 201]. Howe e , con a y o TH1
lymphocy es, TH17 cells exp ess high le els o Bcl-2, which makes
his subse insensi i e o glucoco icoids. Indeed, sho hai pin RNA
(shRNA) media ed silencing o Bcl-2 gene in TH17 cells makes hem
sensi i e o glucoco icoid-induced apop osis [202]. Fu he mo e,
TH17 cells we e augmen ed in he lungs o a mu ine model o se e e
as hma [202].
INTRODUCTION
47
5.3. O he TH subse s implica ed in as hma pa hogenesis: TH9,
TH22 and T H lymphocy es
A leas 3 mo e TH cell subse s ha e implica ions in as hma
pa hogenesis: TH9, TH22 and ollicula helpe T (T H) cells. TH9
lymphocy es di e en ia e om TN cells when IL-4 and TGF-β a e
p esen in he media. Bo h STAT6 and PU.1 a e ansc ip ion ac o s
impo an o he de elopmen o TH9 cells; hey also exp ess
GATA3, bu o a lesse ex en han TH2 cells. Apa om IL-10 and
IL-21, TH9 cells a e a majo sou ce o IL-9, a cy okine which gi es
hem hei name. IL-9 has se e al ele an e ec s in AA pa hogenesis
[203]. Fo example, his in e leukin induces he p oduc ion o
his amine, p o eases and cy okines om mas cells, p omo es TH2-
di e en ia ion and eosinophils de elopmen , induces IgE class-
swi ching in B cells, and a ou s b onchocons ic ion, globe cell
me aplasia, and mucus p oduc ion [203, 204]. In addi ion o TH17
cells, TH9 lymphocy es a e esis an o glucoco icoids and can be
media o s o s e oid- esis an as hma [205].
In he p esence o IL-6 and TNF-α, TN cells can di e en ia e
in o TH22, which a e cha ac e ized by he sec e ion o IL-22 and he
exp ession o CCR6, CCR4, and CCR10 [206]. The unc ion o his
cy okine is con o e sial, as i has been implica ed in bo h an i-
in lamma o y and p o-in lamma o y p ocesses [207]. Indeed, some
wo ks ha e desc ibed highe le els o IL-22 in se um om as hma ics
compa ed o heal hy indi iduals [207], o in se e e as hma compa ed
o mode a e disease [207-209]. In con as , o he in i o o in i o
s udies sugges ha IL-22 ha e a p o ec i e ole in he la e-phase o
alle gic induced ai way in lamma ion by dec easing eosinophils
le els, IL-5, IL-13, and CXCL10 [207].
T H cells a e ano he subse o TH cells impo an o he
main enance o ge minal cen es and he gene a ion o long-li ed
Juan José Nie o Fon a igo
48
plasma cells and memo y B cell esponses [210]. These cells display a
high dependency o IL-2 o i s gene a ion and a e iden i ied by he
high exp ession o ICOS, PD-1, and CXCR5, whose mRNA has been
ound augmen ed in as hma ics compa ed o heal hy subjec s [211].
The ligand o CXCR5, CXCL13, is p oduced by ollicula s omal
cells and helps T H cell o ind he way o ge minal cen es (B-cell
ollicles), whe e soma ic hype mu a ion o BCR and T-B cells
in e ac ion leading o class swi ching and B-cell di e en ia ion akes
place [212]. B-cell lymphoma 6 p o ein (Bcl-6) is a ansc ip ion
ac o exp essed by T H cells ha supp esses he exp ession o
ansc ip ion ac o s in ol ed in he di e en ia ion o o he TH
subse s (e.g., TH1, TH2, TH17) [213]. Mo eo e , T H cells elease
IL-21, which is also inc eased in as hma ic lungs [211]. The e o e,
mo e s udies a e needed o cla i y he unc ion o he di e en TH
lymphocy e subse s, hei ela ionship and hei implica ion in as hma
pa hogenesis.
5.4. The ole o egula o y T cells (T eg) in as hma
The abo e-men ioned e ec o unc ions o human TH lymphocy es
a e coun e ac ed by T eg cells [214, 215]. The e a e wo majo ypes
o T eg cells, wi h di e en biological unc ions: na u al T eg cells
(nT egs) and induced T eg cells (iT egs). The i s sub ype is
esponsible o ole ance o sel -an igens. These
FoxP3+CD25+CD304/Neu opilin-1+ nT eg cells a e gene a ed in he
hymus, whe e hey manage o escape he nega i e selec ion. Lineage-
speci ic ansc ip ion ac o s like FoxP3, Helios, o RUNX a e
impo an egula o s o he de elopmen o nT egs and hei
immunosupp essi e unc ions [215, 216]. In con as , iT egs a e
gene a ed in he pe iphe y om o he TH subse s unde ole ogenic
condi ions: i.e., s ong signalling ia TCR, low co-s imula ion, and
INTRODUCTION
49
high le els o TGF-β and e inoic acid [215]. iT egs appea o con ol
immune esponses o “non-sel ” an igens (e.g., alle gens, commensal
mic obio a, die ), and a e classically di ided in o h ee majo
sub ypes: FoxP3+ iT egs, IL-10+ T 1 cells, and TGF-β+ TH3 cells
[217]. Loss o iT eg cells leads o alle gic- ype-2 in lamma ion a
mucosal in e aces ( o example, in he lung) [218].
Figu e 5. Schema ic ep esen a ion o he mul i ude o egula o y T cells (T eg)
unc ions implica ed in as hma pa hogenesis. T eg cells con ol he e ec o
unc ions o se e al inna e sys em media o s such as mas cells, basophils, and
eosinophils, as well as TH subse s om he adap i e immune sys em. They also
con ol immunoglobulin (Ig) class swi ching h ough he induc ion o IgG4
p oduc ion in de imen o IgE p oduc ion. S imula o y unc ions a e ep esen ed by
a solid a ow, whe eas inhibi o y signals a e ep esen ed by blun a ows.
T eg cells can be dis inguished om Te cells acco ding o he
le els o CD25 (IL-2Rα) and CD127 (IL-7Rα). Thus, con a y o Te
Juan José Nie o Fon a igo
50
cells (CD25lowCD127high), mos o T eg lymphocy es display high
le els o CD25 and low le els o CD127 (CD25highCD127low) [219].
CD25 is conside ed an ac i a ion ma ke eleased by TH lymphocy es
du ing in lamma ion in he o m o soluble CD25 (sCD25) [220-222].
Indeed, sCD25 is augmen ed in BALF o as hma ics compa ed o
heal hy subjec s [221, 222]. Mo eo e , sCD25 is inc eased in se um
du ing as hma exace ba ion [223] and has been co ela ed wi h AA
se e i y [224]. On he o he hand, CD127 is a subuni o bo h IL-7 and
TSLP ecep o s [225]. Toge he wi h IL-15, IL-7 is cy okine
impo an o homeos a ic su i al o TH cells, and he e o e CD127
is also a ma ke o naï e and ce ain memo y subpopula ions (s em-
cell memo y T cells/TSCM and TCM). O he ma ke s exp essed on T eg
cells, like CTLA-4 o GITR, a e also up- egula ed upon ac i a ion
[215]. The e o e, his means ha he T eg pheno ype displays
cha ac e is ics o ac i a ed and TEM cells (see below).
T eg cells play an impo an ole as nega i e egula o s o
in lamma o y diso de s and issue epai [215]. This unc ion equi es
signalling ia TCR and IL-2R, and is exe cised by means o mul iple
cell-con ac dependen o independen mechanisms in ol ing soluble
(e.g., adenosine, g anzyme, TGF-β, IL-10, and IL-35) o memb ane-
associa ed (e.g., CTLA-4, CD39, CD73, CD25, pe o in) molecules
[215]. T eg lymphocy es dep i e he en i onmen o IL-2, educe he
ac i a ion s a us o dend i ic and myeloid cells, and es ain Te
unc ions by di ec ly inhibi (o killing) TH1, TH2, and TH17 cells.
Mo eo e , his T eg subse swi ches immunoglobulin iso ypes om
IgE o IgG4 in B cells and p e en s he ac i a ion and mig a ion
h ough he ai way epi helium o mas cells, eosinophils, and
basophils (Figu e 5) [1, 217]. The e o e, a unc ional de ec in T eg
cells o a change in he e ec o / egula o y equilib ium may be
impo an in as hma pa hogenesis [214]. Indeed, he pe cen age o IL-
4 p oducing cells was augmen ed in alle gic subjec s, whe eas IL-10-
INTRODUCTION
51
p oducing T eg cells we e dec eased in alle gic indi iduals (in
con as , hey we e he dominan alle gen-speci ic subse in heal hy
subjec s) [226]. In he same line, child en wi h as hma display low
le els o T eg cells in BALF compa ed o con ol subjec s.
Fu he mo e, con a y o CD4+CD25high T cells isola ed om con ol
subjec s, T eg cells om as hma ic subjec s ail o supp ess
p oli e a ion and TH2-cy okine p oduc ion by esponde T cells [227].
5.5. Naï e-memo y di e en ia ion o T cells and as hma
As s a ed abo e, he adap i e immune sys em is cha ac e ized by he
gene a ion o long-li ed memo y cells, e en hough i has been ound
ecen ly ha ILC2 cells, NK cells, o monocy es display an „inna e
memo y‟ o “ ained immuni y”; i.e., a long- e m imp o emen o he
unc ion o inna e immune cells a e in ec ion o accina ion due o
epigene ic causes [228].
Following Ag encoun e , TN cells p oli e a e and di e en ia e
in o cells wi h di e en pheno ypes and unc ions ollowing a linea
model (Figu e 6). These six subse s o T cells (TN, s em cell memo y
T cells/TSCM, TCM, ansi ional memo y T cells, TEM, and TEMRA) can
be cha ac e ized acco ding o combina ions o 4 ma ke s: CD45RA o
CD45RO, CD62L o CCR7, CD27 o CD28, and CD95 (Figu e 6)
[229]. TN cells display he highes exp ession o CD45RA and no
exp ession o CD45RO, wo splicing a ian s o he CD45 gene.
CD45RO de ine memo y T cells, whe eas TEMRA cells e-exp ess he
high molecula weigh p o ein CD45RA. CD62L (L-selec in) and
CCR7 allow T cells o mig a e o seconda y lymphoid issues. Thus,
hese wo molecules a e highly exp essed in TN, TSCM and TCM cells.
TSCM cells e ain s em cell p ope ies and hey sha e some genes wi h
TN cells, bu hey display high exp ession o CD95 (as he o he
Juan José Nie o Fon a igo
58
adminis a ion [301]. Mo eo e , a model o OVA-induced
expe imen al as hma in CD26-/- mice showed inc eased le els o IL-4,
IL-5, and IL-13 in BALF, highe ci cula ing le els o eosinophils,
enhanced le els o eo axin and RANTES, as well as augmen ed
exp ession o TH2 chemokine ecep o s (CCR3 and CCR5), compa ed
o WT mice, suppo ing ha CD26/DPP4 could play a “b aking” ole
in expe imen al as hma [281].
F om s udies in he human sys em, Lun e al. ha e shown
highe le els o CD26 on o al lymphocy es, CD4+ T cells, and iNKT
lymphocy es, as well as highe concen a ions o sCD26 in adul
alle gic as hma ics [246]. Mo eo e , he TH2-cy okine IL-13 has been
iden i ied as an impo an induce o CD26/DPP4 exp ession in
b onchial epi helial cells [302]. Howe e , o he wo ks ha e shown no
changes o sCD26 in as hma [303]. These esul s could be explained
because o many po en ial con ounde s: low sample size, male/ emale
p opo ion, age, o lymphocy e coun . Fo ins ance, is well-known ha
bo h CD26 exp ession on lymphocy es and ci cula ing sCD26 le els
a e highe in men han women [303, 304]. The e o e, mo e s udies a e
needed o assess he ela ionship be ween CD26 and as hma, as well
as o ex end his a ea o esea ch o di e en as hma pheno ypes (o
endo ypes) and se e i y deg ees.
**Fo mo e in o ma ion see appendix I.
6. ASTHMA PHENOTYPES AND ENDOTYPES
As hma is a he e ogeneous disease ha g oups di e en clinical
pheno ypes o “obse able p ope ies o an o ganism ha a e
p oduced by he in e ac ion o he geno ype and he en i onmen ”,
INTRODUCTION
59
(Me iam-Webs e ‟s Collegia e Dic iona y (© 2019 Me iam-
Webs e , Inco po a ed 2019)). Mo eo e , below his i s ou e and
isible laye o complexi y lies a second one, named as hma
endo ypes, which ep esen he pa hophysiologic and molecula
mechanisms ha igge and make his disease wo se [2]. Howe e ,
his connec ion be ween speci ic as hma pheno ypes and he
unde lying pa hological ea u es is elusi e [2]. The e o e, mos o he
esea ch is ocused on he sea ch o a a ie y o bioma ke s ha
clea ly de ine he di e en as hma pheno ypes and/o endo ypes.
Those wo ks a e in ended o de elop new biological d ugs, necessa y
o pe sonalized/p ecision medicine in as hma and espi a o y diseases
[45, 305, 306].
The mos ex ended classi ica ion o as hma amongs clinicians
elies on he p esence o absence o alle gic igge s: AA and NAA
(Figu e 7). AA is he mos common pheno ype (~ 60%) [307]. I is
cha ac e ized by he p esence o alle gic sensi iza ion, which is
de ined by he p esence o alle gen-speci ic IgE molecules and a
posi i e skin p ick es agains common en i onmen al alle gens.
Compa ed o he adul onse o NAA, AA o en s a s in childhood.
Mo eo e , he p e alence o AA is highe in males, whe eas non-
alle gic as hma ics a e mos ly women. Addi ionally, AA pa ien s
equen ly espond well o inhaled co icos e oid (ICS) ea men
compa ed o NAA pa ien s, which a e usually mo e he e ogeneous
ega ding he unde lying in lamma o y pa e n (neu ophilic o
paucyg anulocy ic in lamma ion) [4, 308, 309]. Howe e , his
classi ica ion o as hma acco ding o he kind o igge is an
o e simpli ica ion, as o he as hma pheno ypes apa om AA and
NAA ha e been de ined by he As hma Pheno ypes Task Fo ce:
aspi in-exace ba ed espi a o y disease, in ec ion-induced as hma, and
exe cise-induced as hma [307].
Juan José Nie o Fon a igo
60
Figu e 7. Cells and molecules implica ed in he di e en as hma pheno ypes
and endo ypes. Two majo pheno ypes ha e been desc ibed in as hma, he ea ly
onse , eosinophilic and a opic/alle gic as hma, and he la e onse , non-alle gic
as hma. Alle gic as hma mainly i s in o he TH2 high as hma, and i is cha ac e ized
by mas cell and eosinophilic esponse and a TH2 d i en in lamma ion. On he o he
hand, non-alle gic as hma is mo e he e ogeneous, and i could be neu ophilic o
obesi y- ela ed. The non-alle gic pheno ype is cha ac e ized by a TH2 low endo ype,
whe e ype-1 and ype-17 cy okines and he cells hey come om (ILC1/3, γδ-T
cells, o TH1/TH17 cells), as well as neu ophils, ha e an impo an ole.
Unde nea h he pheno ypic classi ica ion o AA and NAA, he
mos common endo ype classi ica ion is based on he p edominan
TH- ype in lamma ion: i.e., TH2-high s TH2-low as hma [310, 311].
The ea ly-onse TH2-high as hma is he mos common endo ype and
almos 100% o pa ien s wi h his endo ype all in o he AA ca ego y.
The TH2-high endo ype is cha ac e ized by a TH2- and mas cells-
INTRODUCTION
61
d i en in lamma ion, sub-epi helial basemen memb ane hickening,
and a cen al ole o IgE and eosinophils (Figu e 7) [2, 312]. This
ype o as hma is also associa ed wi h a opy (i.e., a gene ic
p edisposi ion o su e alle gic diseases). Indeed, diseases such as
alle gic hini is, eczema, o ood and d ug alle gy a e o en
como bidi ies in AA pa ien s [2, 4]. Se e al bioma ke s o TH2-high
as hma ha e been desc ibed: ele a ed spu um eosinophils le els, high
ac ional exhaled ni ic oxide (FeNO), which is a su oga e ma ke o
eosinophilic ai way in lamma ion, and aised le els o pe ios in and
o al IgE in se um [311, 313, 314]. Howe e , he main ea u e o his
endo ype is he good esponse o non-speci ic an i-in lamma o y d ugs
(ICS), esul ing in he down-modula ion o TH2 cy okines (IL-4, IL-5,
and IL-13) and he unde lying in lamma ion [2].
Di e en biological he apies ha e eme ged in he o m o
humanised an ibodies ha a ge se e al AA media o s such as IgE o
TH2-cy okines [311]. The i s humanised monoclonal an ibody
speci ically designed o ea AA was omalizumab (Xolai ®). This
an ibody makes a complex wi h ee IgE and inhibi s i s binding o he
high‐a ini y FcϵR1 ecep o on mas cells and basophils [314]. As a
esul o his blockade, he ypical mas cell ac i a ion/deg anula ion
e en ha occu s in AA is in e up ed, as well as he eosinophilic
in lamma ion [315]. The e o e, se e e pe sis en alle gic as hma ic
pa ien s ea ed wi h omalizumab unde go a educ ion o as hma
exace ba ions [316] and an imp o emen in hei quali y o li e [317].
Beyond omalizumab he apy, he e a e o he humanised monoclonal
an ibodies ha a ge se e al impo an molecules in as hma: a) TH2-
cy okines such as IL-5 (mepolizumab and eslizumab) o IL-13
(leb ikizumab and alokinumab); b) TH2-cy okine ecep o s such as
IL-4Rα (dupilumab and AMG-317) o IL-5Rα (ben alizumab); and c)
epi helial cell-de i ed cy okines such as TSLP (AMG-157) [311, 318].
These al e na i e biologicals ha e yielded p omising esul s in e ms
Juan José Nie o Fon a igo
62
o imp o emen o lung unc ion and educ ion o exace ba ions in
TH2-high AA [318].
TH2-low as hma, o i s pa , is mo e he e ogeneous and
poo ly de ined; indeed, he e a e no speci ic bioma ke s o his
as hma endo ype. O e all, pa ien s wi h TH2-low as hma a e badly-
esponde s o ICS and display a mo e se e e disease. In addi ion,
hese ype o pa ien s a e cha ac e ized by he absence o a TH2-
signa u e, lowe le els o IgE, delayed disease onse (adul hood), and
non-alle gic/a opic disease usually associa ed wi h neu ophilic
in lamma ion (Figu e 7) [310, 319-321]. Obesi y- ela ed as hma,
which is p edominan in women, is also included wi hin TH2-low
as hma [322]. TH1 and TH17 cells and hei de i ed cy okines seem
o be key playe s in he pa hogenesis o his endo ype (Figu e 7). As
commen ed abo e, IL-17 is he majo media o o neu ophils
ec ui men and ac i a ion by inducing he p oduc ion o IL-8 and
o he chemokines (e.g., CXCL1 o CXCL5) by AECs (Figu e 7). IL-
17 has been associa ed wi h AHR, emodelling and as hma se e i y
[188, 323, 324]. Al hough IL-17 is mainly p oduced by TH17 cells,
ce ain subpopula ions o inna e lymphocy es also sec e e his
cy okine. This is he case o ILC3s, which play a cen al ole in
obesi y- ela ed as hma [69], o γδ-T lymphocy es, which a e he majo
p oduce s o ea ly IL-17 (Figu e 7) [134]. On he o he hand, IFN-γ,
he inge p in cy okine o TH1 cells, is also implica ed in he
chemo axis o neu ophils h ough he up- egula ion o CCR1 and
CCR3 on his leukocy e subse (Figu e 7) [325].
Gi en all he abo e commen ed, and in he e a o biological
he apies and pe sonalised/p ecision medicine, i is mo e necessa y
han e e he sea ch o new biological ma ke s, wi h applica ion in
pheno ype/endo ype e inemen and disease
diagnosis/p ognosis/managemen . These new bioma ke s will enla ge
he lis o cu en ly a ailable op ions, which include bo h cells (e.g.,
INTRODUCTION
63
eosinophils, neu ophils) and molecules (e.g., o al and speci ic IgE,
pe ios in, FeNO, cy okines) in di e en kind o samples (e.g.,
pe iphe al blood, se um, plasma, IS) [307].
7. APPLICATION OF BIOFLUID PROTEOMICS FOR
ASTHMA-BIOMARKER DISCOVERY
The eme gence o P o eomics, which is he la ge-scale s udy o
p o eins, has mean a u ning poin in he wo ld o bioma ke s
disco e y. P o eomic s udies o en use samples om issues, in i o
cell cul u es, o bio luids such as u ine, BALF, exhaled b ea h
condensa e (EBC), o ce eb ospinal luid (CSF) [326, 327]. Howe e ,
he mos commonly used samples in clinical s udies a e hose de i ed
om enous pe iphe al blood: i.e., se um and plasma. This easy o
acqui e samples a e challenging in P o eomics because hey a e highly
dynamic and complex (>10,000 p o eins;
h p://www.plasmap o eomeda abase.o g). This is because bo h se um
and plasma samples a e an open window o he p o eomes o all he
issues in he body. This makes hese bio luids ideal o bioma ke
disco e y s udies, especially amongs low molecula weigh (LMW; <
30-40 kDa) o low abundance p o ein (LAP) sub-p o eomes [328,
329].
Se um/plasma includes p o eins ha ul il hei unc ion in he
ex acellula compa men , such as cy okines, ho mones, o g ow h
ac o s. Howe e , hey also con ain “bys ande ” molecules such as
issue leakage p oduc s; i.e., in acellula p o eins eleased by issues
o mul iple easons (some o hem summa ized in Figu e 8) [328,
330, 331]. LMW cellula p o eins (especially he cy osolic ones) each
easily he blood ci cula ion du ing physiological cell u no e o
pa hological plasma memb ane pe meabiliza ion due o hei small
Juan José Nie o Fon a igo
64
p o ein size [332]. Despi e his ease, he se um/plasma LMW
p o eome is made o small p o eins, p o ein agmen s, o pep ides ha
display a low abundance and ele a ed complexi y. The e o e, LAP
and LMW sub-p o eomes om se um/plasma a e di icul o
in es iga e bu ep esen a pa ly o e lapping ich sou ce o
in o ma ion and no el po en ial bioma ke s ela ed o cance [333],
ca dio ascula condi ions [334], in ec ious illnesses [335], o ai way
diseases [336, 337].
Figu e 8. Cell/ issue sou ces and o igin o low abundance se um/plasma
p o eins. The se um/plasma abundance o disease bioma ke s is dependen on he
equilib ium be ween sec e ions by cells and emo al (e.g., kidney o p o eolysis).
Se e al easons a e he esponsible o he inc emen o de imen o issue p o eins
in plasma: 1) cell damage (nec osis, cell memb ane pe meabiliza ion), 2) induc ion
o p o ein syn hesis, 3) inc emen o cellula p oli e a ion/ cell eplacemen (e.g.,
cance ), 4) Reduc ion o p o ein exc e ion by exoc ine cells (e.g., panc ea ic o
p os a e cells) due o obs uc ion o he duc s, o 5) less emo al o p o eins om
ci cula ion due o o example enal inju y.
INTRODUCTION
65
Figu e 9. The dynamic ange o p o eins concen a ions in human plasma.
Human plasma p o eome is composed o housands o p o eins. 22 o hem ep esen
he 99.9% and a e conside ed high abundan p o eins. Be ween hem, only 10
ep esen mo e han 90%, and only albumin cons i u es mo e han 50% o o al
p o ein concen a ion. Low abundance p o eins, also called LMW p o eome is
compound by only he 0.1% o p o eins in he human plasma.
As abo e commen ed, se e al challenges mus be conside ed
when analysing LAP/LMW sub-p o eomes om se um/plasma
samples. The mos impo an one is he ex emely high dynamic ange
o p o ein concen a ions in hose bio luids, o almos 1010 magni ude,
and he ac ha <0.1% o he o al numbe o species ep esen s
almos 99% o he bulk mass o plasma/se um p o eins; ha is, a se o
22 p o ein ha includes albumin, immunoglobulins, ans e in, α-2
mac oglobulin o α1-an i ypsin (Figu e 9). Only albumin i sel
cons i u es ~50% o p o ein con en in se um, wi h concen a ions
anging be ween 35 and 50 mg/mL. In con as , he low abundance
p o eome eaches a sca ce 0.1% o he bulk mass o p o eins, co e s a
concen a ion ange om ng/mL o g/mL, and is made up o
housands o LMW p o eins wi h a speci ic unc ion in plasma (e.g.,
cy okines, g ow h ac o s, and chemokines) o de i ed om issues o
Juan José Nie o Fon a igo
66
cells (Figu e 9) [328, 330, 331, 338, 339]. The e o e, educ ion o
sample complexi y is necessa y o ensu e access o his po en ial
sou ce o disease bioma ke s, coined by ce ain esea che s as he
“deep-p o eome” [339].
7.1. P e- ac ioning me hods o plasma/se um p o eins
In he las yea s, di e en expe imen al app oaches consis ing o
ei he deple ion o high-abundance p o eins o en ichmen o
LAP/LMW species in se um/plasma samples ha e been de eloped in
o de o educe he se um/plasma dynamic ange o p o ein
abundances.
Ul a il a ion is included in he i s g oup o echniques [340].
This echnique consis s o cen i ugal il e de ices whose memb anes
display a speci ic cu -o (e.g., 40 kDa cu -o ), which means ha only
low molecula weigh p o eins will be able o pass h ough. Howe e ,
i has been shown ha high molecula weigh p o eins like albumin
s ill appea in he elu ed ac ion. O he app oaches in ol e he
deple ion o high abundan p o ein by a ini y ch oma og aphy [341].
This is he case o human se um albumin a ini y columns (e.g.
Cibac on Blue-based a ini y ch oma og aphy) [342],
immunoglobulins deple ion (e.g., p o ein A/G) [343], P o eop ep
columns (Me ck Millipo e) o Albumin/IgG deple ion [344], he
Pie ce™ Top 12 Abundan P o ein Deple ion Spin Columns (The mo
Scien i ic™), o he mul iple a ini y emo al sys em (MARS)
(Agilen Technologies). Finally, high-abundance p o eins can be
deple ed om se um by using o ganic sol en p ecipi a ion (e.g.,
ace oni ile p ecipi a ion) [345]. Howe e , i has been desc ibed ha
some LMW p o eins a e ound in associa ion wi h ca ie p o eins
such as albumin [346]. The e o e, deple ion o p ecipi a ion o hese
INTRODUCTION
67
highly-abundan “shippe ” p o eins can esul in he loss o in e es ing
LMW bioma ke s. Mo e impo an ly, emo al o he uppe -laye o
high-abundance p o eins om se um/plasma samples only allows
esea che s o each he medium abundance sub-p o eome.
Figu e 10. The use o combina o ial pep ide ligand lib a ies (CPLL) as an
en ichmen me hod o low abundance p o eins in complex samples. The
P o eoMine TM p o ein en ichmen echnology (BioRad) is based on he use o
combina o ial ligand lib a y o hexapep ides (CPLL) a ached o beads. S ep 1. The
CPLL is added o a complex sample (e.g., se um o plasma) whe e almos all low
abundan p o eins bind o hei speci ic ligand hexapep ides, bu high abundan
p o eins sa u a e hei ligands. S ep 2. The washing o he sample allows he
elimina ion o high abundan p o eins, which had sa u a ed hei ligands. S ep 3. The
p o eins a ached o he beads a e elu ed. The e o e, he elu ed ac ion is en iched in
low abundan p o eins, whe eas high abundan p o eins a e elimina ed in he
low h ough ac ion.
Apa om deple ion me hods, se e al app oaches ha en ich
low-abundance/LMW p o eins ha e been de eloped. This is he case
o a ini y cap u e o low-abundance p o eins by means o magne ic
beads [347], nanopa icles [348], an ibody a ays [349], o he use o
combina o ial pep ide ligand lib a ies (CPLLs) (Figu e 10) [350]. The
las app oach, ma ke ed unde he name o P o eoMine TM p o ein
Juan José Nie o Fon a igo
74
Figu e 12. iTRAQ labelling. Each label o iTRAQ 8-plex is composed o 3
egions. T yp ic pep ides co alen ly bind o he amine-speci ic pep ide
eac i e g oup (NHS). The egion nex o NHS is he balance g oup (184-
192 Da) aimed o conse e a cons an ag mass o 305.1 in MS1. Finally, he
epo e g oup e ains he cha ge a e pep ide agmen a ion, p esen s
a iable mass (113.1-119.1, 121.1 Da), and display an in ense signal in MS2.
Compa ed o label- ee s a egies, which can p ocess an
unlimi ed numbe o samples in he same expe imen , iTRAQ only
allows us o label, analyse, and compa e up o 8 samples (Figu e 13).
Howe e , label- ee s a egies p esen se e al challenges [368, 373]:
1) all samples a e p epa ed and analysed sepa a ely; 2) i is necessa y
o use in e nal s anda ds in o de o a oid a ia ions in he peak
in ensi ies be ween di e en expe imen s; 3) quan i ica ion e o s due
o close mass signals can be p esen , so ha echnical eplica es a e
manda o y (Figu e 13). The e o e, label- ee quan i ica ion esul s in a
huge amoun o da a, which a e di icul and labo ious o p ocess
(Figu e 13). This issue has been sol ed wi h iTRAQ echnology,
which is an easy- o-use me hod ha allows he analysis and
INTRODUCTION
75
compa ison o all samples (up o 8) in he same MS/MS ead ou , as
all labelled samples a e mixed in one common sample (CS) be o e
LC-MS/MS analysis (Figu e 13).
Figu e 13. Compa ison o label- ee and iTRAQ quan i ica ion. In label-
ee quan i ica ion, all samples a e p epa ed and analysed sepa a ely, which
esul s in a huge amoun o da a di icul o in e p e . S able-iso ope labelling
by means o iTRAQ allows he compa ison o p o eins om each sample in
he same LC-MS/MS ead ou . CS, common sample; HAP, high abundan
p o ein; LAP, low abundan p o ein; LC-MS/MS, liquid ch oma og aphy
coupled o andem MS; S, sample; F, ac ion.
Juan José Nie o Fon a igo
76
7.4. P o eomic s udies in as hma
As Table 1 shows, se e al p o eomic s udies ha e been ca ied ou in
he las yea s in o de o sea ch o speci ic bioma ke s o as hma,
as hma exace ba ion, and ea men esponse. The i s analysis was
ca ied ou by Lindahl e al. in 1999, who used 2-D elec opho esis o
show he al e a ion o se e al p o eins (Lipocalin-1, ans hy e in,
cys a in S, and IgBF) in bo h BALF and NLF (nasal la age luid)
om pa ien s wi h as hma compa ed o heal hy subjec s [374].
Following his ini ial wo k, a ew mo e s udies in BALF [375, 376]
and NLF [377, 378] came o ligh . Fo example, Wu e al. in 2005
used a ini y deple ion o op 6 abundan p o eins, 1-DE-LC-MS/MS,
and a label- ee app oach o iden i y 160 BALF p o eins wi h changes
in as hma ics a e alle gen challenge, some o hem wi h high
impo ance such as IGFBPs, AMBP p o ein, CD5L o AGP1 [375].
Some yea s la e , Cede u e al. employed a simila MS app oach o
iden i y a se o galec in binding glycop o eins as po en ial as hma
bioma ke s (e.g., CD59, ke a in 8, HP, RAIG, SBEM, a ac in, CD55,
AGP, o SERPINA1) [376]. In addi ion, FABP5 and VEGF we e
inc eased in as hma ics compa ed o heal hy con ols bo h in NLF and
IS [378]. Apa om BALF and NLF, a ew numbe s o s udies ha e
been pe o med in BB [357, 379], nasal b ushing (NB) [380], o EBC.
Howe e , p o eomics s udies wi h IS [378, 381-386] o
plasma/se um [358, 380, 386-393] samples o look o speci ic as hma
bioma ke s ha e been a mo e equen . Mo eo e , mos o hese
s udies ha e compa ed as hma ics s. heal hy subjec s, coming ac oss
signi ican changes in p o eins such as S100A8/9/12, CC16, cys a in s,
AAT/SERPINA1, α2M, HP, CP, Hpx, I-TAC, EGF, MMP-9, ITIH4,
ORM1, o FGA/B [381, 382, 385, 390, 391]. In con as , o he
p o eomic s udies ha e compa ed as hma wi h COPD [381, 383, 390]
o cys ic ib osis [380], aspi in-induced as hma s. aspi in- ole an
as hma [377, 387], he e ec o exe cise-induced b onchocons ic ion
INTRODUCTION
77
[382], he le el o as hma con ol [384, 386, 394] o exace ba ion
[388], and he e ec o di e en ea men s [357, 358, 379]. Howe e ,
none o hem was aimed o compa e he di e ences be ween as hma
pheno ypes, and only a hand ul epo ed p o eomic changes in pa ien s
wi h di e en as hma se e i ies [384, 386, 388, 394]. Mo eo e , mos
o he s udies in plasma/se um we e pe o med using 2-DE and
MALDI-TOF, only one was ca ied ou by an LC-MS/MS (label- ee
quan i ica ion) app oach [391], and none o hem used he iTRAQ
echnology. The e o e, u he s udies a e needed o compa e he
se um/plasma p o eome o di e en as hma pheno ypes (e.g., AA s.
NAA) and se e i ies (in e mi en -mild s. mode a e-se e e).
Juan José Nie o Fon a igo
78
Table 1. P o eomic s udies in as hma.
Au ho s
Sample
Me hods
Resul s
Re .
Lindahl M,
1999
BALF
NLF
2-DE
LCN1, TTR, cys a in S, and
IgBF a e al e ed in
as hma.
[374]
Wu J,
2005
BALF
AC and 1DE
LC-MS/MS
Label-F ee
160 p o eins we e
di e en ially exp essed
in as hma ic pa ien s
a e2 alle gen challenge
(e.g., IGFBPs, AMBP,
CD5L, o AGP1)
[375]
Lee SH,
2006
Plasma
2-DE
MALDI-TOF
C3a and C4a we e highe
in AIA pa ien s han in
pa ien s wi h ATA.
[387]
G ay RD,
2008
IS
CM10, Q10,
o IMACNi
SELDI-TOF
105 peaks wi h changes
be ween as hma ics and
con ols, and 16 be ween
as hma and COPD.
P o eins iden i ied:
S100A8, 9 and 12, CC16,
lysosyme c, p oline ich
sali a y pep ide, cys a in
s, and HBA1.
[381]
Nishioka T,
2008
Plasma
2-DE (DIGE)
MALDI-TOF
19 p o eins wi h changes
du ing as hma
exace ba ion s. non-
exace ba ion: GP1BB,
CLU, C7, C1s,
AAT/SERPINA1,
SERPINA2 and3, F2, FGB,
KNG1, A1BG, AZGP1, ALB,
HP, AFM, APCS, CD5L,
APOA1, APOA4.
[388]
Con inued on nex page
INTRODUCTION
79
Table 1 (Con inued)
Rhim T,
2009
Plasma
2-DE
MALDI-TOF
C3 inc eases whe eas γ-
ib inogen dec eases in
as hma posi i e
esponde s agains
De ma ophagoides
p e onyssinus inhala ion
in compa ison wi h
nega i e esponde s.
[389]
Gomes-
Al es P,
2010
NB
Se um
CM10 pH 4
Q10 pH 10
SELDI-TOF
91 peaks wi h changes
be ween
con ol/as hma/cys ic
ib osis
P o ein iden i ied:
Hemoglobin subuni -be a
[380]
Bloemen K,
2011
EBC
nano-HPLC
MALDI-
TOF/TOF
A peak pa e n o
pep ides was iden i ied
o disc imina e be ween
heal hy con ols and
as hma ics. Only
cy oke a in 1 was
iden i ied.
[395]
Gha ib SA,
2011
IS
LC-MS/MS
Label-F ee
(spec al
coun ing)
70 p o eins wi h
di e ences be ween
as hma and HC (e.g.,
S100A8/9,
AAT/SERPINA1, SMR3B,
o SCGB1A1).
5 di e en ial p o eins
be ween EIB+ and EIB-
(e.g., C3a and HPX).
[382]
Con inued on nex page
Juan José Nie o Fon a igo
80
Table 1 (Con inued)
Ko YC,
2011
CD4+ T
cells
2-DE
MALDI-TOF
13 p o eins wi h
di e en ial exp ession
be ween con olled and
uncon olled as hma ics:
HSP-70, FGB, TPM3, ATP-
dependen DNA helicase
II, HSP-90, ACTB, VIM,
ARHGDIB, ENO1, CALR
p ecu so , YWHAZ,
PRDX2.
[394]
O'Neil SE,
2011
BB
SCX
LC-MS/MS
iTRAQ
7 di e en ial p o eins
be ween as hma pa ien s
and HC (ANXA5, DPT,
HIST1H2AH, LMNA, PPIA,
RPL7 and 8).
7 modi ied p o eins
be ween budesonide p e
and pos - ea ed
as hma ics (α2M, ALDOA,
ATP5B, DPYSL5, RPS20,
SERPINB3, and VIM)
[379]
Te acciano
R,
2011
IS
MSB
MALDI
TOF/TOF
HNP1, HNP2, HNP3, and
h ee C- e minal
amida ed pep ides wi h
changes be ween
as hma, COPD, and
heal hy olun ee s.
[383]
Ve ills NM,
2011
Plasma
2-DE
MALDI-TOF
20 p o eins wi h
di e ences be ween
as hma, COPD, and
con ols. 4 o hem able
o disc imina e be ween
he h ee g oups: α2M,
HP, CP, and Hpx.
[390]
Con inued on nex page
INTRODUCTION
81
Table 1 (Con inued)
Cede u C,
2012
BALF
LC-MS/MS
Label-F ee
(spec al
coun ing)
Some galec in-binding
glycop o eins we e ound
only in as hma compa ed
o con ols: CD59, ke a in
8, HP, RAIG, SBEM,
a ac in, CD55, AGP, and
SERPINA1
[376]
Izbicka E,
2012
Plasma
LC- MS/MS
Label-F ee
I-TAC, EGF, and MMP-9
we e highe in as hma ics
han in con ols.
[391]
Choi GS,
2013
NLF
2-DE (DIGE)
MALDI-TOF
ApoA1, α2M, and CP
we e inc eased in aspi in-
induced nasal esponde s
compa ed o non-
esponde s.
[377]
Lee TH,
2013
IS
2-DE
MALDI-TOF
HNP-2, S100A9, β-
amylase, NGAL, 4-
aminobu y a e
ansaminase, and
cys a in SA we e
inc eased whe eas plunc
p ecu so , C3, GFAP, IgM
κIIIb SON, MLL-AF4
de (11) usion p o ein,
CK-8, and IgG4 hea y
chain we e dec eased in
se e e uncon olled
as hma compa ed o
con olled as hma.
[384]
Con inued on nex page
Juan José Nie o Fon a igo
82
Table 1 (Con inued)
Mau i P,
2014
BB
SCX-LC-
MS/MS
Label-F ee
(Spec al
coun )
23 p o eins we e
un egula ed in
omalizumab esponde s
and 84 in omalizumab
non- esponde s a e
ea men .
LGALS3, HSPG2, ELN,
TGFB1, VCL, EZR, MYH9,
Myl6, MYH11, ac in,
ACTG2, FBLN5, and
FBLN2 we e only
iden i ied in omalizumab
esponde s.
[357]
Suojaleh o
H, 2015
IS
NLF
2D-DIGE
LC-MS/MS
FABP5 and VEGF we e
inc eased in as hma ics
compa ed o heal hy
con ols
[378]
Hams en C,
2016
Plasma
Suspension
bead a ay
Child en wi h as hma
display less CL5 and
HPGDS le els and high
NPSR1 le els.
[392]
Jiang H,
2016
Se um
2-DE (DIGE)
MALDI-TOF
7 p o eins we e
di e en ially exp essed
in SRA s. SSA: Up
(MTMR9, VDBP, HP
p ecu so , C4a) Down
(ALB p ecu so , MASP2,
ApoA1)
[358]
Xu H,
2016
Plasma
An ibody
a ay
EPO and sGP130 we e
ound augmen ed in
childhood as hma
compa ed o con ols.
[393]
Con inued on nex page
INTRODUCTION
83
Table 1 (Con inued)
Cao C,
2017
IS
LC-MS/MS
23 p o eins inc eased in
as hma compa ed o
con ols: A2M, ANXA1,
APOA2, ELANE, GPI,
S100A8/9/12, AGT, C5,
FGA, FGB, APOA1,
SERPINF2, CHI3L1, HPX,
ITIH4, ORM1, ILRN, PRG2,
B2M, CTSB, FN1.
[385]
Kasaian MT,
2018
Se um
IS
Olink
mul iplex
a ays
27 p o eins wi h changes
be ween con olled and
uncon olled as hma.
Se um: CCL11, CCL19,
CCL25, CDCP1, FGF21,
FGF23, Fl 3L, IL-6, IL-
10Rβ.
IS: ADA, AZU1, PA,
DNER, KLK6, MMP9,
Chi 1, GRN, PGLYRP1,
MPO, HGF, PRTN3, RETN,
PI3, Chi3L1, and OPG.
[386]
AC, a ini y ch oma og aphy; AIA, aspi in-induced as hma; ATA, aspi in- ole an
as hma; BALF, b onchoal eola la age luid; BB, b onchial biopsy; EBC, exhaled
b ea h condensa e; EIB, exe cise-induced b onchocons ic ion; IS, induced spu um;
MSB, mesopo ous silica beads; NB, nasal b ushing; NLF, nasal la age luid; SRA,
s e oid- esis an as hma; SSA, s e oid-sensi i e as hma.
CHAPTER I
91
INTRODUCTION
Du ing as hma a acks alle gens igge lung epi helial cells o elease
cy okines (e.g., Thymic s omal lymphopoie in (TSLP), In e leukin
(IL)-33, IL-25) ha ac i a e inna e leukocy es and d i e he
di e en ia ion o alle gen-speci ic T helpe (TH)2 lymphocy es.1
Inna e de ences also ely on pa e n ecogni ion ecep o s, such as oll-
like ecep o s (TLRs), capable o de ec ing pa hogen-associa ed
molecula pa e ns (PAMPs). Lipopolysaccha ide (LPS) is a PAMP
ha in e ac s wi h CD14 (monocy es, mac ophages, and neu ophils),2
a ecep o whose gene has been linked o as hma/alle gy.3 CD14
ans e s LPS o he TLR4-MD2 complex, which induces he sec e ion
o a se o cy okines (IL-12/IL-18) ha ac as an inna e-adap i e
b idge and a ou he TH1 di e en ia ion.4 In addi ion, IL-12 also
p e en s he de elopmen o alle gen-speci ic TH2 cells, which
amelio a es ai way in lamma ion in alle gic as hma.4 The e o e, a
unc ional de ec in CD14 could al e TH2 di e en ia ion and IgE-
media ed alle gic diseases.5
Memb ane-bound CD14 (mCD14) is eleased o he medium
as soluble CD14 (sCD14),2,6,7 which ei he po en ia es he esponse o
LPS in mac ophages by inducing he elease o p oin lamma o y
cy okines,8 o has a p o ec i e ole by ans e ing LPS o
lipop o eins.9 sCD14 is eleased om monocy es h ough a p ocess
enhanced by LPS, IL-6 and IL-1β,6 bu dec eased by in e e on-γ
(IFN-γ) and IL-4.10 sCD14 in e sely co ela es wi h IL-4,11 bu
posi i ely wi h he numbe o spu um eosinophils.12 Mo eo e , plasma
sCD14 is a bioma ke o ongoing o acu e immune esponses.2,13
Thus, child en wi h as hma exace ba ions display augmen ed sCD14
le els compa ed o he eco e y phase,14,15 whe eas adul as hma ics
ha e highe amoun s in spu um han heal hy subjec s.12 An inc eased
sCD14 concen a ion has also been de ec ed in b onchoal eola la age
Juan José Nie o Fon a igo
92
luid (BALF) upon alle gen exposu e.16 Howe e , o he wo ks
epo ed unal e ed sCD14 le els, like o example in as hma ic
child en.7,15
Single-nucleo ide polymo phisms (SNPs) also cons i u e
modula o y agen s o CD14 le els ha could in luence he isk
ac o s o de eloping as hma o agg a a e disease symp oms.17
Baldini e al. ound an associa ion be ween CD14 (-159 C/T) SNP
( s2569190) and sCD14 in a opic child en, showing TT homozygo es
highe le els han CT/CC geno ypes11 in line wi h he enhanced CD14
ansc ip ional ac i i y in he T allele.6 Since hose landma k s udies,
di e en wo ks ha e con i med hese esul s,18,19 while he C allele
has been associa ed wi h high IgE le els, a opy and as hma.15,17,20
Howe e , his associa ion has no been consis en ly eplica ed.3,13,21-24
These di e en esul s may be a ibu ed o a numbe o ac o s, like
he low sample size, as hma pheno ype (a opic, non-a opic o mixed
as hma), age o s udy popula ion (child en o adul s), e hnici y
(A ican, Caucasian o Asia ic popula ion) o gene-en i onmen
in e ac ions (e.g., endo oxin le els).21,25 The e o e, i seems necessa y
o unde ake new wo ks aimed o he simul aneous measu emen o
bo h memb ane and soluble o ms o CD14 as well as he CD14 (-159
C/T) SNP in adul Caucasian subjec s wi h alle gic as hma and
di e en deg ees o se e i y (in e mi en -mild and mode a e-se e e).
MATERIAL AND METHODS
Subjec s
The s udy popula ion was ec ui ed om Janua y 2009 o Decembe
2012, a he Uni o Pneumology and Alle gy o he USC Uni e si y
Hospi al Complex o San iago de Compos ela (CHUS) and he
Pon e ed a Hospi al Complex (CHOP). This s udy popula ion
CHAPTER I
93
included 277 heal hy con ols (HC) and 277 alle gic as hma ics (AA),
consis ing o 108 in e mi en -mild alle gic as hma ics (IMAA) and
169 mode a e-se e e alle gic as hma ics (MSAA). As hma and alle gy
diagnosis was con i med acco ding o he Global S a egy o As hma
Managemen and P e en ion c i e ia (GINA 2006,
h p://www.seicap.es/documen os/a chi os/GINA2006gene al.pd ).
All pa ien s we e in a s able phase o a leas 4 weeks be o e he s udy
ini ia ion. Fo ced i al capaci y (FVC), o ced expi a o y olume in 1
second (FEV1), and he FEV1/FVC a io, we e measu ed. As hma
diagnosis was con i med by a posi i e b onchodila o es (>12% o
FEV1 change a e salbu amol) o me hacholine challenge. Alle gic
sensi iza ion was con i med h ough a skin p ick es o se um IgE
speci ic o equen alle gens. O he a iables we e measu ed:
smoking, pe s a home, esidence ( u al/u ban), p o ession,
como bidi ies, he age o symp oms onse , as hma con ol, o numbe
o isi s o eme gency uni s, amily doc o s o hospi als du ing he
yea p io o he s udy ini ia ion. HC we e selec ed om pa ien s
scheduled in he hospi al o mino su ge ies such as o hopedic
su ge y o inguinal he nia, and smoke and sys emic diseases o
alle gies we e used as exclusion c i e ia. The esea ch was ca ied ou
acco ding o The Code o E hics o he Wo ld Medical Associa ion
(Decla a ion o Helsinki). The p ojec was also app o ed by he E hics
Commi ee o Clinical Resea ch o Galicia (2011/001), Spain, and all
subjec s signed in o med consen s a emen s.
Flow cy ome y assays
Venous pe iphe al blood was collec ed in e hylenediamine e aace ic
acid (EDTA) ea ed ubes (BD Vacu aine K2E). To analyse he
exp ession o CD14 on pe iphe al blood leukocy es, CD14-FITC
(Mouse IgG2a κ; BD Biosciences) o Iso ype-FITC (Mouse IgG2a;
BD Biosciences) we e added o 100 μL o whole blood (30 min, oom
Juan José Nie o Fon a igo
94
emp.). Then, ed cells we e lysed (BD FACSTM Lysing Solu ion).
Finally, 10 000 e en s we e collec ed and analysed by means o a BD
FACSCalibu low cy ome e . Da a we e examined using WinMDI
2.9 so wa e (Joseph T o e , La Jolla, CA. USA).
Biochemical de e mina ions
Biochemical de e mina ions we e ca ied ou by an ADVIA®1650
analyse (SIEMENS Heal hca e Diagnos ics S.L., Be lin, Ge many)
while neu on-speci ic enolase (NSE) was measu ed wi h
elec ochemiluminescence analyse (MODULAR ANALYTICS
Cobas E-601, Roche Diagnos ics; Mannheim, Ge many). The
nuclea ed cell coun ing was pe o med using an ADVIA®2120
hema ology coun e (SIEMENS Heal hca e Diagnos ics S.L., Be lin,
Ge many).
Se um sCD14 le els we e measu ed by means o an enzyme-
linked immunoso ben assay (ELISA) wi h he Quan ikine®Human
sCD14 Immunoassay ki (R&D Sys ems, MN, USA). Op ical
densi ies we e eco ded a 450 nm and p o ein concen a ion was
calcula ed om s anda d cu es.
Genomic DNA pu i ica ion and CD14 (-159 C/T) p omo e SNP
( s2569190) s udies
Genomic DNA was pu i ied om whole blood (200 µL) wi h he
QIAamp® DNA Mini Ki (QIAGEN, Melbou ne, Aus alia). The
subsequen s udy o he s2569190 SNP in he CD14 p omo e was
conduc ed by he CEGEN-PRB2 USC node using he iPlex® Gold
chemis y and MassARRAY pla o m, acco ding o manu ac u e ‟s
ins uc ions (Agena Bioscience, San Diego, CA). Geno yping assay
(Polyme ase chain eac ion/PCR p ime s and single-base-
ex ension/SBE p ime s) we e designed using he Agena Bioscience
MassARRAY Assay Designe 4.1 so wa e. To a oid con usion in he
CHAPTER I
95
mass spec um, a ag (5-ACGTTGGATG-3) was added o he 5´ end
o each PCR p ime . Bo h SBE and PCR p ime sequences a e shown
in Supplemen a y Table 1. PCR eac ion was se up in a 5 µL olume
and con ained empla e DNA (20 ng), 1× PCR bu e , MgCl2 (2 mM),
dNTPs (500 µM) and PCR enzyme (1 U/ eac ion). A pool o PCR
p ime s was made a a inal concen a ion o each p ime o 100 nM
(Me abion In e na ional AG, Ge many). The he mal cycling
condi ions o he eac ion consis ed o an ini ial dena u a ion s ep a
95°C o 2 minu es, ollowed by 45 cycles o 95°C o 30 seconds,
56°C o 30 seconds and 72°C o 1 minu e, and a inal ex ension s ep
o 72°C o 5 minu es. PCR p oduc s we e ea ed wi h sh imp
alkaline phospha ase (1.5 U) by incuba ion a 37°C o 40 min,
ollowed by enzyme inac i a ion by hea ing a 85°C o 5 min o
neu alize uninco po a ed dNTPs.
The iPLEX GOLD eac ions we e se up in a inal 9 µL
olume and con ained 0.222x iPLEX bu e Plus, 0.222x iPLEX
e mina ion mix and iPLEX enzyme (1.35 U/ eac ion). An SBE
p ime mix was made o gi e a inal concen a ion o each p ime
be ween 0.52 µM and 1.57 µM (Me abion In e na ional AG,
Ge many). The he mal cycling condi ions o he eac ion included an
ini ial dena u a ion s ep a 95°C o 30 seconds, ollowed by 40 cycles
o 95°C o 5 seconds, wi h an in e nal 5 cycles loop a 52°C o 5
seconds and 80°C o 5 seconds, ollowed by a inal ex ension s ep o
72°C o 3 minu es. The nex s ep is o desal he iPLEX Gold eac ion
p oduc s wi h Clean Resin ollowing he manu ac u e ‟s p o ocol. The
desal ed p oduc s we e dispensed on o a 384 Spec ochip II using an
RS1000 Nanodispense and spec a we e acqui ed using he MA4
mass spec ome e , ollowed by manual inspec ion o spec a by
ained pe sonnel using MassARRAY Type so wa e, e sion 4.0. All
assays we e pe o med in 384-well pla es, including nega i e con ols
and a io o Co iell samples (Na10830, Na10831 and Na12147) o
Juan José Nie o Fon a igo
96
quali y con ol. 10% o andom samples we e es ed in duplica e and
he ep oducibili y was 100%.
S a is ics
Desc ip i e da a a e p esen ed as ei he median (in e qua ile ange;
IQR1-3) o pe cen ages. To assess he signi icance o changes
be ween AA and HC, Mann–Whi ney U wo- ailed es o K uskal–
Wallis One Way Analysis o Va iance on Ranks ollowed by Dunn‟s
mul iple compa ison es we e used. Recei e Ope a ing Cha ac e is ic
(ROC) cu es and Spea man´s associa ion es we e also employed,
while di e ences in p opo ions we e assayed by he χ2 es . To
e alua e he associa ion be ween CD14 (-159 C/T) SNP and he isk
o alle gic as hma, Odd´s a ios (ORs) and 95% con idence in e als
(CIs) we e calcula ed acco ding o di e en models: TT+TC s. CC
(dominan model), TT s. TC+CC ( ecessi e model), TT s. CC, TC
s. CC, and T s. C (allelic model). Ha dy–Weinbe g equilib ium
(HWE) was calcula ed by using Pea son χ2 es . All analyses and
g aphs we e conduc ed using G aphPad P ism e sion 6.0 (G aphPad
So wa e, Inc., San Jose, Cali o nia, USA). Da a a e p esen ed in box
and whiske plo s, whe e median, 25 and 75 qua iles, 5-95 pe cen iles
(e o ba s) and anomalous alues a e shown. The s a is ical
signi ica ion was de ined as P < 0.05.
RESULTS
Demog aphic and clinical cha ac e is ics o he s udy popula ion
In his wo k a case-con ol s udy was pe o med, whe e adul pa ien s
wi h bo h in e mi en -mild and mode a e-se e e alle gic as hma we e
ec ui ed. These pa ien s we e in a clinically s able s a e, had a well-
CHAPTER I
97
con olled disease, and he majo i y we e non-smoke s unde
ea men wi h inhaled co icos e oids (Table 1). FEV1 (%) and
FEV1/FVC a io (%) alues a e desc ibed in Table 1, showing
dec eased le els in MSAA (mode a e-se e e alle gic as hma ics)
compa ed o IMAA (in e mi en -mild alle gic as hma ics) (Table 1).
Al hough 66.4% o AA came om u al a eas, mos o hem had no
animals o jus dogs/ca s; only 11% had a m animals.
Table 1. Cha ac e is ics o he s udy popula ion.
AA
HC
ALL
IMAA
MSAA
N
277
108
169
277
Agea
32 (25-42)
31 (22-38)
33 (26-46)
46 (31-60)
Sex (M/F)
119/158
42/66
77/92
105/172
Smoke s (%)
21.3
15.7
24.8
0
Con ol:
Good
219
98
121
-
Pa ial
36
8
28
-
Bad
22
2
20
-
T ea men :
No
66
66
0
-
Inhaled
Co icos e oids
196
34
162
-
O al
Co icos e oids
15
0
15
-
An ileuko ienes
63
6
57
-
Omalizumab
4
0
4
-
FEV1 (%)
97.2 (83.7-
108.0)
105.0 (95.3-
115.7)#
93 (74.5-
102.2)#
-
FEV1/FVC (%)
78.0 (70.7-
85.4)
82.9 (75.9-
88.4)#
74.7 (67.6-
81.9)#
-
Neu ophils (103
cells/μL)a
3.67 (2.77-
4.50)
3.53 (2.99-
4.27)
3.71 (2.90-
4.96)
3.54 (2.77-
4.50)
Lymphocy es (103
cells/μL )a
2.22 (1.85-
2.71)*
2.20 (1.86-
2.79)&
2.23 (1.80-
2.71)&
2.02 (1.62-
2.45)
Monocy es (103
cells/μL )a
0.47 (0.38-
0.59)*
0.47 (0.39-
0.56)&
0.46 (0.37-
0.62)&
0.35 (0.28-
0.43)
Con inued on nex page
Juan José Nie o Fon a igo
98
Acco ding o hei alle gic disease s a e, pa ien s had
signi ican ly augmen ed le els o eosinophils and o al IgE compa ed
o HC, bu he e we e no signi ican changes ega ding disease
se e i y (Table 1). IgE showed a posi i e co ela ion wi h eosinophil,
monocy e and, o a lesse ex en , lymphocy e blood coun , unde lying
he ele ance o hese subse s in alle gic as hma pa hogenesis (Table
2). Since ac i a ion o eosinophils and mac ophages has been
associa ed wi h enhanced NSE le els unde some pa hological
condi ions,26,27 we also unde ook he measu emen o his enzyme in
se um samples. As able 2 shows, i was ound a posi i e co ela ion
o IgE, eosinophils and monocy es wi h NSE (Table 2), which
p omp ed us o examine he u ili y o his pa ame e as an addi ional
ma ke o alle gic as hma. As shown in Figu e 1a, NSE le els we e
augmen ed in AA compa ed o HC and end o be a ound 14.6%
highe in men han in women (Figu e 1a). Mo eo e , he a ea unde
he cu e (AUC) o he ROC plo o NSE le els was close o AUC o
o al IgE and highe han AUC o blood eosinophils (absolu e alues)
(Figu e 1b).
Table 1 (Con inued)
Eosinophils (103
cells/μL )a
0.33 (0.19-
0.51)*
0.29 (0.20-
0.51)&
0.34 (0.18-
0.51)&
0.16 (0.11-
0.23)
Basophils ((103
cells/μL )a
0.04 (0.03-
0.05)
0.04 (0.03-
0.05)
0.04 (0.02-
0.06)
0.04 (0.03-
0.06)
IgE (IU/mL)a
272 (116-
533)*
270 (111-
485)&
276 (122-
668)&
34 (11-102)
AA, alle gic as hma ics; HC, heal hy con ols; IMAA, in e mi en -mild alle gic as hma ics;
MSAA, mode a e-se e e alle gic as hma ics.
a Median alue (IQR1-3); * AA s HC: Mann-Wi ney U S a is ic, P < 0.001; # IMAA s
MSAA: Mann-Wi ney U S a is ic, P < 0.001; & IMAA s MSAA s HC: K uskal-Wallis One
Way Analysis o Va iance on Ranks (P < 0.001), IMAA/MSAA s HC P < 0.05 Dunn's
Me hod.
CHAPTER I
99
Table 2. Spea man co ela ion ma ix o he s udy popula ion.
VARIABLES
NSE
CRP
IgE
mCD14#
sCD14
Age
Leucocy e
coun
0.163*
0.221***
0.192***
-0.021
-0.035
-0.165***
Neu ophil
coun
0.003
0.190***
-0.027
-0.027
-0.034
-0.052
Lymphocy e
coun
0.077
0.107*
0.143***
-0.020
-0091
-0.152***
Monocy e
coun
0.303***
0.154***
0.336***
0.234***P
-0.021
-0.182***
Eosinophil
coun
0.279***
0.054
0.459***
-0.199***
-0.071
-0.273***
Basophil
coun
0.149***
-0.024
0.057
-0.034
-0.005
-0.113*
FEV1%
0.099P
-0.088P
-0.057P
0.140P
-0.177P
-0.209***P
FEV1/FVC
0.106P
-0.071P
-0.051P
0.151
-0.146P
-0.375***P
TNF
-0.022
0.179***
-0.011
0.082
0.119**
0.198***
NSE
-0.004
0.322***
-0.215***
0.087
-0.128**
CRP
-0.033
-0.000
0.158**
*
0.119**
IgE
-0.264***
-0.054
-0.296***
mCD14#
-0.079
0.177***
sCD14
0.102*
CRP, C- eac i e p o ein; IgE, immunoglobulin E; NSE, neu on speci ic enolase; P,
pa ien popula ion; TNF, umou nec osis ac o . *P<0.05, **P<0.01, ***P<0.001. # % o
CD14+ monocy es.
No al e a ions we e app ecia ed o C- eac i e p o ein (CRP),
IgG, IgA, IgM, o umou nec osis ac o (TNF) in AA. The in luence
o age was also aken in o conside a ion, wi h a nega i e co ela ion
wi h FEV1%, FEV1/FVC, IgE and leukocy e sub ypes (mos ly
eosinophils), bu a small posi i e associa ion wi h TNF, CRP, mCD14
and sCD14 (Table 2).
Juan José Nie o Fon a igo
106
Table 4. Associa ion be ween CD14 (-159 C/T) SNP and alle gic
as hma isk.
OR (95% CI)
χ2 (Ya e´s
co ec ion)
P
AA (ALL)
TT+TC s
CC
0.67 (0.45-
0.98)*
3.805
0.0511
TT s
TC+CC
0.68 (0.46-
1.00)
3.484
0.0620
TT s CC
0.55 (0.34-
0.89)*
5.449
0.0196*
TC s CC
0.74 (0.49-
1.11)
1.180
0.1704
T s C
0.74 (0.59-
0.94)*
5.776
0.0162*
IMAA
TT+TC s
CC
0.72 (0.43-
1.20)
1.300
0.2543
TT s
TC+CC
0.79 (0.48-
1.33)
0.566
0.4517
TT s CC
0.66 (0.35-
1.23)
1.346
0.2460
TC s CC
0.75 (0.44-
1.30)
0.781
0.3768
T s C
0.81 (0.59-
1.11)
1.531
0.2159
MSAA
TT+TC s
CC
0.64 (0.42-
0.99)*
3.630
0.0547
TT s
TC+CC
0.61 (0.38-
0.96)*
4.029
0.0442*
TT s CC
0.49 (0.28-
0.85)*
5.767
0.0163*
TC s CC
0.72 (0.46-
1.15)
1.572
0.2100
T s C
0.70 (0.54-
0.92)*
6.091
0.0136*
AA, alle gic as hma ics; CI, con idence in e al; IMAA, in e mi en -mild alle gic as hma ics;
MSAA, mode a e-se e e alle gic as hma ics; OR, odds a io; TC s CC, he e ozygo e; TT s
CC, homozygo e; TT s TC+CC, ecessi e model; TT+TC s CC, dominan model.
∗Signi ican di e ence.
CHAPTER I
107
Rega ding o he o he gene ic models, a signi ican associa ion
o CD14 (-159 C/T) and alle gic as hma isk was ound be ween he
homozygo es TT s. CC (OR = 0.55, 95% CI = 0.34-0.89, P =
0.0196), and almos eached signi icance in a dominan model
(TT+TC s. CC: OR = 0.67, 95% CI = 0.45–0.98, P = 0.0511) (Table
4). Fu he mo e, a e seg ega ing by disease se e i y, i was ound an
associa ion o alle gic as hma isk wi h his SNP only in MSAA
acco ding o a ecessi e model (TT s. TC+CC: OR = 0.61, 95% CI =
0.38-0.96, P = 0.0442) o TT s. CC geno ype compa isons (OR =
0.49, 95% CI = 0.28-0.85, P = 0.0163). The e o e, he esul s
sugges ed ha T allele and TT homozygo e indi iduals ha e
dec eased isk o alle gic as hma compa ed wi h C allele and CC
homozygo e ca ie s, espec i ely.
The in luence o he CD14 (-159 C/T) SNP on CD14 le els
Al hough we ha e shown augmen ed pe iphe al blood monocy e coun
and dec eased le els o mCD14 (Figu es 2a-c), he numbe o
monocy es was no in luenced by he CD14 (-159 C/T) SNP geno ype.
We also ailed o de ec any change in mCD14 ela ed o he SNP
geno ype (da a no shown). In con as (and ega dless o whe he hey
belong o he con ol g oup o o he as hma ic popula ion), CC
geno ypes and o a lesse ex en TC geno ypes had lowe
concen a ions o sCD14 (absolu e alues) han TT ca ie s, while no
signi ican di e ences we e obse ed be ween TC and CC subjec s
(Figu e 3a). This associa ion was main ained be ween TT and CC
ca ie s when sCD14 le els we e no malised by he absolu e coun o
monocy es (Figu e 3b). The e o e, his SNP could be pa ially
esponsible o he educ ion o no malised sCD14 le els in AA and
in luence he se e i y o his disease (Figu e 2e).
Juan José Nie o Fon a igo
108
Figu e 3. Impac o he CD14 (-159 C/T) SNP ( s2569190) on sCD14 le els in
se um. Absolu e (ng/mL) (a) and ela i e (ng/1 x 103 monocy es) (b) sCD14 le els
in se um samples om TT, TC and CC dono s (heal hy and as hma ics). *K uskal-
Wallis One Way Analysis o Va iance on Ranks, P < 0.001; Dunn's Me hod was
used o mul iple compa isons (numbe s on he g aph ep esen P- alues).
DISCUSSION
In he cu en s udy, we epo da a suppo ing an inc ease o NSE and
monocy es and a down-modula ion o hei mCD14 exp ession in
alle gic as hma ega dless o disease se e i y. In addi ion, we de ec a
dec ease o no malised sCD14 alues in se um samples om
as hma ics, sugges ing he expansion o a CD14low monocy e subse
and he in luence o he CD14 (-159 C/T) SNP geno ype. Indeed, we
e idence an associa ion o he T allele and TT geno ype o CD14 (-
159 C/T) polymo phism wi h educed isk o mode a e-se e e alle gic
as hma.
AA in ou s udy ha e an a opic disease wi h eosinophilia,
monocy osis and ele a ed IgE le els. NSE is he neu onal isome o
he glycoly ic enzyme 2-phospho-D-glyce a e hyd olase, and a ypical
bioma ke o small cell lung cance .28 Ne e heless, changes in non-
malignan in lamma o y lung diseases ha e also been ound,27,29-32
CHAPTER I
109
since his enzyme can ansloca e owa ds he cell su ace upon p ope
s imula o y signals o enhance a p oin lamma o y esponse.33 Ou
esul s suppo he p esence o highe NSE le els in men, as
p e iously epo ed by Collazos e al.,29 bu con a y o his wo k ou
as hma pa ien coho displays abo e-no mal NSE le els in se um.
Monocy es/mac ophages appea o be a possible sou ce o NSE,27,33
and inc eased numbe s o monocy es as well as a co ela ion o hem
wi h NSE le els we e demons a ed in ou s udy. Howe e , o he
plausible sou ces o NSE a e eosinophils o inju ed epi helial cells
du ing pulmona y in il a ion,26 while he neu onal dis ess o hypoxia
occu ing in he lung also could play a ole du ing he disease.29
Apa om o he cells as T and B lymphocy es, eosinophils,
basophils o neu ophils, monocy es a e gaining impo ance as
egula o s o in lamma ion in as hma and as key playe s in he
pa hogenesis.34-36 Ou esul s show he expansion o his subse in AA
ega dless o he se e i y o he symp oms (i.e., IMAA and MSAA),
as well as a educ ion o mCD14, a monocy e ma ke associa ed o
as hma.3 In con as , some au ho s ha e desc ibed no di e ences in
he s aining o mCD14,37,38 e en hough hese could be he esul o a
low s a is ical sample size. Reduced le els o mCD14 o TLR4 in AA
makes biological sense,39 as signal ansduc ion h ough CD14/TLR4
leads o IL-12 sec e ion, a powe ul induce o TH1 pola iza ion.13
The e o e, a enua ed mCD14 le els on an igen p esen ing cells
(APCs) could a ou TH2-d i en alle gic as hma.4,5,13,16 This
diminished numbe o mCD14 molecules on monocy es could a ise as
a esul o se e al, and no mu ually exclusi e, mechanisms: a) al e ed
ansc ip ion/ ansla ion a es a ec ing p o ein abundance; b)
expansion o CD14low monocy e subse s; c) a esicle- o enzyma ic-
media ed mechanism ha elease mCD14 om monocy es and should
also a ec sCD14 concen a ion.
Juan José Nie o Fon a igo
110
The deg ee o mCD14 down-modula ion on monocy es
sugges s he p e e en ial expansion o a small CD14low subse and no
a globally al e ed ansc ip ion/ ansla ion a e. This, o example, is in
line wi h he inc eased pe cen age o CD14low/- monocy es upon in
i o cul u e in he p esence o TSLP, a cy okine impo an in alle gic
as hma.40 Monocy es a e he e ogeneous, wi h majo (CD14high) and
mino (CD14low) subse s.33 CD14high (“classical”) monocy es display a
CD16/FcγRIII- pheno ype, while he less equen CD16+ subse
consis s o bo h in e media e (CD14highCD16+) and non-classical
(CD14lowCD16+) subpopula ions.34 CD16+ monocy es, pa icula ly he
in e media e subse , a e expanded in in lamma ion, se e e as hma o
upon alle gen challenge,34,36 in line wi h hei p o-in lamma o y
na u e.41 A majo cons ain o ou s udy is ha we ha e no analysed
CD16, bu ou esul s show a signi ican down-modula ion o mCD14
in alle gic as hma, which appea s o a he suppo he expansion o
CD14lowCD16+ monocy es. These cells (non-classical subse ) exp ess
high le els o CD80, CD86, and CD163, sugges ing a high an igen
p esen ing capabili y.42 Fu he mo e, non-classical monocy es a e in
an ad anced di e en ia ion s age and hey e idence high in ading
abili y o in il a e and di e en ia e in o M2- ype mac ophages,43 a
subse ela ed o alle gic in lamma ion.44
CD14 can be eleased o medium om hepa ocy es as an acu e
phase p o ein.45 Al hough we saw a small co ela ion be ween sCD14
and CRP o TNF, he le els o hese wo las molecules had no
changes be ween AA and HC, and ou pa ien s we e in a s eady-s a e
o he disease. Excluding he hepa ocy e o igin, monocy es a e he
mos likely cell sou ce o sCD14. Down-modula ion o mCD14 in
monocy es om AA does no i wi h ei he i s shedding2,6,7 o he
elease o mCD14-en iched esicles
[h p://exoca a.o g/gene_summa y?gene_id=12475] om hese cells,
because bo h p ocesses should lead o a highe numbe o sCD14
CHAPTER I
111
molecules in he ex acellula compa men , as happen du ing he
acu e phase.15,16,46,47 Howe e , pa ien s in ou s udy a e in a ch onic
phase, whe e he e is no ela ionship be ween monocy e coun s and
sCD14 o mCD14-sCD14 co ela ion.16 The e o e, ou esul s only
make sense conside ing a puzzling scene y wi h an ele a ion o
monocy e numbe s and enhanced equencies o bo h CD14high,34 bu
also CD14low (ou esul s) subse s o monocy es in as hma. Indeed, in
ou s udy only no malised se um le els o sCD14 we e signi ican ly
educed in pa ien s. In ag eemen , sCD14 le els ha e been in e sely
co ela ed wi h IL-4-p oduc ion,11 o al IgE,11 o as hma se e i y.46
Howe e , some au ho s ha e de ec ed highe le els12 o no
di e ences7,15 o baseline sCD14 in pe iphe al blood om as hma ics.
Hence, we canno ule ou he con ibu ion o many po en ial
con ounding ac o s ha explain hese di e en esul s, like gene-gene
o gene-en i onmen in e ac ions.11,15
One o he mos s udied CD14 polymo phisms in as hma is he
CD14 (-159 C/T) SNP ( s2569190).11 P e ious s udies in es iga ing
he associa ion o his SNP wi h alle gic as hma yielded a iable
esul s ega ding he s eng h and di ec ion o he associa ion.3,17,24
These con adic o y esul s can be explained by di e ences in
e hnici y, low sample size, he age o pa ien s o gene-en i onmen
in e ac ion.21,25 We pe o med ou s udy in a well-de ined popula ion
(Caucasian, adul s, alle gic as hma ics and mos ly non- a me s), wi h
a high sample size (277 AA s. 277 HC), and wo di e en disease
se e i y g ades (IMAA and MSAA). In ag eemen wi h o he s,11,17 we
show an associa ion o he equency o he C allele and he CC
geno ype wi h alle gic as hma (whole as hma ics). Mo e in e es ing,
his associa ion is also ela ed o he disease se e i y, as i is only
main ained in MSAA and wi hin his g oup, in se e e as hma ics.
Mo eo e , he isk o ha ing mode a e-se e e alle gic as hma (bu no
in e mi en -mild as hma) is lowe in ca ie s o he T allele (T s. C)
Juan José Nie o Fon a igo
112
and TT geno ype, ollowing ei he a ecessi e model (TT s. TC+CC)
o a e compa ing TT s. CC homozygo es. As o he wo ks ha e
shown,5,11,12,18,19 we e idence augmen ed sCD14 le els in subjec s
ca ying he TT geno ype bu no associa ion o his polymo phism
wi h mCD14 le els on monocy es. This sugges s an ad e se ole o
he C allele, he CC geno ype and he p esence o low le els o
sCD14/mCD14 in alle gic as hma o a opy,11,46 especially among
adul and a opic subjec s exposed o low le els o endo oxin, like ou
coho .13
In summa y, ou indings show an inc emen in he se um
le els o NSE, which could be used as a no el bioma ke o alle gic
as hma. On he o he hand, we also ound a dec ease in he exp ession
o CD14 on monocy es om alle gic as hma ic pa ien s, p obably
ela ed o an inc ease o CD14low monocy e subse . Mo eo e , we
e idence an associa ion o he (-159 C/T) SNP in he CD14 p omo e
wi h alle gic as hma, and a dec eased isk o ha ing mode a e-se e e
alle gic as hma in ca ie s o T allele and TT geno ype. Fu he mo e,
TT geno ype is associa ed wi h highe le els o sCD14, poin ing ou a
p o ec i e ole o he T allele in his disease.
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CHAPTER II
123
INTRODUCTION
As hma is in luenced by gene ic ac o s (eg, dipep idyl pep idase 10
[DPP10] and ADAM me allopep idase domain 33 [ADAM33]) and
en i onmen al ac o s [1]. I s managemen should be based on
endo ypes [1,2]. Du ing as hma a acks, alle gens igge lung
epi helial cells o elease cy okines, which in u n ac i a e inna e
leukocy es and d i e ype 2 helpe T cell (TH2) lymphocy e
di e en ia ion [1]. These cells elease in e leukins (IL-4, IL-5, and IL-
13), s imula e IgE p oduc ion and a o he ac i a ion o eosinophils,
mas cells, and basophils [1]. This e ec o ole is coun e ac ed by
egula o y T cells (T egs) [3], whose numbe and/o unc ion may be
al e ed in as hma [4]. Bo h CD4+ T subse s exp ess di e en ial le els
o in e leukin ecep o 2 (IL-2R). Thus, CD25 (IL-2Rα) is mainly
exp essed by T egs [4], while a CD25–/low pheno ype is p esen in
e ec o CD4+ T lymphocy es (Te ). T-cell ecep o (TCR)– igge ed
Te cells elease soluble CD25 (sCD25), an ac i a ion ma ke [5] ha
is ele a ed in se um/plasma du ing as hma exace ba ions [6] and ha
co ela es posi i ely wi h he se e i y o alle gic as hma [7]. In
addi ion, T egs a e hough o be ano he sou ce o sCD25 [4,8,9].
sCD25 is also inc eased in b onchoal eola la age luid om as hma
pa ien s [8,9].
Ano he in e es ing p o ein in he pa hogenesis o as hma is
CD26/DPP4, a su ace glycop o ein en iched in CD4+ T cells [10]. In
he o m o dipep idyl pep idase 4 (DPP4; EC 3.4.14.5), he enzyme
belongs o he se ine pep idase sub amily S9B, which includes an
as hma suscep ibili y locus (DPP10 [11]), dipep idyl pep idase 8
(DPP8), dipep idyl pep idase 9 (DPP9), and ib oblas ac i a ion
p o ein alpha (FAP) [12-16]. Dipep idyl pep idase 2 (DPP2, DPP7)
om se ine pep idase sub amily S28 also displays DPP4-like ac i i y
a acidic pH. As CD26, he glycop o ein in e ac s wi h adenosine
deaminase, CD45, ca eolin-1, and C-X-C chemokine ecep o ype 4
Juan José Nie o Fon a igo
124
(CXCR4), he eby ul illing ei he inhibi o y o enhancing oles upon
associa ion [17]. CD26 is an ac i a ion ma ke known o be
up egula ed on lymphocy es (especially CD4+) in adul s wi h alle gic
as hma [18]. CD4+ T cells a e majo ac o s in he pa hogenesis o
as hma; howe e , unlike CD25, T eg cells display lowe CD26 le els
han Te lymphocy es. Indeed, CD26 is a nega i e ma ke o T eg
cells and a ma ke o he emaining TH subse s. Thus, exp ession o
CD26 on TH ollows he o de o TH17>>TH1>TH2>T eg [19,20].
Hence, an ele a ed p esence o CD26 on CD4+ T cells in adul alle gic
as hma sugges s an ac i a ed s a us [18,21] and may poin o a speci ic
T-cell pheno ype. Mo eo e , a soluble o m o CD26 (sCD26/sDPP4)
is eleased om T cells in o he bloods eam, ei he shed by
me allop o eases [22] o sec e ed by CD26+ esicles
(h p://www.exoca a.o g). In plasma/se um, sCD26 accoun s o
>90% o o al sDPP4-like ac i i y [23,24], and he emaining 10% is
de i ed om he in acellula pep idases DPP2, DPP8, and DPP9.
These DPP4 homologs a e also in ol ed in he pa hogenesis o as hma
[13] and show di e en ial exp ession in leukocy es [14,15] and
eosinophils [25]. Mo eo e , bo h DPP8 and DPP9 enzymes a e
up egula ed in ac i a ed mac ophages and immed o an igen
p esen a ion [14,15]. Besides, DPP2 is necessa y o main aining he
quiescence o lymphocy es and is downmodula ed upon ac i a ion
[15].
CD26/DPP4 clea es X-P o o X-Ala amino e minal
dipep ides om chemokines (eg, CXCL12a [s omal cell-de i ed
ac o -1α, SDF-1α], CCL11 [eo axin], and CCL5 [ egula ed on
ac i a ion, no mal T cell exp essed and sec e ed, RANTES]), he eby
modula ing hei biological ac i i y and immunological unc ion, as
ecen ly e iewed [17,26,27]. O he subs a es include neu opep ides
and pep ide ho mones such as inc e ins [28,29], whose hal -li e is
p olonged by he DPP4 inhibi o s cu en ly used as an idiabe ic d ugs
CHAPTER II
125
[30]. Vascula subs a es o DPP4 may be clea ed by sDPP4, DPP4
exp essed on leukocy es, and endo helial DPP4 [31]. Inc eased DPP4-
like ac i i y has been obse ed in b onchoal eola la age luid om
as hma ic a lungs and is due mos ly o sDPP4 and, o some ex en ,
DPP8, DPP9, and DPP2 [13,32].
CD4+ T lymphocy es a e he main sou ce o sCD26, as his
subpopula ion displays he highes pe cen age o CD26+ cells
[10,23,33]. The e o e, his molecule could be used as a “ inge p in ”
o es he ac i a ion s a us o di e en ia ion s a us o CD4+ T cells in
as hma. Howe e , he ew s udies ha ha e been unde aken show
augmen ed le els o sCD26 in alle gic as hma ha we e posi i ely
co ela ed wi h eosinophils and IgE [18]. In con as , sCD26 was
in e sely associa ed wi h in lamma ion in ch onic eosinophilic
pneumonia, a disease linked o as hma [34], while no changes we e
obse ed o sCD26 in child en wi h as hma o a opy [35].
To da e, ew s udies ha e moni o ed sCD26 in as hma, and
none conside he po en ial oles o CD26 in he pa hogenesis o
as hma o ake in o accoun he possibili y ha abno mali ies o
ci cula ing bioma ke s (sCD25 and sCD26) may e lec changes in
leukocy e pheno ype such as CD25–/lowCD26+ Te cells and
CD25+/highCD26low T eg cells. The e o e, in he p esen s udy, a
comp ehensi e analysis was ca ied ou o assess he a o emen ioned
immune bioma ke s in pa ien s wi h mode a e-se e e alle gic as hma.
MATERIAL AND METHODS
Subjec s
The s udy was conduc ed be ween 2009 and 2012 and included
pa ien s om hospi al consul a ions o Pneumology and Alle gy in
Juan José Nie o Fon a igo
126
Galicia (Spain). Pa ien s had con i med diagnosis o as hma and
alle gy o a leas one yea acco ding o Global S a egy o As hma
Managemen and P e en ion (GINA 2006,
h p://www.seicap.es/documen os/a chi os/GINA2006gene al.pd )
c i e ia. The esea ch p ojec was app o ed by he E hics Commi ee
o Clinical Resea ch o Galicia (2011/001), Spain, and in o med
consen was ob ained om all indi idual pa icipan s included in he
s udy. A alida ion coho was also ec ui ed om 2014 o 2016
(Neumology Se ice, Uni e si y Hospi al o San iago de Compos ela,
Spain). All pa ien s we e in a s able phase o a leas 4 weeks be o e
sample collec ion. Heal hy Con ols (HC) we e selec ed om pa ien s
scheduled in he hospi al o mino su ge ies such as inguinal he nia
o o hopedic su ge y; hey we e non-smoke s and sys emic diseases
o alle gies we e absen . None o he pa ien s o heal hy con ols we e
on DPP4 inhibi o s ea men .
Sensi iza ion in alle gic as hma ic pa ien s (AAP) was
con i med h ough a skin p ick es o se um IgE speci ic o equen
alle gens. O he a iables we e also accoun ed: body mass index
(BMI), u al-u ban esidence, p o ession, smoking o como bidi ies.
Biochemical de e mina ions we e pe o med using an ADVIA®1650
analyze (SIEMENS Heal hca e Diagnos ics S.L., Be lin, Ge many).
The nuclea ed cells numbe was measu ed using an ADVIA®2120
hema ology coun e (SIEMENS Heal hca e Diagnos ics S.L., Be lin,
Ge many).
Magne ic pu i ica ion o CD4+ T cell subse s and in i o cul u e
Bu y coa s om heal hy subjec s we e dona ed by “Axencia Galega
de Sangue, Ó ganos e Tecidos” (San iago de Compos ela, Spain) and
used o isola e pe iphe al blood mononuclea cells (PBMCs) by
Ficoll® densi y g adien s. Te and T eg cells we e p epa ed om
PBMCs by means o he Dynabeads® Regula o y CD4+CD25+ T cell
CHAPTER II
127
Ki (Li e-Technologies, Spain). Viabili y was always >90% ( ypan
blue exclusion).
Te (CD4+CD25-) and T eg (CD4+CD25+) cells we e cul u ed
in i o o 4 days in 96-mic owell U-bo om pla es wi h
ImmunoCul ™-XF T Cell Expansion Medium (S emCell, G enoble,
F ance) supplemen ed (o no ) wi h soluble e ame ic an ibody
complexes (ImmunoCul ™ Human CD3/CD28 T Cell Ac i a o ;
S emCell). To p omo e a pa ial Te di e en ia ion, he ollowing
cy okines (Pep oTech, London, UK) we e added: 10 ng/mL IL-12
(TH1-like), 10 ng/mL IL-4 (TH2-like), o IL-1β (100 ng/mL), IL-6 (30
ng/ml) and IL-23 (100 ng/mL) (TH17-like). In addi ion, 800 ng/mL o
IL-2 was used o T eg cells main enance.
Flow cy ome y assays
Venous pe iphe al blood was collec ed (BD Vacu aine K2E) i o de
o examine he p opo ion o Te and T eg cells. Leukocy es om
100 μL o whole blood we e s ained (30 min, oom emp.) wi h mouse
IgG1 ĸ iso ype an ibodies (BD Biosciences) labelled wi h FITC, PE-
Cy7, AlexaFluo -647 and PE as nega i e con ols. Al e na i ely, cells
we e s ained wi h mouse IgG1 ĸ speci ic an ibodies (BD Biosciences)
agains CD4 (FITC), CD25 (PE-Cy7) o CD127 (Alexa Fluo -647)
and a mouse IgG2b an ibody speci ic o CD26 (PE; Immunos ep);
hen, ed cells we e lysed (BD FACSTM Lysing Solu ion). The pu i y
o Te /T eg lymphocy es p io (o a e ) in i o cell cul u e was also
assayed wi h he same speci ic (CD4-FITC, CD25-PE-Cy7, CD26-
PE) o iso ype an ibodies (Iso ype-FITC, Iso ype-PE-Cy7, Iso ype-
PE). Finally, samples we e analysed (BD FACSCalibu and FACSo )
and a numbe o 10,000-200,000 e en s collec ed. Da a we e
examined using WinMDI 2.9 so wa e (Joseph T o e , La Jolla, CA.
USA).
Juan José Nie o Fon a igo
128
DPP4 ac i i y measu emen
To al DPP4 enzyma ic ac i i y was colou ime ically assayed by
means o a la -bo om 96-well mic opla e-adap ed and end-poin
p o ocol. Cell cul u e supe na an s (50 μL) we e dilu ed wi h 50 μL
eac ion bu e (0.05 M T is(hyd oxyme hyl)aminome hane (TRIS)-
HCl pH 8.0 bu e ) and 100 μL o 2 mM glycyl-p olyl-
pa ani oanilide (Gly-P o-pNA). Pla es we e incuba ed a 37ºC and
abso bance sequen ially (30-120 min) eco ded a 405 nm
(Labsys ems Mul iscan MS mic o i e pla e eade ). The concen a ion
o pNA was calcula ed om a s anda d cu e, anging om 0 – 2000
μM. One in e na ional uni (IU) was de ined as he amoun o enzyme
ha p ocesses 1 μmol Gly-P o-pNA (o eleases 1 μmol pNA om
his subs a e) pe minu e. Assays we e pe o med in duplica e o
each sample.
De e mina ion o sCD25 and sCD26
Se um sCD25 le els we e measu ed h ough enzyme-linked
immunoso ben assay (ELISA) om R&D Sys ems, MN, USA
(Quan ikine® Human IL-2Rα Immunoassay), and sCD26 was
quan i ied wi h ELISAs pla es om eBioscience®, Vienna, Aus ia
(Human sCD26 Pla inum ELISA). Op ical densi ies we e eco ded a
450 nm and p o ein concen a ion calcula ed om s anda d cu es.
S a is ics
Desc ip i e da a a e p esen ed as ei he median (in e qua ile ange;
IQR1-3) o pe cen ages. To assess he changes be ween AAP and HC
in non-no mally dis ibu ed a iables we used Mann–Whi ney U wo-
ailed es , o he K uskal–Wallis es ollowed by Dunn‟s mul iple
compa ison es o mo e han 2 g oups. Spea man´s es was used o
measu e he associa ion be ween a iables. All analyses we e
conduc ed using G aphPad P ism 6.0 (G aphPad So wa e, Inc., San
CHAPTER II
129
Jose, Cali o nia, USA). The s a is ical signi icance was de ined as P <
0.05.
RESULTS
Cha ac e is ics o he Fi s Coho o Alle gic As hma ic Pa ien s
Table 1. Cha ac e is ics o he S udy Popula iona
Alle gic As hma ic
Pa ien sb
Heal hy Con ols
Male
Female
All
Male
Female
All
No. (%)
33 (40.7)
48 (59.3)
81 (100)
36 (37.1)
61 (62.9)
97 (100)
Age
33 (21-45)
36 (28-48)
35 (26-47)
35 (27-49)
37 (29-52)
35 (29-51)
Smoke s, %c
45.5
18.75
29.6
0
0
0
BMI, kg/m2
26.4 (23.8-
29)
26.7 (22.6-
28)
26 (23.1-
28.4)
-
-
-
As hma
Se e i y:
Mild
2
4
6
-
-
-
Mode a e/High
31
44
75
-
-
-
Con ol:
Good
23
37
60
-
-
-
Bad
10
11
21
-
-
-
T ea men :
No
1
2
3
-
-
-
Inhaled
co icos e oids
32
46
78
-
-
-
O al
co icos e oids
5
3
8
-
-
-
An ileuko ienes
6
19
25
-
-
-
Omalizumab
0
1
1
-
-
-
Con inued on nex page
Juan José Nie o Fon a igo
130
Table 1 (Con inued)
FEV1 (%)
93 (74-
101)
91.8 (73-
102)
93 (74-
102)
-
-
-
FEV1/FVC (%)
74.5 (64-
83)
73.5 (65-
81)
74.2 (65-
82)
-
-
-
Lymphocy es,
cells/μL
2480
(1935-
2835)
2150
(1770-
2680)
2310
(1848-
2765) d
1941.5
(1615-
2372)
1892.3
(1585-
1892)
1912.7
(1594-
2362) d
Eosinophils,
cells/μL
327 (154-
502)d
326 (182-
492)d
327 (175-
494)d
173 (110-
268)d
133 (97-
198) d
152 (103-
218) d
Monocy es,
cells/μL
501 (414-
658)
420 (306-
518)d
444 (349-
575)d
425 (350-
528)
318.8
(231-399)d
361 (271-
451)d
Neu ophils,
cells/μL
4458
(3202-
5617)
3851
(2814-
4859)
4045
(3003-
5232)
3286
(2645-
5685)
3399
(2812-
4472)
3395
(2776-
4809)
IgE, IU/mL
355 (87-
707)d
204 (89-
680)d
241 (90-
682) d
78 (19-
195) d
22 (7-55) d
34 (10-
95) d
CRP, mg/dL
0.09 (0.07-
0.42)
0.16 (0.07-
0.32)
0.15 (0.07-
0.34)
0.19 (0.1-
0.69)
0.16 (0.05-
0.39)
0.17 (0.07-
0.57)
TNF, pg/mL
9.4 (6.6-
12.0)
9.5 (7.6-
12.9)
9.4 (7.2-
12.3)
8.6 (7.3-
10.4)
10.5 (7.6-
13.2)
9.7 (7.4-
12.9)
Lep in, ng/mL
2.9 (1.3-
7.2)e
18.1 (11.2-
27.9)e
11.2 (3.1-
23.9)
2.9 (1.0-
8.0)e
13.0 (5.35-
26.9)e
8.1 (3.3-
20.0)
Abb e ia ions: BMI, body mass index; CRP, C- eac i e p o ein; FEV1, o ced expi a o y olume in he
i s second; FVC, o ced i al capaci y; TNF, umo nec osis ac o .
aValues a e exp essed as median (IQR), unless o he wise speci ied.
bWe eco ded he p o essional ac i i y o ~70% o pa ien s, dis ibu ed acco ding o he ollowing o de :
s uden s (14.8%), cons uc ion p o essionals (9.9%), housewi es (8.6%), adminis a i e o ice s (7.4%),
cleaning se ice (6.2%), heal h p o essionals (6.2%), wai e s (4.9%), educa o s (4.9%), a me s (2.5%),
salespe sons (2.5%), and ex ile wo ke s (2.5%).
cApa om he non egis e ed (8.6%), o me ly smoke (11.1%) and cu en smoke (29.6%) pa ien s,
hey we e mos ly nonsmoke s (50.6%).
dDi e ences be ween AAPs and HCs (Mann-Whi ney, P<.05).
eDi e ences be ween male and emale (Mann-Whi ney, P<.05).
CHAPTER II
131
The cha ac e is ics o his i s coho o pa ien s a e
summa ized in Table 1. In AAPs, he median o ced expi a o y
olume in he i s second (FEV1) (%) was 93 (74.5-102.2), while he
FEV1/ o ced i al capaci y (FVC) a io (%) was 74.2 (65.2-81.9).
As hma was mainly mode a e-pe sis en (71.6%), and pa ien s had an
alle gic disease, wi h posi i e skin p ick es eac ions agains
common alle gens. In addi ion, mos pa ien s li ed in u al a eas
(72%), al hough only a small pe cen age o hem we e a me s (Table
1). As expec ed, a signi ican g oup o AAPs had pe iphe al blood
eosinophilia (45% had >350/μL) and ele a ed o al IgE (Table 1).
The e was a posi i e co ela ion be ween eosinophils and IgE, bu no
be ween FEV1 and se um IgE in AAPs (Table 2). Pa ien s we e unde
di e en ea men s, we e mos ly nonsmoke s, and had well-
con olled as hma (Table 1).
Gi en he well-known associa ion be ween lep in and body
mass index (BMI) and he assumed co ela ion be ween he
de elopmen /wo sening o as hma and BMI, we also s udied hese
pa ame e s. Fi s , BMI in AAPs was posi i ely co ela ed wi h lep in
and C- eac i e p o ein (CRP), bu nega i ely associa ed wi h IgE and
bo h FEV1% and FEV1/FVC (Table 2). Second, lep in le els we e
gene ally 4 o 6– old highe in women, al hough no di e ences we e
de ec ed be ween HCs and AAPs (Figu e S1, Table 1). These indings
unde line he lack o associa ion be ween BMI and as hma. In
con as , some pa ame e s we e mo e ele a ed in men, including IgE,
basophil coun s, and sCD26 (da a no shown). The in luence o age
was also aken in o conside a ion, indica ing a posi i e co ela ion
be ween BMI and TNF and a nega i e in e dependence wi h IgE,
FEV1%, and FEV1/FVC; no associa ion wi h age was de ec ed o
sCD26 o sCD25 (Table 2).
Juan José Nie o Fon a igo
234
ele an as candida e bioma ke s o as hma pheno ypes diagnosis o
assessmen o as hma se e i y. This is he case, o example, o
IGFALS, p o ein AMBP, and HSPG2 in he case o AA, and CFI, o
CFH o NAA.
IGFALS belongs o a amily o p o eins p e iously linked o
as hma pa hogenesis ha includes IGF1 and IGF2 [40]. IGF1 is a key
ac o in as hma pa hogenesis, h ough he p omo ion o subepi helial
ib osis, in lamma ion, hype esponsi eness, and smoo h muscle cell
hype plasia in he ai ways [40]. Indeed, omalizumab (an an i-IgE
an ibody) [41], as well as o al glucoco icoids [42], dec ease he le els
o IGF-I. Bo h IGF1 and IGF2 exe hei biological e ec s h ough
he binding o IGF1R, bu IGF2 can be also seques a ed by
IGF2R/ca ion-independen mannose-6-phospha e ecep o , a high-
a ini y inhibi o y p o ein ha a enua es IGF2 signalling [43].
S ikingly, his las ecep o has been ela ed o CD26/DPP4 [44, 45],
a se ine pep idase in ol ed in as hma pa hogenesis [46]. The e o e,
he e a e many lines o e idence poin ing o an impo an ole o IGF
amily in alle gic as hma.
The IGF sys em is comple ed wi h se e al binding p o eins
(IGFBPs: 1-6) [47], a no el IGFBP3-speci ic ecep o (IGFBP3R)
[48], and IGFALS [47, 49]. Bo h IGFBPs and IGFALS appea o
in luence ee-IGF concen a ion in he ex acellula compa men ,
playing a ole on he bioa ailabili y o IGFs [40]. The o ma ion o a
high molecula weigh complex (IGFALS-IGF1-IGFBP) p e en s he
ex a asa ion o IGF-1, he IGF-1/IGFBPs p o eolysis, and he enal
elimina ion o IGF-1 [50]. Indeed, IGFALS de iciency esul s in a
d ama ic dec ease in IGF-1, IGF-2, and IGFBP3 le els [50, 51]. Apa
om IGF1 [40] and IGFBP3 [40], he p esen esul s show ha
IGFALS is ele a ed in AA, especially mode a e-se e e o ms, bu no
in NAA. Rega ding IGFBP3, his p o ein con ols AA in lamma ion
h ough IGF-dependen and IGF-independen (IGFBPR-media ed)
CHAPTER IV
235
mechanisms ha a ge he HIF/VEGF axis, TGFβ1 and TH2
cy okines p oduc ion, and NF-kB ac i a ion [40]. Ve aldi e al. also
desc ibed a ole in AA o IGFBP-3 h ough he p omo ion o
subepi helial ib osis [52]. Rega ding he ole o IGFALS in as hma,
his emains mos ly unknown, bu oge he wi h IGFs and IGFBPs his
p o ein could be an addi ional he apeu ic a ge o as hma
managemen .
O he possible bioma ke s o AA a e p o ein AMBP and
HSPG2. Al hough he i s one has no been ex ensi ely s udied in
as hma o ai way- ela ed diseases, a s udy o se um p o eome om
pa ien s wi h idiopa hic pulmona y ib osis (IPF) has shown dec eased
le els o AMBP in IPF pa ien s compa ed o HC, and he same
dec ease was obse ed o AHSG, a p o ein wi h a simila pa e n o
changes as AMBP in ou s udy [38]. Al hough he possible unc ion o
p o ein AMBP in AA is no known, his p o ein sha es he same
ch omosomal egion (9q32) as ORM1/AGP, a posi i e acu e phase
p o ein inc eased in AA (Table 3). Thus, i is p obable ha bo h
p o eins coo dina e hei le els and ha AMBP pa icipa es in he
acu e in lamma o y esponse. Indeed, AMBP and ORM1 le els, as
well as o he p o eins augmen ed in AA and R in ou s udy (e.g.,
AHSG o CD5L), ha e been ound inc eased in BALF o as hma ic
indi iduals 24 h a e segmen al alle gen challenge [53], highligh ing
he possible ole o hese se o p o eins in AA.
On he o he hand, oge he wi h hyalu onan (HA) o esican
(VCAN), HSPG2/pe lecan is an ex acellula ma ix molecule which
is deposi ed in he lamina e icula is and gene a es subepi helial
ib osis in he ai ways [54]. Basemen memb ane hickening has been
epo ed in as hma [55-58] and e en alle gic hini is [58, 59].
Mo eo e , HSPG2 is an ups eam egula o o genes (ACAN,
COL10A1, and FGFR3) con aining as hma-associa ed di e en ially
me hyla ed egions [60]. The e is a nega i e co ela ion be ween he
Juan José Nie o Fon a igo
236
le els o HSPG2 and ai way hype esponsi eness (PC20) [61], and his
p o ein has been linked o ib osis as well. Indeed, ma u e ib ocy es
[62] cons i u i ely p oduce VCAN, HA, COL3/5/6, ib onec in, and
HSPG2 [63, 64] and con ibu e o he subepi helial ib osis in as hma
[63]. Aligned wi h hese esul s and ou own da a showing highe
le els o HSPG2 in se um samples om AA and R pa ien s, ib ocy es
om as hma ic pa ien s exposed o TH2- (IL-4, IL-13) bu no TH17
(IL-17A) cy okines show an enhanced exp ession o HSPG2 and a
p o ib o ic pheno ype (i.e., ele a ed exp ession o HA, COL315,
VCAN and HSPG2) [65]. Changes in HSPG2 deposi ion could ha e
mechanis ic e ec s, bu also modula e he bioabili y o g ow h ac o s
(e.g., bFGF) o cy okines (e.g., IL-4, TGF-β, o GM-CSF). Taken
oge he , hese esul s could explain he inc ease o HSPG2 and
p o ein AMBP in ou g oup o MSAA and R pa ien s and hei
po en ial as a opic disease bioma ke s.
As hma and especially i s NAA pheno ype sha e ce ain
cha ac e is ics wi h au oimmune diso de s, like hei p e alence in
emale he p esence o au oan ibodies in highe equency han HC
[66-68]. I is well known ha he complemen sys em plays an
impo an ole in au oimmune diseases [69], bu also appea o ha e a
p oin lamma o y ole in as hma [70-72]. Howe e , he complemen
ac o s in ol ed emain unclea . Acco ding o he ini ial s imuli, h ee
pa hways (classical, al e na i e, and lec in) ha e been desc ibed o
complemen ac i a ion. Ou esul s suppo ha complemen ac o s I
and H, wo egula o y p o eins ha con ol he excessi e ac i a ion o
he al e na i e complemen pa hway [73, 74], a e up egula ed in NAA.
CFI is a se ine p o ease ha a ou s he deg ada ion o C4b,
diminishes he le els o C3-con e ase (C4bC2a) and p e en s
in lamma ion a ising om complemen ac i a ion [75]. Complemen
ac o H, o i s pa , is a co ac o o CFI [73, 76] and i is also known
as ad enomedullin binding p o ein (AMBP-1) [77, 78].
CHAPTER IV
237
Ad enomedullin is an an i-in lamma o y pep ide ha supp esses TH2
in lamma ion and main ains issue in eg i y in an OVA-induced model
o as hma [79]. Mo eo e , Weiszhá e al. ha e ound augmen ed
le els o CFH in as hma ic spu um and a co ela ion wi h se e i y and
he loss o lung unc ion [80]. The inc ease o CFI and CFH in ou
g oup o NAA pa ien s could be ela ed o a mechanism o e ade he
immune esponse, as i happens wi h some pa hogens [81-83], which
could also explain he highe se e i y o NAA.
Addi ionally, he le els o he se ine p o ease MASP1 a e also
inc eased in NAA. MASP1 pa icipa e in he lec in pa hway o
complemen ac i a ion, bu his p o ease has also subs a es ha
belong o he coagula ion cascade [84]; we ha e ound some o hem
al e ed in as hma as well, such as kininogen, FXIII, o F2. Mo eo e ,
ano he subs a e o MASP1 is he p o ease ac i a ion ecep o 4
(PAR4) in endo helial cells, whose ac i a ion leads o he p oduc ion
o IL-6 and IL-8, he las one a chemo ac ic molecule o neu ophils
[84]. This is ele an , as neu ophils ha e been ela ed o some NAA
endo ypes [85] and highe disease se e i y [86, 87]. Besides, C1 and
C1s p o eins (classical complemen pa hway) a e also modi ied in ou
g oup o as hma pa ien s. The e o e, ou esul s poin ou ha all he
complemen ac i a ion pa hways (classical, al e na i e, and lec in)
appea o be in ol ed in as hma, especially in he NAA pheno ype.
Like o he s udies, he p esen wo k has also some limi a ions.
Fi s , no all he p o eins we e de ec ed in all he g oups since i is
known ha in LC-MS/MS assays is necessa y o pe o m mo e han 3
echnical eplica es in o de o de ec all p o eins in a sample
(comple eness o analysis issue) [88]. A second p oblem is ela ed o
he "pooled sample" analysed wi h he isoba ic label 121. This sample
has been in oduced in all analy ical se ies o allow he no maliza ion
o he signals and he adjus men o he in e se ial a iabili y.
Howe e , he use o his ype o e e ence samples makes di icul o
Juan José Nie o Fon a igo
238
de ec low abundance p o eins, which in addi ion makes he
no maliza ion p ocess di icul . Thi d, ano he issue is he whole
numbe o p o eins de ec ed, especially low abundance species,
despi e he use o lipop o ein-deple ion, CPLLs-based en ichmen , and
e e se phase LC. The e o e, an addi ional s ep o pep ides-
ac iona ion using s ong ca ion exchange (SCX) ch oma og aphy
be o e LC-MS/MS analysis would be o bene i . Finally, some o ou
esul s a e s ill p elimina y, especially hose e e ing o he p o eins
de ec ed wi h di e en ial abundance by LS-MS/MS which ha e no
been alida ed by ELISA ye .
In conclusion, a sho gun/bo om-up/non- a ge ed me hodology
has been de eloped o he quan i a i e analysis o he p o eome o
medium-low abundance in se um samples om pa ien s wi h hini is
and a ious as hma ic pheno ypes. This p o ocol is based on he
elimina ion o lipop o eins by he LRA esin, p o ein en ichmen o
medium and low abundance by CPLLs applica ion, labelling wi h
iTRAQ 8plex eagen s, and analysis by LC-MS/MS. Ou app oach
de ec ed se e al di e en ially abundan p o eins, and he e o e
po en ial non-in asi e bioma ke s o as hma pheno ypes (e.g.,
IGFALS, P o ein AMBP, HSPG2 o AA, and CFI o NAA) o
se e i ies (e.g., IGFALS o MSAA). In any case, u u e s udies will
be necessa y o ge a be e unde s anding o as hma pa hophysiology,
o e alua e he diagnos ic pe o mance o hese new bioma ke s, and
o ansla e his knowledge in o he clinic o ge a be e he apeu ic
esponse and p ognosis.
CHAPTER IV
239
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34. Du E, Yu J, K asinska KM, Ca e LA, Ya es JR, Tes a JE, e
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36. Izbicka E, S eepe RT, Michalek JE, Louden CL, Diaz A 3 d,
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39. O'Neil SE, Si kauskiene B, Babusy e A, K isiukeniene A,
S a inskai e-Bieksiene K, Sakalauskas R, e al. Ne wo k
analysis o quan i a i e p o eomics on as hma ic b onchi: e ec s
o inhaled glucoco icoid ea men . Respi Res. 2011;12:124.
40. Lee H, Kim SR, Oh Y, Cho SH, Schleime RP, Lee YC.
Ta ge ing insulin-like g ow h ac o -I and insulin-like g ow h
ac o -binding p o ein-3 signaling pa hways. A no el he apeu ic
app oach o as hma. Am J Respi Cell Mol Biol.
2014;50(4):667-77.
41. Bulu I, Ozseke ZF, Coskun A, Se ese M, Unsal I. P egnancy-
associa ed plasma p o ein-A (PAPP-A) le els in pa ien s wi h
se e e alle gic as hma a e educed by omalizumab. J As hma.
2017;55(10):1116-21.
42. F ys yk J, Schou AJ, Heuck C, Vo um H, Lyngholm M,
Fly bje g A, e al. P ednisolone educes he abili y o se um o
ac i a e he IGF1 ecep o in i o wi hou a ec ing ci cula ing
o al o ee IGF1. Eu J Endoc inol. 2012;168(1):1-8.
43. B own J, Jones EY, Fo bes BE. In e ac ions o IGF-II wi h he
IGF2R/ca ion-independen mannose-6-phospha e ecep o
mechanism and biological ou comes. Vi am Ho m.
2009;80:699-719.
44. Ikushima H, Munaka a Y, Ishii T, Iwa a S, Te ashima M,
Tanaka H, e al. In e naliza ion o CD26 by mannose 6-
phospha e/insulin-like g ow h ac o II ecep o con ibu es o T
cell ac i a ion. P oc Na l Acad Sci U S A. 2000;97(15):8439-44.
45. Ikushima H, Munaka a Y, Iwa a S, Ohnuma K, Kobayashi S,
Dang NH, e al. Soluble CD26/dipep idyl pep idase IV enhances
ansendo helial mig a ion ia i s in e ac ion wi h mannose 6-
Juan José Nie o Fon a igo
250
En ichmen o low abundance p o eins: P o eomine TM.
To en ich samples in low abundance p o eins, P o eoMine ™ P o ein
En ichmen La ge-Capaci y Ki (Bio-Rad, ca 163-3007) was used
ollowing comme cial guidelines. In b ie , 1 mL om each se um
sample was loaded in each column and subsequen ly incuba ed unde
s i ing condi ions (2 h, RT). A e incuba ion, he samples we e
washed 4 imes and hen elu ed by using a bu e p o ided by he ki .
All cen i uga ions we e pe o med a 1000 xg, 1 min, RT. Th ee
ac ions we e collec ed and mixed in a single sample. Then, in o de
o elimina e con aminan s such as he sal s o he P o eomine elu ion
bu e , he 2-D Cleanup Ki (GE Heal hca e, Ca No. 80-6484-51) was
used also ollowing comme cial guidelines. Finally, he pelle
ob ained was dilu ed in o 30 μL o 0.5M TEAB 6M u ea o ul e io
analysis.
P o ein quan i ica ion
Two me hodologies we e used acco ding o he manu ac u e 's
p o ocol. The i s one, Pie ceTM BCA P o ein Assay Ki (The mo
Fishe , # 23225), was used o p o ein quan i ica ion a e he
delipida ion p ocess. A calib a ion line was gene a ed using di e en
concen a ions o bo ine se um albumin (BSA) in phospha e bu e
(10 mM, pH 7.0): 0-2 mg/mL. The es samples we e also dilu ed in
phospha e bu e (1:60 dilu ion). In a 96-well pla e, 25 μL o he blank
(PBS), samples and s anda ds we e added. Each condi ion was
analysed a leas in duplica e. Subsequen ly, 200 μL o wo king
eagen (50 A: 1 B) was added and incuba ed a 37 °C, 20 min.
Finally, he eading was made a 550 nm in a pla e eade (model 680,
Bio-Rad).
The quan i ica ion a e Clean-up equi ed using he CB-X ki
(786-12X, G-Biosciences). Fi s ly, o p ecipi a e p o eins om
CHAPTER IV
251
samples, 1 mL o p e iously cooled (-20 ° C) CB-X ™ eagen was
added o 50 µL o each sample (dilu ed 1:10 in TEAB bu e ) and
mixed by o exing. The samples we e hen cen i uged a 16,000 xg
o 5 min, he supe na an was emo ed, and 50 μL o CB-X ™ I and
50 μL CB-X ™ II solubiliza ion bu e s we e sequen ially added. The
p o eins we e dissol ed by o ex, and 1 mL/sample o CB-X ™ es
dye was added. A e o exing and incuba ion o 5 min a RT, he
samples we e ans e ed o 96-well pla es and he abso bance was
ead a 595 nm on a mic opla e eade (Labsys ems Mul iskan MS). A
s anda d line wi h BSA (0.2-1 mg/mL) dilu ed in 0.5M TEAB bu e
con aining 6M u ea was used o de e mine he p o ein concen a ion.
Reduc ion, alkyla ion, and ypsiniza ion
Fi y μg o p o ein om each sample in 20 μL o 0.5M TEAB 6M
u ea we e educed and alkyla ed. Fo his pu pose, 2 μL o educing
agen (TCEP) we e added o each ube, ollowed by 1 h incuba ion a
RT. Nex , he samples we e alkyla ed by adding 1 μL/sample o
MMTS, ollowed by 10 min incuba ion a RT. Subsequen ly, 123 μL
o 1M TEAB we e added o educe he u ea concen a ion, and
ypsiniza ion was pe o med (1 μg/µL ypsin; 4326682, Sciex) a 37
° C o e nigh . To s op he eac ion (pH < 6.0), 4 μL o ace ic acid was
added. Finally, all samples we e lyophilized.
iTRAQ labelling
The iTRAQ® Reagen -8PLEX Mul iplex Ki (Sigma-Ald ich) was
used acco ding o he manu ac u e 's p o ocol. Fi s o all, he samples
we e dissol ed in 30 μL o 0.5M TEAB. Hal o he con en s o each
ial (10 μL) we e dilu ed in 25 μL o isop opanol, and hen added o
each pep ide samples wi h he ollowing labels: MSAA, 113; IMAA,
114; R, 115; MSNAA, 116; IMNAA, 117; HC, 119; PS, 121. PS
sample is a pool o all he se um samples used o no malize he da a
Juan José Nie o Fon a igo
252
be ween he di e en MS analy ical se ies. A e 2h incuba ion, he
se en labelled pep ide samples we e collec ed in a single ube and
lyophilized. This p ocedu e was pe o med wice, one o each
biological eplica e (pool A and pool B).
nanoLC/MS-MS iden i ica ion and quan i ica ion o pep ides
The samples we e analysed in he S uc u al, P o eomic and Genomic
De e mina ion Se ice, Cac i Mass Spec ome y Uni , Uni e si y o
Vigo. Fi s ly, he pep ide samples we e econs i u ed in solu ion A
(0.1% o mic acid (FA) in wa e ) and desal ed by Zip-Tips C18
(Millipo e). Secondly, pep ides we e dispensed o nano- e e se phase
EASY-Sp ay Columns (PepMap® RSLC, C18, 2µm, 100 Å, 75µm x
500mm, The mo Fishe Scien i ic) moun ed in he P oxeon EASY-
nLC 1000 UHPLC (The mo Fishe Scien i ic), and we e elu ed wi h a
g adien o solu ion B (ACN) o 5-30% (240 min), 30%-90% (10min),
and 90%-5% (17min). The nanoLC sys em is coupled online o an
LTQ-O bi ap ELITE (The mo Fishe Fishe ) used in a da a
dependen and posi i e ion mode. A ull MS scan was ca ied ou
om 380−1600 m/z wi h a esolu ion a 12000. HCD- agmen a ion
was used and he MS/MS scan was accomplished wi h op 15, a 28%
no malized collision ene gy, wi h a esolu ion a 30000, a dynamic
exclusion ime a 30 s, a minimum signal equi ed a 1000, and an
isola ion wid h a 1.50 Da. Th ee echnical eplica es we e pe o med
om each biological eplica e.
MS da a analysis
P o eome Disco e e ( e sion 2.1.1.21) was used o p o ein
iden i ica ion and iTRAQ quan i ica ion. HCD spec a we e analysed
using Seques HT wi h Pe cola o alida ion. Spec a we e sea ched
agains he la es UniP o KB Release and common con aminan
sequences (e.g., ypsin, o ke a ins). The pep ide mass ole ance was
CHAPTER IV
253
10 ppm and he agmen mass ole ance 0.020 Da. As s a ic
modi ica ions, he iTRAQ 8-plex N- e minus, and
ca bamidome hyla ion o cys eine we e speci ied, and as a iable
modi ica ions, he oxida ion o me hionine, he iTRAQ 8-plex o
y osine and lysine, he ca bamyla ion o lysine, and he N- e minus
ace yla ion. The in ensi ies acqui ed in MS/MS we e globally
no malized on p o ein median. Then, all epo e in ensi ies we e
no malized by he epo e in ensi y o he pooled sample (PS, 121).
The abundance o each p o ein was calcula ed as an a e age
abundance o all i s de ec ed pep ides. A 1.3 old change (down-
egula ion, < 0.77; up- egula ion, ≥1.3) in iTRAQ a ios, as well as a
p- alue less han 0.05, was used o iden i y di e en ially exp essed
p o eins be ween he di e en g oups o s udy.
S a is ical and bioin o ma ic analysis o he da a
S a is ical da a analyses we e ca ied ou using G aphPad P ism
e sion 6.00 o Windows (G aphPad So wa e, La Jolla Cali o nia
USA, www.g aphpad.com). Quan i a i e da a compa isons be ween
he di e en sample g oups we e pe o med by K uskal–Wallis one-
way analysis o a iance ollowed by Dunn's mul iple compa ison
es s. The ecei e ope a ing cha ac e is ic (ROC) analysis was also
pe o med wi h G aphPad P ism. A p < 0.05 was conside ed o
s a is ical signi icance. Bioin o ma ic analysis: GO anno a ion, GO
e ms o e ep esen a ion es , and Reac ome pa hway
o e ep esen a ion analyses agains he plasma p o eome da abase
(Ve sion 2015-02-02) (www.plasmap o eomeda abase.o g/) we e
pe o med in PANTHER Classi ica ion Sys em (PANTHER e sion
13.1. Released 2018-02-03). A p ospec i e s udy o all he po en ial
physical o unc ional p o ein-p o ein in e ac ions was also pe o med
by means o he Sea ch Tool o he Re ie al o In e ac ing Genes
(STRING) (Ve sion: 11.0, www.s ing-db.o g)
Juan José Nie o Fon a igo
254
SUPPLEMENTARY TABLES
Supplemen a y Table 1. Cha ac e is ics o he subjec s om pool A.
MSAA
IMAA
MSNAA
IMNAA
R
HC
N
25
26
22
24
22
16
Age (mean
( ange))
39 (21-
64)
37 (20-
64)
53 (24-
68)
53 (29-
72)
31 (18-
46)
45 (22-
58)
Sex (M/F)
11/14
15/11
6/16
2/22
12/10
5/11
Disease con ol:
Yes
18
26
13
22
22
-
No
7
0
9
2
0
-
Baseline
ea men :
ICS-LABA
24
16
18
17
0
-
ICS
0
7
1
0
0
-
OCS
0
0
0
0
0
-
An ileuko ienes
14
8
10
5
3
-
An icholine gic
5
3
13
2
0
-
Ro lumilas
0
0
0
0
0
-
P ednisone
0
0
2
0
0
-
FEV1 (%)
97.0
(85.5-
111.5)$
102.5
(94.5-
110.5)
70.6
(60.0-
88.8)#&
112.0
(98.0-
120.5)
109.5
(99.5-
121.5)
-
FEV1/FVC (%)
76.5
(70.7-
79.6)#
79.6
(72.6-
82.7)
67.6
(58.1-
77.1)#
76.6
(73.7-
80.6)#
84.6
(79.1-
88.3)
-
Neu ophils (103
cells/μL)
3.14
(2.28-
4.19)
3.94
(3.21-
4.45)
3.61
(2.98-
4.77)
3.16
(2.49-
3.53)
3.73
(2.86-
4.90)
3.68
(2.25-
4.34)
Lymphocy es
(103cells/μL)
1.89
(1.37-
2.12)
1.98
(1.74-
2.21)
2.07
(1.64-
2.50)
1.87
(1.49-
2.16)
2.20
(1.95-
2.71)
2.04
(1.48-
2.45)
Con inued on nex page
CHAPTER IV
255
Supplemen a y Table 1 (Con inued)
Monocy es (103
cells/μL)
0.33
(0.29-
0.42)
0.41
(0.31-
0.46)
0.37
(0.32-
0.50)
0.32
(0.29-
0.43)
0.40
(0.34-
0.50)
0.42
(0.33-
0.62)
Eosinophils (103
cells/μL)
0.36
(0.22-
0.55)*
0.24
(0.16-
0.34)
0.31
(0.18-
0.46)
0.25
(0.13-
0.37)
0.22
(0.13-
0.38)
0.20
(0.09-
0.30)
Basophils (103
cells/μL)
0.04
(0.03-
0.05)
0.04
(0.03-
0.05)
0.03
(0.02-
0.05)
0.03
(0.02-
0.04)
0.03
(0.02-
0.06)
0.03
(0.02-
0.05)
ESR (1h; mm)
10.0
(6.5-
17.5)
5.5
(2.0-
17.2)
15.0
(9.7-
25.5)*#
12.0
(7.2-
20.5)
4.5
(2.0-
14.8)
10.0
(7.0-
11.0)
IgE (IU/mL)
122.0
(62.5-
316.0)*$
174
(79-
241)*$
38 (10-
96)
17 (5-
32)
76 (24-
283)
11 (3-
45)
IgG (mg/dL)
1100
(951-
1260)
1075
(952-
1195)
1045
(951-
1313)
1030
(794-
1140)
1005
(943-
1288)
980
(842-
1090)
IgG1 (mg/dL)
630
(548-
807)
681
(594-
732)
658
(495-
761)
550
(447-
605)#
673
(546-
775)
-
IgG2 (mg/dL)
374
(285-
448)
365
(276-
469)
293
(213-
459)
338
(246-
426)
337
(268-
405)
-
IgG3 (mg/dL)
32 (22-
45)
32 (21-
48)
42 (22-
55)
33 (28-
57)
39 (29-
59)
-
IgG4 (mg/dL)
50 (27-
87)
47 (23-
96)
65 (39-
125)
28 (10-
60)
23 (14-
51)
-
IgA (mg/dL)
223
(159-
270)
226
(176-
295)
204
(147-
262)
209
(162-
251)
166
(100-
262)
250
(121-
333)
IgM (mg/dL)
113 (76-
166)
97 (64-
153)
115 (93-
172)
101 (70-
133)
109
(67-
171)
88 (71-
118)
HC, heal hy con ols; IMAA, in e mi en -mild alle gic as hma ics; IMNAA, in e mi en -
mild non-alle gic as hma ics;MSAA, mode a e-se e e alle gic as hma ics; MSNAA,
mode a e-se e e non-alle gic as hma ics; R, hini is pa ien s.
Da a a e p esen ed as median alue (IQR1-3), unless o he wise exp essed.
S a is ical signi icance is shown: *Disease s HC; #As hma s R; $AA s NAA; &Mode a e-
se e e s In e mi en -mild as hma. K uskal-Wallys es ollowed by Dunn´s mul iple
compa ison es . p < 0.05
Juan José Nie o Fon a igo
256
Supplemen a y Table 2. Cha ac e is ics o he subjec s om pool B.
MSAA
IMAA
MSNAA
IMNAA
R
HC
N
24
27
21
23
21
16
Age (mean
( ange))
39 (18-
68)
36 (21-
66)
54 (28-
67)
51 (34-
72)
39 (24-
55)
41 (27-
61)
Sex (M/F)
12/12
10/17
7/14
6/17
8/13
9/7
Disease con ol:
Yes
12
27
17
23
21
-
No
12
0
4
0
0
-
Baseline
ea men :
ICS-LABA
23
17
21
18
18
-
ICS
1
8
0
3
0
-
OCS
1
0
0
0
0
-
An ileuko ienes
11
10
10
4
7
-
An icholine gic
8
0
12
0
0
-
Ro lumilas
0
0
1
0
0
-
P ednisone
0
0
3
0
0
-
FEV1 (%)
89.5
(65.7-
99.2)#
100.0
(92.0-
108.0)
77.0
(66.0-
96.0)#&
105.0
(97.0-
113.0)
102.5
(96.2-
115.0)
-
FEV1/FVC (%)
69.8
(55.9-
78.6)#
78.9
(72.3-
80.7)
67.2
(61.0-
76.9)#
77.0
(72.5-
80.1)
83.2
(78.1-
86.1)
-
Neu ophils (103
cells/μL)
3.90
(2.93-
4.61)
3.55
(3.14-
4.06)
3.56
(3.31-
4.31)
3.78
(3.19-
4.53)
3.54
(2.97-
3.86)
3.02
(2.40-
3.69)
Lymphocy es
(103cells/μL)
1.94
(1.81-
2.81)
2.02
(1.59-
2.29)
1.83
(1.51-
2.67)
1.96
(1.67-
2.28)
2.13
(1.65-
2.48)
1.95
(1.53-
2.57)
Monocy es (103
cells/μL)
0.31
(0.30-
0.47)
0.31
(0.40-
0.50)
0.43
(0.32-
0.52)
0.37
(0.30-
0.51)
0.34
(0.28-
0.46)
0.39
(0.32-
0.47)
Con inued on nex page
CHAPTER IV
257
Supplemen a y Table 2 (Con inued)
Eosinophils (103
cells/μL)
0.35
(0.22-
0.50)*
0.29
(0.20-
0.45)*
0.28
(0.15-
0.42)*
0.24
(0.17-
0.35)
0.22
(0.14-
0.28)
0.12
(0.10-
0.22)
Basophils (103
cells/μL)
0.04
(0.03-
0.06)
0.04
(0.03-
0.05)
0.04
(0.02-
0.06)
0.03
(0.02-
0.05)
0.04
(0.02-
0.05)
0.04
(0.03-
0.05)
ESR (1h; mm)
7.0 (4.0-
13.7)
7.0
(4.0-
12.0)
9.0 (4.0-
20.0)
11.0 (8.0-
20.0)*
11.0
(2.5-
15.5)
3.0
(2.0-
8.5)
IgE (IU/mL)
209.0
(72.5-
523.3)*$
122
(50-
304)$
44 (16-
104)
25 (11-
43)
59 (27-
99)
15 (12-
144)
IgG (mg/dL)
1030
(898-
1100)
1100
(923-
1250)
1065
(915-
1203)
1010
(880-
1175)
1045
(879-
1180)
1080
(904-
1290)
IgG1 (mg/dL)
644
(555-
725)
651
(538-
768)
607
(474-
733)
549
(455-
654)
603
(521-
692)
-
IgG2 (mg/dL)
333
(257-
383)
331
(279-
409)
345
(318-
436)
347
(273-
393)
312
(258-
426)
-
IgG3 (mg/dL)
30 (20-
43)
39 (31-
56)
36 (27-
64)
36 (32-
62)
31 (26-
38)
-
IgG4 (mg/dL)
52 (38-
86)
47 (23-
68)
37 (21-
64)
32 (17-
67)
41 (29-
54)
-
IgA (mg/dL)
200
(170-
279)
254
(172-
315)
238
(170-
296)
197
(147-
270)
214
(135-
275)
332
(148-
375)
IgM (mg/dL)
93 (65-
143)
106
(85-
133)
111 (72-
149)
123 (79-
154)
112
(78-
160)
102
(86-
103)
HC, heal hy con ols; IMAA, in e mi en -mild alle gic as hma ics; IMNAA, in e mi en -
mild non-alle gic as hma ics;MSAA, mode a e-se e e alle gic as hma ics; MSNAA,
mode a e-se e e non-alle gic as hma ics; R, hini is pa ien s.
Da a a e p esen ed as median alue (IQR1-3), unless o he wise exp essed.
S a is ical signi icance is shown: *Disease s HC; #As hma s R; $AA s NAA; &Mode a e-
se e e s In e mi en -mild as hma. K uskal-Wallys es ollowed by Dunn´s mul iple
compa ison es . p < 0.05
Juan José Nie o Fon a igo
258
SUPPLEMENTARY FIGURES
Supplemen a y Figu e 1. T ea men wi h LRA esin educes se um lipop o ein
le els. A olume o 300 μL o se um was ea ed wi h di e en concen a ions o
LRA esin (0-100 mg / mL) o e nigh a 4 ° C in o a ion. An SDS-PAGE (15% T
sepa a ing gel) is shown in which 30 μg o p o ein belonging o he ac ion no
e ained by he esin (channels 2-7) has been loaded. Channel 1 co esponds o
molecula weigh ma ke s, and channel 9 o he e ained ac ion (R).
Juan José Nie o Fon a igo
266
like mCD14, his glycop o ein p esen s a memb ane and a soluble
(sCD26) iso o m. Mo eo e , e en hough CD26 does no ancho o
he plasma memb ane h ough a glycophospha idylinosi ol linkage as
CD14 does, his molecule p esen s an ex emely sho cy oplasmic
egion o 6 esidues ha equi es he pa icipa ion o o he molecules
(e.g., CARMA1) o downs eam signal ansduc ion [252]. Likewise,
CD26 exp ession is a he con ined o CD4+ T lymphocy es in a
manne compa able o CD14 and monocy es. Finally, CD26/DPP4
belongs o a sub amily o se ine p o eases ( he S9B) wi h se e al
membe s showing an associa ion wi h as hma: CD26/DPP4 i sel and
DPP10. Based on all hese poin s, Chap e s II and III we e aimed o
s udy he exp ession pa e n o CD26 in di e en lymphocy e
subpopula ions and hei implica ions in bo h AA and NAA, as well as
disease se e i y. Because o he complexi y o his issue, we ca ied
ou a comp ehensi e e iew (see appendix I) aimed o p o ide a
s uc u ed o e iew o he nume ous unc ions o CD26 and i s
implica ions in as hma pa hogenesis and p og ession. Up un il ha
ime, he e was only one e iew pape speci ically co e ing he
ela ionship be ween CD26, T cells, and as hma [412].
CD26 is a p olyl oligopep idase ha belongs o he se ine
p o ease amily (S9B) [243]. CD26 is widely dis ibu ed o e se e al
cell ypes and issues. Howe e , exp ession o CD26 is especially
associa ed wi h immune cells, such as g anulocy es, monocy es, B
cells, and T lymphocy es (pa icula ly CD4+ T cells) [251]. Wi hin
CD4+ T lymphocy es, i s exp ession is highe in Te cells compa ed
o T eg cells [258]. In addi ion, Bengsch e al. used low cy ome y
da a and FACS so ing o shown ha he exp ession o CD26 amongs
Te cells is qui e a iable, acco ding o he ollowing o de : TH17>>
TH1> TH2 [259, 413, 414]. These esul s ha e been con i med in
i o in Chap e II, whe e CD4+CD25- Te cells we e ac i a ed and
di e en ia ed owa ds a TH1-like (IL-12), TH2-like (IL-4), and TH17-
GENERAL DISCUSSION
267
like (IL-1β, IL-6, and IL-23) pheno ypes. Ou esul s show he same
di e en ial exp ession o CD26 in Te cells (TH17>> TH1> TH2)
ha Bengsch e al. desc ibed p e iously.
As abo e commen ed, CD26 can also be ound in he
ex acellula space (e.g., se um/plasma, BALF, CSF) as sCD26 [286].
This soluble e sion o CD26 is eleased by CD26+ cells h ough
enzyma ic-shedding [291] o p oduc ion o CD26+ esicles
(h p://www.exoca a.com). I has been shown ha isce al a elease
sCD26 as an adipokine [415], bu CD4+ T cells emain as he mos
likely sou ce [286, 293, 294]. Indeed, ou in i o assays (Chap e II)
e eal a s ong posi i e co ela ion be ween he exp ession o CD26
(mean luo escen in ensi y/MFI) on di e en TH lymphocy e subse s
(TH17>>TH1>TH2>T eg) and he soluble DPP4 ac i i y (a bona ide
indica o o sCD26 le els) in cul u e supe na an s. The e o e, i his
was happening in i o, i was expec ed ha se um sCD26 le els we e
in luenced by he numbe o CD4+ T cells o mi o he p edominan
subpopula ion o CD4+ T cells (e.g., TH1, TH2, TH17) in he di e en
as hma pheno ypes/endo ypes. In o he wo ds, we we e expec ing a
coo dina ed and a iable ele a ion o bo h CD26 and sCD26 in
as hma, highe o lowe depending on he disease pheno ype and he
p e ailing TH subse .
Wi h his in mind, we unde ook a sys ema ic analysis o he
exp ession o CD26 on TH lymphocy es om AA, NAA, hini is, and
heal hy indi iduals, as well as an e alua ion o sCD26 le els in se um
samples (Chap e s II and III). Fi s ly, as Lun and cowo ke s [246]
p e iously showed in AA (and as expec ed om an ac i a ion ma ke )
[252, 416, 417], he e was an inc ease in he exp ession o CD26 on
CD4+ lymphocy es om AA an NAA pa ien s (Chap e s II and III).
Howe e , inconsis en ly wi h he highe sCD26 le els de ec ed by Lun
e al. in AA [246], in he i s s udy (Chap e II) i was shown a
diminished concen a ion o sCD26 in se um samples om wo
Juan José Nie o Fon a igo
268
coho s o AA pa ien s compa ed o heal hy subjec s. Mo eo e , he
same happened o sCD25 (Chap e II), ano he soluble iso o m o an
ac i a ion ma ke : CD25/IL-2Rα [220-224]. A simila down-
modula ion o sCD26 was ound in NAA pa ien s (Chap e III) and
eosinophilic pneumonia [418], whe eas o he au ho s did no de ec
any di e ence in sCD26 le els in child en wi h as hma [303]. The
di e en esul s epo ed by Lun and cowo ke s [246] ega ding
sCD26 le els in AA as compa ed wi h ou indings in Chap e II may
ha e se e al explana ions. On he one hand, he di e en male/ emale
p opo ions be ween AA pa ien s and con ols in he pape o
Saman ha Lun, as sCD26 concen a ion is highe in males. On he
o he hand, he mo e ac i e disease s a us o pa ien s in hese s udies
compa ed o ou coho o AA pa ien s, which we e in a s able phase.
Ou in i o expe imen s (Chap e II) suppo ha CD4+CD25-
Te cells p ima ily sec e e CD26 upon TCR igge ing. In he same
line, Te cells up- egula e and elease CD25 a e being ac i a ed
[419]. The e o e, he small educ ion de ec ed o bo h soluble
ma ke s (sCD26 and sCD25) in se um samples om AA pa ien s may
be indica i e o he expansion o a CD25-/lowCD26-/low TH
subpopula ion in his disease. Indeed, in Chap e II we desc ibe, o
AA pa ien s, a signi ican inc ease in he pe cen age o a small
popula ion o CD4+CD25-/lowCD26-/lowCD127-/low TH cells; we ha e
called his subse “ iple low” o Tlow cells. These esul s ha e been
con i med in Chap e III, showing ha CD26-/low TH cells belong o a
g oup o CD4+ T lymphocy es ha ha e los he exp ession o se e al
ma ke s: CD27, CD28, CCR7, and CD127. This ac highligh s he
ad anced di e en ia ion s age (TEM o TEMRA) o his subse . Thus, in
line wi h o he wo ks [262], he numbe o CD26 molecules on TH
cells allows desc ibing lymphocy es wi h a TN (CD26in ;
CD45RA+CCR7+CD28+), a TCM (CD26high; CD45RA-CCR7+CD28+),
o a highly di e en ia ed pheno ype (CD26-/low; CD45RA+/-CCR7-
GENERAL DISCUSSION
269
CD28-): i.e., TEM o TEMRA. Pe haps some o hese cells s op
eci cula ing be ween blood/lympha ic sys em and non-lymphoid
issues and en e he lung issues o become issue- esiden memo y T
cells [420, 421].
Resea ch in as hma is mos ly ocused on CD4+ T cells and he
AA pheno ype. Howe e , CD4- lymphocy es (i.e., B cells, CD8+ T
cells, NKT, γδ-T lymphocy es, and NK cells) could ha e an ac i e
ole in as hma pa hogenesis and be ele an o as hma immune-
pheno yping. Along wi h his line o easoning, he wo k shown in
Chap e III has ocused on he possibili y ha CD4- subpopula ions
may be al e ed amongs pa ien s wi h hini is o di e en as hma
pheno ypes (AA and NAA) o se e i ies. In his way, ha s udy d aws
a pa allel be ween CD4+ T cells in AA and CD4- lymphocy es in
NAA, as i was also ound an expansion o CD26-/low subpopula ions
in CD4- lymphocy es om NAA pa ien s ha explains why se um
sCD26 le els a e also educed in his pheno ype. Mo eo e , in
Chap e III i was also shown ha his expansion in NAA (compa ed
o AA) can be mos ly asc ibed o CD26-CD4- γδ-T lymphocy es.
Mos o ci cula ing γδ-T cells a e V𝛿2/Vγ9+ cells [119], which
display a simila dis ibu ion o naï e-memo y popula ions as CD4+
αβ T cells: TN (CD45RA+CD27+), TCM (CD45RA-CD27+), TEM
(CD45RA-CD27-), and TEMRA (CD45RA+CD27-) [122, 135].
The e o e, hese indings a e compa ible wi h he desc ip ion in
Chap e III o CD26- (TEM/TEMRA), CD26in (TN), and CD26high (TCM)
γδ-T subse s. Fu he mo e, γδ-T cells a e he majo p oduce s o ea ly
IL-17 [134]. The e o e, he inc ease o a popula ion o CD26- γδ-T
lymphocy es wi h a TEM/TEMRA pheno ype in NAA (Chap e III) wi h
p e e en ial p oduc ion o TH17 o TH1 cy okines [422] migh explain
he in e se ela ionship be ween γδ-T cells and B cells (Chap e III)
and he enhanced ai way in lamma ion in NAA [309]. The numbe o
pe iphe al blood γδ-T cells was p e iously ound dec eased in AA
Juan José Nie o Fon a igo
270
[119, 133, 423], whe eas no changes o e en an inc ease was ound in
BALF o b onchial biopsies om AA pa ien s [142, 143]. We did no
measu e he numbe o γδ-T cells in heal hy subjec s, bu we ound in
AA a sligh ly lowe pe cen age (2.9%) han he one p e iously
desc ibed in he li e a u e o heal hy subjec s (4.1%) [119]. In any
case, mo e s udies a e needed o alida e hese indings because, o
ou knowledge, his is he i s wo k add essing he le els o CD26 in
γδ-T cells in pa ien s wi h NAA.
To summa ize, da a om he s udies shown in Chap e II and
III p o ide e idence ha bo h as hma pheno ypes sha e common
immunopa hological mechanisms, wi h expansion o CD26-/low subse s
in AA (CD4+ Tlow) and NAA (CD4- T cells; γδ-T lymphocy es) and
down-modula ion o addi ional su ace molecules (CD27, CD28, IL-
7Rα/CD127, CCR7) o p oduce di e en ia ed e ec o subse s and
ex acellula sCD26 educ ion. Decline in sCD26 and CD26
exp ession in di e en lymphocy e popula ions mus be conside ed in
he ligh o di e en indings such as he educ ion o ca eolin-1 (a
CD26 ligand) in monocy es and b onchial epi helial cells om
as hma ics [424], o he ole o CD26 con olling he bioa ailabili y o
soluble ac o s such as cy okines (e.g., IL-3, GM-CSF) [425] and
chemokines (e.g., eo axin 1/CCL11, RANTES/CCL5, MDC/CCL22)
[274, 286]. Thus, he clea age by CD26/DPP4 o chemokines ha a e
po en chemoa ac an o eosinophils and TH cells such as RANTES
[282] and eo axin [275, 277, 278] plays a key ole in as hma
pa hogenesis. Fo example, he ea men wi h CD26 inhibi o s o he
use o CD26-/- animals esul s in highe le els o eosinophils, highe
in il a ion o hese cells in o he ai ways, and highe disease se e i y
[275, 281]. In he same way, CD26 media ed clea age o SDF-
1α/CXCL12 [426] o IFN-γ induced chemokines (CXCL9-11) [279,
427, 428] educe he chemo axis o TH1 cells owa ds he epi helial
ba ie . The e o e, CD26 o ms pa o a homeos a ic mechanism
GENERAL DISCUSSION
271
aimed o he down-modula ion o ai ways in lamma ion. Thus, he
immunomodula o y po en ial o CD26/DPP4 should be conside ed in
ligh o he clinical usage o CD26 inhibi o s (glip ins) (see appendix
I).
Ano he a ea o a en ion in Chap e III was he CD126/IL-6Rα
molecule. Toge he wi h gp130, CD126 composes he ecep o o IL-
6, a molecule wi h a key ole in he di e en ia ion o TN lymphocy es
owa ds a TH17 (i.e., CD26high) pheno ype [176]. IL-6 signalling is
also essen ial o he gene a ion o unc ionally ac i e memo y CD4+
T cells [429], while CD126 exp ession on CD8+ T cells de ines
di e se naï e-memo y di e en ia ion s ages in hese cells [152], in he
same way ha CD26 [261]. Indeed, we epo a highly posi i e
co ela ion be ween he le els o hese wo ma ke s on ci cula ing
lymphocy es, bu no di e ences in CD126 exp ession be ween he
di e en g oups o pa ien s (heal hy con ols, hini is, AA, NAA). As
well, his pape desc ibes ha CD4- lymphocy es can be seg ega ed in
di e en subpopula ions based on he exp ession o CD126.
The e o e, his molecule could be use ul o naï e-memo y
cha ac e iza ion as well. Mo eo e , CD126-CD4- lymphocy es
(augmen ed in NAA s. AA) downmodula e he le els o CCR7,
CD28, and CD27, bu e ain a high abundance o CD45RA molecules,
highligh ing hei ad anced di e en ia ion s age (TEMRA-like).
As CD26, CD126 can be eleased om he memb ane o
CD126+ cells as sIL-6Rα. This soluble ecep o e ains he capaci y o
bind IL-6, leading o he ac i a ion o CD126- gp130+ cells. This
p ocess is known as ans-signalling [179, 180], and migh be
impo an o as hma h ough he main enance o TH17 cells o he
inhibi ion o T cell apop osis [430]. Fo example, i has been
desc ibed ha CD126 is down-modula ed upon in lamma ion on CD4+
T cells, bu hese cells e ain he IL-6 esponse capaci y h ough ans-
signalling e en s [431]. Indeed, he elease o sIL6Rα has been
Juan José Nie o Fon a igo
272
desc ibed in as hma, and sIL-6Rα le els ha e been di ec ly associa ed
wi h IgE le els. Fu he mo e, sIL-6Rα in se um is nega i ely
co ela ed wi h lung unc ion [432]. The e o e, he down-modula ion
o he numbe o CD126 molecules on monocy es, neu ophils, and
CD4+ cells om mode a e-se e e pa ien s epo ed in Chap e III
highligh s he po en ial ole o IL-6 ans-signalling in as hma
se e i y.
The e ec o unc ion o human TH lymphocy es is
coun e ac ed by he immunosupp essi e b anch o his subse , called
T eg cells [214]. The e o e, a de ec in hei biological unc ion o a
educ ion in hei numbe could esul in highe in lamma ion le els in
as hma. Al hough some au ho s ha e desc ibed a educ ion o he
equency o T eg cells in as hma ics, in Chap e II and III we ail o
de ec such a nume ical al e a ion. Ne e heless, women did show an
inc eased Te /T eg a io compa ed o men (Chap e II), and his could
be behind he g ea e suscep ibili y o his gende o adul as hma.
Ou esul s also sugges an impai ed unc ion o T eg cells in
as hma (Chap e III). Thus, i appea s ha T eg lymphocy es om
as hma ic pa ien s display highe le els o CD26 han hose om
heal hy subjec s. Mo eo e , he pe cen age o CD26+ T eg cells was
highe in mode a e-se e e pa ien s compa e o in e mi en -mild non-
alle gic as hma ics. Why a e CD26 le els so impo an o T eg
unc ion? CD26 is a glycop o ein ha ancho s ADA o he cell-
su ace, and ADA is an ec o-enzyme in ol ed in he ca abolism o
adenosine [264]. Mo eo e , CD39 is ano he ec o-enzyme exp essed
by CD26- T eg lymphocy es in ol ed in adenosine p oduc ion [258,
433]. The e o e, a CD26high pheno ype in T egs om as hma ics could
cause highe as hma se e i y h ough a dec eased local concen a ion
o adenosine, a pu ine nucleoside wi h immunomodula o y unc ions
[434]. On he o he hand, he pe cen age o CD127+ T eg cells was
inc eased in ou as hma ic coho compa ed o heal hy and hini is, a
GENERAL DISCUSSION
273
pa ame e nega i ely co ela ed wi h he supp essi e capaci y o hese
cells acco ding o o he s [435]. The e o e, u u e s udies including he
assessmen o T eg unc ion in NAA and AA will be necessa y.
3. SEARCHING FOR BIOMARKERS IN SERUM SAMPLES
FROM PATIENTS WITH RHINITIS OR DIFFERENT
ASTHMA PHENOTYPES/SEVERITIES (Chap e IV)
Because o he absence o s udies o his kind, he las chap e o he
p esen hesis is de o ed o he sea ch o se um bioma ke s associa ed
o as hma pheno ypes (AA s. NAA) o se e i ies (in e mi en -mild
and mode a e-se e e) by means o mode n p o eomic echniques. The
i s se um/plasma p o eomic s udies we e only able o “sc a ch” he
i s laye o he p o eome (i.e., he high-abundance p o eins) as a
esul o he high dynamic ange o p o ein concen a ions in se um.
Since hen, se e al echniques ha e been de eloped o deal wi h his
p oblem, such as ul a il a ion, a ge ed p o ein-deple ion, o low
abundan p o ein en ichmen . A he same ime, analy ical me hods
ha e e ol ed om classical p ocedu es wi h a low sensi i i y,
p ecision, and h oughpu (e.g. 2-DE), o mo e mode n and high-
pe o mance echnologies (e.g., LC-MS/MS). Howe e , despi e hese
e inemen s, mos o he cu en p o eomic app oaches a e no ye
capable o each wi h ease he medium-low abundan p o eome ( he
so-called “deep p o eome”). The e o e, he i s aim o he wo k
accomplished in Chap e IV was o de elop a p o ocol o he
quan i a i e s udy o he medium-low abundance p o eome o se um
samples, which combines lipop o eins-deple ion, CPLL-based
en ichmen o low abundan p o eins (P o eoMine ®), and iTRAQ-LC-
MS/MS analysis. The second aim was o apply his p o ocol o
di e en se um samples om hini is, alle gic as hma ics, non-alle gic
as hma ics, and heal hy subjec s, in o de o disco e no el
bioma ke s capable o p edic ing di e en as hma pheno ypes o
se e i ies.
Juan José Nie o Fon a igo
274
In he las yea s, a high numbe o p o eomic s udies ha e been
ca ied ou in as hma (summa ized in Table 1 om in oduc ion). In
all hese pieces o wo k, di e en ypes o samples (e.g., BALF, EBC,
NLF, BB, NB, IS, se um/plasma) we e used, and se e al ypes o
bioma ke s pu sued (e.g., diagnosis, exace ba ion, ea men esponse)
[337, 436-438]. Howe e , none o hem was aimed o analyse he
se um p o eome o di e en as hma pheno ypes (AA s. NAA) o
se e i ies, as abo e commen ed. Addi ionally, mos o he s udies in
se um samples used 2-DE, and jus a hand ul was ca ied ou by LC-
MS/MS [391]. Mo eo e , one single p o eomic s udy used iTRAQ-
LC-MS/MS o e alua e b onchial b ushing samples om as hma ics
[379]. The e o e, he i s pa o Chap e IV add esses he
de elopmen o a new quan i a i e p o eomic app oach based on
lipop o eins-deple ion, low abundan p o ein en ichmen
(P o eoMine ), iTRAQ pep ides labelling, and subsequen LC-MS/MS
and iTRAQ quan i ica ion, wi h applica ions in a di e se se o
pa hologies.
On he o he hand, he second pa o he wo k accomplished in
Chap e IV unde akes he iden i ica ion o he se um p o eome
signa u e in di e en g oups o dono s: mode a e-se e e AA,
in e mi en -mild AA, hini is, mode a e-se e e NAA, in e mi en -
mild NAA, and heal hy con ols (HC; e e ence g oup). Due o a la ge
numbe o se um specimens and he abili y o iTRAQ eagen s o
label up o 8 biological samples, each g oup o pa ien s was spli in o
wo subg oups o gene a e wo biological eplica es. In u n, hese
biological samples we e analysed in iplica e ( echnical eplica es),
oge he wi h a pooled se um sample o med by all he samples
(in e nal pool; 121 epo e ion), which was used o no maliza ion.
A e LC-MS/MS analysis, a se o 217 p o eins was iden i ied wi h
high con idence, o which 26 p esen ed changes be ween he g oups
o s udy. P incipal Componen Analysis o he di e en ially abundan
GENERAL DISCUSSION
275
p o eins e ealed he p esence o 5 p incipal componen s (PCs)
explaining 81.6% o he a ia ion. PC1 is made up o p o eins ela ed
o alle gy (AA and hini is), mos o hem implica ed in me abolic
(e.g., p o ein AMBP/alpha-1-mic oglobulin/bikunin p ecu so ;
HSPG2/Hepa an sulpha e p o eoglycan 2/Pe lecan; IGFALS/Insulin-
like g ow h ac o binding p o ein, acid labile subuni ; o
AHSG/alpha-2-HS-glycop o ein) o immune sys em p ocesses (e.g.,
HSPG2, AHSG, ORM1/AGP1/Alpha-1-acid glycop o ein 1, o
CD5L). Mo eo e , PC1 has an en ichmen o he Reac ome pa hway
“Regula ion o Insulin-like G ow h Fac o (IGF) anspo and up ake
by Insulin-like G ow h Fac o Binding P o eins (IGFBPs)” o which
calumenin, IGFALS, AHSG, and p o h ombin belong. Amongs
p o eins in PC1, IGFALS, p o ein AMBP, and HSPG2 we e chosen
o u he con i ma ion by Enzyme-Linked ImmunoSo ben Assays
(ELISAs). So a , only IGFALS ELISA was pe o med wi h iden ical
esul s.
IGFALS is a p o ein p oduced by hepa ocy es, bu also
exp essed in o he issues, including hymus and lungs [439]. This
p o ein can be ound in plasma o as a ee molecule o o ming a
complex wi h IGF1-IGFBP (3 o 5 p o eins) [440]. IGFALS has an
impo an ole on he bioa ailabili y o IGFs [441], p e en ing he
ex a asa ion, p o eolysis o enal elimina ion o IGF-1 [442]. Fo his
eason, IGFALS de iciency esul s in a d ama ic dec ease o IGF-1
le els, bu also a ec s he IGF-2 and IGFBP3 p o eins [442, 443].
IGF1 is impo an o as hma pa hogenesis h ough he p omo ion o
in lamma ion, sub-epi helial ib osis, hype esponsi eness, and
smoo h muscle cell hype plasia in he lungs [441]. Apa om he
p e iously epo ed ele a ion o IGF1 [441] and IGFBP3 [441] in AA,
he wo k p esen ed in Chap e IV shows ha IGFALS is also
augmen ed in his kind o pa ien s, especially hose displaying a high
disease se e i y. IGFBP3 con ols AA in lamma ion h ough
Juan José Nie o Fon a igo
282
esemble memo y/e ec o cells wi h an ad anced
di e en ia ion s age: TEM o TEMRA lymphocy es.
4. The TEM/TEMRA subse s expanded in as hma pa ien s belong o
di e en lymphocy e lineages: CD4+ TH cells in AA and CD4-
γδ-T lymphocy es in NAA.
5. Se um sCD26 le els a e dec eased in bo h AA and NAA
pa ien s; he e o e, hey p obably e lec he “ inge p in ” in
se um p o eome o he expansion o CD26-/lowCD4+ TH in AA
and CD26-/lowCD4- γδ-T subse s in NAA.
6. The educ ion o ci cula ing sCD26 le els as well as CD26
exp ession on e ec o TH lymphocy es om as hma ics migh
be impo an o imp o e hei mig a o y and p oli e a i e
capabili ies. This inding should be conside ed in ligh o he
cu en clinical usage o DPP4 inhibi o s and an i-CD26
an ibodies.
7. The e is a s ong co ela ion be ween he exp ession o CD26
and CD126/IL-6Rα on Te lymphocy es, pa icula ly hose
lacking he CD4 ma ke . The e o e, he TEM/TEMRA subse s
expanded in as hma (especially amongs he NAA pa ien s) a e
also CD126-/low cells.
8. The down-modula ion o CD126/IL-6Rα in mode a e-se e e
pa ien s compa ed o in e mi en -mild as hma ics is a
widesp ead e en , aking place on monocy es, neu ophils, and
CD4+ cells. This highligh s he po en ial ole o IL-6 ans-
signalling in as hma se e i y.
9. Al hough he numbe o T eg cells emains unchanged in
as hma, hei supp essi e capaci y could be impai ed acco ding
o hei highe CD26 le els compa ed o heal hy con ols.
Chap e IV
1. A “sho gun” p o eomics me hodology has been de eloped o
he quan i a i e analysis o medium-low abundance se um
CONCLUSIONS
283
p o eins. This p o ocol is based on dynamic ange comp ession
wi h CPLLs, pep ides-labelling wi h iTRAQ eagen s, and LC-
MS/MS analysis.
2. Twen y-six ou o wo hund ed and se en een p o eins display
abundance changes associa ed wi h di e en g oups o dono s
(HC, R, MSAA, IMAA, MSNAA, and IMNAA). Some o
hese se um p o eins could be use ul bioma ke s o as hma
pheno ypes (e.g., IGFALS, P o ein AMBP, and HSPG2 o
AA; CFI o NAA) o se e i ies (e.g., IGFALS in MSAA).
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ABSTRACT
As hma is a he e ogeneous and ch onic in lamma o y amily o
diso de s o he ai ways wi h an inc easing p e alence ha esul s in
ecu en and e e sible b onchial obs uc ion and expi a o y ai low
limi a ion. These diseases a ise om he in e ac ion be ween
en i onmen al and gene ic ac o s, which collabo a e o cause
inc eased suscep ibili y and se e i y. Many as hma suscep ibili y
genes a e linked o he immune sys em o encode enzymes like
me allop o eases (e.g., ADAM-33) o se ine p o eases. The S9 amily
o se ine p o eases (p olyl oligopep idases) is capable o p ocess
pep ide bonds adjacen o p oline, a kind o clea age- esis an pep ide
bonds p esen in many g ow h ac o s, chemokines o cy okines ha
a e impo an o as hma. Cu iously, wo se ine p o eases wi hin he
S9 amily encoded by genes loca ed on ch omosome 2 appea o ha e
a ole in as hma: CD26/dipep idyl pep idase 4 (DPP4) and DPP10.
The aim o his e iew is o summa ize he cu en knowledge abou
CD26, and o p o ide a s uc u ed o e iew o he nume ous unc ions
and implica ions ha his e sa ile enzyme could ha e in his disease,
especially a e he de ec ion o some seconda y e ec s (e.g., i al
nasopha yngi is) in ype II diabe es melli us pa ien s (a subse wi h a
ce ain isk o de eloping obesi y- ela ed as hma) upon CD26
inhibi o y he apy.
KEYWORDS:
CD26, DPP4, as hma, cy okines and chemokines, sCD26, CD26
inhibi o s.
1.- DEFINITION AND GENETIC FACTORS IMPLICATED IN
ASTHMA
As hma is a he e ogeneous and ch onic disease cha ac e ized by
e e sible expi a o y ai low limi a ion, b onchial
hype esponsi eness, mucous cell hype plasia, highe ascula u e
pe meabili y, ai way emodelling wi h ib osis and in lamma o y cell
in il a ion [1, 2]. As hma is a majo conce n, wi h 334 million people
wo ldwide a ec ed, inc easing p e alence [3] and a high economic
cha ge [4]. I is mo e equen and se e e in boys un il he age o 13
yea s, bu bo h p e alence and se e i y o as hma ise in women a e
pube y, becoming e en mo e p e alen in women [3, 5, 6]. 5-10% o
cases display a highly se e e and ea men - e ac o y disease,
su e ing om ecu en exace ba ions ha h ea en pa ien ´s li e and
inc ease he heal h ca e cos s. This is a complex pa hology, wi h bo h
gene ic and en i onmen al ac o s causing inc eased suscep ibili y and
se e i y. G ea e o s ha e been made o disco e he gene ic bases
(mos ly GWAS), e ealing he in luence o se e al genes encoding
p o eins wi h an impo an ole in he immune sys em (HLA-DQ,
HLA-G, IL1RL1, IL18R, TSLP, PDE, IL-33, LRRC32, SMAD3,
IL2RB, IL6R, IL13) and also p o eases (ADAM33, and DPP10) [7-9].
2.- ASTHMA AND THE PROLYL OLIGOPEPTIDASE
FAMILY OF PROTEASES: DIPEPTIDYL PEPTIDASE 10
(DPP10) AND CD26/DPP4
As commen ed abo e, p o eases a e in ol ed in many physiological
and pa hological p ocesses, including ch onic espi a o y condi ions
like as hma. Amongs all p o eases, he e a e only a ew p oline-
speci ic enzymes, as pep ide bonds adjacen o p oline (p esen in
some g ow h ac o s, chemokines o cy okines) a e esis an o
clea age [10]. These enzymes include se ine p o eases, wi h a se ine
esidue in hei ca aly ic egion (sequence consensus Gly-Xaa-Se -
Xaa-Gly) [11] and co e ing di e en amilies (S1-S81) and
sub amilies. Thus, he S9 amily (p olyl oligopep idases) includes
om S9A o S9D, all wi h he ca aly ic iad Se , Asp, His bu wi h
sligh ly di e en sequence consensus a ound he ca aly ic Se . Fo
example, mos o membe s o he S9B sub amily (EC 3.4.14.5;
CD26/DPP4, DPP8, DPP9, and ib oblas ac i a ion
p o ein/FAP/Sep ase) (h p://me ops.sange .ac.uk/) p esen he
sequence Gly-T p-Se -Ty -Gly-Gly, while he o he addi ional wo
membe s, DPP6 (DPPX; Gly-Lys-Asp-Ty -Gly-Gly) and DPP10
(Gly-Lys-Gly-Ty -Gly-Gly), do no possess he ca aly ic se ine and,
he e o e, DPP4 ac i i y [12]. Cu iously, ou o hese pep idases
(CD26, FAP, DPP6, and DPP10) a e ype II memb ane p o eins
eleased o he ex acellula medium, h ee a e encoded by genes
loca ed on ch omosome 2 (CD26, FAP and DPP10), and only wo
(CD26, DPP10) appea o ha e a ole in as hma [13-16]. Fo example,
DPP10 ha e been linked o as hma suscep ibili y in di e en
popula ions [7, 13, 15], and his p o ein (as well as DPP8 and DPP9)
has been p ima ily loca ed in he achea and he b onchi o he
ai ways in a s [17]. Fo i s pa , he p o ease CD26 is he majo
membe o he S9B amily and also a ecep o o he Middle-Eas
espi a o y synd ome co ona i us (MERS-CoV) [18, 19], a new
co ona i us ha causes se e e lowe espi a o y ac in ec ions ha
could lead o as hma exace ba ions. CD26 has been ound ele a ed in
bo h plasma samples (sCD26) and he su ace o pe iphe al CD4+ T
cells om adul pa ien s wi h alle gic as hma [20, 21], and di e en
animal models [14, 16, 22] sugges a ole o his pep idase in he
pa hogenesis o his disease.
3.- STRUCTURE AND DISTRIBUTION OF CD26
3.1.- S uc u e o he CD26 molecule
CD26 is a single-pass ype II in eg al memb ane glycop o ein o 105-
110 kDa [23]. Only he homodime ic o m o CD26 is biologically
ac i e [24-27]. Homodime iza ion akes place in bo h he Golgi
appa a us [28] and he endoplasmic e iculum [29]. Each monome
displays a highly conse ed and sho (6 amino acids) cy oplasmic ail
a he N- e minus, a 22 amino acid hyd ophobic ansmemb ane
egion and a long ex acellula domain o 738 amino acids. The
hea ily N-glycosyla ed ex acellula domain can be subdi ided, in
u n, in se e al egions. The closes o he amino- e minal pa s a s
wi h a lexible s alk egion and con ains 8 ou o 10 possible N-
glycosyla ion si es, while he in e media e egion is highly en iched in
cys eines (9 ou o 12). Finally, he ac i e cen e is loca ed owa ds he
ca boxyl- e minal end and is ano he highly conse ed egion [30-34].
3.2.- Tissue and cell dis ibu ions o CD26
Human CD26 is b oadly dis ibu ed in a a ie y o cell ypes, issues,
and o gans [35-39]. Lungs display he second highes CD26 ac i i y
amongs o gans, especially in lung pa enchyma [17, 22]. Con a y o
DPP8/9 and DPP10, b onchi almos no exp ess CD26 [17], bu can be
ound in he apical memb ane o epi helial cells, capilla y endo helial
cells, ib oblas s and se osal submucosal glands o human b onchi
[40-42]. In mos o hese places, CD26 le els a e cons i u i e and
highly co ela ed wi h he mRNA con en
(h p://www.p o eina las.o g/ENSG00000197635-DPP4/ issue) [43].
Howe e , IL-13 causes a s ong p oin lamma o y up egula ion o
CD26 in he ai way epi helial cells [44]. Mo eo e , bo h DPP4
ac i i y and CD26 p o ein (bu no mRNA) inc ease a e alle gen
exposu e in lung pa enchyma in a a model o alle gic ai way
in lamma ion [17], sugges ing ha CD26 on lung epi helium could be
impo an in as hma pa hogenesis [45].
CD26 is also exp essed in he Immune Sys em, being de ec ed
in medulla hymocy es, T cell a eas o spleen and lymph nodes,
pe iphe al blood T cells and, a a lesse ex en , B cells, NK cells,
monocy es/mac ophages and g anulocy es [24, 25, 30, 41, 46, 47].
CD26 densi y in hese cells is hea ily con olled and augmen s upon
cell ac i a ion and acquisi ion o a memo y pheno ype, especially in
he T cell lineage [33, 41, 48]. Human T lymphocy es display a iable
basal exp ession o CD26 (CD4+ T >> CD8+ T cells), which depends
on he indi idual and he mAb used. Despi e no all es ing T cells a e
CD26+, mos o hem con ain CD26 mRNA and display CD26
molecules on he su ace 4-8 hou s a e s imula ion [49-51].
Howe e , only a ce ain egula ion on CD26 mRNA le els has been
de ec ed by ei he no he n blo [36, 50, 52-54] o gene a ays [55]
upon ac i a ion. Addi ionally, CD26 is up egula ed by IFNγ on enal
epi helial cells [56] and B-CLL [57] h ough he modi ica ion o
mRNA le els.
Despi e he abo e-desc ibed adjus men o CD26 exp ession
h ough mRNA le els, se e al pieces o e idence suppo he p esence
o ups eam con ol le els. Thus, CD26 is s ongly egula ed a p o ein
le el by se e al soluble ac o s. Fo example, he TH1 cy okine IL-12
(and o a lesse ex en IL-2) enhances he exp ession o CD26 on bo h
ac i a ed T cells [58, 59] and NK cells ( oge he wi h IL-15) [47, 60].
On he con a y, IFNγ (ano he TH1 cy okine) has no e ec on CD26
le els in hese lymphocy es [47, 58]. Rega ding TH2 cy okines, IL-4
p omo es he exp ession o CD26 on human B lymphocy es ac i a ed
wi h S aphylococcus au eus cowan I [61, 62], while his cy okine
mo es om a lack o e ec [63] o a ce ain downmodula ion o
CD26 le els in T cells a high concen a ions [unpublished esul s]. In
addi ion, in i o exposu e o egula o y T cells (T eg)-de i ed soluble
ac o s like TGFβ1 [64, 65] o Ado (adenosine) [66] leads o
diminished CD26 le els.
Mos o pe iphe al blood T eg lymphocy es display an
“ac i a ed-like” (e.g., CD25high), “memo y” (CD45RO+) and
“ane gic/apop osis-p one” pheno ype [67] and we e expec ed o
exp ess o he ac i a ion/memo y ma ke s such as CD26. This seems
o be he case o a s, whe e CD25+ (T eg) and CD25- (e ec o T
cells o Te ) subse s o pe iphe al CD4+ T cells show equi alen
CD26 exp ession [68]. In con as , human T eg cells (CD4+CD25high
o CD4+FoxP3high) display lowe le els o CD26 compa ed o Te
lymphocy es (CD4+CD25-/low o CD4+FoxP3-/low) [69-72]. This likely
explains why cy okines ha a ou “e ec o ” esponses (e.g., IL-12)
cause a s ong up egula ion o CD26 on “bulk” TH cell cul u es, while
o he s impo an o he T eg homeos asis (e.g., IL-2, IL-15) only
induce a sligh inc ease on CD26 le els [58, 73]. I also gi es a clue
on why CD26 is downmodula ed upon exposu e o T cells o TGF-β1
[64, 65].
CD26 exp ession amongs Te lymphocy es is also a iable,
and TH17 cells appea o display he highes le els o CD26 acco ding
o he ollowing o de : TH17>>TH1>TH2 [72, 74-77]. Fu he mo e, wo
subse s we e epo ed amongs TH17 cells on he base o CCR4
exp ession [78], bu only he CCR4- TH17 subse (and no he TGF-β-
sec e ing CCR4+ TH17 subpopula ion) has a CD26high pheno ype [79].
This inding likely indica es ha he deg ee o pheno ypic di e si y
ound in Te cells o CD26 le els is also p obably p esen in T egs
and e lec s he p esence o unc ionally di e en subse s ha mi o
he co esponding Te subse s [80].
The di e en exp ession le els o CD26 in T eg and Te
lymphocy es could depend on a a ia ion in CD26 mRNA le els as i
happens wi h “ egula o y-like” CD4+ T cells in classical Hodgkin´s
lymphoma [81]. Howe e , i is wo h o men ion ha , apa om
egula ing his se o CD26 mRNA molecules, he e is an in acellula
pool o CD26 p o ein main ained by con inuous ansla ion in human
T cells ( ega dless hei CD26+ o CD26- pheno ype) [50] ha can be
mobilized owa ds he plasma memb ane [54] o eleased in o he
ex acellula space. The e o e, he e could be a numbe o addi ional
mechanisms leading o a CD26-/low pheno ype in human T eg cells.
3.3.- Body luid compa men s and dis ibu ion o soluble CD26
CD26 can be ound in he ex acellula space, wi h au oc ine,
pa ac ine o endoc ine e ec s. The soluble e sion o CD26 (sCD26)
has been desc ibed in many biological luids (e.g., se um, plasma,
syno ial luid, ce eb ospinal luid) om di e en o ganisms [82].
B onchoal eola la age con ains DPP4 enzyma ic ac i i y in a s [17]
and humans [83]. In se um o plasma a leas 90% o his ac i i y is
associa ed o a hea ily glycosyla ed 110 kDa CD26 iso o m [84, 85],
whose concen a ion displays a no mal dis ibu ion [86] and high
biological a iabili y [87, 88] ha seems o depend on p e-analy ical
a iables, like age o gende . Thus, se um sCD26 concen a ion
inc eased in up o 10-12 yea s in child en, a e which he alues s a
dec easing [89]. In adul s, i has been also de ec ed a sligh dec ease o
sCD26 o DPP4 ac i i y in se um wi h age [87, 88], bu o he s udies
did no desc ibe such co ela ion [86, 90]. In addi ion, no di e ences
we e de ec ed ega ding gende in some s udies [90], while di e en
au ho s did ind a highe sCD26 concen a ion in se um/plasma
samples [86-89, 91] bu a lowe CD26 exp ession on CD4+ T cells
(ou unpublished esul s) om males.
Se e al s udies ha e ound a posi i e co ela ion be ween
sDPP4 ac i i y and sCD26 in humans [91-93], bu o he s ha e
epo ed a low co ela ion ins ead, wi h di e en explana ions like he
p esence o hype sialyla ed CD26 iso o ms o al e na i e p o eins
wi h DPP4 ac i i y [82]. Thus, plasma samples con ain a soluble
glycop o ein named DPPT-L o a ac in wi hou homology wi h
CD26 bu wi h DPP4 ac i i y [94-96], al hough his ac i i y has been
ques ioned mo e ecen ly [97]. A ac in is accumula ed in he plasma
memb ane upon T cell ecep o (TCR) igge ing (~24h) [95, 96],
especially in CD26high Te cells (ou unpublished esul s), and
eleased in o he plasma a 48-72h [95, 96]. Cu iously, linkage
disequilib ium s udies show an associa ion o as hma wi h a egion
including he a ac in (ATRN) gene, which is ups eam o he gene
encoding he sec e ase/sheddase ADAM-33 (an as hma suscep ibili y
gene) [97].
Release o CD26 could be accomplished by a classical o non-
classical pa hway, and ei he by a cons i u i e o induced mechanism.
The classical pa hway equi es esicula a ic om he endoplasmic
e iculum/Golgi owa ds he plasma memb ane. In a esicula p o eins
a e libe a ed in he ex acellula medium a e memb anes me ging
(exocy osis), while ansmemb ane p o eins appea in he sec e ome
h ough p o eolysis media ed by “sec e ases”/“sheddases”. This las
p ocess ("shedding") deli e s g ow h ac o s, cy okines, ecep o s and
p obably molecules like CD26 [98]. This is addi ionally suppo ed by
he lack o pos ansc ip ional splicing in CD26 mRNA, he absence
o cy oplasmic and ansmemb ane domains in sCD26, o esul s om
pulse chase and ans ec ion expe imen s [25, 82]. Howe e , CD26
has been de ec ed in mic o esicles and exosomes om lymphocy es
(h p://www.exoca a.o g). The e o e, cons i u i e o induced elease
o CD26+ esicles (exosomes, mic o esicles o apop o ic bodies) in o
he medium could also con ibu e o he pool o sCD26 in he
ci cula ion and e lec he cell subse o o igin (“cell lineage
inge p in ").
The cell sou ce o sCD26 is s ill con o e sial [25, 82].
Visce al a , a leas in obese pa ien s, o e exp esses CD26 and
eleases his “adipokine” in o he ci cula ion [88], and he e a e da a
suppo ing he p esence o a posi i e co ela ion be ween as ing
sCD26 le els and body mass indexes/BMI >25 [88, 99]. sCD26 may
also o igina e om o he sou ces, such as endo helial cells (e.g. lung)
o epi helial cells om li e (bile canaliculi) o kidney. Howe e ,
immune cells a e also a likely sou ce [25, 82, 100]. Thus, a
ansplan a ion model in a s has de e mined ha , unde heal hy
condi ions, bone ma ow-de i ed cells ep esen a signi ican sou ce
o sCD26 [101]. The e o e, he concen a ion o sCD26 could also be
in luenced by he numbe o lymphocy es [102] o mi o he
p edominan pheno ype o ci cula ing CD4+ T lymphocy es in a
pa hological si ua ion.
4.- CD26 AND ASTHMA
4.1.- As hma pheno ypes, disease se e i y and CD26 le els in
CD4+ T cells.
CD4+ TH cells play a cen al ole in adap i e immune esponses and
he induc ion/pe sis ence o as hma and o he alle gic/a opic diseases.
They a e plas ic and he e ogeneous lymphocy es, wi h ou main
lineages (TH1, TH2, TH17 and T eg cells) and di e en oles. Fo
example, TH2 (GATA-3+) cells in il a e ai ways in alle gic as hma
and p oduce a se o cy okines (IL-4, IL-5, IL-13) impo an o
ai way emodelling, leucocy osis, eosinophilia, mac ophages/mas
cells ac i a ion, and B-cell dependen IgE ele a ion [103, 104]. In he
same way ha TH lymphocy es a e he e ogeneous, he e a e also a
numbe o as hma pheno ypes as a likely e lec ion o he TH cells
he e ogenei y i sel , wi h bo h TH2high and TH2low pheno ypes sha ing
di e en clinical signs: a) he ea ly alle gic as hma, wi h a s ong
amilial backg ound and media ed by TH2 cy okines (TH2high) and
alle gen-speci ic IgE (a opy); b) he less-alle gic la e-onse as hma
(TH2high), which is cha ac e ized by eosinophilic in lamma ion,
absence o speci ic IgE (i.e., non-a opic) and wo se esponse o
co icos e oids; c) neu ophilic as hma, a TH2low pheno ype e ac o y
o co icos e oids and linked o TH17- esponses, spu um neu ophilia,
and augmen ed le els o IL-8 and IL-17; d) and he obesi y- ela ed
as hma, a TH2low pheno ype p edominan in obese women, wi h highe
le els o TNFα, IL-6 and lep in bu low numbe s o eosinophils [1]
(Table 1). The e o e, al e na i e CD26+/high e ec o TH subpopula ions
a e gaining impo ance in as hma, pa icula ly as he disease becomes
ch onic and e ac o y o ea men . In his con ex , e en hough i was
ini ially desc ibed ha CD26+ TH1 cells we e p o ec i e (“hygiene
hypo hesis”), now i is conside ed ha hese cells could be a ou ing
he in lamma ion o he ai ways [105]. In addi ion, p oin lamma o y
CD26high TH17 cells ha e been de ec ed in biopsies om as hma
pa ien s, and hei cy okines (e.g., IL-17, IL-6, IL-21, IL-22) in ol ed
in eosinophilia/neu ophilia and highe se e i y du ing acu e a acks
[106]. On he o he hand, T eg cells (FoxP3+, CD26-/low) also seem o
play a ele an ole in con olling exagge a ed TH2 esponses and
as hma de elopmen [9, 107, 108]. T eg cells exe hei supp esso
ac i i y by using bo h soluble (e.g., adenosine, TGFβ, IL-10) and
memb ane-associa ed (e.g., TGFβ, CTLA-4) molecules [67], and a
numbe o GWAS in as hma ha e iden i ied some genes (e.g., IL2RB,
SMAD3, GARP/LRRC32) linked o his supp esso ac i i y. Thus, he
lowe o highe p e alence o di e en Te subse s and/o he
dis o ion o he Te /T eg balance could also al e he pheno ype and
se e i y o as hma [104, 109, 110], Mo eo e , depending on he
as hma pheno ype o he se e i y o his disease, he le els and
unc ions o CD26 on he su ace o immune cells and bio luids could
be a he di e en and be a ac o in luencing disease de elopmen .
lymphocy es, eosinophils, neu ophils, endo helial and mas ,
epi helial, endo helial, and smoo h muscle cells) [161, 162]. This ole
o Ado in as hma is a ec ed by binding a ini y, ecep o densi y and
local le els o his nucleoside, which a e in luenced by a delica e
balance whe e di e en mechanisms a e in ol ed (e.g.,
ex acellula /in acellula Ado me abolism) [161, 162]. Amongs hese
mechanisms, he Ado ca abolism media ed by ADA molecules
ancho ed o CD26 is he majo egula ed pa hway ha in luences he
local concen a ion and biological e ec s o Ado. In his espec ,
ele an TH2 cy okines in alle gic as hma (IL-4, IL-13) educe ADA
ac i i y in he lung [161] and ec o-ADA le els on TH lymphocy es
[63]. These indings sugges ha low le els o CD26/ec o-ADA on he
TH2 subse in TH2high (alle gic/a opic) as hma could a ou he local
accumula ion o Ado, he ac i a ion o A1AR, and he pa icipa ion o
p oin lamma o y ARs ha equi e Ado concen a ions o e he
physiological le els (e.g., he low-a ini y A2BAR) in o de o
neu alize he o -signals p o ided by he engagemen o high-a ini y
and as hma-p o ec i e A2AARs. The e o e, i is likely ha ARs-
a ge ed he apy is mo e e ec i e in TH2high as hma han in TH2low o
se e e as hma, whe ein CD26/ec o-ADAhigh TH1 and TH17 cells a e
mo e equen . Howe e , addi ional s udies a e needed o cla i y wha
could be exac ly he ole o T eg cells in as hma, a lymphocy e subse
wi h a CD26/ec o-ADAlow pheno ype ha should a ou he high
p oduc ion o Ado [70]. On he o he hand, CD45 has been in ol ed
in Janus kinases dephospho yla ion [163]. The e o e, he CD26-CD45
associa ion could ha e a nega i e and DPP4-independen ole
impo an o es ain he signal ansduc ion o cy okine ecep o s
[122] (Fig. 1). In his sense, he low PTPase ac i i y in human naï e
CD4+CD45RA+CD26-/low T cells makes hem suscep ible o small
amoun s o IL-4, as men ioned an impo an cy okine in alle gic
as hma, while memo y/e ec o CD4+CD45RO+CD26+/high T cells
sec e e his soluble ac o upon ac i a ion, bu a e less sensi i e o i s
e ec s [164]. Indeed, high le els o CD26 in memo y/e ec o TH
lymphocy es migh ac as a “b ake” o he p oli e a i e esponses o
cy okines ( o example, IL-12; ou unpublished esul s).
The p o ec i e unc ion o CD26 in as hma could also be
linked o he DPP4 ac i i y (Fig. 1). Some CD26 subs a es all
ou side he scope o he immune sys em and a e beyond he scope o
his e iew, like inc e ins (GIP/glucose-dependen insulino opic
pep ide, GLP-1/glucagon-like pep ide-1, GLP-2/ glucagon-like
pep ide-2) [10, 165, 166], glucagon, PACAP (pi ui a y adenyla e
cyclase-ac i a ing polypep ide), GRP (gas in- eleasing pep ide),
pep ide YY, asoac i e pep ides (b adykinin and VIP/ asoac i e
in es inal pep ide), na iu e ic pep ides (BNP/B- ype na iu e ic
pep ide), and neu opep ides (NPY/neu opep ide Y, be a-
casomo phins, endomo phins, subs ance P) [41, 82, 166, 167] (Table
2). Howe e , some o he s ha e an impo an immunomodula o y ole,
such as cy okines (e.g., IL-3, G-CSF, GM-CSF) [168] o chemokines
(e.g., RANTES/CCL5, eo axin/CCL11, MDC/CCL22) [82, 166].
CD26 enzyma ic ac i i y down-modula es he biological unc ion o
mos o hese chemokines and cy okines (Table 3). Consis en wi h
his obse a ion, i has been ound ha CD26 inhibi o s can enhance
some in i o immune esponses depending on he dose, applica ion
ou e, iming o p edominan Te subse [45, 169], which could be
explained by he p esence o o - a ge s e ec s as well [25, 170].
Howe e , animal models o heuma oid a h i is [93], mul iple
scle osis [171], and in lamma o y bowel disease [172] in CD26 KO
mice do suppo an immunosupp essi e ole o bo h CD26 and DPP4
ac i i y. This immunosupp essi e unc ion has also been obse ed o
sCD26 du ing s ong in i o p oli e a i e esponses o immune cells
[143, 145]. Mo eo e , CD26 has been e en ega ded as a umou
supp esso gene in melanomas o neu oblas omas [173, 174]. Fo his
eason, we will ocus his pa o his e iew on he CD26 subs a es
wi h a di ec connec ion wi h he immune sys em like chemokines.
Table 2. Known subs a es p ocessed by CD26/DPP4 ou o he immune
sys em
Subs a e name
and amily
N- e minal
sequence
E ec o DPP4
p ocessing
In i o / in
i o e idence
Re e ence
β-casomo phins
(neu opep ide)
Ty -P oPhe
Inhibi o y
Yes/Yes
[10] [82]
[156]
BNP (na iu e ic
pep ide)
Se -P oLys
Inhibi o y
Change in
ecep o
p e e ence
Yes/Yes
[82] [233]
B adykinin
( asoac i e)
A g-P oP o
Inhibi o y
Change in
ecep o
p e e ence
Yes/Yes
[10] [82]
[156]
Endo mo phins
(neu opep ide)
Ty -P oPhe
Inhibi o y
Change in
ecep o
p e e ence
Yes/Yes
[10] [41] [82]
[156]
En e os a in
(g as oin es inal
ho mone)
Val-P oAsp
Inhibi o y
Yes/Yes
[10] [41]
[156]
GIP (inc e in)
Ty -AlaGlu
Inhibi o y
Yes/Yes
[10] [41] [82]
[154] [156]
GLP (GLP-1 [7-
36]amide, GLP-1
[7-37]& and GLP-2)
(inc e in)
His-AlaGlu
(GLP-1)
His-AlaAsp
(GLP-2)
Inhibi o y
Yes/Yes
[10] [41] [82]
[154] [156]
Glucagon
(gas oin es inal
ho mone)*
His-Se Gln
Inhibi o y
Yes/Yes
[82] [156]
GRH (GRH[1-29]
and GRH[1-44]
(hypo halamic
ho mone)
Ty -AlaAsp
Inhibi o y
Yes/No
[10]
Con inued on nex page
Table 1 (Con inued)
GRP (ho mone
bombesin amily)
Val-P oLeu
No known
Yes/Yes
[82]
NPY
(neu opep ide)
Ty -P oSe
Recep o Y1
Inac i a ion
Change in
ecep o
p e e ence
Yes/Yes
[10] [41] [82]
[156]
PACAP
(PACAP27 and
PACAP38)*
His-Se Asp
P obably
inhibi o y
Yes/Yes
[82] [234]
Pep ide YY
(Panc ea ic
pep ide)
Ty -P oIle
Recep o Y1
Inac i a ion
Change in
ecep o
p e e ence
Yes/Yes
[10] [41] [82]
[156]
Subs ance P
(neu opep ide)
A g-P oLys
Inhibi o y
Ques ionable
Yes/Yes
[10] [41] [82]
[156]
VIP pep ides (VIP,
PHV42, PHM27)
( asoac i e)*
His-AlaAsp
(PHV, PHM)
His-Se Asp
(VIP)
P obably
inhibi o y
Yes/Yes
[10] [41] [82]
[154] [156]
BNP: B- ype na iu e ic pep ide; GIP: glucagon inhibi o y pep ide; GLP: glucagon like pep ide; GRH:
g ow h ho mone- eleasing ho mone; GRP: gas in eleasing pep ide; NPY: neu opep ide Y; PCAP:
pi ui a y adenyla e cyclase-ac i a ing polypep ide; VIP: asoac i e in es inal pep ide; PHV42: pep ide
his idine aline 42; PHM27: pep ide his idine me hionine 27 *: Glucagon, PACAP and VIP ha e a Se in
posi ion 2 and, wi h less e iciency ha P o o Ala, also can be DPP4 po en ial subs a e; &: GLP-1 [7-36]
amide and [7-37] a e he ac i e o ms o GLP-1 and subs a es o DPP4.
4.3.1.- CD26, TH2- ela ed chemokines and TH2high as hma.
E en hough cy okines like IL-1β o IL-2 con ain an app op ia e N-
e minal sequence, he molecula weigh p ecludes hei N- e minal
clipping, wi h excep ions like IL-3, G-CSF (g anulocy e colony-
s imula ing ac o ), GM-CSF (g anulocy e-mac ophage colony-
s imula ing ac o ), o Epo (e y h opoie in) [168]. In con as , CD26-
dependen p ocessing o chemokines (8-10 kDa) like RANTES
( egula ed on ac i a ion, no mal T cell exp essed and sec e ed) is one
o he mos in e es ing aspec s o CD26 biology [175]. Mo eo e , his
unc ion is mos ly dependen on CD26 le els, which is in luenced by
he T cell subse o disease, as we ha e jus seen.
Chemokines a e p oin lamma o y cy okines wi h a ole in
leukocy e ac i a ion and mig a ion. They a e classi ied acco ding o
he posi ion o wo N- e minal cys eine esidues (CC, CXC, C and
CX3C) [176], bu can also be di ided as a unc ion o he cells hey
a ac o he ecep o hey ecognize. As able 3 shows, he majo
b anches o e ec o (TH1, TH2, and TH17) and egula o y T cells
exp ess cha ac e is ic (bu no o ally selec i e) chemokine ecep o s
[76, 103]. Fo example, TH2 lymphocy es and o he leukocy es
impo an in as hma (basophils, mas cells, and eosinophils) exp ess
CCR3, CCR4, CCR8, CXCR4 and, in humans, CRTH2 [103]. CCR3,
CCR4, and CXCR4 in e ac wi h CD26-subs a es, bu only CCR3
and CCR4 a e ac ually speci ic o TH2 cells. Rega ding CXCR4, TH2
cells seem o display a sligh o e exp ession compa ed o TH1
lymphocy es [177], bu o he au ho s desc ibe CXCR4 as a me ely
a icking ma ke [178-180].
Liga ion o CCR3 wi h eo axin/CCL11, RANTES/CCL5, and
MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13
pa icipa es in he ec ui men o basophils, eosinophils and mas cells
[103, 181, 182] (Table 3). Eo axin is an impo an chemokine in
alle gic as hma p oduced by endo helial cells and monocy es in
esponse o IFNγ and TNFα, espec i ely. This chemokine is
ecognized by CCR3 (eosinophils, basophils, mas cells and TH2) and
a lesse ex en by CCR5, a pu a i e TH1-ma ke (see la e ) [183].
Eo axin is a chemoa ac an o eosinophils ha acili a es hei
mobiliza ion om bone ma ow [184]. T unca ion o eo axin by CD26
is cha ac e ized by an in e media e-low e iciency (kca /Km: SDF-1α >
MDC > I-TAC > IP-10 > MIG > eo axin > RANTES > LD78β) [185].
Despi e his, N- e minal clipping by CD26 esul s in an iso o m (3-74)
wi h a educed chemo ac ic ac i i y ha causes CCR3-desensi iza ion
[183, 186, 187]. Indeed, adminis a ion o eo axin o F344 a s leads o
a mobiliza ion o eosinophils, an e ec enhanced in CD26-de icien
animals o wi h CD26 inhibi o s [187]. Addi ionally, CD26-/- mice
challenged wi h o albumin/OVA show highe le els o CCR3 and
CCR3-ligands (eo axin, RANTES) and s onge eosinophils
in il a ion in lungs [16]. Ano he ligand o CCR3 is CCL14 (Table 3),
a chemokine p ocessed by plasmin and u okinase plasminogen
ac i a o (UPA) o yield CCL14 (9-74). This iso o m binds o CCR3
(and also CCR1 and CCR5) o e icien ly a ac eosinophils,
monocy es and TH2 cells, a p ocess also dampened by CD26 [188].
The e o e, a CD26-/low pheno ype in bo h eosinophils and TH2 cells
(CCR3+) seems o a ou he in lamma o y esponse in TH2high-as hma.
CXCR4 is ound in monocy es and B/T (p e e en ially TH2)
cells [177, 189] and p omo es he ec ui men o T-cells in he lungs
du ing alle gic ai way diseases [178, 190]. CXCR4 ecognizes
mac ophage mig a ion inhibi o y ac o (MIF) and s omal cell-
de i ed ac o 1 (SDF1/CXCL12) [189] (Table 3), he las one a small
chemokine syn hesized by endo helial cells and ib oblas s ha
induces T/B cells ac i a ion [142] and egula es he a ic o
lymphocy es, monocy es and dend i ic cells owa d in lamed epi helia
[179, 189, 191, 192]. Amongs he se e al SDF1 iso o ms, a leas wo
(SDF1α and SDF1β) a e e icien ly p ocessed by CD26 in i o [185,
193-195] and in i o [93]. The SDF-1α (3-67) iso o m is unable o
ac i a e CXCR4 and displays an agonis ic ac i i y [185, 193-198].
Mo eo e , bo h CD26 and CXCR4 ha e simila exp ession kine ics
upon ac i a ion [58, 199] and in e ac in lymphocy es [114]. Binding
o SDF1α o CXCR4 ini ia es he CXCR4-CD26 complex endocy osis
[114], a p ocess dis up ed by N- e minal clipping o SDF1α [195].
This inding could no explain howe e he p e e en ial exp ession o
CXCR4 in CD26low cells (e.g., TH2 o naï e TH) [76, 199], o why
TGFβ (a cy okine ha induces CD26 downmodula ion) leads o
augmen ed exp ession o CXCR4 and a po en ia ed SDF1α-CXCR4
axis [200-202]. This CD26-/lowCXCR4+ pheno ype also acili a es a
igo ous esponse o SDF1α in he ec ui men o T cells and
eosinophils in a mouse model o lung alle gic in lamma ion [178]. On
he o he hand, CD26 has also been posi i ely co ela ed wi h he in
i o in asi e capaci y o T cell lines in esponse o SDF1α, and his
posi i e e ec is media ed by CD45 [203], a y osine phospha ase ha
enhances cell mig a ion in esponse o SDF-1α [204].
CCR4 is p esen on monocy es, dend i ic cells, NK cells and
TH cells (TH2, TH17, T eg). CCR4 ecognizes bo h CCL17/TARC and
CCL22/MDC (Table 3), he las chemokine p oduced by
mac ophages, dend i ic cells, NK cells, and B/T lymphocy es [205].
TARC and MDC a e exp essed by epi helial cells in he ai ways, and
hei le els a e up egula ed a e alle gen challenge [103]. TH2
cy okines (e.g., IL-4 and IL-13), LPS, IL-1 and TNFα s imula e he
sec e ion o MDC, whe eas TH1 cy okines (e.g., IFNα, IL-12) inhibi
MDC p oduc ion [205, 206]. Mo eo e , MDC gene a es and ampli ies
TH2 esponses and ec ui s TH2 lymphocy es [205-207], being
impo an in diseases wi h a TH2-cy okine p o ile (e.g., as hma) o
equi alen models in mice [207]. In hese models, CCR4-MDC has a
dominan ole in la e and ch onic s ages o he disease as compa ed
wi h CCR3-Eo axin [181]. Fu he mo e, CD26 p ocesses e y
e icien ly MDC (hal -li e: 2-5 min) o gene a e MDC(3-69) and,
subsequen ly, MDC(5–69) [185]. This las iso o m p ese es he
a ac an powe o monocy es, bu displays educed chemo ac ic
ac i i y o lymphocy es and dend i ic cells [150], wo subse s
impo an in as hma. As T egs, TH2 and CCR4+ TH17 cells a e CD26-
/low cells, while CCR4- TH17 cells a e CD26high [72, 79], he CD26-/low
pheno ype o hese CCR4+ subse s should appa en ly acili a es hei
ec ui men o in lamma o y si es.
4.3.2.- CD26, TH1/TH17- ela ed chemokines and TH2low as hma
Mos o as hma pa ien s p esen a TH2high disease, bu he e a e o he
wo ypes (obesi y-associa ed and neu ophilic as hma) linked o a
TH2low (i.e., TH1/TH17) p o ile [1]. The CD26high T cell subse displays a
CD45RO+CCR7low e ec o -memo y pheno ype and p oduces
TH1/TH17 cy okines [72, 77, 208]. TH1 cells a e CCR5+CXCR3A+
lymphocy es [72, 208], a e essen ial o he p oduc ion o IgM, IgG,
and IgA (bu no IgE) by B-cells, and o ches a e he esponse agains
in acellula mic obes [103]. A ew CCR5 ligands and all he
CXCR3A-speci ic chemokines a e CD26 subs a es (Table 3). Fo
example, CCL5/RANTES is p oduced by endo helial and epi helial
cells, pla ele s, mac ophages, eosinophils and T cells [209]. RANTES
ac i a es CCR1, CCR3 (TH2) and p e e en ially CCR5 (TH1), he eby
a ac ing monocy es, eosinophils and T cells o in lamma o y si es
[209]. This chemokine is clea ed wi h low e iciency (hal -li e o 400
min) by CD26 [100, 185], leading o RANTES(3-68), a chemokine
ha loses i s capaci y o bind CCR1 and CCR3 (monocy es,
eosinophils and TH2 cells), bu no CCR5 [197]. As CCR5 is exp essed
on TH1 and CD45RA-CD45R0+CCR7low e ec o -memo y CD4+ T
cells (bo h CD26+/high) [76, 103, 199], his means ha RANTES(3-68)
a ou s hei a ac ion owa d in lamma o y si es [175, 197, 198,
210].
Ano he TH1-chemokine and CD26-subs a e ha binds CCR5
is LD78β/CCL3L1 [211]. Mac ophage in lamma o y p o ein–1α
(MIP-1α) is encoded by 2 di e en loci: LD78α and LD78β [212].
Bo h chemokines a e agonis s o CCR1, CCR3 and CCR5, bu LD78β
binds wi h high a ini y o CCR5 [213], egula ing he
a ic/ac i a ion o mac ophages/monocy es, NK cells, eosinophils,
basophils, imma u e dend i ic cells, and T lymphocy es. S ikingly,
he ine icien (hal -li e ~ 5 h) [185, 211] clipping o LD78β by CD26
gene a es LD78β(3-70) enhances he chemo ac ic ac i i y o TH1 cells
al eady been highligh ing h oughou his e iew, wi h
p oin lamma o y o an i-in lamma o y e ec s depending on he
p ocessed subs a e: inc e ins o chemokines/subs ance P,
espec i ely.
Obese pe sons a e a isk o a numbe o co-mo bidi ies like
ype 2 diabe es melli us (T2DM), a disease cha ac e ized by impai
p oduc ion o inc e ins, wi h subsequen ly hype glycaemia. Inc e ins
(GIP, GLP-1, GLP-2) a e well-known CD26 subs a es ha lose hei
unc ion when hey a e p ocessed. Inhibi ion o CD26 enzyma ic
ac i i y a oids he deg ada ion o hese ho mones, enhances he
inc e in e ec , imp o es bo h he insulin sec e ion and he glucose
up ake and lowe s he glycosyla ed haemoglobin A1c le els. To
achie e hese goals, a new g oup o an i-hype glycaemic d ugs (CD26
inhibi o s o glip ins) ha e come on he ma ke , wi h si aglip in,
(Janu ia®; Me ck Sha p & Dohme L d) and ildaglip in (Gal us®;
No a is Eu opha m L d) (bo h app o ed by Eu opean Medicines
Agency/EMA in 2007) as he i s ou pos (Table 4). Cu en ly, he e
a e se e al o ally adminis e ed CD26 inhibi o s ha ha e been
app o ed by agencies like he Food and D ug Adminis a ion (FDA)
o EMA and a e being used wi h sa is ac o y esul s as a second o
hi d line medica ion in combina ion wi h o he o al an idiabe ic
d ugs. Comme cially a ailable glip ins include he abo e-men ioned
si aglip in (Janu ia®, Ris aben®), bu also saxaglip in (Onglyza®),
linaglip in (T ajen a®), aloglip in (Vipidia®), and ildaglip in
(Gal us®, Jal a®, Xilia x®) (Table 4). Howe e , he e is a
con o e sial deba e abou he seconda y e ec s on co-mo bidi ies
(e.g., ca dio ascula ou come, enal impai men , acu e panc ea i is)
ha he long- e m ea men wi h hese long hal -li e inhibi o s may
ha e. These conce ns a e as a esul o se e al issues ela ed o he
g ea numbe o CD26 subs a es linked o no o he immune sys em.
Thus, he SAVOR TIMI-53 (saxaglip in) [235] ial de ec ed a
signi ican ly inc eased a e o hospi aliza ion due o hea ailu e, and
a mo e ecen s udy based on FAERS (US-FDA Ad e se E en
Repo ing Sys em) [236] is also consis en wi h his inding. In clea
con as , he TECOS ial (si aglip in) [237] did no ind an inc eased
isk o hea ailu e.
Table 4. DPP4 inhibi o s egis e ed o clinical use in EMA and ad e se
d ugs eac ions iden i ied du ing bo h he clinical and pos -ma ke ing
su eillance s age
Ac i e
subs ance
used as
mono he apy
P op ie a y Name
(Company;
Da e o EMA
au ho iza ion)
Immune sys em &
Respi a o y
diso de s,
in ec ions
(F equency)**
Skin and subcu aneous issue
diso de s (F equency)**
Si aglip in
Janu ia®
(Me ck Sha p &
Dohme L d; 2007)
Ris aben®
(Me ck Sha p &
Dohme L d; 2010)
Hype sensi i i y,
including
anaphylac ic
esponses (no
known)*
In e s i ial lung
disease (no
known)
P u i us (uncommon)*
Angioedema (no known) *
Rash (no known) *
U ica ia (no known) *
Cu aneous asculi is (no
known) *
Ex olia i e skin condi ions
including S e ens-Johnson
synd ome (no known) *
Bullous pemphigoid (no
known) *
Vildaglip in
Gal us®
(No a is
Eu opha m L d;
2007)
Jal a®
(No a is
Eu opha m L d;
2008)
Xilia x®
(No a is
Eu opha m L d;
2008)
Uppe
espi a o y
in ec ion ( e y
a e)
Nasopha yngi is
( e y a e)
U ica ia (no known) *
Bullous pemphigoid (no
known)*
Ex olia i e skin condi ions
(no known)*
Con inued on nex page
Table 4 (Con inued)
Saxaglip in
Onglyza®
(As aZeneca AB;
2009)
Small dec ease
in he absolu e
coun o
pe iphe al blood
lymphocy es
Uppe
espi a o y
in ec ion
(common)
Sinusi is
(common)
P u i us (uncommon) *
Angioedema ( a e) *
Rash (common) *
U ica ia (uncommon) *
De ma i is (uncommon)
Linaglip in
T ajen a®
(Boeh inge
Ingelheim
In e na ional
GmbH; 2011)
Nasopha yngi is
(uncommon)
Hype sensi i i y
(e.g. b onchial
hype eac i i y)
(no known)
Cough
(uncommon)
Angioedema ( a e) *
Rash (uncommon) *
U ica ia ( a e)*
Bullous pemphigoid (no
known)*
Aloglip in
Vipidia®
(Takeda Pha ma
A/S; 2013)
Uppe
espi a o y
in ec ion
(common)
Nasopha yngi is
(common)
Hype sensi i i y
eac ions (no
known) *
P u i us (common)
Rash (common)
Ex olia i e skin condi ions,
including S e ens-Johnson
synd ome, E y hema
mul i o me, Angioedema
and U ica ia (no known) *
*Ad e se d ug eac ions (ADRs) iden i ied based on pos -ma ke ing su eillance **Absolu e
equencies o ADRs; e y common (≥ 1/10), common (≥ 1/100 o < 1/10), uncommon (≥
1/1,000 o 1/100), a e (≥ 1/10,000 o 1/1,000), e y a e (< 1/10,000), o no known (i.e., no
es ima ed).
Mo e in acco dance wi h he scope o his e iew, CD26
e e sible inhibi o s seem o inc ease he equency o isk ac o s o
as hma de elopmen /exace ba ion like a opic sensi iza ion, hini is, o
hino i us in ec ion in T2DM pa ien s [232]. Thus, a apid e iew o
clinical and da a published on he EMA web page
(h p://www.ema.eu opa.eu) suppo s inc eased isk o nonse ious
uppe espi a o y ac in ec ions (e.g., i al nasopha yngi is, hini is,
sinusi is) (Table 4), which would be in line wi h he cos imula o y ole
o CD26. As Willemen and co-au ho s sus ain, his inc eased isk o
in ec ions wi h CD26 inhibi o s (~3% cases) canno be compa ed wi h
he magni ude o he e ec s seen wi h biological agen s like umou
nec osis ac o inhibi o s [238]. Howe e , his e ec should no be
unde es ima ed and mus be added o he immune impai men caused
by T2DM i sel o inc ease he o e all as hma
de elopmen /exace ba ion isk in hese pa ien s.
O he ad e se d ug eac ions (ADRs) epo ed in associa ion
wi h glip ins and mo e associa ed wi h an inhibi o y ole o CD26
would be in e s i ial lung disease, hype sensi i i y eac ions (including
anaphylac ic esponses o b onchial hype eac i i y), and skin and
subcu aneous issue diso de s such as p u i us, angioedema, ash,
u ica ia, cu aneous asculi is, o mo e se e e and a e skin condi ions
(Table 4). Fo example, Bullous pemphigoid [187, 239-241] is an
au oimmune subepide mal disease wi h o e p oduc ion o eo axin and
eosinophilia ha a ec s skin and mucosae. The e o e, he associa ion
be ween glip ins and Bullous pemphigoid is in une wi h in i o
s udies in F344 a s showing ha CD26 limi s he eo axin-media ed
ec ui men o eosinophils [187], o wi h he inding ha o al
adminis a ion o CD26 inhibi o s p omo es he alle gic in lamma ion
o he ai ways [45]. This means ha CD26 could be pa o a
homeos a ic mechanism o downmodula e ai ways in lamma ion
media ed by TH2 and especially TH17/TH1 cells impo an in some
TH2low as hma pheno ypes such as obesi y- ela ed as hma.
Ne e heless, i should be no ed ha besides chemokines he e a e
o he CD26 subs a es wi h implica ions on obesi y and as hma-like
he subs ance P. Inhaled subs ance P, bu no b adykinin enhances he
ai way esponse o b onchocons ic ing agen s in guinea-pigs [242].
Subs ance P also a ou s alle gen sensi iza ion and b onchial
in lamma ion, as obse ed in a mouse model o die -induced obesi y
sensi ized and challenged wi h OVA and ea ed wi h an an agonis o
he subs ance P ecep o (NK1-R) [243]. The e o e, expanded hal -li e
in subs ance P caused by ea men wi h CD26 inhibi o s in T2DM
pa ien s could inc ease ce ain obesi y- ela ed co-mo bidi ies such as
as hma. In he same way, adminis a ion o si aglip in in T2DM
gene a es a empo a y dec ease in he pe cen age o pe iphe al blood
T egs ha also could a ou as hma p e alence in hese pa ien s [244].
In clea con as , GLP-1-based he apies ha e an i-in lamma o y
e ec s in ch onic in lamma o y diseases including T2DM and as hma
[245]. Fo example, in i o s udies ha e desc ibed ha CD26
inhibi o s induce a dec ease o NLRP3 in lammasome, oll-like
ecep o 4 (TLR4) signalling and IL-1β in mac ophages ia GLP-1
ecep o [160]. The e o e, mo e s udies a e needed o cla i y whe he
glip ins ha e an o e all posi i e o nega i e ole in T2DM pa ien s
wi h obesi y- ela ed as hma.
6.- CONCLUDING REMARKS
CD26 is a ele an molecule in as hma o se e al easons. The i s
one because o i s po en ial u ili y oge he wi h pe ios in o es he
e icacy o he ea men wi h in e leukin-13 neu alising monoclonal
an ibodies (e.g., T alokinumab) in pa ien s wi h se e e uncon olled
as hma [246, 247]. Secondly, because o i s mul i unc ionali y na u e,
wi h bo h cos imula o y and inhibi o y oles in he immune sys em,
apa om he high (bu a iable) CD26 le els in a subse o
lymphocy es cen al o as hma: he CD4+ T cells. Di e en au ho s
ha e highligh ed he inhibi o y unc ions o his molecule ela i e o
signalling media ed by cy okines and chemokines. A hand ul o
chemokines impo an o CD26+ TH2 cells du ing ea ly-onse alle gic
o la e-onse eosinophilic as hma includes chemo ac ic ac o s whose
biological ac i i y is educed wi h low-in e media e (eo axin) o high
(MDC, SDF-1α) e iciency, whe eas impo an chemokines o CD26+
TH1 (I-TAC, IP-10, Mig) and CD26high TH17 cells (MDC), wo Te
subse s ele an in obesi y- ela ed as hma, la e-onse neu ophilic
as hma o se e e as hma, a e inac i a ed wi h in e media e-high
e ec i eness. The e o e, CD26 could ac as a “biological b ake” by
educing he a ac ion o Te cells a si es o in lamma ion (e.g., he
b onchi). This means ha CD26 inhibi o s used in T2DM pa ien s
could lead o a highe pe sis ence o unp ocessed subs ance P o
chemokines and exace ba ion o TH2high and especially TH2low as hma.
In conclusion, i is necessa y o p e iously ake in o conside a ion he
many exis ing ques ions ega ding he biological unc ions o his
enzyme be o e he he apeu ic a ge ing o his molecule [248], as
CD26 inhibi o s could enhance he equency o hospi al admission
among people wi h ce ain as hma pheno ypes.
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