Gu Mic obes
ISSN: (P in ) (Online) Jou nal homepage: h ps://www. and online.com/loi/kgmi20
The commensal bac e ium Lac iplan ibacillus
plan a um imp in s inna e memo y-like esponses
in mononuclea phagocy es
Aize Pellon, Diego Ba iales, Ainize Peña-Cea a, Jani e Cas elo-Ca eaga,
Ainhoa Palacios, Ne ea Lopez, Es ibaliz A ondo, Miguel Angel Pascual-
I oiz, I zia Ma ín-Ruiz, Le icia Samped o, Monika Gonzalez-Lopez, Lau a
Bá cena, Te esa Ma ín-Ma eos, Jose Ma ía Lande e, Ra ael P ados-Rosales,
Lau a Plaza-Vinuesa, Rosa io Muñoz, Blanca de las Ri as, Juan Miguel
Rod íguez, Edu ne Be a, Ana M. A ansay, Le icia Abecia, Jose Luis La ín,
Hec o Rod íguez & Juan Angui a
To ci e his a icle: Aize Pellon, Diego Ba iales, Ainize Peña-Cea a, Jani e Cas elo-Ca eaga,
Ainhoa Palacios, Ne ea Lopez, Es ibaliz A ondo, Miguel Angel Pascual-I oiz, I zia Ma ín-Ruiz,
Le icia Samped o, Monika Gonzalez-Lopez, Lau a Bá cena, Te esa Ma ín-Ma eos, Jose Ma ía
Lande e, Ra ael P ados-Rosales, Lau a Plaza-Vinuesa, Rosa io Muñoz, Blanca de las Ri as,
Juan Miguel Rod íguez, Edu ne Be a, Ana M. A ansay, Le icia Abecia, Jose Luis La ín, Hec o
Rod íguez & Juan Angui a (2021) The commensal bac e ium Lac iplan ibacillus�plan a um imp in s
inna e memo y-like esponses in mononuclea phagocy es, Gu Mic obes, 13:1, 1939598, DOI:
10.1080/19490976.2021.1939598
To link o his a icle: h ps://doi.o g/10.1080/19490976.2021.1939598
© 2021 The Au ho (s). Published wi h
license by Taylo & F ancis G oup, LLC.
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RESEARCH PAPER/REPORT
The commensal bac e ium Lac iplan ibacillus plan a um imp in s inna e
memo y-like esponses in mononuclea phagocy es
Aize Pellon
a
#
, Diego Ba iales
a
#
, Ainize Peña-Cea a
a,b
, Jani e Cas elo-Ca eaga
a
, Ainhoa Palacios
a
,
Ne ea Lopez
a
, Es ibaliz A ondo
a
, Miguel Angel Pascual-I oiz
a
, I zia Ma ín-Ruiz
a
, Le icia Samped o
a
,
Monika Gonzalez-Lopez
c
, Lau a Bá cena
c
, Te esa Ma ín-Ma eos
d
, Jose Ma ía Lande e
e
, Ra ael P ados-Rosales
a,k
,
Lau a Plaza-Vinuesa
, Rosa io Muñoz
, Blanca de las Ri as
, Juan Miguel Rod íguez
g
, Edu ne Be a
d
,
Ana M. A ansay
c,h
, Le icia Abecia
a,b
, Jose Luis La ín
i,k
, Hec o Rod íguez
a
, and Juan Angui a
a,j
a
In lamma ion and Mac ophage Plas ici y Labo a o y, CIC bioGUNE-BRTA (Basque Resea ch and Technology Alliance), De io, Spain;
b
Facul y o
Medicine and Nu sing, Uni e sidad Del Pais Vasco (UPV/EHU), Leioa, Spain;
c
Genomic Analysis Pla o m, CIC bioGUNE-BRTA, De io, Spain;
d
Physiopa hology o he Hypoxia-signaling Pa hway Labo a o y, CIC bioGUNE-BRTA, De io, Spain;
e
Depa amen o De Tecnología De Alimen os,
Ins i u o Nacional De In es igación Y Tecnología Ag a ia Y Alimen a ia (INIA), Mad id, Spain;
Labo a o io De Bio ecnología Bac e iana, Ins i u o
De Ciencia Y Tecnología De Alimen os Y Nu ición (ICTAN-CSIC), Mad id, Spain;
g
Depa men o Nu i ion and Food Science, Uni e sidad
Complu ense De Mad id, Mad id, Spain;
h
CIBERehd, ISCIII, Mad id, Spain;
i
Bioin o ma ics Uni , CIC bioGUNE-BRTA, De io, Spain;
j
Ike basque,
Basque Founda ion o Science, Bilbao, Bizkaia, Spain;
k
Cen e o Hos -Mic obiome In e ac ions, Facul y o Den is y, O al and C anio acial
Sciences, King’s College London, Uni ed Kingdom; RPR: Depa men o P e en i e Medicine and Public Heal h and Mic obiology, Uni e sidad
Au ónoma De Mad id, Mad id 28029, Spain; JLL: Applied Ma hema ics Depa men , Bioin o ma ics Uni , NEIKER-BRTA, Pa que Tecnológico De
Bizkaia, De io, Spain
ABSTRACT
Gu mic obio a is a cons an sou ce o an igens and s imuli o which he esiden immune sys em
has de eloped ole ance. Howe e , he mechanisms by which mononuclea phagocy es, speci i-
cally monocy es/mac ophages, cope wi h hese usually p o-in lamma o y signals a e poo ly unde -
s ood. He e, we show ha inna e immune memo y p omo es an i-in lamma o y homeos asis, using
as model s ains o he commensal bac e ium Lac iplan ibacillus plan a um. P iming o monocy es/
mac ophages wi h bac e ia, especially in i s li e o m, enhances bac e ial in acellula su i al and
dec eases he elease o p o-in lamma o y signals o he en i onmen , wi h lowe p oduc ion o
TNF and highe le els o IL-10. Analysis o he ansc ip omic landscape o hese cells shows
down egula ion o pa hways associa ed wi h he p oduc ion o eac i e oxygen species (ROS)
and he elease o cy okines, chemokines and an imic obial pep ides. Indeed, he induc ion o
ROS p e en s memo y-induced bac e ial su i al. In addi ion, he e is a dys egula ion in gene
exp ession o se e al me abolic pa hways leading o dec eased glycoly ic and espi a o y a es in
memo y cells. These da a suppo commensal mic obe-speci ic me abolic changes in inna e
immune memo y cells ha migh con ibu e o homeos asis in he gu .
ARTICLE HISTORY
Recei ed 14 Oc obe 2020
Re ised 14 May 2021
Accep ed 30 May 2021
KEYWORDS
Lac iplan ibacillus plan a um;
Lac obacillus; monocy es;
mac ophages; inna e
immune memo y; ained
immuni y; mic obio a;
immunome abolism
In oduc ion
The eme gence o mic obio a esea ch has
expanded ou knowledge on he ole o commensal
mic oo ganisms in con olling a wide a ie y o
physiological unc ions bo h in he s eady s a e
and in disease. In he gu , whe e he mic obial
load is g ea e han in any o he body si e, mic o-
bio a componen s cons i u e a con inuous sou ce o
s imuli o which he immune sys em has e ol ed
ole ance, leading o he modula ion o immune
esponses.
1
Co-e olu ion wi h he my iad o
mic obes p esen in he gu has led o an equili-
b ium be ween he egula ion o homeos a ic
esponses o ha mless an igens, and he abili y o
e ec i ely elimina e pa hogens. Al hough he ole
o adap i e immune cells in he gu has been ex en-
si ely desc ibed,
2
he egula ion o mononuclea
phagocy ic unc ion (i.e. mac ophages and dend i-
ic cells) by mic obio a membe s emains poo ly
unde s ood.
3
Impo an ly, gu mononuclea pha-
gocy es show abe an an i-in lamma o y esponses
in an ibio ic- ea ed mice. These cells ail o
CONTACT Juan Angui a [email p o ec ed] In lamma ion and Mac ophage Plas ici y Labo a o y, CIC bioGUNE-BRTA (Basque Resea ch and
Technology Alliance), De io 48160, Spain; Hec o Rod íguez [email p o ec ed] In lamma ion and Mac ophage Plas ici y Labo a o y, CIC
bioGUNE-BRTA (Basque Resea ch and Technology Alliance), De io 48160, Spain
#
Equal con ibu ion
Supplemen al da a o his a icle can be accessed on he publishe ’s websi e.
GUT MICROBES
2021, VOL. 13, NO. 1, e1939598 (16 pages)
h ps://doi.o g/10.1080/19490976.2021.1939598
© 2021 The Au ho (s). Published wi h license by Taylo & F ancis G oup, LLC.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use,
dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
egula e T cell popula ions in his o gan,
4
while
some bac e ial species such as Helicobac e hepa i-
cus
5
o Clos idium bu y icum
6
di ec ly con ibu e
o IL-10 homeos asis. Rema kably, small popula-
ions o gu bac e ia ha e been ound o be asso-
cia ed wi h dend i ic cells in he mesen e ic lymph
nodes, inducing he p oduc ion o speci ic sec e o y
IgA and p omo ing an i-in lamma o y esponses,
including he elease o he immuno egula o y
cy okine IL-10.
7
Addi ionally, mic obial me abo-
li es, such as sho -chain a y acids (SCFAs), a e
known o egula e bo h dend i ic cell and mac o-
phage unc ion in he gu , p omo ing an i-
in lamma o y/hypo esponsi e s a es in inna e
immune cells and con ibu ing o he de elopmen
o in es inal homeos asis.
3
Since he disco e y o phagocy ic cells by Ilya
Mechniko ,
8
ou knowledge on monocy e and
mac ophage plas ici y has eno mously e ol ed.
This includes phenomena desc ibed in he las dec-
ade such as he abili y o di e en inna e immune
cell ypes, monocy es/mac ophages among hem, o
gene a e long- e m esponses, namely inna e
immune memo y.
9
This mechanism in ol es an
epigene ic and me abolic ep og amming induced
by he con ac wi h an in ec ious agen o mic obial
componen ha leads o an enhanced ( ained
inna e immuni y) o dec eased ( ole ance) cy o-
kine-media ed esponse o a seconda y s imula ion
wi h a, usually di e en , mic obial componen .
10,11
Consequen ly, inna e immune memo y has been
p o en o be an impo an egula o no only o
an imic obial esponses o inna e immune cells, bu
o hei oles in in lamma o y and neu ological
diseases.
12
Howe e , since all o hese s udies
ha e been pe o med in a pa hological con ex , no
in o ma ion is a ailable o da e ega ding he ole
ha inna e immune memo y plays in coping wi h
he con inuous exposu e o inna e immune cells o
commensal mic obes (i.e. human mic obio a), and
whe he his phenomenon plays a ole in immune
modula ion by commensal bac e ia.
13
Lac obacillus plan a um, ecen ly eclassi ied as
Lac iplan ibacillus plan a um,
14
is a G am-posi i e
species ha has been ex ensi ely cha ac e ized o
i s adap i e abili y o h i e wi hin he human gu
and he bene i s o i s immunomodula o y p ope -
ies o he human hos , wi h some L. plan a um
s ains being widely used as p obio ics.
15
The e o e, we used his species as a model o
a bene icial mic obe o show ha , while acu e expo-
su e o monocy e/mac ophages allows bac e ial
in acellula su i al and induce bo h p o- and
an i-in lamma o y cy okine elease, a p e ious con-
ac wi h L. plan a um ep og ams he ansc ip-
ional and me abolic p o iles o immune cells in he
long e m. This inna e immune memo y-like e en s
e en ually lead o enhanced bac e ial in acellula
su i al and dec eased p o-in lamma o y ea u es
in p e-s imula ed cells.
Resul s
Lac iplan ibacillus plan a um can su i e wi hin
mac ophages om di e en o igins
Recen epo s ha e shown ha dend i ic cells
popula ing he mesen e ic lymph nodes ha bo
small amoun s o gu bac e ia inside, which con-
ibu e o he modula ion o bo h inna e and adap-
i e esponses.
7
Gi en i s genomic simila i y wi h
some in acellula pa hogens, such as Lis e ia
monocy ogenes,
16
we hypo hesized ha
L. plan a um migh also be able o su i e in acel-
lula ly and ha his could be associa ed wi h i s
immunomodula o y e ec s. To un eil he po en ial
o his species o su i e in acellula ly in mac o-
phages we used an ibio ic p o ec ion assays (Fig.
S1A). As con ols, we also used he closely ela ed
species, Lac obacillus casei ( ecen ly eclassi ied as
Lac icaseibacillus casei) as well as he en e ic spe-
cies, Esche ichia coli. Fi s , using a mul iplici y o
in ec ion (m.o.i.) o 10, we obse ed ha all he
L. plan a um s ains used in his s udy we e able
o su i e inside he mac ophage-like cell line,
RAW264.7 o 24 h, wi h s ains o human milk
o igin showing sligh ly highe su i al a es. In
con as , he numbe o L. casei and E. coli inside
hese phagocy es was lowe (Fig. S1B).
In o de o alida e ou esul s, we used wo
p ima y cellula models, mu ine bone ma ow-
de i ed mac ophages (mBMM) and human mono-
cy e-de i ed mac ophages (hMDM). O e all, in a-
cellula su i al o L. plan a um s ains was
obse ed in bo h cell ypes, al hough o a lesse
ex en in compa ison wi h he RAW264.7 cell line
(Figu e 1a, b). Simila o ou obse a ions using he
cell line, in acellula su i al o L. casei was lowe
e1939598-2 A. PELLON ET AL.
han ha o L. plan a um s ains, wi h he p esence
o iable E. coli wi hin he mac ophages being d a-
ma ically dec eased om he beginning o he
expe imen . Simila esul s we e obse ed when
an m.o.i. o 1 was used (Fig. S1C, D). To de ec
he cellula compa men in which L. plan a um
pe sis s inside mac ophages, we incuba ed
mBMMs wi h mChe y-labeled bac e ia and
assessed hei colocaliza ion wi h LAMP-2,
a phagolysosome ma ke
17
, using con ocal mic o-
scopy. No ably, while hea -killed bac e ia we e
obse ed su ounded by LAMP-2 posi i e esicles,
li e bac e ia did no show colocaliza ion wi h he
ma ke sugges ing ha hey a e able o e ade hei
localiza ion wi hin hese deg ada i e o ganelles
(Figu e 1c).
We also pe o med whole blood ex i o in ec ion
assays and analyzed he p esence o bac e ia. We
incuba ed EDTA- ea ed human blood om
heal hy dono s wi h L. plan a um WCFS1 modi ied
o cons i u i ely exp ess mChe y and e alua ed
he in e naliza ion o he bac e ia and i s p esence
o e ime by low cy ome y. L. plan a um was
ound p edominan ly associa ed wi h CD14
+
monocy es (Figu e 1d). In ac , up o 42.2% o
hese cells we e able o in e nalize and main ain
L. plan a um o he du a ion o he assay.
Nex , we assessed he abili y o L. plan a um o
escape om mac ophages a e i s in e naliza ion.
A e adding bac e ia and p omo ing phagocy osis
o 45 min, ex acellula bac e ia we e washed
away, and mac ophages we e cul u ed ei he o 1
o 4 h in medium supplemen ed wi h an ibio ics o
kill emaining ex acellula mic obes (Fig. S1E).
Then, mBMMs we e washed wi h wa m PBS and
incuba ed in an ibio ic- ee medium o 24 h.
Samples om supe na an s and cell lysa es we e
pla ed on o MRS aga pla es o assess bac e ial
iabili y. No ably, high numbe s o iable bac e ia
we e ound bo h in he in acellula (Figu e 1e) and
ex acellula ( igu e 1 ) compa men s. O e all,
L. plan a um s ains su i ed be e in compa ison
Figu e 1. L. plan a um colonize and su i e wi hin mac ophages om di e en o igins. Bac e ia we e co-cul u ed wi h ei he
mouse bone ma ow-de i ed mac ophages (mBMMs) (a) o human monocy e-de i ed mac ophages (hMDMs) (b), and hei in acel-
lula su i al a di e en ime poin s was de e mined using an ibio ic p o ec ion assays (Fig. S1A). A e incuba ing bac e ia wi h
immune cells, wells we e washed, and an ibio ic-con aining medium was added. A e 1, 4 o 24 h cells we e lysed, and suspensions
pla ed o assess bac e ial in acellula su i al. (c) Dis inc colocaliza ion o li e (le mic og aph) and hea -killed, mChe y-exp essing
L. plan a um ( igh mic og aph, ed) wi h he phagolysosome ma ke , LAMP-2 (g een). The nuclei we e s ained wi h DAPI (blue). The
o hogonal p ojec ion o he indica ed poin s (c osshai s) is p esen ed a he bo om and igh side o he mic og aphs. (d)
An icoagula ed whole blood was incuba ed wi h mChe y-labeled L. plan a um WCFS1 and i s associa ion wi h CD14
+
cells was
de e mined by low cy ome y. Rep esen a i e low cy ome y da a is shown. (e, ) Bac e ial abili y o pe sis in acellula ly (e) and
each he ex acellula medium ( ) a e being engul ed by immune cells. A e sho incuba ion imes (1 and 4 hou s) in an ibio ic-
con aining medium, cells we e ex ensi ely washed and incuba ed wi h an ibio ic- ee medium o 24 h. Samples om he supe na an
and immune cell lysa es we e pla ed o check bac e ial iabili y. Da a a e shown as mean ± s.e.m., n ≥ 3. **; p < .01, ***; p < .001, wo-
way ANOVA compa ed o L. plan a um WCFS1.
GUT MICROBES e1939598-3
wi h L. casei bo h inside and ou side o mBMMs.
The e o e, hese da a show ha a e coloniza ion
o mac ophages, L. plan a um can escape om he
phagocy es o he ex acellula medium.
Toge he , hese da a show ha L. plan a um can
be in e nalized by mu ine and human monocy e/
mac ophages and su i e o p olonged pe iods o
ime, compa ed o o he commensal bac e ia.
P e-exposu e o mac ophages o L. plan a um
enhances bac e ial su i al and dec eases
p o-in lamma o y ou pu s
In he las decades, ou knowledge on mac ophage
plas ici y and abili y o display long- e m esponses
has g ea ly inc eased. O no e, esea ch e o s ha e
mainly ocused on s udying pa hogenic/pa hobion
mic obes, pa ially igno ing he epe oi e o long-
las ing esponses de eloped by hese cells in
esponse o di e en s imuli.
18
Thus, we assessed
he abili y o L. plan a um o induce inna e
immune memo y-like esponses in monocy es/
mac ophages in i o (Figu e 2a). P e-s imula ion
o mBMMs wi h a m.o.i. o 1 o li e L. plan a um
(Lp-Lp) enhanced bac e ial su i al in a second
encoun e in compa ison wi h uns imula ed cells
(U-Lp), while p iming wi h hea -killed bac e ia
(HkLp-Lp) did no esul in a signi ican inc ease
in bac e ial su i al (Figu e 2b). O no e, no bac e -
ial su i al was de ec ed in he Lp-Lp g oup be o e
he second s imula ion, sugges ing ha li e bac e ia
om he i s s imulus had been elimina ed.
Rema kably, p e-s imula ion wi h li e bac e ia
also led o dec eased TNF elease a e he second
s imula ion, while hea -inac i a ed bac e ia p o-
mo ed a mo e mode a e educ ion. In u n, cell
cul u e supe na an s o mBMMs p imed wi h li e
bac e ia con ained inc eased le els o IL-10 com-
pa ed o s imula ed naï e cells (Figu e 2c). This
e ec was no es ic ed o he WCSF1 s ain as
Figu e 2. P iming wi h L. plan a um enhances bac e ial in acellula su i al and educes p o-in lamma o y cy okine elease. (a)
Diag am showing he expe imen al se up o p iming expe imen s using mu ine bone ma ow-de i ed mac ophages (mBMMs) and
human monocy es (hMon). P iming mBMMs wi h L. plan a um (m.o.i. = 1), especially in i s li e o m, inc eased bac e ial in acellula
su i al o e ime (b) and educed he immune cell p o-in lamma o y p o ile (c). (d) Dec eased TNF elease by p imed cells depends on
bac e ial m.o.i. used. In acellula su i al and TNF p oduc ion p o iles showed simila pa e ns in p imed hMon a e ei he 24 h (e, ) o
6 d (g, h) es ing ime. P iming wi h ei he L. plan a um o L. casei imp o es bac e ial su i al (i, k) bu does no educe TNF elease (j, m)
i he o he species is used o he second s imula ion. Da a a e shown as mean ± s.e.m., n ≥ 3. *; p < .05, **; p < .01, ***; p < .001, wo-
way (B,E,G,I,K) o one-way (C,D,F,H,J,M) ANOVA.
e1939598-4 A. PELLON ET AL.
TNF dec ease was also obse ed when using as
p iming agen s wo di e en s ains isola ed om
human b eas milk, MP31 (Fig. S2A) and MP33
(Fig. S2B). No ably, induc ion o memo y-like ea-
u es in mBMMs a e L. plan a um exposu e was
dependen on bac e ial dose, showing ha
a minimum o m.o.i. 0.1 used as he i s s imulus
was necessa y o induce a signi ican dec ease in
TNF elease upon a seconda y s imula ion
(Figu e 2d). Finally, o de e mine whe he he
induc ion o memo y was due o sec e ed ac o s
p oduced by L. plan a um, we p e-s imula ed
mBMMs wi h s e ile MRS medium o il e ed con-
di ioned medium ob ained om an o e nigh cul-
u e o L. plan a um WCFS1. No changes in
in acellula su i al o L. plan a um we e obse ed
(Fig. S2C), sugges ing ha soluble ac o s we e no
esponsible o he inc eased su i al o he bac e -
ium upon s imula ion wi h li e mic oo ganisms.
Ou esul s we e ecapi ula ed in human CD14
+
monocy es (hMon) in i o using bo h 24 h o 6 d
o es ing ime a e he i s s imulus, a p o ocol
p e iously used o s udy induc ion o long- e m
esponses in hese cells.
19
While ei he li e o hea -
killed L. plan a um induced he same changes in
bac e ial in acellula su i al and TNF p oduc ion
using a 24 h- es ing ime (Figu e 2e and ), li e
L. plan a um induced highe e ec s when longe
es ing imes we e applied (Figu e 2g and h). These
changes in cy okine elease p o iles sugges ed ha
p e-s imula ion wi h L. plan a um, pa icula ly in
i s li e o m, induced long- e m changes in bo h
mBMMs and hMon ea u ing an an i-in lamma o y
p o ile, which may be in ol ed in he inc eased
bac e ial in acellula su i al obse ed.
We hen es ed whe he p iming wi h o he p o-
bio ic bac e ial species, L. casei, could lead o he
same immunomodula o y e en s obse ed wi h
L. plan a um. O no e, p iming o mBMMs wi h
li e L. casei enhanced bac e ial in acellula su i al
(Fig. S2D) and educed TNF elease (Fig. S2E)
compa ed o unp imed cells, al hough o a lesse
ex en han when L. plan a um was used.
Mo eo e , since ained cells ha e shown ce ain
non-speci ici y in hei esponses o seconda y
challenges (e.g. BCG s imula ion p o ec s om un-
gal and bac e ial in ec ions
12
), we es ed he e ec
o p iming wi h one p obio ic species and use he
o he one as he seconda y s imula ion. Al hough
ends o in acellula su i al a es we e compa -
able o hose p e iously obse ed (Lp-Lp . Lc-Lp,
Figu e 2i; Lc-Lc . Lp-Lc, Figu e 2k), p iming wi h
di e en species han hose used o he second
s imula ion did no ecapi ula e he educ ion in
TNF le els (Figu e 2j and m). These da a show
ha o hese bac e ia, long- e m e ec s on phago-
cy ic cells a e, a leas pa ly, species-speci ic.
L. plan a um p iming induces long- e m changes
in he ansc ip ional p o ile o human monocy es
To del e in o he mechanisms inducing hese long-
e m esponses by L. plan a um, we s udied he
ansc ip ional p o iles o human monocy es by
RNA-seq in he h ee expe imen al condi ions p e-
iously analyzed a e 6 d o es ing ime: U-Lp, Lp-
Lp and HkLp-Lp. The h ee condi ions showed
dis inc ansc ip ional p o iles, as shown by p in-
cipal componen analysis (PCA) (Figu e 3a) and
clus e ing o he mos egula ed genes (Figu e 3b).
O e all, we ound 1030 di e en ially exp essed
genes (using cu o alues o 1 o he absolu e
log
2
Fold Change and p adj <0.05) be ween
unp imed and li e bac e ia-p imed monocy es
(U-Lp . Lp-Lp, 514 up and 516 down; Figu e 3c),
and 326 when unp imed con ols we e compa ed
wi h cells p e-s imula ed wi h hea -killed bac e ia
(U-Lp . HkLp-Lp, 93 up and 233 down; Figu e 3d),
showing ha p e-exposu e o li e L. plan a um cells
induced a g ea e impac on monocy es in he long
e m. Pa hway analysis using Pan he DB
20,21
(Table S1) showed a signi ican en ichmen o se -
e al cy okine and chemokine pa hways among he
down egula ed genes in monocy es p imed ei he
wi h li e o hea -killed bac e ia. Among o he s,
genes as IL1A, IL1B, IL6 o CCL20 we e ound
down egula ed in bo h condi ions compa ed o
uns imula ed cells (Figu e 3e, ). In addi ion, pa h-
way analysis showed down egula ed unc ions
ela ed o o ganism killing and p oduc ion o eac-
i e oxygen and ni ogen species (Table S1). In his
ega d, se e al genes coding o an imic obial pep-
ides/p o eins, such as calp o ec in (S100A8 and
S100A9) and calg anulin (S100A12), we e ound
down egula ed only in he Lp-Lp g oup
(Figu e 3e, ), which may be linked o he inc eased
bac e ial in acellula su i al obse ed in hese
cells. No ably, TNF and o he six membe s o his
GUT MICROBES e1939598-5
cy okine signaling pa hway we e obse ed down-
egula ed only in Lp-Lp monocy es, in addi ion o
he ou ound in monocy es p imed ei he wi h
li e o hea -killed bac e ia (Figu e 3g), which con-
i med he p o iles o TNF elease p e iously
de ec ed by ELISA. Mo eo e , we ound changes
in he exp ession o KAT2A (up egula ed in bo h
Lp-Lp and HkLp-Lp) and HDAC9 (down egula ed
in Lp-Lp), bo h o which a e ela ed o his one
modi ica ions and ac i a ion o ansc ip ion and
migh be in ol ed in epigene ic modi ica ions asso-
cia ed wi h inna e immune memo y.
Nex , we ca ied ou a compa a i e ansc ip ion
ac o en ichmen analysis o he di e en ially
exp essed genes in Lp-Lp and HkLp-Lp using he
HOMER package.
22
Fo up egula ed genes, only
he p53 mo i was ound as egula o o 1.46% o
genes in he Lp-Lp condi ion, while no signi ican
esul s we e ound o he HkLp-Lp gene se
(Figu e 3h). On he o he hand, mo i s associa ed
wi h NFκB-p65 con ibu ed o he highes pe cen-
age o down egula ed genes in bo h li e (Lp-Lp
condi ion) and hea -killed (HkLp-Lp condi ion)
bac e ia-p imed cells, wi h mino con ibu ions
om in e e on- egula ed ansc ip ion ac o s
(IRF2, ISRE) and CBEP unde li e bac e ia p iming
condi ions (Figu e 3h).
T ansc ip ional ep og amming in L.
plan a um-s imula ed monocy es induce changes
in cell me abolism
Analysis o monocy e ansc ip omic p o iles also
allowed us o iden i y he impac o p iming in
U-Lp . Lp-Lp U-Lp . HkLp-Lp
U-Lp Lp-Lp HkLp-Lp
U-Lp
Lp-Lp
HkLp-Lp
PC1: 51% a iance
-10 0 10
PC2: 20% a iance
10
5
0
-5
AB
CD
E
CD22
CD244
KHK
PHGDH
KAT2A
LDHD
TLR5
SLC40A1
SLC45A3
SLC46A1
TRIB3
ACSS
ALDOC
ASNS
ASRGL1
ATP6VOD2
ATP6VOE2
CD180
CERKL
CHDH
CHPT1
CR1
FAAH
FFAR4
FOLR2
GALM
GPT2
IDH2
IL1R2
LY86
ME3
MRC2
PDK2
PDK3
PDK4
PHYH
SLC16A5
SLC22A5
SLC2A1
TLR7
ACO1
ACOD1
ACSL4
ACSL5
CLEC4A
CLEC4E
CLEC4G
CSFRA
CSFRB
GZMB
HDAC9
IL18
IRF1
NLRP7
S100A8
S100A9
S100A12
SGMS2
SLC16A10
SLC2A6
SLC41A2
TLR2
TLR8
TNF
TNFRSF4
TNFRSF6B
TNFRSF9
TNFRSF10A
TNIP1
CCL20
CD55
CLEC5A
CLEC12A
CSF2
IL1A
IL1B
IL6
IL23A
IL24
ITGB3
ITGB8
SLC1A2
SLC28A3
SLC5A3
SOCS3
TGFA
TNFRSF18
TNFSF16
TNIP3
TRAF1
Up egula ed genes
HkLp-LpLp-Lp
Down egula ed genes
HkLp-LpLp-Lp
IL1B
CXCL8
CXCL16
S100A9
CCL3
CXCL9
CCL8
IL1A
CCL4
CCL18
S100A8
CXCR4
CSF3R
HAMP
DEF6
CCL1
IL18
CXCL12
PPBP
CSF2
CXCL11
CXCL6
CCL19
AZU1
S100A12
IL23A
CSF3
CXCR5
CCL23
CCL20
CXCL10
IL6
TNF
CCL3L1
CXCL2
CCL4L2
CCL24
CCL22
IL24
CCL5
CCL7
CXCL3
CTSL
CXCL5
U-Lp Lp-Lp HkLp-Lp
F
TNIP1
TNF
TNFSF13B
TNFSF15
EDA2R
TNFSF18
TNFSF6B
TNFSF4
TNFSF12A
TNFSF8
TNFSF10A
TNFSF9
TNFSF14
CD40
TRAF1
TNIP3
U-Lp Lp-Lp HkLp-Lp
G H
No signi ican esul s
Gene se
Up egula ed in Lp-Lp
Up egula ed in HkLp-Lp
Down egula ed in Lp-Lp
Down egula ed in HkLp-Lp
Mo i
C
T
G
A
A
G
C
G
C
T
A
C
G
A
T
G
G
T
C
A
T
C
G
A
T
C
C
T
A
G
C
A
G
C
A
G
A
C
G
C
T
A
C
A
G
T
C
G
T
A
A
T
G
G
T
G
T
A
G
T
C
G
A
G
C
A
T
C
G
A
T
C
T
T
C
T
C
G
T
A
C
G
A
T
G
T
A
G
A
T
A
A
G
C
T
C
T
A
C
T
C
G
A
C
C
T
A
G
T
C
G
A
C
T
G
A
C
G
T
A
T
A
C
G
G
A
C
T
T
C
A
G
T
C
G
A
G
T
C
A
T
G
C
A
T
A
C
G
A
G
C
T
T
G
A
A
T
C
G
T
C
T
G
T
G
C
C
T
G
A
T
A
C
G
A
T
T
A
C
T
G
T
C
T
G
C
A
T
G
T
T
A
G
G
A
T
C
C
T
A
G
A
T
C
C
A
A
A
G
T
C
C
G
T
A
A
T
G
G
T
G
T
A
G
T
C
G
A
G
C
A
T
C
G
A
T
C
T
T
C
T
C
G
T
A
C
G
A
T
G
T
A
G
A
T
A
A
G
C
T
C
T
A
C
T
C
G
A
C
Rank
1
1
2
3
4
5
1
2
Name
p53
NFκB-p65
NFκB-p65-Rel
IRF2
ISRE
CEBP
NFκB-p65
NFκB-p65-Rel
0.0485
0
0.0008
0.0021
0.0021
0.0021
0.0033
0.0145
(Benjamini)
q- alue
1.46%
16.74%
3.70%
5.22%
3.91%
12.39%
18.01%
4.74%
Ta ge s
% o
0.21%
8.13%
0.95%
1.90%
1.18%
6.89%
8.43%
1.30%
Backg ound
% o
CXCL1
Figu e 3. B oad ansc ip ional emodeling is induced in human monocy es a e L. plan a um p iming. (a) P incipal Componen
Analysis o human monocy es (hMon) unp imed (U-Lp) o p imed wi h ei he li e (Lp-Lp) o hea -killed (HkLp-Lp) L. plan a um. Hea -
map (b) and olcano plo s (c, d) showing di e en ially egula ed genes. Blue do s ep esen up egula ed genes, whe eas ed do s
indica e down egula ed genes. (e) Venn diag ams depic ing up- o down- egula ed genes sha ed be ween Lp-Lp and HkLp-Lp
compa ed o U-Lp. Hea -maps o selec ed di e en ially exp essed genes in ol ed in immune esponses ( ) and belonging o he TNF
signaling pa hway (g). (h) Compa a i e ansc ip ion ac o en ichmen analysis o di e en ially exp essed genes using he HOMER
package. The di e en ial exp ession o genes was se a an absolu e log
2
Fold Induc ion alue o 1 and Padj < 0.05.
e1939598-6 A. PELLON ET AL.
se e al me abolic pa hways o monocy es in com-
pa ison wi h cells acu ely exposed o L. plan a um,
especially in hose monocy es p e-s imula ed wi h
li e bac e ia (Figu e 4a). Al hough we did no ind
g ea changes in he exp ession le els wi hin mem-
be s o cen al me abolic pa hways, we obse ed
ha some adjacen me abolic pa hways we e
en iched in ou unc ional s udy. Indeed, ou da a
showed he up egula ion o olic acid me abolism,
amino acid and ca boxylic acid biosyn hesis, and
monoca boxylic acid ca abolism, and he down e-
gula ion o hyalu onan biosyn hesis, nega i e eg-
ula ion o lipid s o age, and glyce ol anspo
(Table S1). Speci ically, we obse ed he up egula-
ion o h ee py u a e dehyd ogenase kinases genes
(PDK2, PDK3, PDK4) in cells p imed wi h li e
bac e ia, as well as a dec eased exp ession o
ACO1 and ACOD1, coding o aconi ase and aco-
ni a e deca boxylase, sugges ing a educ ion in he
in eg i y o he ica boxylic acid (TCA) cycle and
he i acona e pa hway. We also ound he di e en-
ial egula ion o se e al genes coding o me abo-
li e anspo e s (Figu e 3e), including hose o
glucose (SLC2A1), o he hexoses and monoca -
boxylic compounds (SLC2A6, SLC45A3,
SLC16A5), and amino acids (SLC1A2, SLC16A10),
which possibly con ibu e o changes in cellula
me abolism.
To assess whe he hese al e a ions in he an-
sc ip ional landscape had physiological conse-
quences in p imed cells, we analyzed he
me abolic p o iles o human monocy es by
Figu e 4. L. plan a um p iming p omo es a me abolic ewi ing esul ing in dec eased oxida i e bu s . (a) Hea -map depic ing
selec ed di e en ially exp essed genes wi h unc ions ela ed o cellula me abolism. Seaho se ex acellula lux analyze was used o
de e mine OCR (b) and ECAR (c) p o iles o human monocy es (hMon). Da a a e shown o a ep esen a i e expe imen ou o wo
independen ly pe o med. (d) Phenog am showing OCR/ECAR a io o unp imed hMon, and cells p imed wi h ei he li e (Lp-Lp) o
hea -killed (HkLp-Lp) L. plan a um. (e) ROS p oduc ion a e he second bac e ial encoun e . Pho bol-12-my is a e-13-ace a e (PMA)
was used o inc ease ROS p oduc ion in mBMMs ( ), which induced a dec ease in L. plan a um in acellula su i al in memo y
mac ophages (g). Da a a e shown as mean ± s.e.m., n ≥ 3. *; p < .05, **; p < .01, ***; p < .001, One-way ANOVA (E) and S uden ’s es ( ,
g).
GUT MICROBES e1939598-7
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