The exp ession o immune
esponse genes in pa ien s wi h
ch onic Chagas disease is shi ed
owa d he le els obse ed in
heal hy subjec s as a esul o
ea men wi h Benznidazole
Inmaculada Go
´mez
1
, Ad iana Egui
1
,Ge
´nesis Palacios
1,2
,
Ba olome
´Ca ile o
3
, Celia Benı
´ ez
1
, Ma ina Simo
´n
3
,
Manuel Sego ia
3
, Emma Ca melo
2,4
, Manuel Ca los Lo
´pez
1
*
and M. Ca men Thomas
1
*
1
Depa amen o de Biologı
´a Molecula , Ins i u o de Pa asi ologı
´a y Biomedicina Lo
´pez-Ney a, Consejo
Supe io de In es igaciones Cien ı
´ficas, G anada, Spain,
2
Ins i u o Uni e si a io de En e medades
T opicales y Salud Pu
´blica de Cana ias, Uni e sidad de La Laguna, La Laguna, Spain,
3
Unidad Regional
de Medicina T opical, Hospi al Uni e si a io Vi gen de la A ixaca, Mu cia, Spain,
4
Depa amen o de
Obs e icia y Ginecologı
´a, Pedia ı
´a, Medicina P e en i a y Salud Pu
´blica, Toxicologı
´a, Medicina Legal
y Fo ense y Pa asi ologı
´a, Uni e sidad de La Laguna, La Laguna, Spain
In oduc ion: Chagas disease, caused by he T ypanosoma c uzi pa asi e
in ec ion, is a po en ially li e- h ea ening neglec ed opical disease wi h a
wo ldwide dis ibu ion. Du ing he ch onic phase o he disease, he e exis s a
agile balance be ween he hos immune esponse and pa asi e eplica ion ha
keeps pa ien s in a clinically-silen asymp oma ic s age o yea s o e en decades.
Howe e , in 40% o pa ien s, he disease p og esses o clinical mani es a ions
mainly a ec ing and comp omising he ca diac sys em. T ea men is
ecommended in he ch onic phase, al hough he e a e no ea ly ma ke s o i s
e ec i eness. The aim o his s udy is o iden i y di e en ial exp ession changes in
genes in ol ed in he immune esponse in an igen- es imula ed PBMC om
ch onic pa ien s wi h Chagas disease due o benznidazole ea men .
Me hods: Thus, high- h oughpu eal- ime qPCR analysis has been pe o med o
simul aneously de e mine global changes in he exp ession o 106 genes
in ol ed in he immune esponse in asymp oma ic (IND) and ea ly ca diac
mani es a ions (CCC I) Chagas disease pa ien s p e- and pos - ea men
wi h benznidazole.
Resul s and discussion: The esul s e ealed ha 7 ou o he 106 analyzed genes
we e di e en ially exp essed (4 up- and 3 down egula ed) a e ea men in IND
pa ien s and 15 ou o 106 (3 up- and 12 down egula ed) a e ea men o ea ly
ca diac Chagas disease pa ien s. Pa icula ly in CCC I pa ien s, egula ion o he
exp ession le el o some o hese genes owa ds a le el simila o ha o heal hy
subjec s sugges s a beneficial e ec o ea men and suppo s ecommenda ion
o benznidazole adminis a ion o ea ly ca diac Chagas disease pa ien s. The da a
F on ie s in Cellula and In ec ion Mic obiology on ie sin.o g01
OPEN ACCESS
EDITED BY
Ma in C aig Taylo ,
Uni e si y o London, Uni ed Kingdom
REVIEWED BY
Paula Mello De Luca,
Oswaldo C uz Founda ion (Fioc uz), B azil
Damian Pe ez-Mazliah,
Uni e si y o Yo k, Uni ed Kingdom
*CORRESPONDENCE
M. Ca men Thomas
[email p o ec ed]
Manuel Ca los Lo
´pez
[email p o ec ed]
RECEIVED 28 May 2024
ACCEPTED 02 July 2024
PUBLISHED 23 July 2024
CITATION
Go
´mez I, Egui A, Palacios G, Ca ile o B,
Benı
´ ez C, Simo
´nM,Sego ia M, Ca melo E,
Lo
´pez MC and Thomas MC (2024) The
exp ession o immune esponse genes in
pa ien s wi h ch onic Chagas disease is
shi ed owa d he le els obse ed in
heal hy subjec s as a esul o ea men
wi h Benznidazole.
F on . Cell. In ec . Mic obiol. 14:1439714.
doi: 10.3389/ cimb.2024.1439714
COPYRIGHT
©2024Go
´mez, Egui, Palacios, Ca ile o,
Benı
´ ez, Simo
´n, Sego ia, Ca melo, Lo
´pez and
Thomas. 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 (CC BY). The
use, dis ibu ion o ep oduc ion in o he
o ums is pe mi ed, p o ided he o iginal
au ho (s) and he copy igh owne (s) a e
c edi ed and ha he o iginal publica ion in
his jou nal is ci ed, in acco dance wi h
accep ed academic p ac ice. No use,
dis ibu ion o ep oduc ion is pe mi ed
which does no comply wi h hese e ms.
TYPE O iginal Resea ch
PUBLISHED 23 July 2024
DOI 10.3389/ cimb.2024.1439714
ob ained also demons a ed ha bo h in asymp oma ic pa ien s and in ea ly
ca diac ch onic pa ien s, a e ea men wi h benznidazole he e is a nega i e
egula ion o he p oinflamma o y and cy o oxic esponses igge ed as a
consequence o T. c uzi in ec ion and he pe sis ence o he pa asi e. This
down egula ion o he immune esponse likely p e en s ma ked issue damage
and healing in ea ly ca diac pa ien s, sugges ing i s posi i e e ec in con olling
he pa hology.
KEYWORDS
ch onic ca diac Chagas disease, T ypanosoma c uzi, ansc ip ional p ofile,
high h oughpu RT-qPCR, di e en ial gene exp ession, immune esponse,
immunological pa hways, bioma ke s
1 In oduc ion
Chagas disease, also known as Ame ican ypanosomiasis, is
caused by he p o ozoan pa asi e T ypanosoma c uzi. I is a
po en ially li e- h ea ening neglec ed opical disease ha a ec s
a ound 6–7 million people wo ldwide and causes be ween 10,000
and 14,000 dea hs pe yea (Rassi e al., 2012;O ganiza ion Pan
Ame ican Heal h and Wo ld Heal h O ganiza ion, 2021). The disease
is endemic in La in Ame ica, al hough mig a ion om endemic a eas
o de eloped coun ies has caused he disease o sp ead o o he
egions such as he USA (Manne-Goehle e al., 2016), Canada,
Aus alia, as well as many Eu opean coun ies, and some coun ies in
A ica, he Eas e n Medi e anean, and he Wes e n Pacific
(Schmunis, 2007;Schmunis and Yadon, 2010;An ino i e al., 2017).
A e in ec ion, he disease p esen s wi h an acu e o m
cha ac e ized by high pa asi emia and in ense inflamma o y
infil a es. Indi iduals who su i e heacu ephaseo he
in ec ion de elop a pa asi e-specificimmune esponse ha
e ficien ly educes pa asi e le els in he issues and blood, leading
o he ch onic phase o he disease (Pe ez-Molina and Molina,
2018). Ch onic pa ien s may emain asymp oma ic o yea s, a
phase known as he inde e mina e (IND) o m o he disease.
Howe e , app oxima ely 30%–40% o hem subsequen ly de elop
ca diac and/o gas oin es inal complica ions (Rassi e al., 2010).
The mos se e e cases o ca diac al e a ions lead o he de elopmen
o ch onic chagasic ca diomyopa hy (CCC), wi h which high
mo ali y a es a e associa ed (Mo is e al., 1990;Rocha e al.,
2003). Recen s udies ha e shown ha bo h pa ien s in
inde e mina e and symp oma ic ea ly ca diac phases o Chagas
disease (CCC I) show a di e en ial gene exp ession p ofile in which
se e al ele an immunological pa hways seem o be ac i a ed o
ep essed (Gomez e al., 2021,2023).
Since he beginning o he 1970s, he e ha e been wo d ugs
a ailable o ea ing Chagas disease: benznidazole and ni u imox.
Bo h d ugs a e e ec i e in acu e and pedia ic in ec ions (Al cheh
e al., 2005;Alonso-Vega e al., 2013;Vio i e al., 2014). Howe e ,
apa om causing ad e se e en s (up o 70% o he pa ien s), which
equen ly o ce hem o in e up he ea men cou se, hei
e ficacy in adul ch onic pa ien s is lowe , a iable, and di ficul
o e alua e due o he lack o a ma ke o define cu e.
The Pan-Ame ican Heal h O ganiza ion (PAHO) guidelines o
he diagnosis and ea men o Chagas disease s a e ha only
se ocon e sion by con en ional se ology can be in e p e ed as an
indica o o a pa asi ological cu e (Pan Ame ican Heal h
O ganiza ion, 2018). The limi a ion o se ocon e sion is ha i
akes 10–20 yea s o occu (Vio i e al., 2006;Fabb o e al., 2007).
The de ec ion o pa asi e DNA in he pa ien ’s bloods eam by
qPCR is consis en wi h ea men ailu e. By con as , a nega i e
PCR esul may o may no be consis en wi h a cu e due o he low
pa asi emia du ing he ch onic phase (de Lima e al., 2023).
This si ua ion highligh s he need o elucida e he molecula
mechanisms and signaling pa hways ha may be ac i a ed and
egula ed by ea men , wi h he aim o finding ou and de eloping
ea ly bioma ke s o ea men esponse ha will acili a e pa ien
ollow-up. Di e en s udies ha e epo ed se e al ma ke
candida es, which oge he may ulfill accep able c i e ia o
indica e he e ficacy o a ypanocidal ea men (Pinazo e al.,
2014). Some o he an igens ha e exhibi ed p omising esul s (Egui
e al., 2019;Alonso-Padilla e al., 2021), indica ing he need o
u he and la ge s udies o de e mine hei po en ial ole as
bioma ke s o ea men esponse. Some o hese s udies a e
cu en ly in p og ess (Alonso-Vega e al., 2021).
The pu pose o his s udy is o iden i y exp ession changes in
genes in ol ed in he immune esponse in PBMC es imula ed wi h
pa asi e’s soluble an igens due o benznidazole ea men in ch onic
pa ien s wi h Chagas disease, di e en ia ing hose who a e
asymp oma ic om hose who p esen ca diac mani es a ions.
Thus, genes coding o cy okines, chemokines, and hei ecep o s,
coes imula o y molecules, adhesion molecules, pheno ype ma ke s,
ansc ip ion ac o s, cy o oxic molecules, inhibi o y ecep o s and
hei ligands, molecules in ol ed in apop osis and senescence, and
dend i ic cell ma ke s, among o he molecules wi h immunological
in ol emen , ha e been analyzed. To his end, a high- h oughpu
qPCR pla o m was employed o simul aneously de e mine he
Go
´mez e al. 10.3389/ cimb.2024.1439714
F on ie s in Cellula and In ec ion Mic obiology on ie sin.o g02
exp ession le el o 106 immune sys em- ela ed genes in pe iphe al
blood mononuclea cell (PBMC) samples om ea ed asymp oma ic
pa ien s (pos ea men [IND-Pos]) and compa ed hem wi h hose
who had no ecei ed ea men (IND pa ien s p e ea men [IND-
P e]). Simila ly, his esponse o ea men was also analyzed in
Chagas disease pa ien s a a mo e ad anced s age o he disease,
specifically wi h ca diac mani es a ions (CCC I, no mal ches X- ay
bu abno mali ies in he elec oca diog am, Kushnni 1) (Kuschni
e al., 1985) be o e and a e benznidazole ea men , CCC-P e and
CCC-Pos, espec i ely.
The esul s ha e shown ha a e benznidazole ea men , he
exp ession pa e n o genes in ol ed in he cellula immune
esponse is modula ed in an igen-s imula ed PBMC om pa ien s
wi h ch onic Chagas disease, bo h asymp oma ic and hose wi h
ca diac mani es a ions. In e es ingly, he exp ession pa e n o some
o hese genes eaches an exp ession pa e n simila o ha de ec ed
in heal hy subjec s. The s udy o he di e en ial exp ession o hese
genes and he immune ou es in which hey a e in ol ed imp o es
he comp ehension o he e ec s o ea men a he immunological
le el. These genes could ep esen new po en ial bioma ke s o
he apeu ic e ficacy in ch onic Chagas disease pa ien s.
2 Ma e ials and me hods
2.1 E hical conside a ions
The p o ocols employed in his s udy we e app o ed by he
E hics Commi ees o he Consejo Supe io de In es igaciones
Cien ı
ficas (Spain - Re e ence: 013/2020) and o he Hospi al
Vi gen de la A ixaca (Mu cia, Spain - Re e ence: 2020-1-11-
HCUVA). An in o med consen was signed by all olun a y
pa ien s p io o hei inclusion in he s udy.
2.2 S udy coho
Adul pa ien s wi h ch onic Chagas disease o igina ing om
endemic a eas and esiding in Spain included in his s udy we e
ec ui ed, diagnosed, and clinically e alua ed a he Hospi al Vi gen
de la A ixaca (Mu cia, Spain). Pa ien s we e diagnosed acco ding
o he WHO c i e ia based on se ological es s (ELISA Chagas,
O ho Clinical Diagnosis, and Inmunofluo Chagas, Biocien ı

fica,
A gen ina) and cha ac e ized, acco ding o he Kuschni
classifica ion, as ca diac (CCC, G1 named CCC I) o hose who
had ca diac mani es a ions o IND (G0) due o he absence o
ca diac o diges i e mani es a ions (Gomez e al., 2021,2023).
Simila ly, 24 CCC I and 61 IND pa ien s who had ecei ed
an ipa asi ic ea men (benznidazole; 5 mg/kg body weigh pe
day o 60 days) we e included. Blood samples om ea ed pa ien s
we e collec ed om 6 o 12 mon hs a e ea men . Da a
conce ning he age, gende , and coun y o o igin o each o he
subjec s included in his s udy a e de ailed in Table 1.
2.3 Isola ion o pe iphe al blood
mononuclea cells
PBMCs we e isola ed om 20- o 30-mL blood samples
collec ed asep ically om each subjec in o EDTA-coa ed ubes.
These PBMCs we e pu ified and c yop ese ed ollowing he
p e iously desc ibed p o ocol (Ma añon e al., 2011).
TABLE 1 Epidemiological and demog aphic da a o he s udy coho .
Pa ien s g oup O igin (%) Age (yea s) Sex
[%Female(F) /Male (M)]
Mean (+ SD) Range
Heal hy dono s (n=34)
F om non- endemic
a ea (n=20) Spain (100%) 37.6 (12.8)
37.5 (23) 22-56
60% F
40% M
F om endemic a ea (n=14)
Colombia (28.6%)
37.5 (8.4)
38 (8) 21-54
64.3% FVenezuela (14.3%)
Chile (7.1%)
Panama (7.1%)
35.7% MEcuado (7.1%)
ND (35.7%)
Pos - ea men IND pa ien s (n=61)
Boli ia (96.7%)
37.1 (8.6) 21-57 70.5% F
29.5% M
El Sal ado (1.6%)
Pa aguay (1.6%)
Pos - ea men CCC pa ien s(n=24)
Boli ia (83.3%)
32.8 (9) 19-55 62.5% F
37.5% M
Pa aguay (4.2%)
ND (12.5%)
ND, no da a.
Go
´mez e al. 10.3389/ cimb.2024.1439714
F on ie s in Cellula and In ec ion Mic obiology on ie sin.o g03
2.4 Isola ion o T ypanosoma c uzi-
soluble an igens
Soluble an igens om T. c uzi (TcSA) SOL s ain (MHOM/ES/
2008/SOL; DTU V) isola ed om an in an ( e e ed o as in ac 1)
in ec ed by congeni al ansmission in Spain, we e ex ac ed
ollowing he p o ocol p e iously desc ibed (Egui e al., 2017;
Gomez e al., 2021) and employed o in i o s imula ion o
pa ien s PBMCs. The p o ein concen a ion o he ex ac s was
de e mined using a mic obicinchoninic acid (BCA) p o ein assay
ki (The mo Fishe Scien ific Inc., Wal ham, MA, USA), wi h he
p o ein p ofile analyzed by SDS-PAGE and subsequen Coomassie
blue s aining.
2.5 Thawing and s imula ion o pe iphe al
blood mononuclea cells
C yop ese ed PBMCs we e hawed and s imula ed ollowing a
p e iously desc ibed labo a o y p o ocol (Egui e al., 2018;Pe ez-
An on e al., 2018;Gomez e al., 2021). B iefly, PBMCs we e apidly
hawed in a 37°C wa e ba h, ans e ed o a ube con aining 10 mL
o RPMI-1640 (supplemen ed wi h 2 mM L-glu amine, 10% iFBS,
and 50 µg/mL o gen amicin), cen i uged a 453 c o 10 min, and
suspended in 2 mL o supplemen ed RPMI-1640 medium. Cell
iabili y was analyzed in each sample by cell s aining wi h ypan
blue, exhibi ing a mean iabili y o 80%. Viable PBMCs we e seeded
a a concen a ion o 7.5–8.5 × 10
6
cells/mL in 12-well pla es in a
maximum olume o 3.5 mL/well, cul u ed o 4 h a 37°C in 5%
CO
2
, and subsequen ly s imula ed wi h TcSA (10 mg/mL) and
cul u ed o 14–14.5 h a 37°C in 5% CO
2
.
2.6 RNA isola ion, quan ifica ion, and
quali y analysis
To al RNA isola ion om s imula ed PBMCs was pe o med
using he RNeasy Plus Mini ki (QIAGEN Inc., Valencia, CA, USA),
elimina ing he genomic DNA and ob aining mRNA-en iched
samples, acco ding o he manu ac u e ’s indica ions. Due o he
quan i y o RNA equi ed o ca y ou cDNA syn hesis o high-
h oughpu RT-qPCR and he limi ed numbe o cells isola ed om
he small olume o blood samples o pa icula pa ien s, in some
cases, i was necessa y o blend cells om some pa ien s. In he
coho o CCC I pa ien s, 18 samples co esponded o indi idual
pa ien s and h ee o a mix u e o wo pa ien s. In he case o IND
pa ien s, 35 samples co esponded o indi idual subjec s, se en o a
mix u e o wo pa ien s, and ou o a mix u e o h ee pa ien s. The
NanoD op 1000 spec opho ome e (The mo Fishe Scien ific Inc.,
Wal ham, MA, USA) and Qubi fluo ome e (In i ogen, Ca lsbad,
CA, USA) we e employed o quan i y and de e mine RNA pu i y.
The quali y o he ex ac ed mRNA was de e mined by analyzing i s
in eg i y wi h he Bioanalyze 2100 (Agilen Technologies, San a
Cla a, USA) using he RNA 6000 Nano ki (Agilen Technologies,
San a Cla a, USA). All RNA samples included in his s udy had high
quali y and in eg i y, wi h an RNA in eg i y numbe (RIN) anging
om 7.4 o 10.
2.7 Re e se ansc ip ion and high-
h oughpu eal- ime quan i a i e PCR
A High-Capaci y cDNA Re e se T ansc ip ion Ki (Applied
Biosys ems, Fos e Ci y, CA, USA) was used o pe o m he e e se
ansc ip ion as p e iously desc ibed (Gomez e al., 2021). B iefly,
eal- ime qPCR was pe o med using OpenA ay®pla es wi h
TaqMan p obes (The mo Fishe Scien ific) o he amplifica ion o
106 specific immune- ela ed genes and six endogenous e e ence
genes. All p ime s and p obes we e comme cially designed by
The mo Fishe Scien ific(Gomez e al., 2023) and a e lis ed in
Supplemen a y Table S1. The PCR mix u e was p epa ed acco ding
o he manu ac u e ’s ins uc ions. Each amplifica ion eac ion was
pe o med in iplica e. The he mal cycle (95°C o 15 s, 60°C o
1min, o 40cycles)andfluo escence de ec ion we e pe o med wi h
he Quan S udio™12K Flex Real-Time PCR Sys em (The mo Fishe
Scien ific) acco ding o he manu ac u e ’s p o ocol. The quan i a i e
cycle (Cq) alues p oduced by his pla o m a e al eady co ec ed o
he e ficiency o he amplifica ion (He nandez-San ana e al., 2016).
2.8 Da a p ocessing
The a i hme ic a e age Cq alues o each qPCR un we e
employed o da a analysis a e hey had been expo ed om
Quan S udio™12K Flex Real-Time PCR Sys em. To iden i y he
genes wi h he highes exp ession s abili y, GeNo m (Vandesompele
e al., 2002), No mFinde (Ande sen e al., 2004), and Re Finde
(Xie e al., 2012)(hea cu e.com.au), which in eg a e he ou
algo i hms GeNo m (Vandesompele e al., 2002), No mFinde
(Ande sen e al., 2004), Bes Keepe (P a fle al., 2004), and DC
me hod (Sil e e al., 2006), we e applied o he 112 analyzed genes.
STAT3,IL10RA,andIFNAR we e he h ee genes ha showed a mo e
s able exp ession, all wi h geNo m M- alues < 0.5 (which is he
s anda d cu o o e e ence gene selec ion). These genes we e
selec ed as e e ence genes and used o da a se no maliza ion
employing GenEx so wa e ( .6, Mul iD), ob aining no malized
ela i e quan i y (NRQ) alues. The six cons i u i e and candida e
e e ence genes included in he panel (ACTB,B2M,GAPDH,HPRT1,
PGK1,andTBP) we e dis ega ded as hey had wo se s abili y alues
han STAT3,IL10RA,andIFNAR in all he algo i hms employed. Fo
compa a i e analyses, he Cq alues o each qPCR and he
co esponden NRQ alues ob ained om heal hy dono s (HD)
we e aken in o conside a ion (Gomez e al., 2021).
2.9 En ichmen analysis
Gene se en ichmen analysis was ca ied ou on he NRQ
alues o each g oup o pa ien s as a whole, employing GSEA 4.1.0
so wa e (Moo ha e al., 2003;Sub amanian e al., 2005). Fo he
Go
´mez e al. 10.3389/ cimb.2024.1439714
F on ie s in Cellula and In ec ion Mic obiology on ie sin.o g04
en ichmen analysis, canonical pa hways gene se s om he
BioCa a pa hway da abase included in C2: cu a ed gene se s
collec ion in Molecula Signa u es Da abase (MSigDB)
(Sub amanian e al., 2005;Libe zon e al., 2011) we e selec ed.
GSEA pa ame e s: pe mu a ions = 100,000, pe mu a ion ype:
pheno ype (sample n> 7), en ichmen s a is ic: weigh ed, me ic
o anking genes: - es , max size: 500, min size: 2.
2.10 S a is ical analyses
All s a is ical analyses we e pe o med using SPSS 25 so wa e
(IBM Co p., A monk, NY, USA) and G aphPad P ism 8 (G aphPad
So wa e, San Diego, CA, USA). Kolmogo o –Smi no and Shapi o–
Wilk es s (a= 0.05) we e employed o check da a no mali y, and a
wo- ailed unpai ed - es o a wo- ailed Mann–Whi ney U es ,
depending on whe he da a had o no a no mal dis ibu ion,
espec i ely, was used o de e mine he s a is ical significance,
conside ing p< 0.05 as s a is ically significan . Di e en ially
exp essed genes be ween ch onic IND- ea ed and un ea ed
pa ien s, as well as be ween ea ly ca diac ea ed and un ea ed
pa ien s, we e iden ified using wo pa ame e s: he old-change o
gene exp ession (FC) and he s a is ical significance (p- alue). FC was
calcula ed as he a io be ween he a e age gene exp ession o he
g oups o pa ien s (IND-Pos/IND-P e, CCC-Pos/CCC-P e),
indica ing how many imes a specific gene is exp essed in one
g oup e sus ano he . To be able o de e mine he FC when he
a e age gene exp ession le el was 0 in one o he compa ed g oups,
he numbe 0 was eplaced by 0.000001 ( his was applied o IL17A in
he CCC-Pos g oup). To isualize changes, olcano plo s we e made
om he –log
10
p- alue (de e mined by an unpai ed - es ) plo ed on
he y-axis and log
2
o FC on he x-axis. Genes passing bo h biological
significance (log
2
o FC > 0.6 o ≤0.6, equi alen o FC > 1.5 o <
0.66) and s a is ical significance (–log
10
p>1.3,equi alen op=0.05)
h esholds we e shown in ed and blue, a ending o hei up- and
down egula ion, espec i ely. STRING 11 (Szkla czyk e al., 2019)
was employed o gene a e in e ac ion ne wo ks be ween di e en ially
exp essed genes. Ac i e in e ac ion sou ces, including expe imen s,
da abases, cooccu ence, gene usion, and neighbo hood, we e
applied o cons uc p o ein–p o ein in e ac ion ne wo ks, limi ing
species o Homo sapiens and in e ac ion sco es > 0.7. P incipal
componen analysis (PCA) was pe o med o mul i a ia e analysis
on NRQ alues o de e mine he s uc u e o he da ase . Di e ences
in sco es o plo ed p incipal componen s be ween he g oups we e
confi med by an unpai ed - es o a Mann–Whi ney U es , as
app op ia e, employing SPSS 25 so wa e.
3 Resul s
To analyze he immune esponse o pa ien s wi h Chagas disease
a e benznidazole ea men and i s ela ionship wi h disease con ol
o p og ession, compa a i e analyses ocusing on he global changes
in he exp ession o 106 genes in ol ed in he immune esponse ha e
been pe o med in pa ien s wi h Chagas disease p e- and
pos ea men wi h benznidazole. The s udy has been ca ied ou in
pa ien s in he IND asymp oma ic phase and pa ien s wi h ca diac
symp oma ology (Kuschni I) (CCC I), gi en ha he e ficacy o he
ea men may a y acco ding o he phase o he disease a he ime
o d ug adminis a ion. The compa a i e analyses we e ca ied ou in
all cases, conside ing he NRQ alues o PBMC cells om hese
pa ien s s imula ed wi h T. c uzi an igens and also hose om heal hy
subjec s (Gomez e al., 2021). Despi e he compa a i e analyses
pe o med among g oups o pa ien s being ca ied ou as a whole
and no as indi idual subjec samples (un ea ed e sus ea ed IND
and un ea ed e sus ea ed CCC I), he po en ial influence on he
gene exp ession pa e n o samples ob ained om indi idual samples
o om blended samples was analyzed. Thus, PCA we e pe o med
conside ing samples om indi idual IND- ea ed pa ien s as a
di e en g oup o he blended samples o ea ed IND pa ien s and
also indi idual ea ed CCC I pa ien s as a di e en g oup o he
ea ed CCC I pa ien s blended samples. The ob ained esul s showed
ha samples om he IND and CCC- ea ed pa ien s conse ed he
same dis ibu ion independen ly, whe he hey came om indi idual
samples o had been blended (Supplemen a y Figu e S1). The same
le el o gene exp ession in bo h he indi idual and blended samples
om IND and CCC I- ea ed samples was suppo ed by a wo- ailed
unpai ed - es , which showed ha he e we e no s a is ically
significan di e ences be ween he sco es ob ained in he wo
g oups o each p incipal componen .
3.1 Analysis o di e en ially exp essed
genes in ch onic pa ien s in he
inde e mina e phase be o e and
a e ea men
To de e mine he dis ibu ion o he gene exp ession da a se
[NRQ alues o IND-P e (n= 39) e sus IND-Pos (n= 46)], a
p incipal componen analysis was pe o med as a fi s
app oxima ion. As shown in Figu e 1, p incipal componen 1
(PC1) and p incipal componen 2 (PC2) explained he highes
pe cen ages o o al a iance wi h alues o 22.4% and 11.8%,
espec i ely (Figu e 1A). Also, a 3D ep esen a ion o PCA
including p incipal componen 3 (PC3) showed PC3 explains
9.2% o he o al a iance (Figu e 1B). The esul s showed ha
he sco es ob ained o PC1 in he p e ea men IND pa ien s we e
mos ly posi i e, whe eas in he pos ea men pa ien s, hey we e
dis ibu ed along he en i e x-axis whe e his componen was
ep esen ed. The obse ed di e ences in PC1 sco es be ween he
wo g oups o subjec s we e confi med by a wo- ailed unpai ed -
es , which indica ed ha he e was a s a is ically significan
di e en ial exp ession p ofile be o e and a e ea men in IND
pa ien s (p= 0.025). Addi ionally, his same s a is ical es was
applied o co obo a e ha p incipal componen s 2 and 3 we e no
esponsible o he di e ences obse ed be ween he wo g oups
(PC2 p= 0.945, PC3 p= 0.354). The 24 genes co ela ed wi h PC1
wi h high ac o loadings a e lis ed in Supplemen a y Table S2.
Subsequen ly, o de e mine he influence o ea men , he gene
exp ession le el o he 106 genes unde s udy in IND pa ien s
un ea ed and ea ed wi h benznidazole was compa a i ely
analyzed. The esul s e ealed ha se en ou o he 106 analyzed
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genes we e di e en ially exp essed be ween ea ed and un ea ed
IND pa ien s wi h s a is ical significance (p< 0.05), as obse ed in
he olcano plo cons uc ed om he FC and s a is ical significance
o each gene (Figu e 2A). Specifically, ou genes (IDO1,IL13,
PDCD1LG2, and TGFB2) we e up egula ed in IND-Pos pa ien s
e sus IND-P e pa ien s (FC > 1.5; o ange do s). Con e sely, h ee
ou o he se en genes (CCL5,GZMH,andIL17A)we e
down egula ed in he IND-Pos pa ien s compa ed wi h IND-P e
(FC < 0.66; blue do s).
The di e ences de ec ed in he gene exp ession le el o he
genes whose exp ession was modified by he ea men we e
quan ified by ep esen ing he log
2
o old change alues ob ained
o each gene in a ba g aph (Figu e 2B). The esul s indica ed ha
up- and down egula ed genes (log
2
FC > 0.6 (o ange ba s) o ≤0.6
(blue ba s), espec i ely) had ai ly simila log
2
FC alues acco ding
o he same le el o exp ession change, wi h he excep ion o he
IL17A gene. Thus, IL17A showed a log
2
FC alue below −1.5, which
is equi alen o a leas a h ee old lowe exp ession le el in he
pos - ea men IND pa ien s compa ed o he p e- ea men
IND pa ien s.
3.2 Iden ifica ion o di e en ially exp essed
genes in ch onic ca diac pa ien s (CCC I)
be o e and a e ea men
The e ec o benznidazole ea men on he global changes in
he exp ession le el o he 106 genes unde s udy was ex ended o
A
B
FIGURE 2
Di e en ial gene exp ession le els in pos ea men e sus p e ea men inde e mina e Chagas disease pa ien s. (A) Volcano plo showing he
di e en ial exp ession le el o he 106 analyzed genes in inde e mina e Chagas disease pa ien s p e ea men (IND-P e, n= 39) and pos ea men
(IND-Pos, n= 46). The x-axis ep esen s log
2
o exp ession old change be ween IND-Pos and IND-P e (log
2
FC). The y-axis co esponds o he
s a is ical significance, exp essed as he nega i e loga i hm o he p- alue (–log
10
p- alue). The pu ple ho izon al lines indica e he cu o s o he
s a is ical significance (co esponding o p= 0.05, p= 0.01, p= 0.001, and p= 0.0001). The g ay e ical lines ep esen he log
2
FC o −0.6 and 0.6
(co esponding o FC o 0.66 and 1.5, espec i ely) used as biological h esholds o iden i y di e en ially exp essed genes. Nega i e alues
co espond o down egula ed genes (blue do s) and posi i e alues o up egula ed genes (o ange do s) in IND-Pos compa ed o IND-P e. Black do s
ep esen nondi e en ially exp essed genes. (B) Exp ession le el o se en di e en ially exp essed genes wi h s a is ical significance in IND-Pos e sus
IND-P e pa ien s. Each plo ba co esponds o he gene e e ed o a he bo om o he g aph (x-axis). The y-axis ep esen s he log
2
o exp ession
old change (log
2
FC) o each gene. Posi i e alues (o ange ba s) indica e up egula ed genes, and nega i e alues (blue ba s) indica e down egula ed
genes in IND-Pos compa ed o IND-P e subjec s.
A
B
FIGURE 1
The p incipal componen analysis applied o he NRQ alues o 106 genes om IND-P e and IND-Pos pa ien s. (A) PCA sco e plo o p incipal
componen s 1 (PC1) and 2 (PC2) on he x-axis and y-axis, espec i ely. (B) 3D sca e plo showing he h ee p incipal componen s 1 (PC1), 2 (PC2),
and 3 (PC3). The p opo ion o a iance explained by each p incipal componen is indica ed as a pe cen age on he axis nex o he co esponding
p incipal componen .
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F on ie s in Cellula and In ec ion Mic obiology on ie sin.o g06
ch onic Chagas disease pa ien s a he ea ly s age o he ca diac
phase (CCC I). A p incipal componen analysis was again
pe o med using he NRQ alues o pa ien s wi h un ea ed CCC
Chagas disease (CCC-P e, n= 18) as well as he g oup o CCC
pa ien s who had ecei ed ea men (CCC-Pos, n= 21) (Figu e 3).
PCA esul s indica ed, as shown in Figu e 3A, ha PC1 and PC2
explained 22.3% and 12.8% o he o al a iance, espec i ely. PC3,
which explained 8.4% o he a iance, was also plo ed oge he wi h
PC1 and PC2 in a 3D sca e plo (Figu e 3B). This analysis
indica ed ha mos samples o each g oup ended o clus e
oge he along he axis whe e PC2 was ep esen ed. These
obse ed di e ences in acqui ed sco es o PC2 be ween p e- and
pos ea men CCC pa ien s we e confi med by a wo- ailed
unpai ed - es , which indica ed ha he e was a s a is ically
significan di e en ial exp ession p ofile be ween he wo g oups
o pa ien s o 13 genes co ela ed wi h PC2 (p= 0.003). A wo-
ailed unpai ed - es was also applied o he sco es acqui ed o PC1
and PC3, which showed ha hey did no pa icipa e in he
obse ed di e ences (PC1 p= 0.641; PC3 p= 0.914). The 13
genes ou o he 106 analyzed co ela ed wi h high ac o ial
loading wi h PC2 a e lis ed in Table 2.
Analysis o he genes di e en ially exp essed in he p e- and
pos ea men CCC pa ien s indica ed ha 15 ou o he 106 genes
unde s udy we e di e en ially exp essed be ween he wo g oups o
pa ien s wi h s a is ical significance (Figu e 4A). Thus, 12 ou o
hese 15 di e en ially exp essed genes (CCL5,CCR1,FCER2,
GZMA,GZMH,HAVCR2,IL10,IL17A,IL1B,IL2RA,ITGAX, and
PRF1) we e down egula ed (log
2
FC ≤0.6) and h ee genes
(LGALS9,STAT1, and TNFSF10) up egula ed (log
2
FC > 0.6) in
CCC-Pos compa ed o CCC-P e, as seen in he olcano g aph
plo ing he log
2
FC o each gene e sus s a is ical significance.
The significance o he di e ences obse ed in he exp ession
le el o each gene eached a –log
10
p- alue g ea e han 1.3,
equi alen o a p< 0.05. To easily isualize he in ensi y o he
changes, he log
2
FC alues o he biologically and s a is ically
di e en ially exp essed genes (log
2
FC > 0.6 o ≤0.6 and p- alue
< 0.05) in pos ea men CCC pa ien s e sus p e ea men CCC
pa ien s we e ep esen ed in a ba g aph. As shown in Figu e 4B,
fi e genes we e pa icula ly down egula ed in CCC-Pos pa ien s
(CCR1,GZMA,IL17A,ITGAX, and PRF1), exhibi ing a log
2
FC
alue lowe han –1, which esul ed in a educ ion o a leas hal
he amoun o exp ession e sus CCC-P e pa ien s. In he case o
IL17A, no exp ession was de ec ed in any o he samples om he
CCC-Pos pa ien g oup.
To p edic he specific biological unc ion in which he 12
down egula ed genes in he CCC-Pos e sus CCC-P e pa ien s
may be in ol ed, a p o ein–p o ein in e ac ion (PPI) ne wo k was
cons uc ed using he STRING pla o m, only conside ing a high
le el o confidence o he p edic ed in e ac ions. The esul s
e ealed ha 12 p o eins encoded by he se o di e en ially
A
B
FIGURE 3
P incipal componen analysis (PCA) was applied o he NRQ alues o 106 genes om CCC-P e and CCC-Pos. (A) PCA sco e plo o p incipal
componen s 1 (PC1) and 2 (PC2) on he x-axis and y-axis, espec i ely. (B) 3D sca e plo showing he h ee p incipal componen s 1 (PC1), 2 (PC2),
and 3 (PC3). The p opo ion o a iance explained by each p incipal componen is indica ed as a pe cen age on he axis nex o he co esponding
p incipal componen .
TABLE 2 PC2-co ela ed genes.
Gene Fac o loading o PC2
FASLG 0.879
CCL5 0.787
B3GAT1 0.747
HAVCR2 0.741
PRF1 0.729
GZMH 0.708
GZMA 0.680
TGFBR1 0.642
IL10RA 0.633
NCAM1 0.625
GZMB 0.625
LAG3 0.623
STAT1 −0.680
Genes wi h ac o loading o p incipal componen 1 (PC1) highe han 0.6 o lowe han −0.6
om he p incipal componen analysis (PCA) applied o he no malized ela i e quan i ies
(NRQ) o ca diac Chagas disease pa ien s p e- and pos ea men .
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down egula ed genes in CCC-Pos e sus CCC-P e had a high
deg ee o in e ac ion, eaching up o 11 in e ac ions (edges) wi h
a PPI en ichmen p- alue < 1.0e−16. In addi ion, he analysis
showed ha no in e ac ion would be expec ed o a andom se o
p o eins o he same size by chance. The ole o hese p o eins in
ele an immunological pa hways was also analyzed in he ob ained
STRING ne wo k. As obse ed in Figu e 5, he esul s showed ha
some p o eins pa icipa ed in mo e han one pa hway. Thus, fi e
p o eins (CCL5, CCR1, FCER2, IL10, and IL1B) we e p edic ed o
pa icipa e in he “IL-10 signaling”pa hway (HSA-6783783; FDR =
5.00e−08; ed nodes), fi e p o eins (FCER2, IL10, IL17A, IL1B, and
ITGAX) in he “IL-4 and IL-13 signaling”pa hway (HSA-6785807;
FDR = 2.33e-06; blue nodes), and ou p o eins (CCL5, IL10, IL1B,
and IL2RA) in he “IL-18 signaling pa hway”(WP4754; FDR =
0.0029; yellow nodes).
To gain insigh in o he immunological pa hways a ec ed by
benznidazole ea men in CCC I, a gene se en ichmen analysis
(GSEA) was applied o he NRQ alues o un ea ed and
benznidazole- ea ed CCC I. The e o e, a hea map was ob ained
showing he op 100 genes ha unde go he g ea es exp ession
changes be ween bo h g oups o samples om CCC pa ien s
(Figu e 6A). In gene al e ms, his g aph allowed us o obse e
ha he exp ession o se e al genes unde s udy was down egula ed
in he majo i y o he samples o CCC I pa ien s a e ea men . By
con as , he exp ession o o he genes was highe in he samples o
pa ien s a e ea men compa ed o hose collec ed be o e
ea men . In addi ion, o ob ain en ichmen plo s able o
illus a e a posi i e o nega i e co ela ion be ween he specific
gene se s and he samples om CCC pa ien s p e- and
pos ea men , a Molecula Signa u es Da abase (MSigDB)
BioCa a gene se collec ion (Sub amanian e al., 2005;Libe zon
e al., 2011) was employed (Figu es 6B,C). As shown in Figu e 6B,
GSEA e ealed a posi i e co ela ion be ween CCC-P e and he
immune pa hways “CTL media ed immune esponse agains a ge
cells (BIOCARTA_CTL_PATHWAY)”and “Blymphocy ecell
su ace molecules (BIOCARTA_BLYMPHOCYTE_PATHWAY)”
wi h a no malized en ichmen sco e (NES) o 1.17 and 1.02,
espec i ely. The exp ession o he genes in ol ed in hese wo
en iched pa hways is shown in GSEA hea maps (Figu e 6C). Thus,
he ICAM1,PRF1,FASLG,ITGB2,GZMB,ITGAL,CD3E, and FAS
A
B
FIGURE 4
Di e en ial gene exp ession le els in pos ea men e sus p e ea men ca diac Chagas disease pa ien s. (A) Volcano plo showing he di e en ial
exp ession le el o he 106 analyzed genes in ca diac Chagas disease pa ien s p e ea men (CCC-P e, n= 18) and pos ea men (CCC-Pos, n= 21).
The x-axis ep esen s log
2
o exp ession old change be ween CCC-Pos and CCC-P e (log
2
FC). The y-axis co esponds o he s a is ical significance,
exp essed as he nega i e loga i hm o he p- alue (–log
10
p- alue). The pu ple ho izon al lines indica e he cu o s o he s a is ical significance
(co esponding o p= 0.05, p= 0.01, p= 0.001, and p= 0.0001). The g ay e ical lines ep esen he log
2
FC o −0.6 and 0.6 (co esponding o FC
o 0.66 and 1.5, espec i ely) used as biological h esholds o iden i y di e en ially exp essed genes. Nega i e alues co espond o down egula ed
genes (blue do s) and posi i e alues o up egula ed genes (o ange do s) in CCC-Pos compa ed o CCC-P e. Black do s ep esen nondi e en ially
exp essed genes. (B) Exp ession le el o 15 di e en ially exp essed genes wi h s a is ical significance in CCC-Pos e sus CCC-P e pa ien s. Each plo
ba co esponds o he gene e e ed o a he bo om o he g aph (x-axis). The y-axis ep esen s he log
2
o exp ession old change (log
2
FC) o
each gene. Posi i e alues (o ange ba s) indica e up egula ed genes, and nega i e alues (blue ba s) indica e down egula ed genes in CCC-Pos
compa ed o CCC-P e.
FIGURE 5
STRING p o ein–p o ein in e ac ion analysis ep esen ing he
p o eins encoded by down egula ed genes in CCC-Pos e sus
CCC-P e. The linkage o each node ep esen s he in e ac ions
(edges) epo ed o hese molecules in di e en immunological
p ocesses/pa hways; PPI en ichmen p- alue < 1.0e−16. Ne wo k
edges ep esen confidence (line hickness indica es he s eng h o
da a suppo ). PPI ne wo k was cons uc ed, se ing he confidence
sco e h eshold a he high le el (0.7) and ac i e in e ac ion sou ces,
including da a om published expe imen s, da abases,
cooccu ence, gene usion, neighbo hood and coexp ession, and
species limi ed o Homo sapiens. P o eins a e desc ibed o be
in ol ed in specific pa hways: IL-10 signaling (HSA-6783783; FDR =
5.00e−08) ( ed), IL-4 and IL-13 signaling (HSA-6785807; FDR =
2.33e−06) (blue), and IL-18 signaling pa hway (WP4754; FDR =
0.0029) (yellow).
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genes a e included in he BIOCARTA_CTL_PATHWAY pa hway.
The co e en ichmen o his pa hway includes ICAM1,PRF1,
FASLG,andITGB2. Simila ly, ICAM1,ITGB2,CD80,ITGAL,
CD40,andCR2 genes pa icipa e in he BIOCARTA_BLYM
PHOCYTE_PATHWAY pa hway, including he co e en ichmen
o his pa hway o ICAM1,ITGB2,CD80, and ITGAL genes.
3.3 Benznidazole ea men modula es
gene exp ession in Chagas disease pa ien s
owa d he exp ession le el o heal hy
dono s, depending on he se e i y o
he disease
To iden i y he changes p oduced in he DEG ela ed o he
e ec o he ea men and he p og ession and se e i y o he
disease, he a e age exp ession o each DEG, shown as NRQ alues,
was compa ed be ween inde e mina e and ca diac Chagas’disease
pa ien s be o e and a e ea men , aking also in o conside a ion
he heal hy subjec s (Gomez e al., 2021). As shown in Table 3, he
exp ession o ce ain genes such as CCL5,FCER2,GZMH,IDO1,
IL10,IL13,IL1B,IL2RA,LGALS9,PDCD1LG2,PRF1,STAT1,
TGFB2,andTNFSF10 was inc eased in IND-P e and also in
CCC-P e pa ien s wi h espec o HD (wi h he excep ion o he
LGALS9 gene). Mos o hese genes we e e en mo e o e exp essed
in CCC-P e pa ien s wi h espec o IND-P e (FCER2,GZMH,
IDO1,IL10,IL13,IL1B,IL2RA,PDCD1LG2,PRF1, and TGFB2).
Howe e , a dec ease in he exp ession o he majo i y o hese genes
(wi h he excep ion o IDO1,TGFB2,andLGALS9,whose
exp ession inc eased) was obse ed in CCC pa ien s a e
ea men (CCC-Pos). This dec ease eached an exp ession alue
close o he exp ession alues de ec ed in heal hy dono s in he
cases o CCL5,FCER2, GZMH,IL10,IL13,IL1B,IL2RA,
PDCD1LG2, and PFR1. In con as , he exp ession le el o
LGALS9 in CCC-P e was lowe han ha de ec ed in HD and
IND-P e pa ien s, al hough i s exp ession inc eased in CCC-Pos
pa ien s, eaching an exp ession le el close o ha de ec ed in HD
and IND-P e pa ien s.
The s udy was subsequen ly ocused on he exp ession o he nine
genes ha in CCC pa ien s showed a dec ease (o an inc ease in he
case o LGALS9) in hei exp ession a e ea men , eaching alues
simila o hose in HD o IND. The esul was ep esen ed in ba
A
B
C
FIGURE 6
Gene se en ichmen analysis (GSEA). (A) Hea map o he op-100 genes de e mined by GSEA in ca diac Chagas disease pa ien s p e- (CCC-P e) and
pos ea men (CCC-Pos). The alues o he exp ession le el o each gene a e ep esen ed as colo s, anging om da k ed o da k blue, based on
he highes and lowes NRQ alues o each gene, espec i ely. The genes ep esen ed in e ical o de om op o bo om a e as ollows: ICAM1,
GZMA,ITGAX,IL2RA,IL1B,HAVCR2,CCL5,PRF1,IL10,FASLG,TGFBR1,B3GAT1,ITGB2,KLRG1,GZMH,PDCD1,TNFRSF1A,IL17A,CD86,FCER2,
CD8A,NCAM1,GZMK,IL4R,CCR1,GZMB,CD80,CXCR3,PDCD1LG2,IL7,ITGAL,IL10RA,IL6R,IL23R,TGFB1,GZMM,CD19,FCER1A,LAG3,IL7R,IL13,
IL12RB1,IL6,CD274,FNAR1,IL17RA,CD160,IL2,IFNGR1,IL5RA,STAT1,FAS,IL2RG,IL12B,CSF2,BTLA,CASP3,ICOS,CD28,CD40LG,CR2,CD48,
TNFRSF14,LGALS9,IL12A,IL18,GATA3,BCL2,CD4,IL12RB2,CD2,TNFSF10,CD27,CD3E,SELL,STAT3,IFNG,CD58,FOXP3,ITGA4,CD83,CSF1,
CD40,IL23A,IFNGR2,TGFBR2,XCR1,IL10RB,IL27,CD69,NOS2,ICOSLG,CLEC9A,TBX21,CCR7,IL5,TNF,CTLA4,IL18R1, and TGFB2. Pa ien s
whose samples we e me ged ha e been ma ked wi h an as e isk a he op o he hea map. (B) En ichmen plo s o gene se s om “CTL media ed
immune esponse agains a ge cells (BIOCARTA_CTL_PATHWAY)”and “B Lymphocy e Cell Su ace Molecules
(BIOCARTA_BLYMPHOCYTE_PATHWAY)”pa hways in CCC-P e and CCC-Pos. The g een cu e deno es he en ichmen sco e (ES) cu e. (C) GSEA
hea maps o gene se s om BIOCARTA_CTL_PATHWAY and BIOCARTA BLYMPHOCYTE PATHWAY. Pa ame e s se o GSEA we e: Molecula
Signa u es Da abase (MSigDB) BioCa a gene se collec ion, pe mu a ions = 100,000, pe mu a ion ype: pheno ype, en ichmen s a is ic: weigh ed,
me ic o anking genes: - es , max size: 500, min size: 2.
Go
´mez e al. 10.3389/ cimb.2024.1439714
F on ie s in Cellula and In ec ion Mic obiology on ie sin.o g09
P a fl, M. W., Tichopad, A., P gome , C., and Neu ians, T. P. (2004). De e mina ion
o s able housekeeping genes, di e en ially egula ed a ge genes and sample in eg i y:
Bes Keepe - Excel-based ool using pai -wise co ela ions. Bio echnol. Le . 26, 509–
515. doi: 10.1023/B:BILE.0000019559.84305.47
Pinazo,M.J.,Thomas,M.C.,Bua,J.,Pe one,A.,Schijman,A.G.,Vio i,R.J.,e al.(2014).
Biological ma ke s o e alua ing he apeu ic e ficacy in Chagas disease, a sys ema ic e iew.
Expe Re . An i In ec . The . 12, 479–496. doi: 10.1586/14787210.2014.899150
Rassi, A., Rassi, A., and Ma condes de Rezende, J. (2012). Ame ican ypanosomiasis
(Chagas disease). In ec . Dis. Clin. No h Am. 26, 275–291. doi: 10.1016/j.idc.2012.03.002
Rassi, A., Rassi, A., and Ma in-Ne o, J. A. (2010). Chagas disease. Lance 375, 1388–
1402. doi: 10.1016/S0140-6736(10)60061-X
Reddy, V. S., Ha skamp, R. E., Van Ginkel, M. W., Calhoon, J., Baisden, C. E., Kim, I.
S., e al. (2008). In e leukin-18 s imula es fib onec in exp ession in p ima y human
ca diac fib oblas s ia PI3K-Ak -dependen NF-kB ac i a ion. J. Cell Physiol. 215, 697–
707. doi: 10.1002/jcp.21348
Rezende-Oli ei a, K., Sa men o, R. R., and Junio , V. R. (2012). P oduc ion o
cy okine and chemokines by human mononuclea cells and whole blood cells a e
in ec ion wi h T ypanosoma c uzi. Re . Soc. B as. Med. T op. 45, 45–50. doi: 10.1590/
S0037-86822012000100009
Rocha, M. O. C., Ribei o, A. L. P., and Teixei a, M. M. (2003). Clinical managemen
o ch onic Chagas ca diomyopa hy. F on . Biosci 8, e44–e54. doi: 10.2741/926
Schmunis, G. A. (2007). Epidemiology o Chagas disease in non-endemic coun ies:
The ole o in e na ional mig a ion. Mem Ins Oswaldo C uz 102, 75–85. doi: 10.1590/
S0074-02762007005000093
Schmunis, G. A., and Yadon, Z. E. (2010). Chagas disease: A La in Ame ican heal h
p oblem becoming a wo ld heal h p oblem. Ac a T op. 115, 14–21. doi: 10.1016/
j.ac a opica.2009.11.003
Sil e , N., Bes , S., Jiang, J., and Thein, S. L. (2006). Selec ion o housekeeping genes
o gene exp ession s udies in human e iculocy es using eal- ime PCR. BMC Mol.
Biol. 7, 33. doi: 10.1186/1471-2199-7-33
Sousa Oli ei a, C. V., Mo eno-Loaiza, O., Figuei edo-Vanzan, D., Pe oba Ramos, I.,
Ma a-San os, H., To es Bozza, M., e al. (2022). IL-1bis no c i ical o ch onic hea
dys unc ion in mice wi h Chagas disease. F on . Immunol. 13. doi: 10.3389/
fimmu.2022.1010257
Sub amanian, A., Tamayo, P., Moo ha, V. K., Mukhe jee, S., Ebe , B. L., Gille e, M.
A., e al. (2005). Gene se en ichmen analysis: A knowledge-based app oach o
in e p e ing genome-wide exp ession p ofiles. P oc. Na l. Acad. Sci. 102, 15545–15550.
doi: 10.1073/pnas.0506580102
Szkla czyk, D., Gable, A. L., Lyon, D., Junge, A., Wyde , S., Hue a-Cepas, J., e al.
(2019). STRING 11: P o ein-p o ein associa ion ne wo ks wi h inc eased co e age,
suppo ing unc ional disco e y in genome-wide expe imen al da ase s. Nucleic Acids
Res. 47, D607–D613. doi: 10.1093/na /gky1131
Tesme , L. A., Lundy, S. K., Sa ka , S., and Fox, D. A. (2008). Th17 cells in human
disease. Immunol. Re . 223, 87–113. doi: 10.1111/j.1600-065X.2008.00628.x
Vandesompele, J., de P e e , K., Pa yn, F., Poppe, B., an Roy, N., de Paepe, A., e al.
(2002). Accu a e no maliza ion o eal- ime quan i a i e RT-PCR da a by geome ic
a e aging o mul iple in e nal con ol genes. Genome Biol. 3, esea ch0034.1–0034.11.
doi: 10.1186/gb-2002-3-7- esea ch0034
Velasquez, C. V., Russomando, G., Espı

nola, E. E., Sanchez, Z., Mochizuki, K., Roca,
Y., e al. (2019). IL-17A, a possible bioma ke o he e alua ion o ea men esponse
in T ypanosoma c uzi in ec ed child en: A 12-mon hs ollow-up s udy in Boli ia. PloS
Negl. T op. Dis. 13, e0007715. doi: 10.1371/jou nal.pn d.0007715
Villa ino, A. V., Gallo, E., and Abbas, A. K. (2010). STAT1-ac i a ing cy okines limi
Th17 esponses h ough bo h T-be -dependen and -independen mechanisms. J.
Immunol. 185, 6461–6471. doi: 10.4049/jimmunol.1001343
Vio i, R., Ala con De Noya, B., A aujo-Jo ge, T., G ijal a, M. J., Guhl, F., Lopez, M.
C., e al. (2014). Towa ds a pa adigm shi in he ea men o ch onic Chagas disease.
An imic ob. Agen s Chemo he . 58, 635–639. doi: 10.1128/AAC.01662-13
Vio i, R., Vigliano, C., Lococo, B., Be occhi, G., Pe i, M., Al a ez, M. G., e al.
(2006). Long- e m ca diac ou comes o ea ing ch onic Chagas disease wi h
benznidazole e sus no ea men : A non andomized ial. Ann. In e n. Med. 144,
724–734. doi: 10.7326/0003-4819-144-10-200605160-00006
Wagne , L., Yang, O. O., Ga cia-Zepeda, E. A., Ge, Y., Kalams, S. A., Walke , B. D.,
e al. (1998). Be a-chemokines a e eleased om HIV-1-specific cy oly ic T-cell
g anules complexed o p o eoglycans. Na u e 391, 908–911. doi: 10.1038/36129
Xie, F., Xiao, P., Chen, D., Xu, L., and Zhang, B. (2012). miRDeepFinde : A miRNA
analysis ool o deep sequencing o plan small RNAs. Plan Mol. Biol. 80, 75–84.
doi: 10.1007/s11103-012-9885-2
Go
´mez e al. 10.3389/ cimb.2024.1439714
F on ie s in Cellula and In ec ion Mic obiology on ie sin.o g16