The expression of immune response genes in patients with chronic Chagas disease is shifted toward the levels observed in healthy subjects as a result of treatment with Benznidazole

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 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 g05 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 . 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 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 . 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 g07 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). 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 g08 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. 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