cytokine polymorphisms, their influence and levels

8/10/2019 Cytokine Polymorphisms, Their Influence and Levels

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Hindawi Publishing Corporationuberculosis Research and reatment

Volume , Article ID , pageshttp://dx.doi.org/ . / /

Clinical Study Cytokine Polymorphisms, Their Influence and Levelsin Brazilian Patients with Pulmonary Tuberculosis during Antituberculosis Treatment

Eliana Peresi,1,2 Larissa Ragozo Cardoso Oliveira,1 Weber Laurentino da Silva,3rika Alessandra Pellison Nunes da Costa,1 Joo Pessoa Araujo Jr.,4Jairo Aparecido Ayres,5 Maria Rita Parise Fortes,6 Edward A. Graviss,7

Ana Carla Pereira,3

and Sueli Aparecida Calvi1

ropical Disease Department, Botucatu School of Medicine, UNESP, Sao Paulo State University, Botucatu, SP, Brazil Departamento de Doencas ropicais e Diagnostico por Imagem, Faculdade de Medicina de Botucatu, UNESP, Rubiao J unior S/N,Botucatu - , SP, Brazil Lauro de Souza Lima Institute, Bauru, SP, Brazil Microbiology and Immunology Department, Bioscience Institute, UNESP, Sao Paulo State University, Botucatu, SP, Brazil Nursing Department, Botucatu School of Medicine, UNESP, Sao Paulo State University, Botucatu, SP, Brazil Dermatology and Radiotherapy Department, Botucatu School of Medicine, UNESP, Sao Paulo State University,Botucatu, SP, Brazil Te Methodist Hospital Research Institute, Houston, X, USA

Correspondence should be addressed to Eliana Peresi; [email protected]

Received December ; Revised February ; Accepted February Academic Editor: Carlo Garzelli

Copyright Eliana Peresi et al. Tis is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cytokinesplayan essential roleduringactive tuberculosis disease andcytokinegenes havebeen described in associationwithalteredcytokine levels. Tere ore, the aim o this study was to veri y i IFNG, IL B, NF, IL A, IL , and GFB gene polymorphismsin uence the immune responseo Brazilianpatients with pulmonary tuberculosis (P B)at different time points o antituberculosistreatment ( , , and ). Our results showed the ollowing associations:IFNG + allele and IFNG + A allele with higherIFN- levels; IL B + C allele with higher IL- levels; NF A allele with higher NF- plasma levels in controls andmRNA levels in P B patients at ; IL A A allele at rs with higher IL- levels; IL allele with higher IL- levels;and GFB + CC genotype higher GF- plasma levels in P B patients at . Te present study suggests that IFNG + /A,IFNG + A/G, IL B + A/C, IL C/ , and GFB + C/ are associated with differential cytokine levels in pulmonary

tuberculosis patients and may play a role in the initiation and maintenance o acquired cellular immunity to tuberculosis and inthe outcome o the active disease while on antituberculosis treatment.

1. Introduction

Mycobacterium tuberculosis ( M. tuberculosis) is an intracel-lular obligate aerobic pathogen which has a predilection orthe lungs [ ]. Macrophages initiate phagocytosis o M. tuber-culosis bacilli and regulate immune responses mediated by proin ammatory cytokines such as NF- . Effector lym-phocytes ( cells) and natural killer (NK) cells secrete IFN-

which activate alveolar macrophages to produce reactive

intermediates rom nitrogen and oxygen, inhibiting growthand promoting mycobacteria death [ ]. IL- , producedmainly by macrophages and dendritic cells, has a key rolein the immune response to M. tuberculosis, bridging innateand adaptive immunity. Moreover, IL- induces cells andNK cells to produce proin ammatory cytokines such as IFN-

and NF- while also regulating the production o IL-[ , ]. A synergistic response o IFN- , IL- , NF- and IL-

activates macrophage, stimulating these cells to eliminate


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uberculosis Research and reatment

the intracellular pathogen, acting as a major effector mecha-nism o the cellular immune response [ ].

Despite the protective effect o T responses against M. tuberculosis, certain cytokines, such as NF- , are cor-related with the immunopathogenesis o the disease [ ].

o prevent tissue damage, active tuberculosis is associated

with decreased T and increased production and actiono suppressing cytokines produced by T and regula-tory ( reg) cells, IL- , and GF- , respectively, which actby deactivating macrophages, modulating proin ammatory cytokines, and reducing the antigen presenting unction o cells [ ]. GF- also participates in the induction o brosis,a hallmark presentation o tuberculosis disease [ ].

Te dynamics o tuberculosis ( B) disease is complex,as various aspects o the parasite-host interaction contributeto the occurrence and presentation o the outcome. In thisscenario there is an important contribution o human geneticsusceptibility to disease afer exposure to M. tuberculosis[ ]. Cytokines have a key role in the de ense against

mycobacteria and theirgenesmightbe considered candidatesor host susceptibility to the onset o active B disease.Te association between cytokines polymorphisms and

human B susceptibility has been reported in studies withex vivo cytokine production in response to mycobacterialantigens and their correlation with variant genotypes; how-ever ew studies have investigated their role in modulatingthe overall cytokine response during P B treatment [ ].We hypothesized that studying the actual overall immunecytokine pattern o P B patients could be important to ourunderstanding o active disease and possible establishmento biomarkers o recovery and anti- B treatment efficiency.Tere ore the aim o this study was to veri y the in uence

o IFNG, IL B, NF , IL A, IL , and GFB gene polymor-phisms on the overall cytokine response o Brazilian patientswith P B under anti- B treatment.

2. Material and Methods

. . Study Population. Te study group enrolled Brazil-ian patients attending the In ectious and Parasitic DiseasesServices at Botucatu Medical School University Hospital,UNESP, Botucatu eaching Health Centre and Primary Healthcare Units o Botucatu and surrounding region withP Bdiagnosiscon rmedby sputum smearorculture positiv-ity or M. tuberculosis or by clinical-epidemiologic data withlaboratory and image exams (radiography or computerizedtomography (C )) compatible with active B. Patients withP B concurrent with other active granulomatous diseases orHIVwere excluded.All patients diagnosed with P Breceivedtreatment or six months, using the our rst-line drugs:isoniazid, ethambutol, pyrazinamide, and ri ampicin. For theevaluation o immunologic unction, patients samples werecollected based on the anti- B treatment timeline, de ned as

: afer diagnosis and with no more thanone month o treat-ment; : with three months o treatment; and : with sixmonths o treatment. Patient-specimen distribution or eachtime point included ( = 5); ( = 1); ( = 2); withtwo time points and ( = 4); and with the three time

points , , and ( = 19). For normal controls (C), westudied healthcare workers rom Botucatu Medical School(Botucatu, SP, Brazil), males (mean age . years), and

emales (mean age . years), without clinical complaintsand with no history o B disease, autoimmune disease, andother in ectiousdisease. All controls were BCG vaccinated in

childhood andtuberculin skin test ( S )positive (induration mm).All patients andcontrols agreed to participate in thestudy, afer study clari cation and written in ormed consent.

. . Single Nucleotide Polymorphism (SNP) Genotyping. Inthe current study seven SNPs were analyzed: IFNG + /A;IFNG + A/G; IL B + A/C; NF G/C; IL Ars ; IL C/ ; and GFB + C/ . Tese poly-morphisms were selected afer veri ying their presence inhigh requency ( %) in the Brazilian population.

For the current study, mL o peripheral blood wasdrawn in an ED A blood tube (by standard phlebotomy procedures), rom P B patients ( = 31) and controls( = 20) at base line ( 0 ), and genomic DNA was extracted

rom leukocytes employing a DNAzol commercial reagent(Invitrogen, Carlsbad, CA, USA), according to the manu ac-turers instructions. Quanti cationandpurity o theextractedDNA were determined on a spectrophotometer (NanoDrop

Termo FisherScienti c). Ampli cation o thegenomicregions o interest was per ormed by PCR using to ngo DNA, recombinant aq DNA polymerase, . mM o each dN P (deoxy-nucleotide-adenine, guanine, thymine orcytosine-triphosphate), . to mM concentration o eacho the speci c primers, appropriate buffer, and ultrapurewater. Te IFNG + /A was genotyped by PCR-ARMSas described by Pravica et al. [ ]. Te variation at IFNG

+ A/G creates a restriction site or the enzyme AciI andwas genotyped by a previously reported PCR-RFLP method[ ]. Te IL B + genotyping was per ormed using aPCR-RFLP method in accordance with Garca-Gonz alez etal. [ ]. Fluorescence-based aqMan technology (AppliedBiosystems, Foster City, CA, USA) was applied to producegenotypes o IL A and GFB polymorphisms according tothe manu acturers instructions. Te NF A/Ggenotypewas de ned using a PCR-RFLP method in accordance withWilson et al. [ ].

. . Cytokines Gene Expression by Reverse ranscriptase Real ime PCR (R -qPCR). o evaluate IFN- , IL- , NF- , IL-, IL- , and GF- mRNA expressions, mL o peripheral

blood was drawn by a standard procedure in heparinizedblood tubes, at a single time point rom controls ( = 20) andat three serial time points or P B patients ( = 31), basedon the antituberculosis treatment timeline ( , , and ),as previously de ned. Peripheral blood mononuclear cells(PBMCs) were isolated by a Histopaque gradient separationmethod [ ]. Te layer rich in lymphocytes and monocyteswas aseptically removed and washed twice with PBS or

min at RPM. Te cell suspension was resuspended inmL o PBS and the identi cation and viability o cells were

determined by counting with urk solution ( L aliquotso cell suspension with L o the dye solution at %).


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: Primers sequence or qPCR.

Primers Forward sequence Reverse sequenceIFN- -AAAAGAG CCA A CCGC ACA C- -G GGG C C C GGC G A-IL- -ACC CCACC GCCGAGAA - -CA GG GGA GCCG CA-

NF- -GG GC ACAACA GGGC ACA- -CCCCAGGGACC C C C AA C-

IL- - AGGC ACA GG GGACAA CGG- -A GAC CC GGGAAGACC CA G-IL- -C GA G C GGG C GG C - -GC GGAGGAC AAGGG AACC -GF- -AGGGCCAGGACC GC G- -CAAGGGC ACCA GCCAAC --actin -GC GGAAGG GGACAGCGA- -GGCA CG GA GGAC CCG-

otal RNA extraction rom PBMC ( 6 cells/mL) wasprepared using rizol Reagent (Invitrogen, Carlsbad, CA,USA), according to manu acturers instructions. Te relativepurity, concentration, and quality o the isolated RNA weredetermined by spectrophotometry and the ratio o A A nm exceeded . or all preparations (NanoDrop Spectrophotometer, Termo Scienti c). o ensure complete

removal o traces o genomic DNA, g o total RNA wasincubated with DNase I (Amp Grade).First-strand cDNA synthesis was per ormed with g o

total RNA per L o reaction using Reverse ranscriptaseSuper Script II (Invitrogen, Carlsbad, CA, USA) and randomprimers ( g/ L) (Invitrogen, Carlsbad, CA, USA) accord-ing to manu acturers instructions. Within minutes aferRNAse H (Invitrogen, Carlsbad, CA, USA) was added andincubated at C or minutes.

Relative quanti cation o each target mRNA was per-ormed using a standard curve-based method or relative

real-time PCR data processing with a Real- ime PCR Systems (Applied Biosystems, USA) and Power SYBR Green

PCR Master Mix [ ]. Primers sets used in the qPCR (ampli ying cytokine ragments o mRNA and o the human-actin mRNA, endogenous mRNA control) are presented inable . Each q-PCR was set in duplicate in a total o mL

each, which contained . mM o each orward and reverseprimer, mL o template cDNA, L qPCR master mix,and . mL nuclease- ree water. In addition, a no templatecontrol was included in duplicate on each plate to veri y that amplicon contamination was absent. PCR conditionswere as ollows: initial denaturation at C or min and

cycles at C or s and C or s, ollowed by a melting curve. Ampli cation o speci c transcripts wascon rmed by melting curve pro les generated at the endo each run. Control samples expression mean received therelative value o . and concentrations in all other sampleswere normalized proportionately.

. . Plasma Cytokine Levels. Plasma samples were obtainedrom the same peripheral blood used or the genetic expres-

sion o cytokines rom controls ( = 20) and at three serialtime points rom patients with P B, based on the antitu-berculosis treatment timeline ( , , and ), as previously de ned. Samples were maintained rozen ( C) until useand then thawed at room temperature on the day they wereused. Quantikine ELISA kits (R & D Systems) were used,according to manu acturers instructions, to measure IFN- ,

IL- , NF- , IL- , IL- , and GF- plasma levels andmethod sensitivity was in accordance with each kit. Cytokineanalysis was not possible in all P B patients becausesamples were also used in other experiments; there ore thedistribution o individuals among cytokines was IFN- ( =30): ( = 5); ( = 1); ( = 2); with two time points

and ( = 4); with two time points and ( = 1);

and with the three time points , , and ( = 17); IL- ,IL- , and IL- ( = 30): ( = 7); ( = 1); ( = 2);with two time points and ( = 4); and with the threetime points , , and ( = 16); NF- ( = 30): ( = 5); ( = 1); ( = 2); with two time points and

( = 4); and with the three time points , , and ( = 18); GF- ( = 29): ( = 5); ( = 1); ( = 2);with two time points and ( = 4); and with the threetime points , , and ( = 17).

. . Statistical Analysis. Comparisons between different gen-otypes in the control and patient groups were made usingMann-Whitney est with two-tail value. For the ana-lytical comparison between the three time points o thetreatment in the P B case group ( , , and ), a Friedmantest was used to veri y which time point differed rom theother, a Dunns multiple comparisons test was applied as aposttest. Results were considered signi cant when < 0.05.

ests were per ormed using GraphPad Prism version .or Windows, GraphPad Sofware (San Diego, CA, USA),

http://www.graphpad.com/ .

3. Results. .Demographic Characteristics of P B Patients. Gender and

agedistributionamong P B patients was males (mean ageo . years) and emales (mean age o . years) andall patients were BCG (Bacillus Calmette-Guerin) vaccinatedin childhood. Despite the large differences o age and sexin controls and P B patients groups, these variables had noeffect on the cytokines expression andplasma levels (data notshown).

P B patients diagnosis was con rmed by sputum smearor culture positivity or M. tuberculosis ( = 2), sputumsmearorculture positive or M. tuberculosis, andimageexams(radiography or C ) compatible with active B ( = 1 9)or by image exams (radiography or C ) compatible withactive B ( = 10) alone with diagnosis con rmation withclinical response afer thebeginning o theanti- B treatment.


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0

50

100

150

C T1 T2 T3

AA

AT/TT

I F N - (

p g / L

)

+874

(a)

C T1 T2 T3

0

1000

2000

3000

4000

AA

AT/TT

G e n

i c e x

p r e s s

i o n I

F N -

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(b)

F : In uence o IFNG + /A gene SNP on IFN- plasma levels (a) and mRNA expression (b) in the control group (C) and P Bpatients group at three time points o the treatment: (afer diagnosis and rst month), (three months), and (six months). Data areshown as median levels. < 0.05.

All patients had respiratory symptoms consistent with dys-pnea, cough, and ventilation-dependent pain, and mostpresented with constitutional symptoms, consistent withweight loss, ever, and weakness at the beginning o the anti-

B treatment ( ). Te symptoms were less requent alongtreatment timeline ( ) and at the end ( ) all patients wereconsidered recovered. Studies classi y pulmonary tuberculo-sis by its severity as minimal, moderate, or advanced disease,depending on the characteristics o clinical-epidemiologic

data and/or image exams (radiography or C ) [ , ]. In ourstudy medical evaluation o these characteristics showed thatall o our patients had a moderate orm o active B disease.

. . General Immune Response during Anti- B reatment.In general, when compared to controls, plasma levels o IFN- , IL- , and IL- were similar among P B patientsduring anti- B treatment. IL- and GF- plasma levelswere increased among P B patients only at the beginningo anti- B treatment ( ) and plasma levels or NF- werelower among P B patients during anti- B treatment ( ,

, and ) (data not shown). When compared to controls,mRNA expression levels o IL- , NF-, and IL- were

similar among P B patients during anti- B treatment ( ,, and ). IFN- and IL- were increased among P Bpatients during anti- B treatment ( , , and ) and GF-

was increasedonly at the time point amongP Bpatients(data not shown).

. . In uenceof IFNG + /A and + A/G Gene Polymor- phisms on IFN- Plasma Level and mRNA Expression. Te allele carriers or IFNG + /A (A / ) in P B patientswere associated with signi cantly higher plasma and mRNAexpression levels o IFN- when compared to individualswith AA genotype at ( = 0.04; = 0.03, resp.) and

( = 0.04; = 0.03) time points o the treatment

(Figures (a) and (b)). When we compared the three timepoints, the AA genotype in P B patients at presentedsigni cant higher plasma levels than ( < 0.05) and

( < 0.05) (Figure (a)) and no differences or mRNAexpression ( Figure (b)). allelecarriers at presentedwithsigni cantly higher mRNA expression levels than at ( A single nucleotide polymorphism is associated withleprosy among Brazilians: a genetic epidemiology assessment,meta-analysis, and unctional study, Te Journal of InfectiousDiseases, vol. , no. , pp. , .

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G. W. Duff, Effects o a polymorphism in the human tumornecrosis actor alpha promoter on transcriptional activation,Proceedings of the National Academy of Sciences of the United States of America, vol. , no. , pp. , .

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