Sathler-Avelar et al. BMC Infectious Diseases 2012, 12:123

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1 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 RESEARCH ARTICLE Open Access Blood leukocytes from benznidzole-treted indeterminte chgs disese ptients disply n overll type-1-modulted cytokine profile upon short-term in vitro stimultion with trypnosom cruzi ntigens Rento Sthler-Avelr 1,2,3,5,6, Dnielle Mrquete Vitelli-Avelr 1,5, Silvn Mri Elói-Sntos 4,5, Eline Dis Gontijo 4,5, André Teixeir-Crvlho 1,5 nd Olindo Assis Mrtins-Filho 1,5 Abstrct Bckground: Benznidzole (Bz)-chemotherpy is recommended to prevent Chgs disese progression, despite its limited efficcy during chronic disese. However, the host mechnisms underlying these benefits still remin to be elucidted. Methods: In this study, we hve used short-term whole blood cultures to describe the cytokine profile of Bztreted Indeterminte Chgs disese ptients-(indt) s compred to untreted ptients-(ind). Results: Our findings showed tht IND presented incresed levels of + neutrophils, + nd + monocytes nd IFN-γ + NK-cells. Moreover, IND showed slight increse of + CD4 + T-cells nd enhnced levels of + CD8 + T-cells nd B-cells. Additionl nlysis of cytokine Low nd High producers lso highlighted the presence of High cytokine producers within IND, including from CD4+ T-cells nd IFN-γ from CD8 + T-cells, s compred to NI. The Bztretment led to n overll cytokine down-regultion in the innte nd dptive comprtments, including low levels of + nd + neutrophils nd monocytes, IFN-γ + NK-cells, +,TNF-α +,IFN-γ + nd IL-5 + CD4 + T-cells nd + B-cells, long with bsl levels of cytokine-expressing CD8 + T-cells in INDt s compred to IND. The in vitro ntigen stimultion shifted the cytokine profile towrd type 1-modulted profile, with incresed levels of + nd + monocytes, IFN-γ + nd + NK-cells long with TNF-α + nd IFN-γ + CD8 + T-cells. Anlysis of Low nd High cytokine producers, upon in vitro ntigen stimultion, further confirm these dt. Conclusion: Together, our findings showed tht the Bz tretment of Indeterminte Chgs disese ptients shifts the cytokine ptterns of peripherl blood monocytes, NK-cells nd CD8 + T-cells towrds long-lsting Type-1-modulted profile tht could be importnt to the mintennce of non-deleterious immunologicl microenvironment. Keywords: Chgs disese, Benznidzole, Immune response, Cytokines, Leucocytes subsets Sponsorships: CNPq, FAPEMIG, FIOCRUZ Correspondence: velr@cpqrr.fiocruz.br 1 Lbortório de Biomrcdores de Dignóstico e Monitorção, CPqRR- FIOCRUZ, Belo Horizonte, MG, Brzil 2 Unicentro Newton Piv, Belo Horizonte, MG, Brzil Full list of uthor informtion is vilble t the end of the rticle 212 Sthler-Avelr et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 2 of 12 Bckground An outstnding triumph of the Brzilin medicine ws the discovery of Chgs disese by Crlos Chgs t the beginning of the 2 th century. One century fter the discovery, severl spects of Chgs disese still remin uncler, including those regrding to the multiple events of pthogenesis underlying the clinicl progression, s well s the mechnisms implicted in the successful etiologicl tretment. Chgs disese is cused by the hemoflgellte protozon Trypnosom cruzi nd is considered one of the most importnt public helth problems in Ltin Americ, ffecting pproximtely 8 to 1 million people nd further 1 million people re considered t risk of infection [1]. The mjor mechnism ssocited with Chgs disese pthology is the continuous inflmmtory infiltrte observed minly in the crdic nd digestive trct orgns chrcterized by dmges in the neuronl conductive systems nd lso by muscle cytolysis [2]. There is growing consensus tht complex co-dpttion between the persistent prsite infection nd the blnced host immune response underlies the estblishment of Indeterminte clinicl form of Chgs disese. On the hnd the persistence of prsite long with unblnced immune response leds to sustined inflmmtory response tht supports these chrcteristic lesions of chronic Chgs disese [2]. In this immunopthogenesis prdigm the erdiction of T. cruzi my be prerequisite to control the disese progression nd prevent the irreversible long-term consequence of Chgs disese. Therefore, Chgs disese must be treted primrily s n infectious disese, nd not s utoimmune disorder, in contrst to long-held views [3]. Specific chemotherpy is recommended for the tretment of Chgs disese pplying the generl ssumption tht the erlier the specific tretment is initited greter re the chnces of prsitologicl cure nd clinicl benefits to the host [4]. At present, Chgs disese chemotherpy is restricted to two drugs, Benznidzole (Bz) nd nifurtimox (Nfx). In Brzil, only Bz hs been used to etiologicl tretment of Chgs disese [5] with higher cure rtio observed during cute, congenitl nd erly chronic Indeterminte Chgs disese [4-6]. Despite the low cure rtes observed in most ptients submitted to tretment during chronic Chgs disese, severl studies hve suggested tht the Bz-tretment should be still recommended during chronic Chgs disese, since it prevents the disese progression, regrdless of lck of complete prsite clernce [7-9]. The mechnism of ction of benznidzole is not completely cler. It hs been proposed tht the BZ-medited trypnocidl mechnism is effective becuse the nitro group is reduced by prsite enzymes to produce free rdicls, superoxide nions, nd hydrogen peroxide [1]. One of the mjor fctors potentilly influencing the prsite clernce s well s the morbidity control following the tretment for Chgs disese is the coopertive ction between the drug effects nd the host immunologicl response [11-14]. However little is known bout the chnges in the host immune following Bz tretment, specificlly during chronic Indeterminte Chgs disese tht could synergisticlly ct with the BZ-therpy. The coherent understnding of these chnges will certinly support the development of new protocol for Bz therpy, intervention nd mngement of chronicchgsdisese.webelievethtthenlysisofhost immunity sttus pre- nd post-tretment is essentil to further elucidte the impct of Bz intervention s well s support the rtionl development of new trypnosomicidl gents. We hve previously shown tht erly fter the end of the Bz therpy, the NK-cells nd CD8 + T-lymphocytes re importnt sources of IFN-γ nd tht produced by CD4 + T-cells nd B lymphocytes re puttive key element to modulte the immune response nd control tissue dmge might inducible by the pro-inflmmtory response. In the current investigtion we would like to test the hypothesis tht the Bz-therpy modultes the immune response inducing long-lsting impct on the immunologicl sttus of treted ptients, inducing similr Type-1 modulted cytokine profile s observed erly fter the end of the tretment [13,14]. Our findings bring new insights to the hypothesis tht lter on fter the end of Bz-tretment, the ptients presented down-regulted cytokine pttern long with the cpcity to build Type-1 modulted cytokine response, upon T. cruzi ntigen stimuli, supported by proinflmmtory cytokine from monocytes, NK nd CD8 + T-cells, counterblnced by from monocytes. Methods Study popultion This cross-sectionl study involved 47 subjects clssified into three groups referred s IND untreted Indeterminte Chgs disese ptients (n = 15; 3 mles nd 12 femles; men ge = 42.5 ± 5.5 yers, rnging from yers); INDt Bz-treted Indeterminte Chgs disese ptients (n = 14; 4 mles nd 1 femles; men ge = 44. ± 8.7 yers, rnging from yers) nd NI non-infected individuls (n = 18, 7 mles nd 11 femles, men ge = 29. ± 9.7 yers, rnging from yers). Indeterminte Chgs disese ws dignosed by t lest two positive results on serologicl tests (indirect hemgglutintion, indirect immunofluorescence, or enzyme-linked immunosorbent ssy) nd no ltertion in the clinicl nd physicl exmintion, besides norml electrocrdiogrm, chest X-ry, Holter, nd echodopplercrdiogrphy profiles. The IND ptients were recruited

3 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 3 of 12 t the Ambultório de doenç de Chgs, Hospitl ds Clínics, Universidde Federl de Mins Geris (Belo Horizonte, Mins Geris, Brzil). Benznidzole (N-benzyl-2-nitro-1-imidzolcetmide) tretment ws crried out in dily doses of 5 mg/kg of body weight, twice dy for 6 dys, ccording to the Brzilin Ministry of Helth [15]. The INDt ptients were recruited seven yers fter the end of etiologicl tretment t the Ambultório de doenç de Chgs, Hospitl ds Clínics, Universidde Federl de Mins Geris (Belo Horizonte, Mins Geris, Brzil). The non-infected subjects showing negtive serologic tests for ntibodies ginst T. cruzi were contcted t the Centro de Hemtologi e Hemoterpi de Mins Geris (Belo Horizonte, Mins Geris, Brzil). All study prticipnts provided written informed consent following the guidelines of Ethics Committee of the Mins Geris Federl University. The study protocol complied with the regultions 196/1996 of Brzilin Ntionl Council on Reserch in Humns nd ws lso pproved by the Ethicl Committee of Mins Geris Federl University under protocol # COEP-ETIC 7/. Blood smples Five milliliters of whole peripherl blood were collected in EDTA nticogulnt for hemogrm nlysis. Ten milliliters of heprinized peripherl blood were collect from ech prticipnt nd used for short-term in vitro culture of whole blood. Heprin whole blood smples were mintined t room temperture for up to 12 hours prior processing. Trypnosom cruzi epimstigote ntigen preprtion Soluble Epimstigote Antigen (EPI) ws prepred from sttionry phse Y strin T. cruzi epimstigotes grown in LIT-medium. After the third or fourth in vitro pssge, epimstigotes were hrvested, wshed in 15 mm phosphte-buffered sline (PBS), ph 7.4, nd resuspended to 1 8 cells/ml in 15 mm PBS, ph 7.4. The suspension ws rpidly frozen t 7 C nd thwed t 37 C three times, with soniction procedure between ech step. The crude lyste ws centrifuged (37, g) for 9 min nd the superntnt collected, dilysed overnight ginst 15 mm PBS, ph 7.4, sterilized by filtrtion through.22-μm pore membrne (Filter millex, Milipore Products Division, Billeric, MA, USA) nd stored t 7 C until use. The protein content ws ssyed by the method described by Lowry et l. [16]. Short-term in vitro culture of whole blood Short-term cultures in vitro were crried out s previously described by Sthler-Avelr et l., [14]. Briefly, 5μL liquots of heprinized whole blood smples were trnsferred into 14 ml polypropylene tubes (Flcon W, BD Phrmingen, Sn Jose, CA, USA) nd incubted in the presence of 5μL of RPMI-164 (GIBCO, Grnd Islnd, NY, USA) Control Culture ; 5μL liquots of heprinized whole blood smples in the presence of EPI soluble ntigen t finl concentrtion of 2 μg/ml in RPMI-164 Stimulted Culture or Phorbol 12- Myristte 13-Acette t 25 ng/ml plus ionomycin t 1 μg/ml (Sigm, St Louis, MO, USA) in RPMI-164 PMA stimulted culture. The Control nd Stimulted cultures were pre-incubted for 1 h t 37 C, 5% CO 2 in humidified incubtor. Afterwrd, ll cultures were incubted for 4 h in t 37 C, 5% CO 2 in humidified incubtor in the presence of BFA t 1 μg/ml. Following incubtion, ll cultures were treted with 1μL of EDTA 2 mm in PBS for 15 min (Sigm, St Louis, MO, USA) in order to block the ctivtion process. Immunophenotyping for cell subsets nd intrcellulr cytokines nlysis Following the short term in vitro stimultion, ll cultures were wshed once with 3 ml of FACS buffer prepred s PBS supplemented with.5% bovine serum lbumin nd.1% sodium zide (Sigm, St Louis, MO, USA). Smples were resuspended into 1 ml of FACS buffer. Aliquots of 4μL were immunostined in the drk for 3 min t room temperture with 1μL of pre-diluted TriColor-lbelled monoclonl ntibodies (mabs) nti- CD4, CD8, CD14, CD16 or CD19 (Cltg, Burlingme, CA, USA). Following incubtion, the erythrocyte lysing nd cell fixtion procedures were performed by dding 2 ml of FACS lysing solution (BD, Sn Diego, CA, USA) nd incubtion for 3 min t room temperture. Membrne-stined leucocytes were permebilized with 3 ml of FACS perm-buffer for 1 min t room temperture (FACS buffer supplemented with.5% sponin) nd wshed once with 2 ml of FACS buffer. Cell suspension liquots of 3μL were trnsferred to 96wells round bottom micropltes (Flcon W ; BD Phrmingen, Sn Jose, CA, USA) were stined for 3 min t room temperture, in the drk in the presence of 2μL ofpelbelled nti-cytokine mabs (, TNF-α, IFN-γ,, IL-5 nd ). All ntibodies were diluted 1:5 in FACS perm-buffer nd were purchsed from BD Phrmingen (Sn Diego, CA, USA). After intrcytoplsmic cytokine stining, the cells were wshed twice with 2μL of FACS perm-buffer nd FACS buffer, respectively, nd then fixed in 2μL of FACS FIX Solution (1 g/l of prformldehyde, 1.2 g/l of sodium ccodilte nd 6.63 g/l of sodium chloride, ph 7.2, ll from Sigm, St Louis, MO, USA). The smples were stored t 4 C prior flow cytometry cquisition nd nlysis. Flow cytometry cquisition nd nlysis Flow cytometry cquisition of 3, immunestined cells/smples were performed in FACSclibur W flow

4 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 4 of 12 cytometer equipped with CELLQUEST TM softwre (Becton Dickinson, Sn Jose, CA, USA). Distinct gting strtegies were used to nlyze the cytokine-expressing leucocyte subpopultions from the innte (monocytes, neutrophils nd NK-cells) nd dptive immunity (lymphocytes s well s T- nd B-cell subsets). The monocytes were selected s CD14 High+ cells nd the neutrophil s SSC High CD14 Low+ cells on FL3/nti-CD14- TC versus SSC/lser side-sctter dot plots. The lymphocyte popultions were selected on FSC/lser forwrd-sctter versus SSC/lser side-sctter dot plots. Following the initil gte selection, the frequencies of cytokine + cells were quntified by qudrnt sttistics pplied on FSC/lser forwrd-sctter versus FL2/nticytokine-PE dot plots for monocytes nd neutrophils nd FL3/nti-CD16, CD4, CD8 or CD19 TC versus FL2/ nti-cytokine-pe dot plots for NK-cells, T-lymphocyte subsets nd B-cells. Anlysis of low nd high cytokine-producers The nlysis of cytokine-producers within IND nd INDt groups were performed by first tking the medin vlue of cytokine + cells from ech leucocyte subpopultion (neutrophils, monocytes, NK-cells, CD4 + nd CD8 + T-cells nd B-cells) from the NI group s the cut-off edge to ctegorize the subjects s Low nd High cytokine-producers. Gry-scle digrms were used to ssemble the prevlence of Low (empty rectngle), High inflmmtory (blck rectngle for, TNF-α nd IFN-γ) or High regultory (gry rectngle for, IL-5 nd ) cytokine-producers mongst IND nd INDt groups. Br chrts were pplied to compile the frequency of Low nd High cytokine-producers within ech leucocyte subset. Sttisticl nlysis Sttisticl nlysis ws performed using the GrphPd Prism 5 softwre pckge (Sn Diego, CA, USA). As ll dt files ssume non-gussin distribution, sttisticl comprisons were crried out using the nonprmetric Kruskl-Wllis test followed by Dunns multiple comprison test to evlute the cytokine profiles mong NI, IND nd INDt groups. The comprtive nlyses between control culture nd stimulted culture were performed by Wilcoxon mtched pirs test. The frequency of high nd low cytokine-producers ws compred by contingency tble nlysis by Chi-squre test. The differences were considered significnt when p-vlues were <.5. Results Cytokine profile of innte immunity in indeterminte chgs disese ptients The number of inflmmtory nd regultory cytokines, expressed by the innte immunity cells, from Indeterminte Chgs disese ptients is shown in Figure 1. Dt from the control culture showed prominent nti-inflmmtory cytokine pttern in neutrophils from IND s compred to NI, represented by enhnced levels of + cells nd bsl levels of + cells (Figure 1A, left pnel). Upon in vitro ntigen stimultion, similr nti-inflmmtory cytokine profile ws observed in IND, with incresed number of + neutrophils s compred to the NI nd control culture (Figure 1A, right pnel). The nlyses of monocytes reveled mixed inflmmtory/nti-inflmmtory cytokine pttern in IND s compred to NI, with incresed levels of + nd + cells (Figure 1B, left pnel). This mixed profile ws preserved fter in vitro ntigen stimultion, s shown by the incresed number of + nd + monocytes in IND s compred to NI (Figure 1B, right pnel). Anlysis of NK-cells showed predominnt inflmmtory cytokine pttern in IND, with higher number of IFN-γ + cells nd bsl levels of + cells s compred to NI (Figure 1C, left pnel). Upon in vitro ntigen stimultion, despite no difference in the number of IFN-γ + cells, the lower levels of + cells count to the predominnt inflmmtory cytokine pttern of NK-cells observed in IND s compred to NI (Figure 1C, right pnel). Impct of bz-tretment on the cytokine pttern of innte immunity of indeterminte chgs disese ptients In order to investigte the impct of etiologicl tretment on the cytokine profile of innte immunity of Indeterminte Chgs disese ptients, we hve compred the cytokine profile of neutrophils, monocytes nd NK-cells from INDt s compred to IND. These differences re highlighted by connecting lines. Dt nlyses showed n overll cytokine downregultion in the innte immunity comprtment fter benznidzole therpy, s shown by the lower levels of +, + neutrophils nd monocytes, s well s reduced levels of IFN-γ + NK-cells in INDt s compred to IND (Figure 1A, B nd C, left pnel). However, the in vitro ntigen stimultion ws ble to shift the cytokine expression by monocytes nd NK-cells towrd type 1-regulted immune profile, with incresed levels of nd IFN-γ respectively counterblnced by from monocytes (Figure 1B nd C, right pnel), despite no chnges in the cytokine profile of neutrophils (Figure 1A, right pnel). Cytokine profile of dptive immunity in indeterminte chgs disese ptients The levels of inflmmtory nd regultory cytokineexpressing cells in the dptive immunity comprtment from Indeterminte Chgs disese ptients re shown in Figures 2, 3 nd 4.

5 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 5 of 12 (A) # Cytokine + NEUTROPHILS/mm Control Culture # Cytokine + NEUTROPHILS/mm Stimulted Culture (B) # Cytokine + MONOCYTES/mm # Cytokine + MONOCYTES/mm (C) # Cytokine + NK-CELLS/mm # Cytokine + NK-CELLS/mm Figure 1 Anlysis of intrcytoplsmic cytokine profile of neutrophils. (A), monocytes (B) nd NK-cells in the peripherl blood from non treted Indeterminte chgsic ptients (IND gry rectngle), Bz-treted Indeterminte chgsic ptients (INDt blck rectngle) s compred to non-infected individul (NI empty rectngle). Dt re expressed s medin bsolute counts plus the interqurtile rnge of cytokine + cells observed fter short-term in vitro Control Culture (left pnels) nd T. cruzi epimstigote ntigen Stimulted Culture (right pnels) of wholeblood smples. Cytokine + neutrophils, monocytes nd NK-cells were quntified by dul color immunophenotyping (nti-cd14-tc or nti-cd16-tc plus nti-cytokine-pe mabs) using specific gte strtegies to select ech leucocyte subset, s described in Mteril nd Methods. The significnt differences t p<.5 for comprisons with NI re indicted by the letter. The significnt differences t p<.5 for comprisons with INDt re indicted by connecting lines. The significnt differences t p<.5 for comprtive nlysis between Control nd Stimulted cultures re indicted by. Dt from the control culture showed smll shift towrds nti-inflmmtory cytokine pttern in CD4 + T-lymphocytes from IND s compred to NI, represented by slight increse in the number of + long with the bsl numbers of TNF-α +, +,IFN-γ +,IL-5 + nd + cells (Figure 2A). Upon in vitro ntigen stimultion despite the generl bsl levels of cytokines observed for CD4 + T-cells, the smll decrese in the + cells could support the smll shift towrds nti-inflmmtory cytokine pttern in CD4 + T-lymphocytes. A typicl nti-inflmmtory cytokine profile ws observed for both CD8 + T-lymphocytes nd B-cells from IND s compred to NI, represented by incresed levels of + cells long with lower number of TNF-α + B-cells

6 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 6 of 12 (A) 6 Control Culture # Cytokine + CD4 + T-CELLS /mm IL-5 (B) 6 Stimulted Culture # Cytokine + CD4 + T-CELLS /mm IL-5 Figure 2 Anlysis of intrcytoplsmic cytokine profile of CD4 +. T-lymphocytes in the peripherl blood from untreted Indeterminte chgsic ptients (IND gry rectngle), Bz-treted Indeterminte chgsic ptients (INDt blck rectngle) s compred to non-infected individul (NI empty rectngle). Dt re expressed s medin bsolute counts plus the interqurtile rnge of cytokine + cells observed fter short-term in vitro Control Culture (top pnel) nd T. cruzi epimstigote ntigen Stimulted Culture (bottom pnel) of whole-blood smples. Cytokine + CD4 + T-cells were quntified by dul color immunophenotyping (nti-cd4-tc plus nti-cytokine-pe mabs) using specific gte strtegy, s described in Mteril nd Methods. The significnt differences t p <.5 for comprisons with NI re indicted by the letter. The significnt differences t p <.5 for comprisons with INDt re indicted by connecting lines. The significnt differences t p <.5 for comprtive nlysis between Control nd Stimulted cultures re indicted by. (Figure 3A nd 4A). Upon in vitro ntigen stimultion, despite the incresed number of IFN-γ + CD8 + T-lymphocytes nd TNF-α + B-cells observed in comprison to the control culture, nti-inflmmtory cytokine profile still remin prepondernt in both CD8 + T-lymphocytes nd B-cells from IND s compred to NI (Figures 3B nd 4B). Impct of bz-tretment on the cytokine pttern of dptive immunity of indeterminte chgs disese ptients The comprtive nlysis of cytokine-expressing cells in the dptive immunity comprtment fter the etiologicl tretment is shown in Figures 2, 3 nd 4. Dt from the control culture showed n overll cytokine down-regultion in CD4 + T-cells from INDt s compred with IND nd NI, with decresed levels of +,TNF-α +, IFN-γ + nd IL-5 + cells, long with bsl numbers of + nd + cells (Figure 2A) with no chnges observed fter the in vitro ntigen stimultion (Figure 2B). On the other hnd, despite the bsl levels of cytokine-expressing CD8 + T-cells observed in the control cultures from INDt s compred to IND (Figure 3A), the in vitro ntigen stimultion ws ble to drive the CD8 + T-cells towrds inflmmtory cytokine profile, with incresed numbers of TNF-α + nd IFN-γ + CD8 + T-cells in INDt s compred to IND nd NI (Figure 3B). Dt nlysis from the control cultures lso showed cytokine down-regultion in B-cells from INDt s compred with IND nd NI, with decresed levels of + cells (Figure 4A) which remin fter in vitro ntigen stimultion, regrdless of slight increse in TNF-α + B-cells observed in comprison to the control culture (Figure 4B). Anlysis of low nd high cytokine-producers mongst indeterminte chgs disese ptients As previously report by Bhi-Oliveir et l. [17] nd Vitelli-Avelr et l. [18], the nlysis of Low nd High

7 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 7 of 12 (A) 75 Control Culture # Cytokine + CD8 + T-CELLS /mm IL-5 (B) # Cytokine+ CD8 + T-CELLS /mm Stimulted Culture IL-5 Figure 3 Anlysis of intrcytoplsmic cytokine profile of CD8 +. T-lymphocytes in the peripherl blood from untreted Indeterminte chgsic ptients (IND gry rectngle), Bz-treted Indeterminte chgsic ptients (INDt blck rectngle) s compred to non-infected individul (NI empty rectngle). Dt re expressed s medin bsolute counts plus the interqurtile rnge of cytokine + cells observed fter short-term in vitro Control Culture (top pnel) nd T. cruzi epimstigote ntigen Stimulted Culture (bottom pnel) of whole-blood smples. Cytokine + CD8 + T-cells were quntified by dul color immunophenotyping (nti-cd8-tc plus nti-cytokine-pe mabs) using specific gte strtegy, s described in Mteril nd Methods. The significnt differences t p <.5 for comprisons with NI re indicted by the letter. The significnt differences t p <.5 for comprisons with INDt re indicted by connecting lines. The significnt differences t p <.5 for comprtive nlysis between Control nd Stimulted cultures re indicted by. cytokine-producers represents powerful tool to chrcterize distinct immunologicl profiles in Chgs disese ptients (Figure 5A nd B, top pnels). The frequencies of High cytokine-producers in the IND nd INDt groups were obtined considering the medin of given cytokine + cells from the NI group s the cut-off edge to ctegorize the subjects s Low nd High cytokine-producers. We hve used gry-scle digrms to ssemble the frequency of Low (empty rectngle), High inflmmtory (blck rectngle) nd regultory (gry rectngle) cytokine-producers mongst IND nd INDt groups. Using this concept, our dt confirmed the presence of type-1 modulted cytokine profile within innte immunity cells, with enhnced frequency of High cytokine producers, including from neutrophils, nd from monocytes nd IFN-γ from NK-cells. The nlysis of the dptive comprtment lso confirmed the presence of predominnt nti-inflmmtory immune response in CD4 +, CD8 + nd B-cells nd dditionlly pointed out the presence of High cytokine producers including from CD4 + T-cells nd IFN-γ from CD8 + T-cells (Figure 5A top pnel). No differences were observed in the frequency of Low nd High producers between control nd stimulted cultures from IND (Figure 4A nd B, top pnel). Impct of bz-tretment of the frequency of low nd high cytokine-producers mongst indeterminte chgs disese ptients Aiming to further pply the concept of Low nd High cytokine producers, we investigte the cytokine profile fter Bz-chemotherpy in ptients with Indeterminte Chgs disese (Figure 5A nd B, bottom pnels). Dt nlysis further confirmed the brod cytokine down-regultion in both innte nd dptive immunity comprtment fter Bz-tretment (Figure 5A bottom pnel). Upon in vitro ntigen stimultion, the leucocytes from INDt were ble to shift the down-regulted cytokine

8 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 8 of 12 (A) 6 Control Culture (B) 6 Stimulted Culture # Cytokine + B-CELLS/mm Figure 4 Anlysis of intrcytoplsmic cytokine profile of B-cells in the peripherl blood from untreted. Indeterminte chgsic ptients (IND gry rectngle), Bz-treted Indeterminte chgsic ptients (INDt blck rectngle) s compred to non-infected individul (NI empty rectngle). Dt re expressed s medin bsolute counts plus the interqurtile rnge of cytokine + cells observed fter short-term in vitro Control Culture (left pnel) nd T. cruzi epimstigote ntigen Stimulted Culture (right pnel) of whole-blood smples. Cytokine + B-cells were quntified by dul color immunophenotyping (nti-cd19-tc plus nti-cytokine-pe mabs) using specific gte strtegy, s described in Mteril nd Methods. The significnt differences t p <.5 for comprisons with NI re indicted by the letter. The significnt differences t p <.5 for comprisons with INDt re indicted by connecting lines. The significnt differences t p <.5 for comprtive nlysis between Control nd Stimulted cultures re indicted by. # Cytokine + B-CELLS/mm 3 profile towrd type 1 modulted pttern, chrcterized by enhnced frequency of High cytokine producers including IFN-γ from NK-cells nd TNF-α nd IFN-γ from CD8 + T-cell long with nd from monocytes (Figure 5A nd B, bottom pnels). Discussion The first insight to use trypnocidl chemotherpeutic drugs in ptients with Chgs disese strted lmost eighty yers go [19]. However, t the present, only Nfx nd Bz re cliniclly vilble worldwide for the etiologicl tretment of Chgs disese, with the ltter been the min nti-prsitic drug used in Brzil. Despite the effectiveness of Bz to prsite erdiction during cute phse of T. cruzi infection, the limited efficcy during chronic Chgs disese, besides the high frequency of undesirble side effects hve contributed to refuse its use to tret over 8 million ptients with chronic Chgs disese [5]. Previous reports hve shown tht, despite the lck of prsite erdiction, the etiologicl tretment contribute to reduce the prsitemi nd rerrnge the host immune response, leding to blnced inflmmtory response crucil to control Chgs disese morbidity [7,9,13,14,2]. Regrdless these insights, there re still few studies focusing on the immunologicl chnges following Bz-tretment during chronic Chgs disese [13,14,21-23]. A coherent understnding of the immunologicl following Chgs disese etiologicl tretment will certinly contribute to determine new perspectives for immunoprevention, therpy, intervention nd mngement [24,25]. In this context, it is importnt to mention tht the development of longitudinl studies would be the choice to void possible experimentl bis due to prticulr fetures of the individul immune response. However, longitudinl interventions presents besides the difficulty to estblish long-term follow-up of ptients, lrge gp between the first nd the second evlution, especilly when focusing on long-lsting fetures, tht would introduce relevnt vrible to the experimentl pproches, minly due to the regent lots nd equipment performnces. In this study, we hve performed cross-sectionl investigtion to verify whether the Bz-therpy would induce long-lsting impct on the immunologicl sttus of treted ptients leding to Type-1 modulted cytokine profile likely tht observed erly fter the end of the tretment [13,14]. For this purpose, we hve first chrcterized the cytokine profile of circulting leucocytes from untreted IND ptients in order to estblish bseline to evlute the impct of etiologicl tretment during chronic T. cruzi infection. Our dt showed tht untreted IND ptients presented in the ex vivo nlysis (Control Culture) n overll type-1 regulted cytokine profile in the innte immune comprtment ( from neutrophils, nd from monocytes nd IFN-γ from NK-cells) nd predominnt type-2 profile in the dptive immunity cells ( from CD4 + T-cells, from CD8 + T-cells nd B-lymphocytes). It ws interesting to notice tht the cytokine profile of innte nd dptive immunity cells ws generlly preserved upon in vitro ntigenic stimultion.

9 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 9 of 12 #1 #2 #3 #4 #5 #6 #7 #8 #9 #1 #11 #12 #13 #14 #15 #1 #2 #3 #4 #5 #6 #7 #8 #9 #1 #11 #12 #13 #14 (A) IND INDt Frequency (%) Control Culture (CC) #1 #2 #3 #4 #5 #6 #7 #8 #9 #1 #11 #12 #13 #14 #15 #1 #2 #3 #4 #5 #6 #7 #8 #9 #1 #11 #12 #13 #14 (B) IND INDt Frequency (%) Stimulted Culture (SC) IL-5 IL-5 IL-5 IL-5 (C) Neu Mon NK CD4 + T-cells CD8 + T-cells B-cells Neu Mon NK CD4 + T-cells CD8 + T-cells B-cells CC SC CC SC CC SC CC SC CC SC INDt IND CD14 CD14 CD16 CD8 CD8 Figure 5 Frequency of low cytokine-producers (empty rectngle). High inflmmtory cytokine-producers (blck rectngle) nd high regultory cytokine-producers (gry rectngle) for ech leukocyte subset in the peripherl blood from untreted Indeterminte chgsic ptients (IND) nd Bz-treted Indeterminte chgsic ptients (INDt) using the medin percentge of cytokine + cells from the group of non-infected individuls s the cut-off edge. Color digrms (left pnels) were first ssembled for individul smples to further compile the resultnt frequency (right pnels) of cytokine producers in the Control Culture (A) nd T. cruzi epimstigote ntigen Stimulted Culture (B) of whole-blood smples. Sttisticl significnce t p <.5 for comprisons between Control nd Stimulted cultures re represented by. (C) Representtive dot plots illustrting the overll cytokine down-regultion fter Bz tretment, s well s the in vitro ntigen stimultion shifted the cytokine profile towrd type 1-modulted profile. These findings showed tht untreted IND ptients re ble to mount pro-inflmmtory cytokine response supported by enhnced levels of + monocytes nd IFN-γ + NK-cells. However, this inflmmtory response seems to be regulted in Indeterminte clinicl form, to consequently prevent the disese progression, by the estblishment of modultory mechnisms tht involve the prticiption of from neutrophils, monocytes, CD8 + T-cells nd B-cells. Our findings showed n incresed frequency of inflmmtory-like monocytes nd NK-cells, together with high levels of regultory-like monocytes nd modulted dptive immunity tht could be importnt to the estblishment/mintennce of symptomtic chronic Chgs disese. Previous studies hve shown tht the estblishment of n inflmmtory immune response medited by monocytes nd NK-cells re essentil to control T. cruzi infection [26,27] nd tht the bility to mount immunoregultory mechnisms in both innte [28] nd dptive immunity [19] re essentil to control the inflmmtory immune ctivity nd re pprently necessry to prevent deleterious effect of nti-t. cruzi immune response during symptomtic chronic Chgs disese. The nlysis of High nd Low cytokine producers hs been proposed by Bhi-Oliveir et l. [17] nd further pplied by Vitelli-Avelr et l. [18] to evlute the cytokine profile of Chgs disese ptients. Using this pproch, our dt further confirmed the cytokine profile described bove. Additionlly, our findings pointed out the presence of incresed frequency of High producers within CD4 + T-cells, but still lower thn the frequency of High producers within monocytes. These dt re-inforce previous reports form Gomes et l., [28] demonstrting tht in IND the mjority of the -producing cells re monocytes. Moreover, our dt showed tht enhnced frequency of High IFN-γ producers could be identified within CD8 + T-cells in IND, not detectble by the conventionl nlysis. This finding is lso in greement with those presented by Gomes et l. [28] showing tht round 6% of the IND could be identified s High IFN-γ producers. These uthors suggested tht the IND ptients tht re high IFN-γ producers re cndidtes to develop crdiomyopthy sooner thn those lower producers. On the other hnd, it is possible tht IND ptients who re ble to lwys keep high levels of

10 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 1 of 12 secretion hs less chnce to develop crdic disese [28]. Humn longitudinl studies under evlution by our group will be ble to vlidte this hypothesis. As IND is not necessrily permnent clinicl form, with pproximtely 36% of them eventully developing severe crdic disese [1], ny effort tht could mke to mintin the IND clinicl sttus, prt from prsitologicl cure, would be relevnt to control Chgs disese morbidity. It hs been suggested tht the etiologicl tretment could be employed to mintin the stble Indeterminte clinicl form nd prevent the disese progression, despite the prsitologicl cure [7-9,21]. Since impirment in the cytokine network hs been pointed out s one of the determining fctors driving disese morbidity [2,18], it is possible tht if the etiologicl tretment would be ble to introduce chnges in the cytokine network towrds immunodultory mechnisms, it could contribute to prevent the disese progression. In this study, we hve chrcterized the cytokine profile of circulting leucocytes from treted INDt ptients, seven yers fter the end of Bz chemotherpy nd compred with tht observed in untreted IND ptients. Our dt showed tht treted INDt ptients presented, in the ex vivo nlysis (Control Culture), n overll downregulted cytokine profile in both, innte immunity ( nd from neutrophils nd monocytes besides IFN-γ from NK-cells) nd dptive immune comprtment (, TNF-α, IFN-γ nd IL-5 from CD4 + T-cells nd from B-cells), long with bsl levels of cytokine-expressing cells within CD8 + T-lymphocytes. The overll down-regultion in the cytokine synthesis observed seven yers fter the end of Bz-tretment is in greement with previous reports the immunomodultory effect of Bz in experimentl models [21,29,3]. This modulted profile my ccount for the generl hypothesis tht the Bz-tretment during chronic Chgs disese my hs supportive impct, modulting the inflmmtory cytokine profile of peripherl blood leucocytes from IND reducing the chnces of progression to crdic disese [13,14,21]. However, this down-regultion in the cytokine profile my shortly fvor the prsite repliction from residul inflmmtory foci, considering the low effectiveness of Bz-tretment to promote prsite clernce during chronic Chgs disese [31-33]. To investigte the possible impct tht residul T. cruzi ntigens would hve on the immune response of Bz-treted INDt ptients, we hve chrcterized the cytokine pttern triggered upon in vitro T. cruzi ntigen stimultion. Interestingly, our dt showed tht the cytokine profile of innte nd dptive immunity cells ws gretly impcted by the ntigenic stimultion. In fct, the in vitro ntigen stimultion ws ble to emerge the cytokine synthesis minly in monocytes ( nd ), NK-cells (IFN-γ) nd CD8 + T-cells (IFN-γ), chrcterizing n overll type 1-modulted immune profile. The nlysis of High nd Low cytokine producers further confirmed the impct of T. cruzi ntigens on the cytokine pttern of INDt. This modulted pro-inflmmtory cytokine pttern resemble tht observed in children treted during erly Indeterminte Chgs disese (E-INDt), by the bility of NK-cells nd CD8 + T-cells to produce IFN-γ [14]. However, there ws mjor difference regrding the source of modultory in INDt (monocytes) nd E-INDt (CD4 + T-cells nd B-lymphocytes) [14]. The bility of the immune system to generte stble memory CD8 + T-cell fter pthogen clernce during cute nd chronic infection hve been lredy shown in experimentl T. cruzi infection [21,34]. These uthors hve shown tht Bz-induced cure drives conversion of the immune response to stble nd protective CD8 + T-cell centrl memory response, cpble of showing effector function nd providing protective immunity upon re-chllenge [21,34]. In greement with our dt, the Bz-tretment does not result in the full retention of CD4 + T-cells ble to recll cytokine production upon ntigenic stimultion [21]. It hs been shown tht Bz-tretment of infected hosts leds to selective expnsion of effector nd memory CD8 + T-cells ble to protect the hosts ginst re-infection. These findings llow these uthors to hypothesize tht besides the direct role in blocking the prsite repliction in vivo, Bztretment ppers to positively ffect the ntigen-driven host immune system nd thus mintin the prticiption of immuneprotective effector mechnism in the ntipthogen response. Therefore, the effectiveness of ny chemotherpy protocols should consider not only the trypnocidl effect but lso its impct on the host immune response. Additionl studies re still needed to better understnd the impct of Bz-tretment on the immune system of ptients t distinct clinicl forms of Chgs disese. As previously reported, we re currently investigting the impct of Bz-tretment on the immunologicl sttus on crdic Chgs disese ptients [14]. Preliminry dt hve pointed out the beneficil impct of Bz-tretment in the immunologicl profile in crdic ptients incresing the frequency of + monocytes, n importnt immunomodultory event to control deleterious ntiprsite immune-medited inflmmtory mechnisms [18]. The bility of Bz to induce the persistence of ntigenspecific memory CD8 + T-cells in crdic ptients re currently under evlution by our group. Conclusion In summry, our findings showed tht the Bz tretment of Indeterminte Chgs disese ptients shifts the cytokine ptterns of peripherl blood monocytes, NK-cells nd CD8 + T-cells towrds long-lsting Type-1-modulted profile tht could be importnt to the mintennce of non-deleterious immunologicl microenvironment.

11 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 11 of 12 Competing interests The uthors hve no competing interests to declre. Authors contributions SMES nd EDG were responsible for the medicl screening of ptients. OAMF, RSA, DMVA nd ATC were involved in the design, cquisition dt nd nlysis. All uthors contributed to the interprettion dt. OAMF, RSA, DMVA, SMES nd ATC drfted the mnuscript nd ll uthors revised the finl drft criticlly for importnt criticl content. All uthors hve given finl pprovl of the version to be published. Acknowledgements We re thnkful to FAPEMIG (APQ ), CNPq (#47834/26-7) grnt gencies nd FIOCRUZ (PAPES V - # 43518/28-3) for the finncil support. We re lso thnk the Progrm for Technologicl Development in Tools for Helth-PDTIS-FIOCRUZ for use of its fcilities. RSA, ATC nd OAMF re grteful for FAPEMIG nd CNPq reserch fellowship (PDJ, PQ nd PQ, respectively). Author detils 1 Lbortório de Biomrcdores de Dignóstico e Monitorção, CPqRR- FIOCRUZ, Belo Horizonte, MG, Brzil. 2 Unicentro Newton Piv, Belo Horizonte, MG, Brzil. 3 Universidde Vle do Rio Verde, UninCor, Belo Horizonte, MG, Brzil. 4 Deprtmento de Medicin Preventiv e Socil, Fculdde de Medicin, Universidde Federl de Mins Geris, Belo Horizonte, Mins Geris, Brzil. 5 Deprtmento de Propedêutic Complementr, Fculdde de Medicin, Universidde Federl de Mins Geris, Belo Horizonte, Mins Geris, Brzil. 6 Lbortório de Biomrcdores de Dignóstico e Monitorção, R, Lbortório de Biomrcdores de Dignóstico e Monitorção, CPqRR-FIOCRUZ, Av. Augusto de Lim 1715, 319 2Belo Horizonte, MG, Brzil. Received: 13 Februry 212 Accepted: 1 My 212 Published: 24 My 212 References 1. World Helth Orgniztion: New globl effort to eliminte Chgs disese. 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12 Sthler-Avelr et l. BMC Infectious Diseses 212, 12:123 Pge 12 of 12 synthesis of nitrite nd cytokines by murine stimulted mcrophges. Clin Exp Immunol 1999, 118: Cmndrob EL, Reis EA, Gonçlves MS, Reis MG, Andrde SG: Trypnosom cruzi: susceptibility to chemotherpy with benznidzole of clones isolted from highly resistnt Colombin strin. Rev Soc Brs Med Trop 23, 36: Cnçdo JR: Long term evlution of etiologicl tretment of chgs disese with benznidzole. Rev Inst Med Trop 22, 44: Toledo MJ, Bhi MT, Veloso VM, Crneiro CM, Mchdo-Coelho GL, Alves CF, Mrtins HR, Cruz RE, Tfuri WL, Ln M: Effects of specific tretment on prsitologicl nd histopthologicl prmeters in mice infected with different Trypnosom cruzi clonl genotypes. J Antimicrob Chemother 24, 53: Olivieri BP, Cott-De-Almeid V, Arujo-Jorge T: Benznidzole tretment following cute Trypnosom cruzi infection triggers CD8 + T-cell expnsion nd promotes resistnce to reinfection. Antimicrob Agents Chemother 22, 46: doi:1.1186/ Cite this rticle s: Sthler-Avelr et l.: Blood leukocytes from benznidzole-treted indeterminte chgs disese ptients disply n overll type-1-modulted cytokine profile upon short-term in vitro stimultion with trypnosom cruzi ntigens. BMC Infectious Diseses :123. Submit your next mnuscript to BioMed Centrl nd tke full dvntge of: Convenient online submission Thorough peer review No spce constrints or color figure chrges Immedite publiction on cceptnce Inclusion in PubMed, CAS, Scopus nd Google Scholr Reserch which is freely vilble for redistribution Submit your mnuscript t