Inhibition of caspase-8 activity promotes protective Th1- and Th2-mediated immunity to Leishmania major infection

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1 Article Inhibition of caspase-8 activity promotes protective Th1- and Th2-mediated immunity to Leishmania major infection Wânia F. Pereira-Manfro,*,1,2 Flávia L. Ribeiro-Gomes,*,,1 Alessandra Almeida Filardy,* Natália S. Vellozo,* Landi V. C. Guillermo,*,3 Elisabeth M. Silva,*,4 Richard M. Siegel, George A. DosReis,*, and Marcela F. Lopes*,5 *Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil; Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, and Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA; and Instituto Nacional para Pesquisa Translacional em Saúde e Ambiente na Região Amazônica, Conselho Nacional de Desenvolvimento Científico e Tecnológico/Ministério da Ciência e Tecnologia, Rio de Janeiro, Brazil RECEIVED SEPTEMBER 20, 2012; REVISED AUGUST 27, 2013; ACCEPTED AUGUST 30, DOI: /jlb ABSTRACT We investigated how apoptosis pathways mediated by death receptors and caspase-8 affect cytokine responses and immunity to Leishmania major parasites. Splenic CD4 T cells undergo activation-induced apoptosis, and blockade of FasL-Fas interaction increased IFN- and IL-4 cytokine responses to L. major antigens. To block death receptor-induced death, we used mice expressing a T cell-restricted transgene for vflip. Inhibition of caspase-8 activation in vflip mice enhanced Th1 and Th2 cytokine responses to L. major infection, even in the Th1-prone B6 background. We also observed increased NO production by splenocytes from vflip mice upon T cell activation. Despite an exacerbated Th2 response, vflip mice controlled better L. major infection, with reduced lesions and lower parasite loads compared with WT mice. Moreover, injection of anti-il-4 mab in infected vflip mice disrupted control of parasite infection. Therefore, blockade of caspase-8 activity in T cells improves immunity to L. major infection by promoting increased Th1 and Th2 responses. J. Leukoc. Biol. 95: ; Introduction The outcome of immunity versus disease progression in Leishmaniasis is multifactorial and depends on Leishmania species Abbreviations: 7-AAD 7-aminoactinomycin D, B6 C57BL/6, CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico, DAF diaminofluorescein, dpi days postinfection, FasL Fas ligand, FLIP FADD-like interleukin-1 converting enzyme inhibitory protein, Gr- 1 granulocyte differentiation antigen, hi high, imfi integrated mean fluorescence intensity, MFI mean fluorescence intensity, neg negative, UFRJ Universidade Federal do Rio de Janeiro, vflip viral FADD-like interleukin-1 converting enzyme inhibitory protein, zietdfmk benzyloxycarbonyl Ile-Glu-Thr-Asp-fluoromethylketone, zlehd-fmk benzyloxycarbonyl Leu-Glu-His-Asp-fluoromethylketone The online version of this paper, found at includes supplemental information. and development of innate and adaptive immune responses [1 3]. Early recruitment and death of neutrophils, which interact and are phagocytosed by infected macrophages and DCs [4], may affect resistance versus susceptibility upon infection with Leishmania major [5 10]. Control of intracellular infection is mantained by CD4 Th1 cells that secrete IFN- and activate NO production by macrophages. By contrast, IL-4 and IL-10 cytokines, produced by CD4 Th2 and regulatory T cells, promote parasite persistence in susceptible mice [1 3, 11, 12]. In the course of immune responses, interactions between FasL and Fas trigger T cell apoptosis upon activation of caspase-8 and effector caspases [13]. It remains controversial, however, whether killing of Th1 and Th2 subsets is differentially regulated by Fas-mediated apoptosis or other cell-death pathways [14 17], as discussed previously [18]. Apoptosis of T lymphocytes occurs in human cutaneous lesions as a result of infection with Leishmania braziliensis [19], whereas splenocytes from patients with visceral Leishmaniasis up-regulate expression of Fas and FasL [20]. In addition, increased Fas/FasL expression was detected in cells infiltrating cutaneous lesions in murine infection [21]. Moreover, systemic neutralization of FasL and TRAIL reduces skin ulceration in experimental Leishmaniasis [22]. Previous studies have also addressed whether apoptosis of lymphocytes and Fas-death pathway correlate with susceptibility versus resistance to Leishmania infection. Increased apoptosis in spleen and draining LNs parallels disease progression in susceptible BALB/c mice 1. These authors contributed equally to this work. 2. Current address: Faculdade de Ciências Médicas, UERJ, Rio de Janeiro, RJ, Brazil. 3. Current address: Instituto Biomédico, UNIRIO, Rio de Janeiro, RJ, Brazil. 4. Current address: Instituto Biomédico, Universidade Federal Fluminense, Niterói, RJ, Brazil. 5. Correspondence: Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Ilha do Fundão, Rio de Janeiro, RJ, , Brazil. marcelal@biof.ufrj.br /14/ Society for Leukocyte Biology Volume 95, February 2014 Journal of Leukocyte Biology 347

2 infected with L. major. By contrast, early onset and limited apoptosis follows infection in resistant CBA mice [23]. In humans with visceral Leishmaniasis [24] and in resistant B6 mice infected with Leishmania donovani [25], T cell apoptosis correlates with Fas expression and defective Th1 responses. Nonetheless, disruption of Fas-death pathway increases susceptibility to L. donovani [26] and L. major [27, 28] infections in Fas-defective lpr or FasL-mutant gld mice, in spite of increased Th1 responses. Killing [26, 27] or activation [29] of infected macrophages by CD4 T cells expressing FasL might promote resistance to Leishmania infection in mice bearing an intact Fasdeath pathway. Although these studies indicate that apoptosis regulates immunity to Leishmania infection, the control of T cell cytokine responses by apoptosis has not been addressed directly. Here, we used transgenic inhibition of T cell caspase-8 activation to investigate how Th2 and Th1 responses to L. major infection were affected in Th1-prone B6 mice. We used T cellrestricted transgenic mice expressing the MC159 vflip, which can block cell death by apoptosis and necroptosis [30 33]. It is noteworthy, however, that caspase-8 activity is also required for other aspects of T cell activation and development of immune responses to viral and parasite infections [13, 31, 34 37]. As we found previously that vflip expression negatively affects the production of cytokines by CD8 T cells and CD8 T cell-mediated immunity [31, 35], we chose the L. major infection model to explore the effects of caspase-8 inhibition on CD4 T cells. Despite expressing increased Th2 cytokines along with a robust Th1 response, vflip mice exerted a better control over L. major infection, with reduced lesions and parasite loads, than WT mice. We also addressed whether resistance was associated with a protective Th2 response in vflip mice. MATERIALS AND METHODS Animals Female B6 mice were obtained from the UFRJ and Fundação Oswaldo Cruz (Rio de Janeiro, Brazil) animal facilities. Transgenic mice expressing vflip in T cells (CD2-vFLIP) were obtained from the U.S. National Institutes of Health (Bethesda, MD, USA) [31], housed and bred at the Taconic (Germantown, NY, USA) and UFRJ transgenic animal facilities. All animal experiments were approved by the Ethics Committee for Use of Animals of UFRJ, in accordance with national regulations. For PCR detection of vflip transgene, DNA was extracted from mouse tails with Extract-N- Amp kit (Sigma, St. Louis, MO, USA). The following pairs of primers were used to detect vflip MC159 transgene: forward, GAC TAC GCA TCC GAC TCC AAG GAG GTC CCT AGC; reverse, CGG AAT TCT CAA GTC GTT TGC TCG GGG CT, as described previously [31]. IL-2 gene, forward, CTA GGC CAC AGA ATT GAA AGA TCT; reverse, GTA GGT GGA AAT TCT AGC ATC ATC C, was used as a control. L. major infection L. major LV39 parasites isolated from BALB/c mice were cultured in Schneider s medium (Invitrogen Life Technologies, Carlsbad, CA, USA) and used at the stationary phase of culture. Female vflip mice and their WT littermates (age 7 8 mo) or 7- to 9-week-old B6 mice were infected in the left hind footpad with s.c. injection of parasites/30 l. Lesions were expressed as the difference between the left (infected) and the right (control) footpads, measured with Mitutoyo vernier caliper. To neutralize IL-4 in vivo, WT and vflip mice were infected with L. major and treated i.p. at 17, 21, and 26 dpi with 0.5 mg/injection/mouse anti-il-4 (mab 11B11) or rat IgG1 isotype control mab (GL 113) from Bio X Cell (West Lebanon, NH, USA). Parasite load Draining (popliteal) LN cells from infected mice were cultured in Schneider s medium (Sigma), supplemented with 2 mm glutamine, 2% human urine, plus 10% FBS, at 28 C for 72 h. Parasite loads were determined by serial dilution assay, and viable promastigotes were counted on a Neubauer chamber. Alternatively, LN cells were serially diluted in 96-well flat-bottom plates containing biphasic medium, prepared using 50 l Novy-MacNeal- Nicolle medium containing 20% defibrinated rabbit blood (Spring Valley Laboratories, Woodbine, MD, USA) and overlaid with 100 l M199 (Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 20% heatinactivated FCS (Hyclone, Logan, UT, USA), 100 U/ml penicillin, 100 g/ml streptomycin, 2 mm L-glutamine, 40 mm Hepes, 0.1 mm adenine (in 50 mm Hepes), 5 mg/ml hemin (in 50% triethanolamine), and 1 mg/ml 6-biotin. The number of viable parasites was determined from the highest dilution, at which promastigotes could be grown out after 7 10 days of incubation at 26 C [38]. Cell suspensions and T cell culture Red blood cell-depleted splenocytes, LN cells, and T cell-enriched suspensions obtained by nylon wool filtration of splenocytes were resuspended in DMEM (Invitrogen Life Technologies), supplemented with 2 mm glutamine, M 2-ME, 10 g/ml gentamicin, 1 mm sodium pyruvate, 0.1 mm MEM nonessential amino acids, and 10 mm HEPES (culture medium), plus 10% FBS (Invitrogen Life Technologies). For evaluation of apoptosis, T cells ( /0.5 ml) from normal and infected mice were cultured in triplicates and stimulated with 10 g/ml plate-bound anti-cd3 (mab 2C11; BD PharMingen, San Diego, CA, USA) in the presence or absence of 10 g/ml anti-fasl mab (MFL3 clone, hamster IgG1), anti-trail (N2B2, rat IgG2a), anti-tnf- (G , rat IgG1), or respective control IgG mab (all from BD PharMingen) or in the presence of 40 M caspase-8 inhibitor zietd-fmk, caspase-9 inhibitor zlehd-fmk (Enzyme Systems Products, Livermore, CA, USA), or control diluent (0.4% DMSO) [35] in 48-well vessels for 24 h. Apoptosis was evaluated in gated CD4 cells by flow cytometry as described below. All cultures were set at 37 C and 7% CO 2 in a humid atmosphere. Magnetic selection of T cells Splenocytes from normal and infected B6 mice were enriched in CD4 T cells and APCs by incubation with anti-b220, anti-cd8, and anti-pannk (CD49b; BD PharMingen), followed by negative selection with magnetic Dynabeads M-450 (sheep anti-rat IgG; Dynal Biotech, Milwaukee, WI, USA). Selected cells (65 80% CD4 T cells) were cultured ( /well) in triplicates and stimulated with L. major antigen (50 g/ml frozen-thawed parasites) in the presence of 10 g/ml anti-fasl, anti-trail, anti-tnf-, or respective control IgG mab in 96-well round-bottom vessels for 24 h. Apoptosis was evaluated in gated CD4 T cells by flow cytometry. For evaluation of cytokines (by ELISA) and proliferation (by scintillation spectroscopy), splenocytes or splenocytes, enriched in CD4 T cells ( /well), were stimulated in triplicates with L. major antigen in the presence or absence of 10 g/ml anti-fasl, anti-trail, or respective control IgG mab in 96-well round-bottom vessels for 48 h. Flow cytometry Fresh cells were washed in FACS buffer (containing 2% FBS) and incubated with anti-cd16/cd32 for Fc blocking, followed by staining with allophycocyanin, PE, PerCP-Cy5.5, or FITC-labeled anti-cd8, anti-cd4, anti- CD44, anti-cd11b, and anti-gr-1 for 30 min at 4 C. All mab are from BD PharMingen, except the PerCP-Cy5.5-labeled anti-cd8 (ebioscience, San Diego, CA, USA). For detection of NO, splenocytes were first incubated with 2.5 M of the DAF-diacetate probe (Molecular Probes, Invitrogen, 348 Journal of Leukocyte Biology Volume 95, February

3 Pereira-Manfro et al. Th2-mediated immunity to L. major in vflip mice Carlsbad, CA, USA) for 15 min, followed by surface staining. For apoptosis detection, cells were stained with FITC-Annexin V (BD PharMingen) for 20 min in annexin buffer; 7-AAD was added just prior to acquisition. Cells were acquired on a FACSCalibur system by using CellQuest software (BD Biosciences, San Jose, CA, USA). For analysis, FlowJo software was used (TreeStar, Ashland, OR, USA). Nitrites For NO assay, splenocytes ( /well) from individual, infected WT and vflip mice were cultured in triplicates in medium only or stimulated with 10 g/ml plate-bound anti-cd3 in 96-well flat-bottom vessels for 48 h. Supernatants were collected and mixed with an equal volume of Griess reagent to detect nitrites [39]. Cytokines LN cells or T cell-enriched splenic suspensions ( /well) from individual, infected WT and vflip mice were cultured in triplicates in medium only or stimulated with 10 g/ml plate-bound anti-cd3 in 96-well flat-bottom vessels or with 50 g L. major antigen in 96-well round-bottom vessels for 48 h. Cytokines IFN-, IL-4, and IL-10 (BD PharMingen or ebioscience) and IL-13 (ebioscience) were measured in culture supernatants by a sandwich ELISA, according to the manufacturer s instructions. Splenocytes ( /well) were cultured in triplicates in medium only or stimulated with 10 g/ml plate-bound anti-cd3 or with 50 g L. major in 48-well vessels for 48 h. Supernatants were collected for ELISA, and cultures were stimulated further with 1 g/ml ionomycin (Sigma) and 50 ng/ml PMA (Sigma) in the presence of 1 g/ml brefeldin A (GolgiPlug; BD Biosciences) during 4 h for intracellular cytokine staining. Cells were stained for surface expression with anti-cd4 (PeCy7) and anti-cd8 (Alexa Fluor 780) and for intracellular labeling with anti-il-4 (allophycocyanin) and anti- IFN- (FITC) from ebiosciences or BD Biosciences. Results were collected using FACSDiva software on a FACSCanto flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar). Forward- and side-scatter width parameters were used to exclude cell doublets from analysis. Dead cells labeled with Live/Dead Fixable Aqua (Invitrogen) were also excluded. Statistics Results were expressed as average and sem. Data were tested for normality by Kolmogorov-Smirnov test and then analyzed by Student s t-test. Results that failed to pass normality test (or n 4 mice/group) were also analyzed by Mann-Whitney test and passed parametric and nonparametric tests. Each individual mouse was subjected to multiple analyses, an analysis with three determinations. The number (n) of animals/group was indicated in figure legends and the symbol (*) denoted for significant differences in Student s t-test with a P value For in vitro experiments, data were expressed as average of triplicates/treatment, and significant differences in Student s t-test were indicated for P 0.05 (*). Legends also contain the number of repeat experiments represented in each figure. RESULTS Figure 1. CD4 T cell activation and death in the course of L. major infection. B6 mice were infected s.c. in the hind footpad with L. major parasites. (A) Footpad lesions develop in B6 mice upon 2, 6, and 11 weeks postinfection., Change. (B) Absolute numbers of CD4 T cells in draining LN (Œ) and spleens (Œ) from normal and infected mice upon 2, 6, and 13 weeks postinfection. (C) Splenocytes from normal (o) and infected (Œ) mice were analyzed for T cell activation and cell death. Activation of CD4 T cells was determined by high expression of CD44. Cell death was assessed by staining with 7-AAD and analyses within the CD4 T cell subset. Significant differences (*P 0.05) between normal and 6-week-infected mice were indicated in figure. Apoptosis in CD4 T cells during L. major infection Following infection with L. major, draining LNs underwent hypertrophy, with numbers of CD4 T cells peaking 6 weeks after infection, paralleling development of footpad lesions (Fig. 1A and B). An increase in CD4 T cells also occurred in spleens at 6 weeks (Fig. 1B), either as a result of activation or trafficking of activated cells [40] through this organ (Fig. 1C, left). In addition, increased cell death, as assessed by 7-AAD staining, was detected in splenic CD4 T cells (Fig. 1C, right). We reasoned that the onset of apoptosis in CD4 T cells could affect the production of cytokines upon infection with L. major. To test this idea, T cells from normal and 6-week-infected mice were activated with anti-cd3 in the presence of anti-fasl, anti-trail, or control mab (Fig. 2). Treatment with anti-fasl, but not anti-trail, increased the recovery of viable (Annexin- V neg, 7-AAD neg ) CD4 T cells in anti-cd3-stimulated T cell cultures from infected but not control mice (Fig. 2A, upper). Likewise, inhibition of caspase-8 but not caspase-9 activity improved viability of CD4 T cells from infected mice (Fig. 2A, lower). Blockade of FasL (Supplemental Fig. 1A) but not treatment with anti-trail or anti-tnf- (Supplemental Fig. 1B) also reduced apoptosis of CD4 T cells stimulated with L. major antigen (Fig. 2B, upper). Moreover, treatment with anti-fasl increased antigen-induced proliferation of CD4 T cells from infected mice (Fig. 2B, lower) and secretion of IFN- (Fig. 2C, upper) and IL-4 (Fig. 2C, lower) by total splenocytes stimulated with L. major antigen. Intact immunity to L. major infection in vflip mice To block apoptosis in vivo and investigate whether caspase-8 activation controls cytokine responses and immunity to L. major, we studied the course of infection and immune responses in mice expressing the caspase-8 inhibitor vflip in T cells. Volume 95, February 2014 Journal of Leukocyte Biology 349

4 Figure 2. Activation-induced T cell death upon L. major infection. Splenocytes enriched in T cells (A), CD4 T cells (B), or total splenocytes (C) from normal or 6-week-infected mice were stimulated with (A) anti- CD3 or (B and C) L. major antigen and treated or not with anti-fasl, anti-trail, control mab, caspase-8 (zietd) or caspase-9 (zlehd) inhibitors, or control vehicle (DMSO). (A and B) After 24 h, cells from stimulated and treated cultures were stained with anti-cd4, Annexin V, and 7-AAD. (A) Bars represent viable (AnnexinV neg, 7-AAD neg ) CD4 cells. (B) Bars represent apoptotic (Annexin V, 7-AAD ) CD4 cells (upper). Proliferation of splenocytes enriched in CD4 T cells was assessed by incorporation of 1 Ci [H 3 ] thymidine after 48 h (lower). (C) After 48 h, supernatants from antigen-stimulated splenocytes were tested for IFN- (upper) and IL-4 (lower) by ELISA. Lines represent antigen-stimulated splenocytes from control mice. Significant differences between cells stimulated in the presence of control IgG and anti-fasl or DMSO and zietd are indicated as *P Results represent at least three independent experiments. Compared with WT mice, vflip mice developed reduced lesions, days after infection, when lesions usually peak at the site of L. major infection in WT mice (Fig. 3A). We accessed the number of parasites in LNs upon 6 weeks of infection and observed that vflip mice better controlled infection compared with WT mice (Fig. 3B). In a repeat experiment, infection was followed until resolution of lesions. Reduced peak lesions were observed in vflip mice, which remained as resistant as WT mice during the resolution phase of infection (data not shown). To investigate improved resistance of vflip mice, we studied changes in T cell-mediated immunity caused by inhibition of caspase-8. Total splenocytes from infected mice were stimulated with anti-cd3 to induce T cell activation, cytokine secretion, and cooperation between T cells and splenocytes for NO production. Despite the higher level of IL-4 produced by total splenocytes ( vs ng/ml from vflip and WT mice, respectively), we detected increased NO production by anti-cd3-activated splenocytes from vflip mice (Fig. 3C). We identified by flow cytometry a population of splenocytes expressing CD11b and Gr-1 that could be a source of NO, as assessed by staining with a DAF-diacetate probe (data not shown). We also detected striking differences in production of cytokines by splenic T cells from infected vflip and WT mice (Fig. 3D). Both splenic T lymphocytes and LN cells from vflip mice had enhanced IL-4 responses to anti-cd3 (Fig. 3D, right, and E), whereas IFN- responses were high but similar to infected WT mice (Fig. 3D, left). No consistent differences were found for IL-10 secretion in cultures from vflip and WT mice (data not shown). Paradoxically, immunity to L. major infection is increased in vflip mice and correlates better with NO and IL-4 production by activated splenocytes. Enhanced Th1 and Th2 responses to L. major antigens We further addressed cytokine responses to antigen and polyclonal stimuli in vflip mice. We found an improved IFN- response to L. major antigen and anti-cd3 in spleen and draining LNs from infected vflip mice (Fig. 4A and B). Whereas CD4 T cells comprise 20% of splenocytes in vflip and WT mice upon infection, there is a significant decrease in percentages and absolute numbers (not shown) of CD8 T cells in vflip (10.18% 2.12 sd) versus WT (16.30% 1.36 sd) mice. As CD4 and CD8 T cells secrete IFN- in response to anti-cd3 in supernatants from total splenocytes (Fig. 4A, upper), we addressed IFN- -expressing T cell subsets by flow cytometry (Fig. 4B). Whereas the numbers of CD4 T cells expressing IFN- increased (Fig. 4B, upper), the numbers of IFN- -expressing CD8 T cells were reduced significantly in vflip mice (not shown). Previous studies indicate that an integrated parameter, which takes into account percentage of events and MFI (imfi), correlates well with protective responses to vaccination against L. major infection [41]. Figure 4B (lower) shows that CD4 T cells from vflip mice express enhanced IFN- (imfi) responses to L. major antigen and anti-cd3 stimuli. Th2 cytokines IL-4 and IL-13 were also accessed in spleen and LNs of vflip mice (Fig. 4C and D). Cells from infected vflip mice secreted more IL-13 in response to L. major antigen and IL Journal of Leukocyte Biology Volume 95, February

5 Pereira-Manfro et al. Th2-mediated immunity to L. major in vflip mice Figure 3. Transgenic vflip mice are resistant to L. major infection despite an exacerbated Th2 response. (A) Transgenic vflip mice ( ; n 4) develop reduced footpad lesions compared with WT mice (o; n 6). (B) After 41 dpi, lower parasite loads were detected in LNs from vflip mice compared with WT mice (n 4 mice/ group). Parasite loads were normalized as log parasites/ml for statistic analyses. (C) Splenocytes, (D) splenic T cells, and (E) draining LN cells from individual-infected vflip and WT mice were cultured with medium only or stimulated with anti-cd3. After 48 h, culture supernatants were collected and tested for NO (C), IFN- (D) and IL-4 (D and E). Significant differences between infected WT and vflip mice (n 4 mice/ group) are indicated as *P Results represent two independent experiments. and IL-13 cytokines upon activation with anti-cd3, whereas Th2 cytokines were not enhanced in infected WT mice above control levels (Fig. 4C and D, basal lines). Increased IL-4 response (Fig. 4D, upper) and CD4 T cells expressing IL-4 (Fig. 4D, lower) were also detected in anti-cd3-stimulated cultures from infected vflip mice. Therefore, vflip mice developed higher Th1 and Th2 responses during L. major infection, in spite of a B6 Th1-biased background. A Th2 cytokine response protects vflip mice to L. major infection To address the role of IL-4 in immunity to L. major infection, we injected infected WT and vflip mice with a neutralizing anti-il-4 antibody (Fig. 5). Although lesions started to increase in WT mice, 2 weeks upon L. major infection, a delayed development of lesions was observed in two untreated groups of infected vflip mice before antibody injection (Fig. 5A, upper). Following antibody treatment, however, increased lesions developed in vflip mice injected with anti-il-4 (Fig. 5A, lower; 32 dpi). In addition, more cells were detected in draining LNs (Fig. 5B, upper) and spleen (Fig. 5C) from vflip mice treated with anti-il-4. Moreover, compared with infected vflip mice treated with control mab, injection of anti-il-4 increased the number of parasites in infected vflip mice (Fig. 5B, lower). IFN- -producing CD4 T cells expanded in response to antigen and parasite burden in vflip mice treated with anti-il-4 (Fig. 5C). Therefore, IL-4 seems to play a protective role, helping vflip mice to control L. major infection. DISCUSSION We studied whether cell death and caspase-8 pathways could affect cytokine responses by CD4 T cells in L. major infection. In B6 mice, kinetics of LN enlargement, T cell activation, and cell death followed progression and resolution of footpad lesions. Increased CD4 T cell death occurred at the peak of development of lesions. Inhibition of caspase-8 but not caspase-9 activity improved CD4 T cell viability in stimulated cultures from infected but not normal mice. Activation-induced T cell death was blocked by anti-fasl, which also increased CD4 T cell proliferation, as well as IFN- and IL-4 production, in response to L. major antigen. By contrast, anti- TRAIL or anti-tnf- did not reduce apoptosis at the conditions studied. These results are compatible with a role for FasL-Fas upstream to caspase-8 activation and apoptosis signaling in activated CD4 T cells from L. major-infected mice, although the involvement of other death receptors in vivo cannot be discarded. Moreover, an intact Fas-death pathway is likely to down-regulate not only Th1, as suggested previously [26, 28], but also Th2 responses to L. major infection in B6 mice. To block death receptor-induced cell death in vivo and investigate the role of caspase-8 in T cell-mediated immunity to L. major infection, we compared WT (B6) mice with transgenic vflip mice, which expressed a marked increase in IL-4 production in spleen and LNs after 6 weeks of infection. IL-13 and IFN- cytokines were also increased in response to L. ma- Volume 95, February 2014 Journal of Leukocyte Biology 351

6 Figure 4. Improved Th1 and Th2 responses to L. major infection in infected vflip mice. Splenocytes and cells from draining LNs from individualinfected vflip and WT mice (40 dpi) were cultured with medium only or stimulated with anti-cd3 or L. major antigen. Dashed (for L. major antigen) and continuous (anti-cd3) basal lines represent results with cells from control (B6) mice. (A, C, and D) After 48 h, culture supernatants were tested for IFN-, IL-4, and IL-13 by ELISA. (B and D) Splenic cells were stimulated further with ionomycin and PMA and processed for intracellular cytokine staining. Cells were stained and analyzed for CD4 and IFN- (B) or IL-4 (D) expression. Significant differences between infected WT (n 4 mice/group) and vflip mice (n 5 mice/group) are indicated as *P Results represent two independent experiments. jor antigens. Although possible negative effects of caspase-8 inhibition on TCR signaling, T cell proliferation, and IFN- production should be taken into account [31, 35, 42], these effects may have been overcome by rescuing of CD4 Th1 cells from Fas-induced apoptosis in infected vflip mice, as observed previously in FasL/Fas-defective mice [26, 28]. Similarly, inhibition of caspase-8 can favor Th2 responses in vflip mice by improving survival of CD4 T cells, as supported by Figure 5. A protective role of IL-4 in infected vflip mice. WT (o) and vflip ( ) mice were infected with L. major and treated i.p. at 17, 21, and 26 dpi with anti- IL-4 (n 6 WT mice, and n 5 vflip mice/group) or control IgG mab (n 4 WT mice, n 7 vflip mice/ group). (A) Lesions were measured in footpads, 2 weeks postinfection, at the time WT and vflip mice were grouped for antibody injection (upper) and at 32 dpi (lower) just before experiment. (B) Numbers of cells (upper) and parasite loads (lower) were accessed at draining LNs at 32 dpi. Parasite loads were normalized as log parasites/ml for statistical analyses. (C) CD4 T cells (upper) and antigenspecific IFN- -producing CD4 T cells (lower) were detected in the spleens. Basal lines represent uninfected WT mice (upper) or unstimulated cells from infected WT (solid line) or vflip (doted line) mice (lower). Significant differences between vflip mice treated with control and anti- IL-4 mab are indicated as *P Journal of Leukocyte Biology Volume 95, February

7 Pereira-Manfro et al. Th2-mediated immunity to L. major in vflip mice comparable effects of blocking FasL and caspase-8 activity in vitro. Nonetheless, other effects of caspase-8 inhibition on cell signaling and/or IFN- production might also promote Th2 responses [37, 43, 44] and deserve further investigation. We have reported previously that CD4 T cells from vflip or FasL-mutant gld mice also secrete increased amounts of IL-4 and IL-10 upon infection with Trypanosoma cruzi [35, 45]. Although alterations in cytokine expression correlated with increased susceptibility to T. cruzi infection [35, 45], resolution of L. major infection in vflip mice was not negatively affected by increased IL-4 production. Remarkably, reduced lesions and parasite loads were observed in infected vflip compared with WT mice. Likewise, transgenic mice, expressing a long form of cellular FLIP in T cells, showed an intact resistance to L. major infection [46]. Moreover, a biased Th2 response could be suggested by increased IgG1 production after reinfection, although increased Th2 cytokine responses were not evidenced [46]. The role that IL-4 plays in immunity to L. major infection varies during the course of infection and on distinct cell targets [47 51]. In BALB/c mice, the role of IL-4 also depends on the strains of L. major used or different protocols of infection [52 55]; i.e., IL-4-deficient mice resist better to infection with L. major IR173 but remain susceptible to the LV39 strain [54]. Constitutive expression of IL-4 turns otherwise resistant into susceptible mice [56, 57], whereas the outcome of IL-4 administration in susceptible BALB/c mice depends on timing of treatment [47 49]. Injection of IL-4 at the moment of T cell priming increases lesions and susceptibility to infection [48, 49], but treatment after lesions have already developed helps resistance and resolution of lesions [47, 48]. Supporting this idea, neutralization of IL-4 after 2 weeks of infection in Th2-prone vflip mice disrupted immunity to L. major parasites. Therefore, in our infection model, IL-4 might play a protective rather than a deleterious role in immunity to L. major. We also observed an improved Th1 response to L. major antigens in vflip mice, considering IFN- secretion and an imfi cell parameter that correlates with protective Th1 responses to L. major infection upon vaccination [41]. It has been suggested that IL-4 synergizes with IFN- to induce NO-dependent L. major killing by macrophages [58 60]. Likewise, CD11b hi IL- 4R myeloid cells from spleens of tumor-bearing mice produce higher levels of NO in response to a combination of IFN- and IL-13, which shares IL-4R signaling with IL-4 [61]. In line with this, we observed increased NO production by CD11b hi splenocytes from Th2-prone vflip mice during infection with L. major (data not shown). Th2 cytokines IL-4 and IL-13 also promote development of monocytes from myeloid precursors [62, 63]. Immature monocytes expressing the Gr- 1 hi CD11b hi phenotype were recognized recently as host/effector cells in immunity to L. major infection [64, 65]. Gr- 1 hi CD11b hi monocytes induced by L. major infection kill intracellular parasites by a NO-dependent mechanism stimulated by synergic effects of treatment with IL-4 and IFN- [65]. Importantly, there is enough evidence that a Th2 response is not protective to L. major in the absence of IFN- production in the B6 genetic background [66]. Therefore, cooperation between Th1 and Th2 responses might promote immunity to L. major infection. Here, we show that inhibition of caspase-8 by vflip improves CD4 T cell responses by up-regulation of Th1 and Th2 cytokine responses during L. major infection. Moreover, neutralization of IL-4 in infected vflip mice abrogates parasite control and reveals a protective effect of IL-4 in a Th1-prone genetic background. Therefore, a mixed Th2/Th1 cytokine environment upon blockade of caspase-8 activity might increase overall immunity to L. major infection. AUTHORSHIP W.F.P-M. and F.L.R-G. performed and designed experiments and analyzed data. A.A.F., L.V.C.G., N.S.V., and E.M.S. performed experiments. R.M.S. contributed with transgenic vflip mice. G.A.D. contributed to interpretation of data. M.F.L. designed the research, supervised the experiments, analyzed data, and wrote the manuscript. ACKNOWLEDGMENTS This investigation received financial support from United Nations Children s Fund (UNICEF)/United Nations Development Programme (UNDP)/World Bank/World Health Organization (WHO) Special Program for Research and Training in Tropical Diseases (TDR; grant A60281 to M.F.L.), Howard Hughes Medical Institute (grant to G.A.D.), Brazilian National Research Council (CNPq), Rio de Janeiro State Science Foundation (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro; FAPERJ), and National Institutes of Science and Technology-Instituto Nacional para Pesquisa Translacional em Saúde e Ambiente na Região Amazônica (INCT-INPeTAm)/CNPq/Ministério da Ciência e Tecnologia (MCT). W.F.P-M. received a Ph.D. fellowship from CNPq. M.F.L. and G.A.D. are research fellows at CNPq, Brazil. We thank Dr. David Sacks for supporting an experiment with vflip mice in his lab at U.S. National Institutes of Health and Dr. Françoise Meylan for genotyping mice. 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