EBI-3 Chain of IL-35 Along With TGF-β Synergistically Regulate Anti-leishmanial Immunity

Immunosuppression is a characteristic feature of chronic leishmaniasis. The dynamicity and the functional cross talks of host immune responses during Leishmania infection are still not clearly understood. Here we explored the functional aspects of accumulation of immune suppressive cellular and cytokine milieu during the progression of murine visceral leishmaniasis. In addition to IL-10 and TGF-β, investigation on the responses of different subunit chains of IL-12 family revealed a progressive elevation of EBI-3 and p35 chains of IL-35 with Leishmania donovani infection in BALB/c mice. The expansion of CD25 and FoxP3 positive T cells is associated with loss of IFN-γ and TNF-α response in advanced disease. Ex-vivo and in vivo neutralization of TGF-β and EBI-3 suggests a synergism in suppression of host anti-leishmanial immunity. The down-regulation of EBI-3 and TGF-β is crucial for re-activation of JAK-STAT pathway for induction as well as restoration of protective immunity against L. donovani infection.


INTRODUCTION
Maintenance of immunological self-tolerance and homeostasis by restraining disproportionate and detrimental immune responses is primarily mediated by regulatory cytokine secreting lymphocytes (1). Conversely, expansion of regulatory cellular and cytokine milieu may lead to compromised immunity against certain infections such as Brucella, HIV, helminthes, and Mycobacteruim tuberculosis including antitumor host immune responses (2)(3)(4)(5). However, the correlation between effector and regulatory cell populations especially in terms of sensing and secretion of cytokines during diseased condition is still not well understood (6).
Visceral leishmaniasis (VL) is a potentially lethal disease caused by parasitisation of cellular components of innate immune system by Leishmania donovani/Leishmania infantum (7). A dysfunctional cell mediated immune response is one of the characteristic features of chronic VL (8,9). Several studies have suggested the role of IL-10 and TGF-β in subversion of proinflammatory response in active VL (10,11). Despite crucial evidences of the role of these cytokines in augmenting VL pathology, the mode of action of these immunosuppressive cytokines is not clearly understood (12). Apart from IL-10 and TGF-β, the role of other immunosuppressive cytokines in VL is yet to be established. IL-35 has been reported for its immunosuppressive activity in autoimmunity and infectious diseases (13)(14)(15). IL-35 is a heterodimeric cytokine with two polypeptides "α" and "β" chains. These polypeptides may participate in the construction of two or more cytokines for example the α-chain, p35, is shared by IL-12 and IL-35, and the β-chain EBI-3 is shared by IL-27 and IL-35 (15). The roles of the subunit chains of IL-35 in immuno-regulation during VL are not known.
In the present study, we show the dynamicity of different subsets of CD4 + T cells at different stages of L. donovani infection of BALB/c mice and their contribution to different cytokine responses. In consistence to the previous reports of IL-10 and TGF-β, we additionally report the effect of the subunit chains of IL-35. We demonstrated the mechanistic action of TGF-β and EBI-3 in inhibition of CD4 + T cell-mediated Th1 response and its consequent effect on VL pathogenesis.

Determination of Hepatic and Splenic Parasite Burden
Spleens from infected hamsters were aseptically removed and the splenocytes were cultured in Schneiders' insect medium containing 10% FBS and 1% penicillin streptomycin to allow the infected amastigotes to transform into promastigotes at 22 • C for 2 weeks. The freshly transformed promastigotes were then cultured in M199 medium containing 10% FBS and 1% penicillin streptomycin and passaged in fresh medium maintaining a parasites density of 10 6 promastigotes/ml. The promastigotes of 2nd or 3rd passage were washed several times in 0.02 M phosphate-buffered saline (PBS) of pH 7.2 and observed in the microscope before injecting (2.5 × 10 7 cells/0.2 ml PBS) i.v., into BALB/c mice as described previously (16). At different mentioned time points of infection, spleen and liver were removed from the mice and multiple impression smears were prepared and stained with Giemsa. Organ parasite burdens expressed as Leishman-Donovan units (LDU) were calculated as the number of parasites per 1,000 nucleated cells × organ weight (mg). To determine whether the spleen and liver contained live parasites, the parasite burden was quantified in these tissues by Limiting Dilution Assay (LDA) as previously described (17)(18)(19). Briefly, a weighed small piece of spleen or liver from experimental mice was first homogenized in Schneider's medium supplemented with 10% FCS, and then diluted with the same medium to a final concentration of 1 mg/ml. Five-fold serial dilution of the homogenized tissue suspensions were plated in 96-well plates and incubated at 22 • C for 2-3 weeks. Wells were examined for viable and motile promastigotes after 15 days, and the reciprocal of the highest dilution positive for parasites was considered as the parasite concentration per milligram of tissue. The total organ parasite burden was calculated using the weight of the respective organs.

Cell Proliferation and Cytokine Assays
For cell proliferation and cytokine assays, single cell suspensions of infected and control mice splenocytes were prepared in RPMI 1640 supplemented with 10% FBS, l00 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate and 50 µM β-mercaptoethanol (complete medium). RBCs were removed by lysis with 0.14 M Tris-buffered NH 4 Cl. The remaining cells were washed twice with culture medium and viable mononuclear cells were counted in a haemocytometer by Trypan blue exclusion method. The cells were then cultured in a 96 well flat bottom plate at a density of 2 × 10 5 cells/well in a final volume of 200 µl complete medium and stimulated with Leishmanial-antigen (LAg) (10 µg/ml). The cells were incubated for 72 h at 37 • C in a humified chamber containing 5% CO 2 as described earlier with little modifications (20). For cytokine analysis, culture supernatants were collected after 72 h of incubation and the concentration of IL-12p40, TNF-α, IFN-γ, IL-12p35, IL-4, IL-10, TGF-β, EBI-3 (USCN Life Science), and IL-27p28 (eBioscience) were quantified by ELISA in accordance with the manufacturer's instructions. All the kits were purchased from Beckton Dickinson (BD Pharmingen), unless mentioned. Sera were collected from various groups of mice, as required and cytokine ELISA was performed according to manufacturers' instructions.
In vivo Neutralization of TGF-β and IL-35

Western Blot
Splenic cells were processed for cell lysate preparation, as described earlier (24). Firstly cells were lysed in cell lysis buffer (1X TBS containing 1% TritonX-100, 1 mM DTT, protease and phosphatase inhibitor cocktail), and the protein concentration in the cleared supernatants obtained after centrifuging at 1000 g were estimated using Lowry's method. The cell lysates were then resolved by 10-12% SDS-PAGE and transferred to nitrocellulose membrane (Millipore). The membranes were then blocked with 5% BSA in TBS for 1 h at room temperature and probed with primary Ab diluted in 5% BSA in TBS by incubating at room temperature for 2 h. After washing the membranes with TBST (Tween 0.5%) thrice for 5 min each, they were again probed with secondary antibody in TBST for 1 h. The membranes were then washed again three times each with TBST and finally detected by chemiluminescence.

Statistical Analysis
All data comparisons were tested for significance by One-way analysis of variance (ANOVA) and Tukey's multiple comparisons using Graph Pad Prism version 5.0 (GraphPad Software, v. 5.0, (or Student's T test were employed were ever indicated) San Diego, CA). Results with p < 0.05 were considered to be statistically significant.

Progression of L. donovani Infection and Associated Cytokine Responses in BALB/c Mice
Sets of BALB/c mice infected at different time points with L. donovani including uninfected controls were sacrificed to evaluate organomegaly, parasite burden and infection induced immunomodulations. Progressive increase in weight of liver and spleen was apparent during the course of infection ( Figure 1A). No apparent organomegaly were observed in age matched control groups. Infection induced hepato-splenomegaly was directly correlated to the parasite burdens in the respective organs as estimated by LDU as well as LDA methods (Figures 1B,C). A non-resolving infective pattern was observed until 4 months of infection (liver LDU = 2558 ± 374.9; log 10 values of LDA = 11.94 ± 0.36 and spleen LDU = 949.9 ± 123; log 10 values of LDA = 8.84 ± 0.68). Cytokine responses in infected mice were assayed from culture supernatants of leishmanial-antigen stimulated splenocyte cultures. Like natural infection, a gradual surge in the levels of IL-4, IL-10, and TGF-β were observed, while the levels of IFN-γ and TNF-α increased gradually at early infection but were reduced to basal level at chronic infection ( Figure 1D). Among various Chains of IL-12 family, the expressions of IL-12 (p35) and EBI-3 were progressively elevated with infection, whereas, the expression of IL-27 (p28) chain was found unaltered ( Figure 1E). Besides, IL-12 the expression of IL-12 (p40) chain was down-regulated at chronic infection.
The study further reveals that while production IFN-γ and TNFα are dominantly restricted to CD4 + FoxP3 − and CD4 + CD25 − T cells, the sources of IL-10, TGF-β, and EBI-3 are from both positive and negative populations of FoxP3 and CD25 (Figures 2B-E).

Increased Proportion of CD4 + CD25 + T Cells ex vivo Leads to Loss of Cell Proliferation and Induction of Regulatory Cytokines
To evaluate how CD4 + cells and their cytokine responses are associated with the disease progression, splenic CD4 + CD25 − and CD4 + CD25 + T cells of 4 months infected mice were enriched ( Figure 3A). Co-cultures of CD4 + CD25 − T cells with CD4 + CD25 + T in various proportions in presence of the anti-CD3 and anti-CD28 stimulation were assayed for CFSE labeled cell proliferation as well as cytokine responses in culture supernatants. A significant loss in the cell proliferative responses were observed with increasing proportions of CD4 + CD25 + T cells (Figure 3B). At equal concentration of CD4 + CD25 − and CD4 + CD25 + T cells (1:1 ratio), T cell proliferation was suppressed to ∼12%. Study of the cytokine response in the co-culture supernatants showed that IL-10, TGF-β and EBI-3 increased while IFN-γ and TNF-α levels decreased with the increasing CD4 + CD25 + T cells (Figures 3C,D). Moreover, IL-10, TGF-β, and EBI-3 secretion was found to be elevated with the increase in CD4 + CD25 + T cells in co-culture. However, there was unnoticeable deflection in the levels of IL-4 at various ratios of CD4 + CD25 − and CD4 + CD25 + T cells.

Neutralization of Regulatory Cytokines ex vivo Restores Cell Proliferation and Proinflammatory Cytokine Response
The co-cultures of CD4 + CD25 − and CD4 + CD25 + T cells were treated with neutralizing monoclonal antibodies (mAbs) against CD25, IL-27 (p28), IL-10, TGF-β, and EBI-3 either singly or in indicated combinations. Neutralization with αCD25 antibodies in the co-culture of CD4 + CD25 + and CD4 + CD25 − T cells from infected mice leads to restoration of cellular proliferation ( Figure 4A). Administration of αIL-10, αEBI-3 or αTGF-β resulted in partial gain in cell proliferation as compared to IgG controls. Moreover, there was no supplementary effect of adding various combinations of αIL-27 (p28), αIL-10, and αTGF-β. Interestingly, neutralization of EBI-3 either alone or in combination with TGF-β show proportionate restoration of cell proliferation in the stimulated co-cultures.
Effects of neutralization of CD25, IL-10, TGF-β, EBI-3, and IL-27 (p28) on disease promoting cytokine response were also examined. While homologous suppression following neutralization was observed for all the cytokines, cross regulation of cytokine response could be observed for TGF-β and EBI-3 neutralization on IL-10 production (Figures 4D-F). Production FIGURE 2 | Role of CD4 + T cell subsets in cytokine production during L. donovani infection. Infected and control mice splenocytes were stimulated with Leishmanial membrane antigens (LAg) (10 µg/ml) overnight and then cells were processed for flow cytometric studies. CD3 + T cells were gated from single cell population of splenic lymphocytes, followed by CD4 and CD8 cells (A). Subsequently, sub-sets of CD4 cells were gated based on Fluorescence Minus two controls for the identification of IFN-γ, TNF-α, IL-10, TGF-β and EBI-3 positive cells among CD4 + Foxp3 + (B) as well as CD4 + CD25 + (C) as representative dot plots. Data are expressed as means ± SE of percentage of cytokine producing CD4 + Foxp3 + / CD4 + Foxp3 − T cells (D), and similarly of CD4 + CD25 + / CD4 + CD25 − T cells (E), for six mice per group with three experimental repeats. Student's T-tests were applied to compare the differences in percentage proportion of cytokine producing cells as compared to uninfected controls. *P < 0.05; **P < 0.01; ***P < 0.001. FIGURE 3 | CD4 + CD25 + T cells suppressed protective immune response when co-cultured with CD4 + CD25 − T cells. Splenic CD4 + CD25 − T cells (labeled with CFSE) and CD4 + CD25 + T cells, were enriched through magnetic sorting from 4 months infected mice (A), were stimulated with anti-CD3 and anti-CD28 and co-cultured in 96 U bottom plate at various indicated ratios. After 96 h cells were collected for CFSE based CD4 + CD25 − proliferation assay through flow cytometry. Histograms and graph show percent proliferation of CD4 + CD25 − cells co-cultured with CD4 + CD25 + T at various ratios (B). Supernatants of the co-culture were used for cytokine profiling (C,D). Data expressed as means ± SE for at least five mice per group are representative of three independent experiments with similar results. **P < 0.01; ***P < 0.001 as assessed by one-way ANOVA and Tukey's multiple comparison tests. (Figures 4D-F).

Administration of αEBI-3 Along With αTGF-β in the Infected Mice Boosted Host Protective Response Concurrent With a Decline in the Disease Promoting Cytokines
For investigation of the involvement of EBI-3 and TGF-β with the disease progression, monoclonal αEBI-3 and αTGF-β antibodies were administered into the peritoneal cavity of the mice either individually or in combination. The first dose was administered 24 h prior Leishmania infection. Subsequent doses were given weekly until the mice were sacrificed. One group of infected mice was also administered with the isotype control (IgG) and another group was treated with αCD25 antibodies. After 3 months of infection, mice were sacrificed and LAg stimulated splenocytes were cultured for immunophenotyping and cytokine response. IFN-γ and TNF-α producing cells are found to be low in control (IgG) treated mice (∼1 and 0.5%, respectively), as compared to αTGF-β treated (∼2-3%) and αEBI-3 (∼4-5%) treated group in mice (Figures 5A,B). Administration of αEBI-3 in combination with αTGF-β increased the IFNγ and TNF-α producing cells significantly (∼7.5 and 6%, respectively), compared to controls. There was no prominent FIGURE 4 | Proliferative and cytokine responses of CD4 + CD25 − T cell, co-cultured with CD4 + CD25 + T, in the presence of neutralizing antibodies. Indicated neutralizing antibodies were added (20 µg/ml each) in the co-culture of splenic CD4 + CD25 − T cells (CFSE labeled) and CD4 + CD25 + T cells (1:1 ratio) obtained from 4 months infected mice (as described in Materials and Methods). After 96 h cells were processed for CFSE based cell proliferation assay (A) and the culture supernatants were collected for estimation of cytokines, IFN-γ (B) TNF-α (C) IL-10 (D), TGF-β (E) and EBI-3 (F). Data represented as means ± SE for five mice per group are representative of three independent experiments with similar results. *P < 0.05; **P < 0.01; ***P < 0.001 between the neutralized and IgG control groups were assessed by two tailed Student's T-test. difference in IL-4 producing CD4 cells post αCD25 or αTGF-β treatments ( Figure 5C).
We estimated various cytokines from the culture supernatants of splenocytes of αEBI-3, αTGF-β, and αEBI-3 mAbs treated mice through Cytometic Bead Assay (CBA). We found that infected mice treated with IgG produced low amounts of IL-12 (p40), IFN-γ, and TNF-α, while the mice treated with αEBI-3 or αTGF-β showed a significant increase in these cytokines. Administration of αEBI-3 alone or in combination with αTGF-β resulted in significant production of these proinflammatory cytokines (Figures 6A-C). Moreover, there was significant suppression of IL-4 and IL-10 responses along with TGF-beta and EBI-3 (Figures 6D-G). However, introduction of αEBI-3 along with αTGF-β not only resulted in the maximum production of these immuno-protective cytokines but also led to inhibition of disease promoting IL-4 and IL-10. No significant alteration in IL-27 (p28) levels in the culture supernatants of various indicated groups of mice ( Figure 6G) were found. The effect of combined neutralization of TGFβ and EBI-3 on signaling cascade was also investigated. Previous reports showed that Leishmania infection is associated with enhanced expression of Arginase 1 and diminished production of nitric oxide synthase thereby low generation of the key anti-leishmanial molecule nitric oxide (25). The expression of arginase 1 from the splenic cell lysates derived from mice treated with the neutralizing antibodies for EBI-3, TGF-β, and CD25, was found to be down regulated as compared to control IgG-treated ( Figure S1A). Mice treated with αCD25 only also showed a substantial reduction in arginase 1 expression compared to naive but the mice treated with αTGF-β+ α EBI-3 antibodies showed the best results with lowest expression of arginase 1. The antagonistic effect of arginase 1 to inducible nitric oxide synthase (iNOS) was found with significant loss of iNOS expression in the αTGF-β and αEBI-3 treated mice ( Figure S1A). To investigate whether the reduction of arginase 1 and concurrent up regulation of iNOS is dependent on the STAT6 as reported by Osorio et al. during L. donovani infection (26) in vitro blocking of EBI-3 in the culture of splenocytes of infected mice was assayed. Neutralization of EBI-3 of infected splenocytes resulted in down regulation in the expression of both STAT6 as well as arginase 1 (Figure S1B).

Neutralization of EBI-3 Along With TGF-β Limits Progression of L. donovani Infection in BALB/c Mice
Since EBI-3 and TGF-β suppressed the host protective anti-leishmanial response, the effects of EBI-3 and TGF-β neutralization on disease progression in BALB/c mice were studied. A significant fall in the parasite burden of spleen and liver in the all treatment groups was observed when compared to control (Figures 7B,C,E,F). However, the most effective protection was observed in mice treated with a combination of FIGURE 5 | Administration of neutralizing antibodies promotes proinflammatory cytokine producing CD4 + T cells. Infected mice treated with various neutralizing mAbs were sacrificed and their splenocytes were stimulated with LAg (10 µg/ml) and cultured. After 24 h, cells were processed for FACS studies as described in Materials and Methods. Percent population CD4 + T cells producing IFN-γ (A), TNF-α (B), and IL-4 (C) were studied post acquisition of the data. Data expressed as means ± SE for five mice per group are representative of two independent experiments with similar results. **P < 0.01; ***P < 0.001 as assessed by one-way ANOVA and Tukey's multiple comparison tests.
αTGF-β + α EBI-3 mAbs. The reduction in parasite burden was concomitant to the resistance to hepato-and splenomegaly in the treatment groups (Figures 7A,D).

DISCUSSION
An impaired cellular immunity is a distinctive feature of active visceral leishmaniasis. Despite advances, the functional role of regulatory cells and their cytokines in suppression of antileishmanial immunity is not well elucidated. In this present study we have investigated the dynamicity of cellular and cytokine responses during experimental visceral leishmaniasis, and to determine the functional effects of the infection induced cytokines on progression of L. donovani infection in BALB/c mice.
Chronic infection of L. donovani in the mammalian phagocytes results in adenopathy of liver and spleen with loss of cell mediated immune responsiveness (19,27,28). A clear understanding of the immunology of VL primarily suffers due to limitations of appropriate disease models for immunological studies. Previous studies on infection kinetics of L. donovani AG83 in BALB/c mice indicate that similar to natural infection the disease is chronically progressive peaking up to 16 weeks post challenge (29,30). Therefore, the same infection model was selected for the present study. The kinetics of parasite burden post challenge indicates a steep increase in liver infection at 2 months compared to splenic infection. Possibly, a complex cellular organization of spleen (12) contributes to initial resistance as observed during several disease models of acute VL infection (31). The progression of infection is suggestive of an acute pre-established infection at 1 month, established infection following 2 months and a chronic phase post 3 months of challenge with virulent L. donovani in BALB/c mice. Therefore, for studies related to comparisons of immunomodulations at pre-established (1 month) and extensively chronic (4 months) of infection time points were evaluated. However, in vivo neutralization studies were performed at 3 months of optimum chronic phase of infection. It is noteworthy that at early infection there was an enhanced induction of pro-inflammatory cytokine response of TNF-α and IFN-γ, which gradually reduced to basal levels with the surge of regulatory cytokines, IL-4, IL-10, and TGF-β in later infection. Studies on different subunit chains of IL-12 family revealed that p35 chain of IL-12 and EBI-3 were gradually up regulated, while p28 of IL-27 and p40 of IL-12 remained restricted during the course of infection. IL-12 (p35) and EBI-3 constitute IL-35, a cytokine known for its immunosuppressive role in cancer and other chronic infectious diseases, but is crucial for deterrence of autoimmune complexities (32)(33)(34). Previous reports implicate the involvement of IL-10 and TGF-β in restriction of the protective cytokine response in murine VL (27,35,36). The roles of IL-35 in VL pathology alone or in combination with other regulatory Immuno-phenotypic characterization of splenic T cells demonstrated a significant surge in proportions of CD4 + CD25 + and CD4 + FoxP3 + T accompanied with loss of cellular proliferative and proinflammatory cytokine response as chronic infection was established. Interestingly, at early infection a significant proportion of IFN-γ and TNF-α producing cells predominantly from CD4 + CD25 − and CD4 + FoxP3 − T cell were observed. However, with progression of infection the level of the proinflammatory cytokine producing cell populations were significantly reduced. The observations were in consistence to previous reports of anti-leishmanial response at acute phase of infection gradually progressing to T cell anergic state in chronic VL (37,38). The drop in IFN-γ and TNF-α producing cells at chronic infection were accompanied with surge in the IL-10, TGF-β and EBI-3 producing CD4 + T cells. The functional association of cytokine response was analyzed by a combination of neutralization of IL-27 (p28), IL-10, TGF-β, and EBI-3 in vitro in the co-cultures of enriched CD4 + CD25 + on CD4 + CD25 − cells. Restoration of the effector functions and proliferation of co-cultured T cells following neutralization of IL-10, TGF-β and EBI-3 revealed the immuno-suppressive effect of the cytokines. IL-10 and TGF-β producing CD4 + T cell have been previously reported to dampen host inflammatory responses and to be involved in the pathogenesis of L. donovani infection (11,12,39,40). In consistence with previous reports of induction of Th1 type immune response following IL-10 neutralization (41,42), similar immunomodulatory effects were found in the ex vivo experiments. Our study extended the findings with EBI-3. Moreover, combined neutralization of TGF-β and EBI-3 resulted in the most effective restoration of proliferation and IFN-γ and TNF-α response. Unaltered immuno-repressed milieu post IL-27 (p28) blocking, either alone or in combination, indicated that IL-27 (p28) may not have a role in suppression of anti-leishmanial immunity. Therefore, it appears that EBI-3 dominantly from IL-35 plays a crucial role in immune suppression.
Earlier studies reported that IL-35, similar to IL-10, to be crucial for suppressing inflammatory responses during auto-immunity and cancer (13,43,44). Therapy with IL-35 during collagen-induced arthritis helped in the expansion of Tregs (45). During viral infection, targeting EBI-3 led to cure (14). The suppressive activities of IL-10 (46, 47) and TGF-β (48)(49)(50) and their contribution in murine VL are well established (51). Subsequently, Murray and colleagues proposed targeting IL-10 by neutralizing antibodies as a promising immunotherapy against murine VL (22,46). To ascertain whether blocking the effect of TGF-β and EBI-3 in vivo can similarly resist Leishmania infection induced immune suppression, immunomodulatory effect and parasite burden of infected BALB/c mice treated with combinations of α-TGF-β and α-EBI-3 were evaluated. The in vivo neutralization led to significant increase in IL-12 (p40) and IFN-γ production post EBI-3 blocking along with a reversal in immune suppressed milieu by simultaneous neutralization of TGF-β and EBI-3 in infected mice. Blocking of these cytokines not only enhanced the pro-inflammatory cytokines but also increased the IFN-γ and TNF-α producing CD4 + T cells. Moreover, whereas, single neutralization of TGF-β or EBI-3 helped in partial resistance to the infection, their combined neutralization led to ∼5-fold reduction in parasite load in both liver and spleen.
The resistance to parasite burden was also associated with a down regulation of arginase 1 expression, along with enhanced nitric oxide production. Suppression of arginase 1, an inhibitor of nitric oxide production (24) may result in NO mediated parasite killing. Moreover, this reduction of arginase 1 level was dependent on the expression of STAT6, which indicates that EBI-3 promotes L. donovani mediated arginase 1 expression in STAT6 dependent manner to down regulate NO production. Our studies, therefore, propose that EBI-3 is crucial for immunosuppression in VL. Moreover, EBI-3 together with TGF-β is the major contributor to the immunologically subverted milieu conducive for parasite survival and immune evasion.