All animal experiments were duly approved by the Institutional Animal Ethics Committee of the National Institute of Immunology, New Delhi (IAEC/AQ/2015/134, serial no. IAEC#372/15). Use of human peripheral blood monocytes (PBMCs) was approved by the Institutional Human Ethics Committee of the National Institute of Immunology, New Delhi (Project no. IHEC #84/14). The work involving human subjects was also approved by the ethical committee of the Institute of Medical Science, Banaras Hindu University, Varanasi, India, and the informed consents were obtained from the patients or their guardians.

Subjects with symptoms of VL were recruited to Kala-Azar Medical Research Centre, Muzaffarpur, Bihar, India, where they were confirmed to be positive for VL by detection of amastigotes in the splenic aspirates and/or by rK39 positive test. All patients included in the study were HIV negative and over 6 years of age. Splenic aspiration is the gold standard method for diagnosis of VL; splenic aspirates were collected before the start of antileishmanial treatment and after cure for examining parasitological status of subjects. Subjects with low hemoglobin, platelet count less than 40,000/μl and prothrombin time <5 s were excluded from the study. Venous blood from VL patients and endemic controls (EC) was collected in heparin. All EC were healthy household members of the patients.

For in vivo experiments, inbred BALB/c mice were used. For in vitro experiments, mouse peritoneal macrophages (mPMs) were obtained from inbred mice of BALB/c strain, wild-type, and TLR-2 knockout mice of C57/BL6 background of either sex at 6–10 weeks of age.

THP-1 cells, an acute monocytic leukemia-derived human cell line (ATCC TIB-202TM) were maintained in RPMI-1640 (Sigma-Aldrich, Cat. No. R8005) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Cat. No. 10082147) and penicillin (100 U/ml), streptomycin (100 U/ml), and gentamycin (20 μg/ml) at 37°C in humidified air containing 5% CO₂. For differentiation into macrophages, they were incubated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Cat. No. P1585) as described previously. Human PBMCs were isolated using a Ficoll-Paque density gradient (Sigma-Aldrich, Cat. No. GE-17-5442-02) from whole blood. Briefly, 2 × 10⁶ monocytes were incubated in the presence of recombinant human M-CSF (300 ng/ml) (Shenandoah Biotechnology Inc., Cat. No. 0114-100-03AF). Non-adherent cells were periodically removed followed by addition of fresh media containing recombinant human M-CSF (300 ng/ml) and 10% human serum every 12 h. Cells were allowed to differentiate into human monocyte derived macrophages (hMDMs) for 96 h.

Mouse peritoneal macrophage monolayers were prepared as described previously (17). Briefly, peritoneal exudate cells were harvested from BALB/c or C57/BL6 mice using chilled serum-free RPMI 1640 medium, incubated for 2 h, non-adherent cells were removed and the adherent cells were further incubated in complete RPMI 1640 medium to form macrophage monolayer.

The parasite, L. donovani (BHU1260), was derived from the primary culture obtained directly from the splenic aspirate of a human VL patient and was maintained in M-199 medium (Sigma-Aldrich, Cat. No. M5017) supplemented with 10% heat-inactivated FBS at 23°C as described previously. The primary cultures were used only up to fourth passage and exhibited good infectivity in vitro.

For siRNA transfection, THP-1 MDMs, or hMDMs were transfected with gene-specific siRNAs (Bcl-2, Mcl-1, TLR-2, IL-13R) or scrambled negative control siRNA at a final concentration of 25 nM. Transfection was performed using N-TER™ Nanoparticle siRNA Transfection System as per manufacturer’s instructions (Sigma Aldrich, Cat. No. N2913). Plasmid containing Bcl-2 clone was transfected using Lipofectamine LTX reagent with PLUS reagent (Thermo Fisher Scientific, Cat. No. 15338100). During the entire course of study, the Bcl-2 selective small molecule inhibitor ABT-199 (10 nM) and in selective experiments, another Bcl-2 inhibitor ABT-263 (10 nM) was used to inhibit Bcl-2 activity. For inhibition of specific cell signaling pathways, pharmacological small molecule inhibitors of various signaling molecules were added 30 min prior to the start of the experiments. The inhibitors used were: 1, 4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene monoethanolate (U0126) (2 μM) (Sigma Aldrich, Cat. No. U120) and 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) (10 μM) (Sigma Aldrich, Cat. No. P215) for MEK1/2; 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) (30 μM) (Sigma Aldrich, Cat. No. L9908) and wortmannin (100 nM) (Sigma Aldrich, Cat. No. W1628) for phosphatidylinositol 3-kinase (PI3-K); and Tyrphostin (AG490) (10 μM) (Calbiochem, Cat. No. 658401) for STAT-3.

Late stationary phase promastigotes were used to challenge macrophages at multiplicity of infection (MOI) of 10 parasites per macrophage. After 2 h of incubation, the unbound parasites were removed by gently shaking and washing the wells with incomplete RPMI-1640 medium three times and the infected macrophage monolayers were incubated in the complete medium for 12 or 24 h. To estimate the intracellular parasitic burden, cells were fixed in 4% paraformaldehyde (PFA) followed by staining with the DNA fluorescent probe DRAQ5 (5 μM) (Thermo Fisher, Cat. No. 62251) for 10 min. After washing three times with sterile PBS to remove the excess stain, cells were mounted in Vectashield anti-fade mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA) and were used to acquire images using Leica TCS SP5 II (Leica Microsystems, Wetzlar, Germany) confocal microscope at 633 nm. Infecting the cells at the mentioned MOI resulted in a situation where 98–99% cells were infected with at least one parasite. At least 200 cells observed from a minimum of 15 randomly selected fields were counted manually for each condition to determine the average number of parasites per macrophage. Similarly, for estimating parasite uptake, a 2-h infection period was used after which cells were processed, stained with DRAQ5, and average parasite counts per macrophage were made. The parasite counting was performed by a person in the laboratory blinded for the experimental conditions, other than the person performing the experiment.

Cells fixed with 2% PFA were permeabilized with 0.1% Triton X-100 and incubated overnight with primary antibody against Bcl-2 (1:200) (Millipore, Cat. No. 05-729) or TLR-2 (1:250) (Novus Biologicals, Cat. No. NBP1-77843) at 4°C. Secondary antibody conjugated to Alexa 488 (1:400) (Invitrogen, Cat. No. A11001) was used to detect staining at excitation/emission wavelength: 490/525 nm. DNA staining was performed using DAPI (10 μg/ml) (excitation/emission wavelength: 647/681 nm) (Thermo Fisher, Cat. No. 62251). Images were captured using Leica TCS SP5 II (Leica Microsystems, Wetzlar, Germany) confocal microscope and analyzed using an integrated Leica LAS AF software program.

Cells were lysed in cell lysis buffer as described earlier (18). Cell lysates (30–80 μg) electrophoresed on 12% SDS polyacrylamide gels were transferred onto nitrocellulose membranes. Membrane blocking was performed in 5% fat free milk (Santa Cruz Biotechnology, Cat. No. sc-2325) followed by incubation with any of the primary antibodies like, Bcl-2 (1:1000) (BD Biosciences, Cat. No. 51-6511GR), Bcl-xL (1:1000) (Cell Signaling Technologies, Cat. No. 54H6), Mcl-1 (1:1000) (BD Biosciences, Cat. No. 559027), Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-492), p-Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-271963), TLR-2 (1:1000) (Novus Biologicals, Cat. No. NBP1-77843), pERK-1/2 (1:1000) (Cell Signaling Technologies, Cat. No. 9101), p-STAT-3 (1:1000) (Cell Signaling Technologies, Cat. No. D3A7), p-PI3K (1:1000) (Cell Signaling Technologies, Cat. No. 4428), p-Akt (1:1000) (Cell Signaling Technologies, Cat. No. 9271), iNOS (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-8310), and β-tubulin (1:4000) (Thermo, Cat. No. RB-9249-P1). Subsequently, post wash, blots were incubated with corresponding secondary antibodies conjugated to horseradish peroxidase. Protein bands were observed on X-ray film using femtoLUCENT™ PLUS-HRP reagent (G-biosciences, Cat. No. 786-003). To monitor for equal loading of proteins in each well, Western blotting using antibody against β-tubulin was performed for each experiment.

The concentration of nitrite, the stable end product of NO, in culture supernatants or serum was determined by Griess’ reaction as described by Biswas et al. with slight modifications (19). Briefly, 10⁶ cells were plated in the wells of a 12-well culture plate, and after the completion of the experiments the cell-free supernatant from individual wells were collected and stored until use. About 100 μl of the sera or culture supernatants were incubated with equal volume of Griess’ reagent for 20 min at room temp. Spectrophotometric readings were taken at 540 nm in a microplate reader (μQuant, BioTek Instruments Inc., VT, USA). Nitrite content was quantified by extrapolation from a sodium nitrite standard curve for each experiment.

IL-13-specific enzyme-linked immunosorbent assay (ELISA) was performed to detect level of secreted IL-13 in the cell-free supernatant obtained from different in vitro/ex vivo experiments or serum of mice using human/mouse-specific IL-13 ELISA kits (e-Biosciences, Cat. Nos. 88-7439-22, human; 88-7137-22, mouse) as per manufacturer’s instructions.

Inbred BALB/c mice were grouped as untreated non-infected control, infected, infected vehicle control, and infected drug treated. Mice in last three groups were inoculated with Leishmania promastigotes through intra-cardiac injection with 50 μl bolus containing 1 × 10⁷ parasites on day 0 of the study. Drug (ABT-199, ABT-263 both at 15 mg/kg body weight) or vehicle (DMSO) was injected through tail vein. In the drug or vehicle control groups, injections were given for first 3 weeks, once every week. Mice were humanely euthanized after 5 weeks and spleens were dissected out, weighed, and impression smears were made. Parasite burden was estimated by counting splenic smears prepared after euthanasia. Blood was collected from mice and plasma was extracted for detection of nitrite and IL-13 levels in circulation.

Standard one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test was performed to derive the statistical significance between the test groups while comparing densitometric data of Western blots. For comparing nitrite concentration, parasitic burden, and mice data between different test groups, where information about a Gaussian distribution could not be conclusively derived, Mann–Whitney test was performed. Wilcoxon signed-rank test was applied to analyze the data from the paired samples. The statistical analysis was performed using GraphPad Prism, version 5.01 (GraphPad, San Diego, CA, USA). P-value of less than 0.05 was considered significant. The error bars of the values represent ± SEM from the replicates.

For investigating the changes in the expression of Bcl-2 family proteins after infection with L. donovani parasite, THP-1 monocyte-derived macrophages (THP-1 MDM) were challenged at MOI of 1 macrophage:10 parasites (Figure S1A in Supplementary Material) in vitro, and the levels of Bcl-2 family proteins were measured. A time-dependent increase in Bcl-2 was observed post infection (Figure 1A). The increase in Bcl-2 dominated from 2–12 h following which there was a gradual decline. Mcl-1 protein expression also increased following infection and the elevated levels were sustained. Bcl-xL levels did not change significantly during the course of infection (Figure 1A). Likewise, when primary human macrophages derived from blood monocytes, i.e., hMDMs were infected with the parasites (Figure S1A in Supplementary Material), similar changes were observed. Both Bcl-2 and Mcl-1 levels increased with almost identical pattern and kinetics as that of THP-1 MDMs (Figure 1A). An investigation into intracellular Bcl-2 immunostaining demonstrated predominantly perinuclear localization of the protein in THP-1 MDM (Figure 1B).

Concomitant increase in the levels of the antiapoptotic proteins Bcl-2 and Mcl-1 and decrease in the level of the pro-apoptotic protein Bad implied enhanced survival of the parasite-infected macrophages. Infected cells by default experience higher levels of cellular stress and therefore are at much higher risks of undergoing apoptosis. Increasing expression of antiapoptotic proteins or reducing expression of pro-apoptotic proteins of the host cells are among well-documented pro-pathogenic strategies and can be critically effective during establishment of infection (1). To explore if increased Bcl-2 and Mcl-1 served any pro-parasitic intent by imparting higher survival advantage to the infected cells, macrophages were knocked down for Bcl-2 or Mcl-1 (Figure 1C), and cell viability was checked at different time points post infection. Macrophages knocked down for either Bcl-2 or Mcl-1 showed no significant impact on viability upto 48 h of infection (Figure S1B in Supplementary Material). However, Bcl-2 knocked down macrophages showed increased cell death as compared with control cells at 72 h post-infection. Similar to Bcl-2 downregulated cells, Mcl-1 knocked down macrophages showed reduced viability at 72 h, however to a lesser extent (Figure S1B in Supplementary Material). To look for any potential impact on parasite internalization introduced due to knocking down of Bcl-2/Mcl-1, macrophages in different groups were infected for 2 h and average parasite numbers were counted. No significant differences in the average number of internalized parasites were found among different groups, negating any possible effect of Bcl-2/Mcl-1 knock down on parasite uptake (Figure 1D). To look for possible role of Bcl-2 and Mcl-1 in the sustenance of the parasite inside macrophages, parasite burden was estimated under Bcl-2/Mcl-1 knock-down conditions at 12 h of infection. Cells treated with Bcl-2 siRNA showed a pronounced reduction in the number of parasites/cell as compared to the scrambled siRNA control. However, unlike Bcl-2 knocked down cells, Mcl-1 knocked down macrophages did not show any significant reduction in parasite number (Figure 1D). To further look if this effect was specific to Bcl-2, Bcl-2 overexpression (Figure 1C) was carried out in Bcl-2 lowered cells before they were challenged with parasites. Bcl-2 overexpressing macrophages showed parasite burden comparable to control cells (Figure 1D). These observations suggested that elevated presence of Bcl-2 but not Mcl-1 favored parasite survival. The siRNA-mediated downregulation of Bcl-2 in hMDM also resulted in lesser infection rates as compared to scrambled siRNA-treated cells (Figure 1D). Consequently, to reaffirm the role of Bcl-2 and further establish its functional relevance, we used Bcl-2 inhibitors ABT-199 and ABT-263 to block Bcl-2 function through binding site interference. Parasite challenge during ABT-199 or ABT-263 mediated inhibition of Bcl-2 in both THP-1 MDM and hMDM showed lower infection as compared to vehicle-treated controls (Figure 1D, ABT-199, ABT-263). Having obtained positive correlation between Bcl-2 expression and parasite burden in human macrophages, we considered it relevant to check this hypothesis in an animal model. Before moving onto a mouse model, which is one of the two established animal models for VL, it was important to look at the status of Bcl-2 family proteins in mouse macrophages. Infection experiments with mPMs reflected similar alterations in the profile of Bcl-2 family proteins in vitro, albeit, with a slightly different time kinetics. Here, the Bcl-2 increase peaked at 8 h followed by a decline and Mcl-1 showed an increase from 8–16 h with Bcl-xL showing no noteworthy change (Figure 1E). Similar to human macrophages, mPMs with Bcl-2 knock down or in the presence of Bcl-2 functional inhibitors did not show reduced parasite uptake, but demonstrated lower parasite burden (Figure 1E). Among the pro-apoptotic members of the Bcl-2 family, only Bad protein showed significantly altered expression. There was a gradual decline in Bad levels pertaining to its rapid phosphorylation post infection (Figure 1F). Taken together, the above data clearly provided evidence for a supportive role of Bcl-2 to parasite survival within the host cells.

Leishmania parasites are sensitive to macrophage generated NO (13). Prior reports in other systems attribute NO regulation to Bcl-2 (16). With this background, we hypothesized an inhibitory role of excess Bcl-2 on NO production, as Bcl-2 knock down or its functional inhibition resulted in lower parasite burden (Figures 1D,E). Treatment with ABT-199 or the vehicle used for this inhibitor, DMSO did not have any effects on iNOS expression (Figure 2A). Infection of THP-1 MDMs resulted in a very low level induction of iNOS enzyme, which was also reflected in nitrite levels (Figures 2A,B). To look for a role of Bcl-2 protein in NO production during L. donovani infection, either Bcl-2 expression was downregulated using gene-specific siRNAs or Bcl-2 protein function was inhibited using ABT-199/ABT-263. A siRNA-mediated downregulation of Bcl-2, but not Mcl-1, resulted into a significant increase in iNOS levels (Figure 2C). Similarly, functional inhibition of Bcl-2 by ABT-199 or ABT-263 led to a significant induction of iNOS post infection (Figure 2D). Such interference with Bcl-2 expression or function also resulted in significantly higher nitrite production (Figures 2C,D), indicating higher iNOS activity. These data clearly demonstrated a negative effect of Bcl-2 on the NO generating machinery. Since higher iNOS level generally corresponds to increased NO production, to further confirm if increased iNOS expression, and consequently higher NO production was responsible for parasite killing in the presence of Bcl-2 inhibitors, cells were treated with an iNOS inhibitor, LNMA. Both NO production as well as parasite killing was abrogated upon iNOS inhibition (Figures 2E–G). Bcl-2 inhibition through ABT-199 or ABT-263 increased NO levels, but in the presence of LNMA this increase was not observed (Figure 2E). When parasite burden was estimated, a lower parasite burden was observed with both ABT-263 and ABT-199 treatment but this protection was significantly lost in the presence of LNMA (Figures 2F,G). Similar results were obtained with hMDMs (Figure 2H); the primary human macrophages demonstrated increased iNOS expression and nitrite levels on downregulation of Bcl-2 but not when Mcl-1 was lowered (Figure 2H).

To see if the parasite clearance seen upon Bcl-2 manipulation corresponded with NO levels in mPMs as observed in case of human macrophages, NO levels were quantified both during Bcl-2 downregulation and in the presence of Bcl-2 inhibitors (Figure 2I). Macrophages knocked down for Bcl-2 or treated with Bcl-2 inhibitors produced significantly more NO as compared to control cells. Both, NO production as well as increased parasite clearance achieved was abrogated upon iNOS inhibition (Figure 2I).

Collectively, the data from both human and mouse macrophages showed that parasite influenced NO production by modulating Bcl-2 expression. Functional inhibition of Bcl-2 protein augmented parasite killing owing to increased NO production.

Prior data suggest that Leishmania binds to macrophage surface through TLR-2 mediated by parasite surface lipophosphoglycans (20). TLR-2 is a pattern recognition receptor whose expression is reported to change during infection (21). A time-dependent upregulation of TLR-2 in THP-1 MDM post infection was observed (Figure 3A). This was confirmed by immunocytochemical staining for TLR-2 showing elevated immunoreactivity (Figure 3A). Based on prior knowledge of Bcl-2 regulation by TLR-2 in certain cell types, we explored the status of Bcl-2 under conditions of downregulated TLR-2. Significantly lesser induction of Bcl-2 was observed when TLR-2 was downregulated by siRNA transfection prior to infection (Figure 3A). As Leishmania parasite physically interacts with TLR-2 on the macrophage surface, downregulating TLR-2 expression may affect parasite internalization. To check if parasite uptake was affected in TLR-2 downregulated cells, unbound parasites were washed after 2 h of infection and a count of internalized parasites showed a minor reduction in the parasite uptake in TLR-2 siRNA-treated cells (Figure 3A). Despite being statistically insignificant, this minor reduction in the parasite uptake was consistently observed in multiple experiments. Similar to THP-1 MDM, hMDM showed an increase in TLR-2 expression post infection (Figure 3B), and TLR-2 downregulation prior to infection (Figure 3B) prevented the increase in Bcl-2 levels (Figure 3B). Unlike THP-1 MDM, statistically significant reduction in the parasite uptake in TLR-2 downregulated group was observed (P = 0.0161) (Figure 3B). The above observations were verified in cells from TLR-2 knockout mice. Wild-type mPM showed a gradual increase in TLR-2 expression post infection (Figure 3C) with an increase in Bcl-2 levels (Figure 3C). On the contrary, the peritoneal macrophages isolated from TLR-2 knockout mice of the same strain showed no significant increase in Bcl-2 levels post infection (Figure 3C). The mPMs derived from TLR-2 knockout mice also showed significantly reduced parasite uptake verifying that Leishmania parasites engaged TLR-2 for entry into cells (Figure 3C). Taken together, the data indicate involvement of TLR-2 in the regulation of Bcl-2 expression and also suggest a possible role of TLR-2 in parasite internalization.

Searching for downstream effectors of TLR-2, changes in pPI3K, pAkt, and pERK1/2 were determined. Parasite challenge caused a time-dependent increase in phosphorylation of PI3K, Akt, and ERK1/2 (Figure 4A). Therefore, to determine the relationship between pPI3K, pAkt, pERK1/2, and Bcl-2 induction during infection, specific pharmacological inhibitors were used to block the kinase activities of the proteins leading to phosphorylation of PI3K, Akt, and ERK1/2. Inhibition of ERK 1/2 phosphorylation with U0126 resulted in an observable reduction in the infection-induced Bcl-2 increase (Figure 4B). Both PD98059 and U0126, MAPK/ERK kinase inhibitors significantly prevented Bcl-2 induction (Figure 4B) suggesting regulation of Bcl-2 through the MEK/ERK pathway. The inability of wortmannin and LY294002, the PI3K inhibitors to prevent the increase in Bcl-2 post infection (Figure 4B) ruled out the involvement of the PI3K pathway in Bcl-2 changes. NO levels were found to be significantly increased in the presence of ERK inhibitors U0126 and PD98059 during parasite invasion (Figure 4C) suggesting higher iNOS activity, whereas, in the presence of wortmannin and LY294002, nitrite levels remained at par with only parasite-infected cells (Figure 4C). In consonance with the above data, presence of both U0126 and PD98059 showed reduced infection (Figure 4D) suggesting higher NO levels to be effective against the parasite, whereas, the presence of wortmannin did not interfere with parasite burden (Figure 4C). The hMDMs also produced significantly higher NO in the presence of ERK inhibitors (Figure 4E) and showed significantly reduced parasite burden (Figure 4F). In summary, this part of the data showed participation of MEK/ERK signaling pathway in L. donovani-induced increases in Bcl-2 levels, lowering of NO production and higher infection rates. Clearly, MEK/ERK acted as the downstream effectors of TLR-2 after infection with the L. donovani parasite leading to Bcl-2 increase favoring parasite survival.

Modulation of cytokines is an important event during infection. From our in vitro studies, it was observed that challenge with L. donovani parasite caused a time-dependent increase in IL-13 levels (Figure 5A). A report involving patient data has shown increased level of IL-13 in the VL sera (22) that has been also confirmed in our study with human VL subjects (unpublished data). Therefore, our in vitro observations were similar to the previously reported patient data. IL-13 is responsible for inducing a host of different functions on different kind of cell types including macrophages (23). As it is known that Bcl-2 levels are regulated by IL-13 via STAT-3 in certain cell types (24), we sought to explore if Bcl-2 was responding to IL-13 signals. THP-1 MDMs were exposed to recombinant IL-13 (rIL-13) and a time-dependent increase in Bcl-2 levels and increase in the phosphorylation of STAT-3 was recorded (Figure 5B). STAT-3 phosphorylation is a downstream event of IL-13 exposure, as is known in other cell types of the immune lineage. To further establish the role of IL-13 in Bcl-2 induction, IL-13 signaling was perturbed at different levels: at the levels of IL-13 receptor (IL-13R) and JAK-2, which is the upstream kinase responsible for phosphorylating STAT-3. THP-1 MDMs with IL-13R downregulation using siRNAs to IL-13R or pretreated with AG490, an inhibitor of JAK-2 displayed reduced phosphorylation of STAT-3 (Figure 5C) upon treatment with rIL-13. These conditions also prevented Bcl-2 increases during exposure to rIL-13 (Figure 5C). Importantly, infection-induced STAT-3 phosphorylation and Bcl-2 increase declined when cells were challenged with parasites in IL-13R downregulated conditions or in the presence of AG490 (Figure 5D). Reduced phosphorylation of STAT-3 was also observed in the presence of PD98059, an ERK inhibitor (Figure 5D), indicating an involvement of pERK 1/2 in STAT-3 phosphorylation in addition to IL-13-JAK-2 axis during infection. This finding also suggested convergence of TLR-2-ERK and IL-13-JAK-2 axes at the level of STAT-3. In all, the data suggested that infection-induced induction of Bcl-2 was predominantly mediated through STAT-3.

Infection of hMDMs with L. donovani parasites generated a similar response like that of THP-1 MDMs by inducing a time-dependent increase in IL-13 (Figure 5E). The rIL-13 administration to naive hMDMs induced a Bcl-2 increase similar to THP-1 MDMs (Figure 5E). Under IL-13R downregulated conditions, both rIL-13 and the Leishmania parasites were unable to induce significant STAT-3 phosphorylation and Bcl-2 increase (Figure 5E) in hMDMs. In summary, in both THP-1 MDMs and hMDMs, IL-13 contributed to Bcl-2 induction via STAT-3 phosphorylation.

It was important to test the occurrence and relevance of the in vitro findings in an animal model of VL. To test the hypothetical role of Bcl-2 inhibition on the disease outcome in vivo, L. donovani infection was established in BALB/c mice and the role of Bcl-2 small molecule inhibitors, ABT-199 and ABT-263, was investigated. It should be noted that the inoculums used to infect mice were derived directly from the primary cultures of the parasites in the splenic aspirate of a VL patient. Splenomegaly is a common feature of VL where the size of the spleen is in general an indication of the parasitic burden and, therefore, the active disease. Mice treated with the Bcl-2 small molecule inhibitors showed significantly lesser spleen size and weight as compared to that from mice without the inhibitor treatments (Figure 6A). In support of the above data, the number of infected macrophages per spleen was observed to be significantly higher in control and only vehicle-treated groups as compared to ABT-199- and ABT-263-treated animals (Figure 6A), suggesting reduction in the number of infected macrophages in low Bcl-2 conditions. The in vitro observation of increased cellular NO production when Bcl-2 was inhibited was confirmed in vivo where ABT-199- and ABT-263-treated mice showed significantly higher serum nitrite levels as compared to vehicle-treated controls (Figure 6B). Infection-induced increase in IL-13 observed in vitro was also visible in the in vivo model where serum IL-13 levels were consistently higher in the infected animals (Figure 6C). On the other hand, ABT-199- and ABT-263-treated groups showed significantly lesser serum IL-13 (Figure 6C). When lysates from mouse spleen were checked for Bcl-2 protein, infected mice from all groups showed elevated Bcl-2 levels as compared to the uninfected control (Figure 6D). ABT-199 treatment apparently had no effect on Bcl-2 expression in vivo. While lysates from uninfected control mice showed no expression of the iNOS protein, barely detectable iNOS levels were visualized from L. donovani-infected group (Figure 6D). Interestingly, higher iNOS levels were found in the lysates from ABT-199-treated group mice (Figure 6D), possibly accounting for higher nitrite levels in the sera of ABT-199 treated. The in vivo data showing decreased parasite burden when infected mice were treated with Bcl-2 inhibitors clearly indicated an antileishmanial effect of the drugs. The confirmation of higher nitrite levels in vivo corroborated the data obtained at the cellular level suggesting NO-mediated killing of the parasites. Taken together, the data from in vitro experiments were reproducible in the animal model.

Sera from VL patients show significantly elevated levels of IL-13 (22), which may result into its downstream biological effects in the cells of monocyte lineage. Our in vitro data showed increased expression of Bcl-2 in response to IL-13, therefore to look at the status of Bcl-2 in the human subjects, PBMCs were isolated and Bcl-2 expression was checked in the lysates. Six out of nine VL patients presented with increased Bcl-2 expression as compared to the healthy controls (Figure 7A). Based on our in vitro data on human macrophages, increased Bcl-2 expression corresponded to deficient NO response. Therefore, it was important to look at the same in the VL patients. PBMCs from VL patients expressed barely detectable levels of the iNOS protein, which was also reflected in the nitrite levels in the corresponding sera (Figure 7B). When paired patients samples were checked for iNOS, a substantial increase in the expression was observed posttreatment which was again reflected in the level of serum nitrites (Figure 7B).