Combination of Mycobacterium indicus pranii and Heat-Induced Promastigotes Cures Drug-Resistant Leishmania Infection: Critical Role of Interleukin-6-Producing Classical Dendritic Cells

The major issues in available therapeutic modalities against leishmaniasis are cost, toxicity, and the emergence of drug resistance. The aim of this work was to develop a successful therapeutic adjuvant against drug-resistant Leishmania donovani infection by means of combining Mycobacterium indicus pranii with heat-induced promastigotes (HIP). One-month postinfected BALB/c mice were administered subcutaneously with M. indicus pranii (10⁸ cells) and HIP (100 μg) for 5 days.

ABSTRACT

The major issues in available therapeutic modalities against leishmaniasis are cost, toxicity, and the emergence of drug resistance. The aim of this work was to develop a successful therapeutic adjuvant against drug-resistant Leishmania donovani infection by means of combining Mycobacterium indicus pranii with heat-induced promastigotes (HIP). One-month postinfected BALB/c mice were administered subcutaneously with M. indicus pranii (10⁸ cells) and HIP (100 μg) for 5 days. Spleens were harvested for flow cytometric and reverse transcriptase PCR analysis. The antileishmanial effect of the combination strategy was associated with induction of a disease-resolving Th1 and Th17 response with simultaneous downregulation of CD4⁺ CD25⁺ Foxp3⁺ (nTreg) cells and CD4⁺ CD25⁻ Foxp3⁻ (Tr1) cells in the spleen. The significant expansion of CD4⁺ T_CM (CD4⁺ CD44^hi CD11a^hi CD62L^hi) cells was a further interesting outcome of this therapeutic strategy in the context of long-term protection of hosts against secondary infection. Toll-like receptor 2 (TLR2) was also found instrumental in this antiparasitic therapy. Induced interleukin-6 (IL-6) production from expanded CD11c⁺ CD8α⁺ (cDC1) and CD11c⁺ CD11b⁺ (cDC2) dendritic cells (DCs) but not from the CD11b⁺ Ly6c⁺ inflammatory monocytes (iMOs), was found critical in the protective expansion of Th17 as evidenced by an in vivo IL-6 neutralization assay. It also promoted the hematopoietic conversion toward DC progenitors (pre-DCs) from common dendritic cell progenitors (CDPs), the immediate precursors, in bone marrow. This novel combinational strategy demonstrated that expansion of Th17 by IL-6 released from CD11c⁺ classical DCs is crucial, together with the conventional Th1 response, to control drug-resistant infection.

INTRODUCTION

Leishmaniasis constitutes a group of human diseases and has been recognized as the second most critical parasitic disease in the contemporary world after malaria (1). Among the three different clinical forms, visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis (MCL), VL is highly endemic to the Indian subcontinent and East Africa and claims maximum lives if left untreated (1). More than 90% of new VL cases occurred in 7 countries: Brazil, Ethiopia, India, Kenya, Somalia, South Sudan, and Sudan (2). Available drugs were not found to be effective over an extended time due to either toxicity or drug resistance (3). Designing a single uniform vaccine/adjuvant against leishmaniasis is a difficult task, given the presence of several virulent strains with variable genetic makeups and the emergence of prevalent drug resistance (3). Several experimental vaccines/adjuvants, ranging from attenuated live parasites to soluble/crude proteins, recombinant proteins, and DNA vaccines, have been tried, but failed to provide long-lasting and steady immunity (3, 4). Well-acclaimed vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) was an initial choice against leishmaniasis, and a mixed BCG-Leishmania major killed vaccine was also considered for clinical trials in Iran, but found less effective (4). Genetic and biochemical differences among substrains of BCG available worldwide are the other concerns for the failure of vaccination strategies (5). Overattenuated BCG is found defective in the production of two lipid virulence factors, phthiocerol dimycocerosates and phenolic glycolipids, and hence evokes poor immunogenicity (5). Among the other classical adjuvants, aluminum salts and tetanus toxoid (TT) have been reported to persuade sturdy Th2 with little or no Th1 response (6, 7). Thus, the current challenge is to develop an immunotherapeutic adjuvant against leishmaniasis which can promote a strong antiparasitic response specifically against the drug-resistance cases, which are the major determinants of the treatment failure. Recently, Mycobacterium indicus pranii, a nonpathogenic strain of Mycobacterium, was found more efficient against tuberculosis than BCG (8). M. indicus pranii is commercially available as a heat-killed vaccine (Immuvac/Cadi-05) for leprosy (8). It was also found effective against drug-sensitive L. donovani infection (9). However, the efficacy of M. indicus pranii against drug-resistant Leishmania infection has remained unexplored. It has been shown that L. infantum HSP70 and HSP83 recombinant proteins stimulated the proliferation of spleen cells and elicited gamma interferon (IFN-γ) in vitro (10); however, denaturation of heat shock proteins (HSPs) either by heat or by specific antibodies failed to induce the Th1 response (10). This may be the reason for the failure of vaccine trial with the autoclaved parasite against cutaneous leishmaniasis (CL) caused by L. mexicana (11) or L. tropica (12), or protection against L. donovani infection (13). In contrast, sudden heat shock (37°C to 40°C) maintains the HSPs in a native stage in Leishmania (14). As the increase in temperature marks stress in Leishmania and induces the expression of HSPs (15), in this study we utilized these HSPs in our therapeutic strategy through low-cost lysates of heat-induced promastigotes (HIP) in combination with recently introduced M. indicus pranii as an effective adjuvant against miltefosine-resistant L. donovani infection. The strategy promoted the upregulation of lineage-committed dendritic cell progenitors (pre-DCs) (Lin⁻ CD11c⁺ Flt3⁺ MHCII⁻) in bone marrow, leading to the expansion of interleukin-6 (IL-6)-producing CD11c⁺ classical DCs (cDC1, CD11c⁺ CD11b⁻ CD8α⁺ and cDC2, and CD11c⁺ CD11b⁺ CD8 α⁻) significantly in the spleen. We also demonstrated that the prominent release of IL-6 guided the Th17 expansion at the cost of the immunosuppressive nTreg (CD4⁺ CD25⁺ FoxP3⁺) and Tr1 (CD4⁺ CD25⁻ FoxP3⁻) population. We established that the combination is effective against drug-resistant L. donovani infection.

RESULTS

Effective dose of combination successfully inhibited the drug-resistant L. donovani.

The antileishmanial effect of the combination of M. indicus pranii and HIP against drug-resistant amastigotes of L. donovani strain (named as HePC-R) in vitro has been confirmed in comparison to IC₅₀ or half of the IC₅₀ doses of miltefosine (HePC) on the basis of L. donovani-kDNA expression in phosphate-buffered saline (PBS)-treated infected macrophages which carry the highest-possible replicating L. donovani in respect to the equalized murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Similar observations were obtained from the calculation of amastigotes/100 macrophages (data not shown). The 50% inhibitory concentration (IC₅₀) (2.56 ± 0.68 μM) or half of the IC₅₀ (1.22 ± 0.44 μM) doses of miltefosine against the drug-sensitive L. donovani (MHOM/IN/1983/AG83) were found ineffective against the miltefosine-resistant amastigotes (HePC-R), which could only be inhibited by 26.57% ± 0.51% and 4.17% ± 0.05%, respectively (Fig. 1A). However, similar doses of miltefosine, as expected, inhibited the drug-sensitive amastigotes by 56.94% ± 1.43% (P < 0.001) and 24.04% ± 0.09% (P < 0.05), respectively (Fig. 1B). The combination treatment inhibited the resistant amastigotes by 77.65% ± 2.18% (P < 0.01, Fig. 1A) at the dose of M. indicus pranii (2 × 10⁷) + HIP (50 μg/ml), in comparison to infected macrophages. The significant effect was further reflected in inducible nitric oxide synthase (iNOS) expression in macrophages infected with both drug-sensitive (35.6-fold, P < 0.001 versus PBS-treated infected macrophages; Fig. 1B) and drug-resistant parasites (8.6-fold, P < 0.001 versus PBS-treated infected macrophages; Fig. 1A). Dose-dependent inhibition of amastigotes was observed in spleen and liver by 89.44% ± 0.9% (P < 0.001 versus PBS treated infected animals) and 86.03% ± 2.1% (P < 0.001 versus PBS treated infected animals), respectively, at the dose of M. indicus pranii (10⁸) + HIP (100 μg) in the case of drug-resistant infection with simultaneous upregulation of nitric oxide (NO) (8-fold, P < 0.001 versus PBS-treated infected animals; Fig. 1C). The same dose could inhibit the drug-sensitive amastigotes by 94.32% ± 1.84% (P < 0.001 versus PBS-treated infected animals) and 88.25% ± 0.20% (P < 0.001 versus PBS treated infected animals), respectively, in the spleen and liver of the treated mice (Fig. 1D). It deserves mentioning that the IC₅₀ dose (2.5 mg/kg body weight) of miltefosine (HePC) against the drug-sensitive L. donovani (MHOM/IN/1983/AG83) could only inhibit drug-resistant parasites (HePC-R) by 17.79% ± 3.36% in the spleen (Fig. 1C and D). Comparative dose analysis revealed that the therapeutic efficacy of M. indicus pranii (10⁸) + HIP (50 μg) against drug-resistant infection provided the equal therapeutic effect of M. indicus pranii (10⁸) + HIP (40 μg) against drug-sensitive infection (data not shown). In that sense, the effective IC₅₀ combination required against drug-resistant Leishmania infection in vivo has been estimated a little higher than that of the drug-sensitive infection. Inactivation of the miltefosine transporter LdMT and its putative subunit LdRos3 causes miltefosine resistance in L. donovani (16, 17). We confirmed that the defects rendering resistance in field isolate-derived L. donovani (HePC-R) were due to the lower responsiveness of the promastigote and amastigote toward miltefosine because of constitutive low expression of LdMT (promastigote: 3.2-fold; amastigote: 2.7-fold) and LdRos3 (promastigote: 2-fold; amastigote: 3.7-fold) but a high expression of MDR1, an ATP-binding cassette P-glycoprotein responsible for the drug efflux in Leishmania (promastigote: 3.2-fold; amastigote: 3.3-fold) (P < 0.001 for all the comparisons, Fig. 1E). It is important to note that the IC₅₀ of miltefosine (5 μM) against the drug-sensitive promastigotes could kill the drug-resistant promastigotes only by 12.5% (Fig. 1F).

FIG 1.

In vitro and in vivo antileishmanial activities of the combination therapy involve the upregulation of iNOS mRNA expression and induction of NO secretion in both drug-resistant and drug-sensitive strains of L. donovani. (A and B) Bone marrow-derived macrophages were infected with both strains of L. donovani (1:10 macrophages to promastigotes) and, after established infection, cells were treated with either the IC₅₀ dose or half the IC₅₀ dose of miltefosine or M. indicus pranii + HIP (M. indicus pranii [2 × 10⁷ cells/ml] + HIP [50 μg/ml]) or PBS (untreated control). In vitro therapeutic efficacy and induction of iNOS2 were measured on the basis of expression of Leishmania-kDNA (Ld-kDNA, gray bars) and murine iNOS2 mRNA (line) with respect to equalized murine GAPDH in the case of the HePC-R (A) and AG83 (B) strains of L. donovani, respectively. (C and D) BALB/c mice were infected with isolated amastigotes (5 × 10⁶ per mouse) and 1-month postinfected animals were treated s.c. with either M. indicus pranii (10⁸/100 μl) or HIP (100 μg) or M. indicus pranii (10⁸/100 μl) + HIP (D1 = 25, D2 = 50, D3 = 75, and D4 = 100 μg) at day 1, day 3, day 5, day 7, and day 14. The splenic (×10⁷, black bars) and hepatic (×10⁸, gray bars) parasitemia was assessed at day 16 and the NO from splenocyte cultures of experimental animals infected with HePC-R (C) and AG83 (D) strains of L. donovani (line) was estimated by the Griess reagent assay. (E and F) The resistance of HePC-R strain to miltefosine was confirmed in comparison to L. donovani strain AG83 by estimating (E) the mRNA expressions of the resistance-specific genes MDR1, LdRos3, and LdMT in promastigotes and intracellular amastigotes with respect to equalized L. donovani kDNA and also by the MTT micro method (F). The densitometry (E) was analyzed by Image Lab software and the relative fold changes were expressed as mean ± SEM; ***, P < 0.001 versus the AG83 strain. For in vivo and in vitro experiments, data from at least 5 animals per group (C and D) and data from at least 3 independent experiments (A, B, E and F) were taken in duplicate and are expressed as mean ± SEM. The differences in means among various groups were considered significant as per one-way analysis of variance using GraphPad Prism v 6.0 software; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Combination therapy induces the proinflammatory responses in vivo: cytokines and chemokines.

The Th1/Th2 balance is crucial during VL, as the disease progression is associated with the upregulation of Th2 whereas the cure requires the Th1 responses (6, 10). A significant increase in proinflammatory cytokines IL-6 (5.1-fold), IL-12 (4.8-fold), IFN-γ (5.6-fold), and tumor necrosis factor alpha (TNF-α) (10.3-fold) was observed (P < 0.001 for all the comparisons) in supernatants collected from the crude soluble antigen (CSA)-pulsed splenocytes (48 h) of the mice treated with the combination of M. indicus pranii (10⁸/100 μl) and HIP (100 μg) by cytometric bead array assay. The expression of the anti-inflammatory cytokine IL-10 (1.8-fold; P < 0.05) and the Th2 cytokines IL-4 (1.9-fold; P < 0.001), IL-5 (1.7-fold; P < 0.001), and IL-13 (1.5-fold; P < 0.01) was downregulated (Fig. 2A), confirming the activation of Th1 and suppression of Th2 responses (18). The same combination therapy induced the key chemokines CCL3 (9.2-fold; P < 0.001), CCL4 (22-fold; P < 0.0001), CCL5 (10.9-fold; P < 0.0001), and CXCL10 (11.3-fold; P < 0.0001) and downregulated CCL2 (8-fold; P < 0.05) in spleen (Fig. 2B and C). CXCL10, CCL3, CCL4, and CCL5 promote Th1 responses, whereas CCL2 supports Th2 polarization (19, 20). The upregulation of CXCL10 and CCL5 leads to increases in CD3⁺ CD4⁺ IFN-γ ⁺ (1.9-fold; P < 0.0001) and CD3⁺ CD8⁺ IFN-γ⁺ (2.5-fold; P < 0.0001) cells and decreases in CD3⁺ CD4⁺ IL-10⁺ (2-fold; P < 0.001) and CD3⁺ CD8⁺ IL-10⁺ (1.8-fold; P < 0.001) cells in combination therapy (Fig. 3A and B; Fig. S1 in the supplemental material).

FIG 2.

In vivo antileishmanial effect of combination therapy significantly induced the proinflammatory cytokines, chemokines, and TLRs and downregulated the anti-inflammatory responses in the spleen. (A) Splenocytes were harvested from uninfected, infected (PBS treated), and treated (M. indicus pranii [10⁸] + HIP [D4 = 100 μg]) animals and incubated with CSA (25 μg/ml) at 37°C for 48 h for antigenic recall. Proinflammatory cytokines (TNF-α, IL-6, IFN-γ, and IL-12p70) as well as anti-inflammatory cytokines (IL-10, IL-4, IL-5, and IL-13) and chemokine (CCL2) were quantified from the culture supernatants by the cytometric bead array mouse inflammation kit, analyzed in FCAP Array software v 3.0 and presented in scatterplots as the cumulative data of the experimental groups. (B and C) Representative expression of TLRs and chemokine genes with respect to equalized murine GAPDH, measured by semiquantitative PCR from the CSA-pulsed splenocytes (incubated with 25 μg/ml of CSA at 37°C for 6 h) of experimental animals. The densitometric quantification was analyzed by Image Lab software and represented as mean ± SEM. For in vivo experiments, data from at least 5 animals per group were taken in duplicate. The differences in means among various groups were considered significant as per one-way analysis of variance using GraphPad Prism v 6.0 software; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

FIG 3.

In vivo combination therapy significantly induced the expansion of IFN-γ⁺ T cells and the subsets of antigen experienced T memory cells. (A and B) Viable CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T cells (excluding the B220⁺ NK1.1⁺ TER-119⁺ CD11b⁺ cells) were acquired in a flow cytometer for induction of IFN-γ and IL-10 from splenocytes (incubated with 25 μg/ml of CSA at 37°C for 12 h) of each group of experimental animals (isotype, uninfected, infected and treated [M. indicus pranii 10⁸] + HIP [D4 = 100 μg]). BD GolgiPlug protein transport inhibitor (containing brefeldin A) was added ex vivo (1 μg/ml for 6 h) to in vivo-stimulated cells for specifically blocking their intracellular transport processes for staining of intracellular cytokines. For in vivo experiments, data from at least 5 animals per group were taken in duplicate. (A) The flow cytometric gating strategy of Lin⁻ CD3⁺ CD4⁺ and Lin⁻ CD3⁺ CD8⁺cells. (B) Statistical significance analyses of IFN-γ⁺/IL-10⁺ T cells in scatterplots as the cumulative data of the experimental groups. The representative presentation of flow cytometric contour plots of IFN-γ⁺/IL-10⁺ Lin⁻ CD3⁺ CD4⁺ T cells of the experimental groups can be found in Fig. S1 in the supplemental material. (C to K) Expansion of subsets of splenic T memory phenotypes and activation of CD62L⁺ cells in the M. indicus pranii + HIP treated animals. (C and D) Representative presentation of flow cytometric contour plots and histograms of splenic T memory cell compartment of each group of experimental animals. Lin⁻ CD3⁺ CD4⁺ and Lin⁻ CD3⁺ CD8⁺ T cells were further analyzed and the antigen-experienced memory cells were designated CD4⁺ CD44^hi CD11a^hi and CD8⁺ CD44^hi CD11a^hi T cell populations. (E and F) Expression of CD62L was evaluated within these cell populations and T effector memory and T central memory cell populations were represented in flow cytometric contour plots as CD62L^lo CD11a⁺ (T_EM) and CD62L^hi CD11a⁺ (T_CM), respectively. (G) Expression of CD62L was also estimated on Lin⁻ CD3⁺ CD4⁺ CD44^hi CD11a^hi cells in the experimental animals and (K) the data (MFI) are presented as mean ± SEM. (H to J) Statistical significance analyses in scatterplots as the cumulative data of the experimental groups for Lin⁻ CD3⁺ CD4⁺ CD44^hi CD11a^hi and Lin⁻ CD3⁺ CD8⁺ CD44^hi CD11a^hi cells (H), CD62L^lo CD11a⁺ (T_EM), and CD62L^hi CD11a⁺ (T_CM) populations, gated on the viable Lin⁻ CD3⁺ CD4⁺ CD44^hi CD11a^hi (I) and Lin⁻ CD3⁺ CD8⁺ CD44^hi CD11a^hi T cells (J). The differences in means among various groups were considered significant as per one-way analysis of variance using GraphPad Prism v 6.0 software; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

Expansion of CD4⁺ T central memory population in the spleen.

To determine whether this novel combination therapy induces long-term protective immune responses against Leishmania donovani infection, we studied the expansion of specific T memory cells. CD11a is an integral part of the lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18) that mediates T cell emigration into the sites of tissue pathogenesis during infection (21). CD11a expression is also essential for the development and maintenance of the tissue specific memory T lymphocytes (21). CD44^hi CD11a^hi marks the population of antigen-experienced T cells (22, 23). We observed an expected increase of the CD44^hi CD11a^hi population of CD4⁺ T cells (1.4-fold; P < 0.001 versus uninfected animals) and of CD8⁺ T cells (2.2-fold; P < 0.05 versus uninfected animals) in infection (Fig. 3C, D, and H; Table S2E) (22, 23). However, the therapy enhanced the proliferation in spleen of the CD44^hi CD11a^hi population of CD4⁺ T cells (1.7-fold, P < 0.001; versus infected animals) over CD8⁺ T cells (1.5-fold, P < 0.05; versus infected animals) (Fig. 3C, D, and H; Table S2E). Considering the chances of reinfection, if any, the expansion of CD4⁺ T_CM (CD4⁺ CD44^hi CD11a^hi CD62L^hi) cells is more beneficial for host protection as it does not require live parasites to remain in homeostasis, unlike CD4⁺ T_EM (CD4⁺ CD44^hi CD11a^hi CD62L^lo) cells (24–26). We observed that the combination therapy increased the pool of CD4⁺ T_CM cells in treated mice (3-fold; P < 0.001 versus uninfected and 2-fold; P < 0.0001 versus infected), which may be instrumental in the future for protecting animals during reinfection (Fig. 3E to K; Table S2E). The expansion of CD4⁺ T_EM (2.5-fold; P < 0.01 versus uninfected and 1.7-fold; P < 0.05 versus infection) cells cannot be ignored, as these cells take shelter in tissues and rapidly produce IFN-γ (26). The role of CD8⁺ T memory cells has received little attention with respect to Leishmania infection (26); however, we observed a steady increase in CD8⁺ CD44^hi CD11a^hi CD62L^hi central memory cells (1.8-fold; P < 0.01 versus uninfected and 1.4-fold; P < 0.001 versus infected) in treated animals.

Enhanced expression of TLR2 on splenic CD11c⁺ and CD11b⁺ cells: correlation with proinflammatory cytokines and chemokines.

As the induction of proinflammatory cytokines is linked with the activation of Toll-like receptors (TLRs), we have also analyzed the involvement of TLRs in combination therapy against resistant infection. Initially, TLR2 was found to be increased (21.2-fold; P < 0.0001 versus infected) in treated spleen and the expression was significantly higher than TLR4 (6-fold; P < 0.01 versus infected) at the mRNA level (Fig. 2B and C). Assessed more precisely, the expression of TLR2 on splenic CD11c⁺ (1.4-fold; P < 0.05) and CD11b⁺ (1.8-fold; P < 0.01) cells was demonstrated to be higher than that of TLR4 (Fig. 4A; Fig. S2A and B). Enhanced expression of MHC-II has also been observed on splenic CD11c⁺ (5.7-fold; P < 0.001) and CD11b⁺ (4.1-fold; P < 0.001) cells (Fig. S2A to C). To identify the involvement of TLR2 in combination therapy, we simulated our strategy in vitro using parasite-infected murine bone marrow-derived dentritic cells (BMDCs) as a tool. The first observation confirmed that the combination could increase TLR2 (4.7-fold; P < 0.0001) but not TLR4 in BMDCs in vitro, in keeping with what was observed in vivo in CD11c⁺ splenic cDCs (Fig. 4B; Fig. S2D). The next observation reconfirmed the contribution of TLR2, as we observed that the addition of TLR2 agonist PAM3Csk4 (27) induced the expression of IL-6, IL-12, TNF-α, and CXCL10 in BMDCs in vitro compared to M. indicus pranii + HIP alone (Fig. 4C). Either in vitro or in vivo, there was a precise and significant increase of the two proinflammatory cytokines IL-6 and IL-12 and the observation agreed with the previous studies showing that TLR2 activation can be influenced by Leishmania HSPs, lipophosphoglycan, and M. indicus pranii itself or in combination to induce these cytokines (28–30). As the key sources of IL-6 and IL-12 during Leishmania infection are either inflammatory monocytes (iMOs) (31, 32) or dendritic cells (31–34), we were curious to know whether the cytokines came from Ly6c⁺ CD11b⁺ iMOs (31) or from CD11c⁺ splenic cDCs (32, 33).

FIG 4.

TLR2 on splenic CD11c⁺ dendritic cells is instrumental in combination therapy. (A) The expressions of TLR2 and TLR4 on splenic CD11c⁺ and CD11b⁺ cells were estimated in a flow cytometer in isotype (black), uninfected (blue), infected (red) and treated (green; M. indicus pranii [10⁸] + HIP [D4 = 100 μg]) animals. For the in vivo experiments, data from at least 5 animals per group were taken in duplicate and are presented as the mean ± SEM in Fig. S2A and B in the supplemental material. (B) The increase in TLR2 in L. donovani-infected bone marrow-derived DCs by the combination therapy (M. indicus pranii [2 × 10⁷] + HIP [50 μg/ml]) in vitro was also analyzed by estimating the expressions of TLR2 and TLR4 by flow cytometry. For the in vitro study, data were taken from at least 3 independent experiments in duplicate and expressed as the mean ± SEM in Fig. S2D. (C) To reconfirm the involvement of TLR2, the induction of proinflammatory mediators IL-6, IL-12, TNF-α, and CXCL10 was measured in the bone marrow-derived DCs at the mRNA level in the presence of TLR2 agonist PAM3Csk4 (10 μg/ml). The densitometry of results obtained from at least 3 independent experiments in duplicate was performed by Image Lab software and expressed as the mean ± SEM; *, P < 0.05; **, P < 0.01; and ****, P < 0.0001.

Expansion of CD11c⁺ CD11b⁻ CD8α⁺ and CD11c⁺ CD11b⁺ CD8α⁻ cDCs: source of IL-6 and IL-12.

cDC2 (CD11c⁺ CD11b⁺ CD8α⁻) cells are the most abundant myeloid DCs, whereas cDC1 (CD11c⁺ CD11b⁻ CD8α ⁺) cells are less prevalent, are the key producers of IL-12, and develop strong Th1 responses (35–37). The therapy reduced the expansion of Ly6c⁺ CD11b⁺ iMOs (2.3-fold; Fig. S3C) in drug-resistant infection. Moreover, both the expressions of IL-6 and IL-12 from iMOs were subdued by the combination therapy compared to the infected animals (IL-6, 1.9-fold; P < 0.001 and IL-12, 1.4-fold; P < 0.05 versus infection) (Fig. S3C).The observations ruled out the probability of functioning Ly6c⁺ CD11b⁺ iMOs as the source of IL-6 and IL-12. Next, we confirmed that the therapy induced the significant expansion of subsets of splenic cDCs, including CD11c⁺ CD11b⁻ CD8α⁺ (2.8-fold; P < 0.0001) and CD11c⁺ CD11b⁺ CD8α⁻ (3.4-fold; P < 0.0001) cells in vivo (Fig. 5B; Fig. S3A and B).

FIG 5.

Combination therapy induced the functional expansion of CD11c⁺ CD11b⁻ CD8α⁺ (cDC1) and CD11c⁺ CD11b⁺ CD8α⁻ (cDC2) splenic dendritic cells in vivo and the higher release of IL-6 from the cDC1 population. (A and B) Splenocytes were harvested from uninfected, infected, and treated (M. indicus pranii [10⁸] + HIP [D4 = 100 μg]) animals and incubated with CSA (25 μg/ml) at 37°C for 12 h. The expansion pattern of IL-6- and IL-12-releasing splenic cDC1 and cDC2 cells was monitored in a flow cytometer. (A) Representative presentation of flow cytometric contour plots of each group of experimental animals and (B) statistical significance analyses in scatterplots as the cumulative data of the experimental groups for IL-6⁺ and IL-12⁺ cDC1 and cDC2 populations. (C) Statistical significance analyses of IL-6- and IL-12-releasing splenic cDC1 and cDC2 cells in scatterplots as the cumulative data of the experimental groups. For the in vivo experiments, data from at least 5 animals per group were taken in duplicate. The flow cytometric gating strategy can be found in Fig. S3A and B. The differences in means among various groups were considered significant as per one-way analysis of variance, using GraphPad Prism v 6.0 software; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

IL-6 and IL-12 have been shown to be the critical cytokines in promoting host protection (32). Leishmania infection can downregulate the dendritic cell-signaling pathways, leading to suppressed cytokine production (34). We also observed the functional impairment of cDCs in infected mice in comparison to uninfected animals (Fig. 5A to C; Fig. S3A and B; Table S2E). The combination therapy induced the expressions of IL-6 and IL-12 from both subsets of cDCs as follows: CD11c⁺ CD11b⁻ CD8α⁺ (IL-6, 3.3-fold; P < 0.0001 and IL-12, 2.8-fold; P < 0.001 versus infection) and CD11c⁺ CD11b⁺ CD8α⁻ (IL-6, 2.5-fold; P < 0.0001 and IL-12, 1.9-fold; P < 0.01 versus infection), which could be correlated with the enhanced host protection against resistant infection. Interestingly, the release of IL-6 from cDCs was found to be prominent over IL-12 and, more precisely, the release of IL-6 from cDC1 (3.3-fold versus infected) was found to be higher than the cDC2 (2.5-fold versus infected) cells with respect to the untreated infected mice (Fig. 5A to C; Fig. S3A and B).

Expansion of lineage-committed DC progenitors.

Consequently, our next question was to investigate the DC expansion pattern, asking whether the differentiation was restricted to the pathological site (spleen) or resulted from lineage commitment in bone marrow by the influence of therapy. Hematopoiesis is a sequential process involving the generation of common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), macrophage-dendritic cell progenitors (MDPs), and common dendritic cell progenitors (CDPs) (38). CDPs (Lin⁻ CD115⁺ Flt3⁺) yield pDCs (CD11c^int B220⁺ PDCA1⁺) and precursors of cDCs (pre-DCs; Lin⁻ CD11c⁺ Flt3⁺ MHCII⁻) which preferentially differentiate in to the CD8α⁺ cDC1 and CD11b⁺ cDC2 in spleen (38, 39). We found that the resistant infection restricted the conversion of CDPs into pre-DCs. However, the therapy significantly increased the conversion of pre-DCs from CDPs (infected: CDP = 7.43% ± 0.64%, pre-DC = 5.43% ± 0.58% and treated: CDP = 4.68% ± 0.43%, pre-DC = 10.55% ± 0.53%; P < 0.001) in bone marrow (Fig. 6A to D). This may be associated with the influx or expansion of CD11c⁺ CD11b⁻ CD8α⁺ and CD11c⁺ CD11b⁺ CD8α⁻ cDCs (Fig. 5B) in spleen. The therapeutic strategy also promoted the expansion of pDCs (treated 2.54% ± 0.38% versus infected 1.07% ± 0.31% cells; P < 0.01) (Fig. 6F and G). We have also observed the enhanced expression of IRF-8 (4.5-fold versus infected; P < 0.05) (Fig. 6E) in the bone marrow of treated mice, which undoubtedly shows commitment-specific differentiation of dendritic cells in our therapeutic strategy.

FIG 6.

Combination therapy prominently modulated the expansion pattern of common dendritic cell progenitors (CDPs) and predendritic cell progenitors (pre-DCs) in the bone marrow. (A to D) Bone marrow cells were harvested from experimental animals and the expansion pattern of CDPs and pre-DCs was monitored in a flow cytometer. Representative presentation of downregulation of CDPs (B) and simultaneous upregulation of pre-DCs (C) based on lineage negative hematopoietic bone marrow cells (A) in contour plots as visualized in flow cytometric analyses. (D) Statistical significance analyses of significant hematopoietic conversion of CDPs to pre-DCs are represented in scatterplots as the cumulative data of the experimental groups. For the in vivo experiments, data from at least 5 animals per group were taken in duplicate. The flow cytometric gating strategy of viable cells for analysis of pre-DCs and CDPs in bone marrow can be found in Fig. S4A. (E) Estimation of IRF8 mRNA in bone marrow by reverse transcriptase PCR as a proof of lineage commitment development of dendritic cells and the densitometry of results obtained from at least 5 animals per group in duplicate was performed by Image Lab software and expressed as the mean ± SEM. (F and G) Representative contour plots of flow cytometric analyses of plasmacytoid dendritic cells (CD11c^int B220⁺ PDCA1⁺) in spleens of experimental groups, based on viable cells (F), and statistical significance analyses of expansion of the pDCs in scatterplots as the cumulative data of those groups (G). The differences in means among various groups were considered significant as per one-way analysis of variance using GraphPad Prism v 6.0 software; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

IL-6 is crucial for the protective balance between Th17 and Treg cells leading to a successful therapeutic strategy.

Apart from Th1 cytokines in Leishmania infection, the role of IL-17 has also been critically established in several studies (40, 41). We observed a clear activation of CD4⁺ IL-17⁺ cells (4.7-fold; P < 0.0001) in spleen (Fig. 7A). IL-6 also controls the Th17 immunity by inhibiting the conversion of conventional T cells into FoxP3⁺ regulatory T cells (Treg) (42, 43). Accordingly, we found reductions in the elevated populations of natural Treg (nTreg) (CD4⁺ CD25⁺ FoxP3⁺) cells (2.3-fold; P < 0.0001), along with decreases in IL-10 (1.7-fold; P < 0.05) and TGF-β (2-fold; P < 0.001) (Fig. 7B to E; Fig. S5A and B). Tr1 cells (CD4⁺ CD25⁻ FoxP3⁻) were reported as the major producers of IL-10 in VL (44) and our strategy successfully restricted this population (1.75-fold; P < 0.001), which correspondingly reduced the fraction of CD4⁺ CD25⁻ FoxP3⁻ IL-10⁺ cells (2.32-fold; P < 0.001) (Fig. 7B to E; Fig. S5A and B). The therapy also reduced the total IL-10⁺ (3.2-fold; P < 0.05) and TGF-β⁺ (1.5-fold; P < 0.001) cells in the spleen (Fig. 7F; Fig. S5C). IL-6 was found to be a crucial factor for parasite clearance, as in vivo neutralization of IL-6 curbed the efficiency of the therapeutic strategy by allowing the amastigote replication in the spleen (4-fold; P < 0.05) and liver (3.5-fold; P < 0.05) (Fig. 8A). This failure of the therapy might be explained by the nonavailability of IL-6, as it restricted the expansion of CD4⁺ IL-17⁺ cells (3.3-fold; P < 0.0001) and upregulated the release of IL-10 and TGF-β from both nTreg (IL-10, 2-fold and TGF- β, 2.5-fold; P < 0.05) and Tr1 (IL-10, 2.8-fold and TGF-β, 2-fold; P < 0.01) cells in comparison to control IgG (Fig. 8C; Fig. S5A and B). However, IL-6 neutralization could not slow down the Th1 response in treated animals (Fig. 8B and C).

FIG 7.

The antileishmanial effect of the combination therapy maintained the balance between Th17 and Treg cells leading to protection against L. donovani infection. (A and B) Splenocytes were harvested from uninfected, infected, and treated (M. indicus pranii [10⁸] + HIP [D4 = 100 μg/100 μl]) animals and were incubated with CSA (25 μg/ml) at 37°C for 12 h for antigenic recall. (A) Representative presentation of flow cytometric contour plots of expansion pattern of CD4⁺ IL-17⁺ (Th17) cells in spleens of each group of experimental animals and statistical significance analyses in scatterplots as the cumulative data of the experimental groups for Th17 cells. (B and C) Representative presentations of expansion pattern of CD4⁺ CD25⁺ Foxp3⁺ (nTreg) cells and CD4⁺ CD25⁻ Foxp3⁻ (Tr1 cells) in spleens of each group of experimental animals are represented in flow cytometric contour plots and statistical significance analyses of those cell populations in scatterplots as the cumulative data of the experimental groups. (D to F) Statistical significance analyses of IL-10 and TGF-β releasing splenic CD4⁺ CD25⁺ Foxp3⁺ (nTreg) cells (D), CD4⁺ CD25⁻ Foxp3⁻ (Tr1) cells (E), and viable splenocytes (F) are presented in scatterplots as the cumulative data of the experimental groups. Gating strategies for viable CD4⁺ T cells and the representative flow cytometric contour plots of expansion pattern of IL-10⁺/TGF-β⁺ viable splenocytes, nTreg, and Tr1 cells in the different experimental groups can be found in Fig. S4B and Fig. S5, respectively. For the in vivo experiments, data from at least 5 animals per group were taken in duplicate and are presented in scatterplots as the cumulative data of the individual animals. The differences in means among various groups were considered significant as per one-way analysis of variance using GraphPad Prism v 6.0 software; *, P < 0.05; ***, P < 0.001; and ****, P < 0.0001 versus infected animals.

FIG 8.

IL-6 is crucial for the protective induction of Th17 leading to successful antileishmanial therapy by M. indicus pranii + HIP. BALB/c mice were infected with isolated amastigotes (5 × 10⁶ per mouse) and 1-month postinfected animals were treated with the combination treatment (M. indicus pranii [10⁸] + HIP [D4 = 100 μg]) as per described for Fig. 1. For in vivo IL-6 neutralization, the mice were injected with purified mouse anti-IL-6 antibody (100 μg/mouse; i.v.) or control IgG. (A) Antileishmanial effect of combination therapy both in spleen and in liver as identified from the ratio of the parasite burden of the different groups of the experimental animals (infected, M. indicus pranii + HIP treated, and M. indicus pranii + HIP + α-IL-6 treated mice). (B) Splenocytes were harvested from each group of experimental animals and were incubated with CSA (25 μg/ml) at 37°C for 6 h. The induction of cytokines in the spleen at the mRNA level was measured by reverse transcriptase PCR. The densitometry was analyzed by Image Lab software and expressed as the mean ± SEM. (C) The effect of serum IL-6 neutralization on the outcome of cytokine profiles of splenic Th1, Th17, nTreg, and Tr1 cells as identified from the ratio of treated to infected and treated + α-IL-6 to infected in harvested splenocytes (cultured at 37°C for 12 h in the presence of CSA) from each group of experimental animals. For the in vivo experiments, data from at least 5 animals per group were taken in duplicate and are expressed as the mean ± SEM. The differences in means among various groups were considered significant as per one-way analysis of variance using GraphPad Prism v 6.0 software; *, P < 0.05; **, P < 0.01; and ****, P < 0.0001.

DISCUSSION

Resistance to available drugs and asymptomatic cases are the major obstructions affecting the elimination program for VL in the Indian subcontinent. The battle against drug-resistant infection is a challenge, as it affects host physiology more severely than drug-sensitive infection (45–47). Antimony-resistant Leishmania was found to impair host-signaling cascades that counter the oxidative burst to establish infection (45). It could also upregulate IL-10 to overexpress MDR1 and thus enhance infection (46). Exposure of antimony-resistant Leishmania to healthy peripheral blood mononuclear cells (PBMCs) in vitro resulted in the increase of proparasitic IL-10-producing CD4⁺ CD25⁺ CD127^low/− iTreg cells (47). Therefore, a potential therapeutic approach is urgently needed to combat resistant infections and must act to enhance an alternative antileishmanial immune axis, leading to the deactivation of T-regulatory cells and activation of Th17 responses in coordination with the conventional Th1 response. Initially, we observed that the therapeutic combination of M. indicus pranii + HIP favorably reestablished the proinflammatory responses in resistant Leishmania-infected macrophages, by an increase in inducible nitric oxide synthase (iNOS) in vitro and release of nitric oxide (NO) in vivo. Our results are consistent with the fact that the upregulation of CCL5 chemotactically induces the proliferation of Th1 cells and secretion of IFN-γ, which consequently activates macrophages and enhances parasite killing (48). Concurrent with the previous report (49), the increase in CXCL10 by the combination therapy may be correlated with the release of IL-12, followed by the expansion of CD4⁺ IFN-γ⁺ T cells and decrease of IL-10 and TGF-β. Dendritic cells (DCs) are the most potent antigen-presenting cells and are also indispensable for the initiation of adaptive immunity against infections by activating the protective cytokine milieu (32, 50, 51). The enhanced and partial expression of TLR2 and TLR4, respectively, on splenic CD11c⁺ and CD11b⁺ cells can be linked with the concept of premeditated use of M. indicus pranii in combination with heat induced promastigotes as the source of HSPs, as exogenous HSPs have been reported to induce IL-6 by engaging TLR2 and TLR4 in HEK293 cells (30). In our therapeutic strategy, TLR2 has been established as one of the key executors, as its expression was found to be increased on splenic CD11c⁺ cDCs in vivo and BMDCs in vitro. Induction of TLR2 by its agonist in a simulation study in vitro confirmed its involvement, as it induced the expression of IL-6, IL-12, TNF-α and CXCL10 mRNA in a manner similar to that of in vivo proinflammatory networks stimulated by the combination strategy, where lipophosphoglycans of heat-induced Leishmania promastigotes (HIP) and Mycobacterium indicus pranii have also been reported to possess a central role in TLR2 activation as agonists (28, 29). It has also been noted that the expansion of DCs is not a peripheral incidence, occurring in the spleen, as this novel strategy could promote the hematopoietic conversion toward DC progenitors, the pre-DCs formed from the immediate precursor CDPs in the bone marrow. IRF-8 has been reported to repress the lineage-committed development of neutrophils and promote DC expansion from lymphoid and myeloid progenitors (52) and our therapeutic strategy-enhanced expression of IRF-8 in bone marrow undoubtedly established the fact of commitment-specific differentiation of DCs. This is the first key point that we observed in our study, as the promotion of CDPs toward pre-DCs is essential for effective immunity in VL, where leukopenia is the most common occurrence. Apart from that, the therapeutic strategy promoted the expansion of antigen-experienced CD44^hi CD11a^hi memory CD4⁺ and CD8⁺ T cells, responsible for long-term immunity. More specifically, the therapy induced the amplification of Ag-specific CD4⁺ T_CM in favor of host protection, as it can expand flexibly toward either Th1 or Th2 during secondary infection and can also provide long-term immunity in the absence of live parasites (24–26). Our therapeutic strategy can thus provide initial protection against the challenge of further infection by Leishmania, if that were to occur.

This is a fundamental report highlighting the role of IL-6-producing splenic classical DCs along with their role as key producers of IL-12. Recently, iMOs have been reported to promote the Leishmania infection under the influence of IL-10, which selectively reduces the expression of CD11c⁺ DCs and blocks the development of Tip DCs, resulting in downregulation of TNF-α and iNOS (53). The interesting therapeutic advantage of our combination strategy may be suitable for restricting functional iMOs and inducing the expansions of the two major types of cDC populations in the treated spleen.

The second key point of our work is the prominent release of IL-6 from splenic cDCs, as we observed in the immunotherapeutic strategy. The distinct role of IL-12 in mediating host-protective immune responses is well characterized in drug-sensitive L. donovani infection (16, 32), but the role of IL-6 in VL is quite controversial, as it plays a dual role in T cell differentiation (54). Interestingly, IL-17 also has a role in positive regulation of IL-6 production (40). In the combination therapy, splenic cDC-derived IL-6 can be correlated with the expansion of Th17, as we observed that depletion of IL-6 downregulated the expression of IL-17 mRNA in the treated spleen. Expansion of IL-6-dependent CD4⁺ IL-17⁺ T cells by the combination therapy is the third major point to be mentioned.

Neutralization of IL-6 was also involved in upregulation of IL-10 and TGF-β mRNA in the spleen; however, the expression of IFN-γ remained unchanged. Tr1 and nTreg cells are the producers of proparasitic IL-10 in the spleen (42, 55). Our therapeutic strategy inhibited the expansion of both nTreg and Tr1 cells in spleen and limited the secretion of IL-10 and TGF-β, a reported proparasitic cytokine that activates the FoxP3 translation leading to Treg differentiation (42). IL-6 predominantly prevents the conversion of naive T cells into Foxp3⁺ Treg cells in vivo in spite of the presence of these two cytokines (40–42, 56). Earlier it was shown that IL-6-producing dendritic cells could inhibit the CD4⁺ CD25⁺ regulatory T cell functions in the congenic mouse model of lupus (57). Here, we observed that apart from IFN-γ-secreting CD4⁺ Th1 cells, both IL-6-producing CD11c⁺ dendritic cells and IL-17-producing Th17 cells are instrumental in our therapeutic strategy. The present study systematically demonstrated that the immunotherapeutic effects of the combination strategy could be initiated quite early at hematopoietic conversion of pre-DCs from its immediate precursor CDPs in bone marrow and the splenic CD11c⁺ classical dendritic cell-derived IL-6 can work as one of the key cytokines in therapeutic strategy against drug-resistant L. donovani infection, not only by promoting Th17 cells in vivo, but by diminishing the immunosuppressive activities of T regulatory cells.

MATERIALS AND METHODS

Materials and equipment.

M. indicus pranii was a kind gift from B. M. Khamar, Cadila Pharmaceuticals Ltd., India. Tissue culture reagents (M-199 and RPMI 1640 media and penicillin-streptomycin solution), Aqua Live/Dead stain kit and/or Green Live/Dead stain kit were procured from Invitrogen. Fetal calf serum (FCS) was purchased from Gibco. Reagents for flow cytometry (as given in supplementary list 1) were acquired from BD Biosciences (San Jose, CA) and BioLegend (San Diego, CA). Recombinant mouse monocyte colony-stimulating factor (rmMCSF) and recombinant mouse granulocyte monocyte colony-stimulating factor (rmGMCSF) were obtained from BioLegend. Moloney murine leukemia virus reverse transcriptase and Taq DNA polymerase enzyme were procured from Bangalore Genei. All other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), Merck Millipore (MA, USA), and Invitrogen (CA, USA), unless otherwise specified. iMark microplate reader and Gel imaging system were purchased from Bio-Rad, USA. The quantification of nucleic acids and proteins was done by UV Vis-spectrophotometer (Eppendorf, Hamburg, Germany). Eppendorf Master Cycler Pro gradient thermal cycler system was used for amplification of desired gene products. Flow cytometric acquisition and analyses were performed in BD FACSVerse using BD FACSuite software.

Animals and Leishmania parasites.

Male BALB/c mice (25 to 30 grams) were obtained from NCLAS, India, and maintained at a standard temperature (25°C ± 2°C) in a 12-h light-dark cycle and provided with food pellets and water ad libitum (58).

To develop the miltefosine-resistant L. donovani strain (named HePC-R), the bone marrow aspirates from a relapsed miltefosine-treated VL patient from West Bengal, India, were transformed and maintained in stepwise drug pressure in our laboratory as per the approval (reference number WBSU-IEC/06, dated 24 April 2012) and described earlier (16). For parasite culture, complete M199 with 10% FCS medium was used. For in vivo infection, BALB/c mice were injected with Leishmania amastigotes (5 × 10⁶ per mouse) via an intravenous (i.v.) route.

Ethics statement.

All the experiments with animals and parasites (L. donovani strain MHOM/IN/1983/AG83 and L. donovani strain HePC-R) were approved by the IAEC (reference number CP-SM/WBSU/2010-11/3, dated 23 February 2012) and Biosafety Committee, of West Bengal State University, Barasat. We made all efforts to minimize animal suffering. Protocols regarding the VL patient–related work have also been approved (reference number WBSU-IEC/06, dated 24 April 2012), as per the guidelines of the Indian Council of Medical Research, Government of India. All adult subjects voluntarily participated in the study and provided written informed consent. No child participant was enrolled in the study.

In vitro and in vivo antileishmanial effect.

Macrophages (BMDMs) and dendritic cells (BMDCs) were generated from bone marrow cells of wild-type BALB/c mice and cultured in complete RPMI 1640 medium in the presence of 30 ng/ml recombinant mouse monocyte colony-stimulating factor (rmMCSF) (BioLegend) and 30 ng/ml recombinant mouse granulocyte monocyte colony-stimulating factor (rmGMCSF) (BioLegend), respectively. L. donovani stationary-phase promastigotes were used at a cell to promastigote ratio of 1:10 and the cultures were continued in complete RPMI 1640 medium for 48 h for the establishment of infection (58). The cells were treated with phosphate-buffered saline (PBS), with the IC₅₀ dose of miltefosine, or with the half IC₅₀ dose of miltefosine, or with the respective doses of the combinations of M. indicus pranii and HIP (as described in Results) and were kept for another 48 h. To check the antileishmanial effect, cells were fixed in methanol and Giemsa-stained micrographs were prepared to calculate the numbers of amastigotes per hundred macrophages (58), and, more precisely, the expression of Leishmania-kDNA (Ld-kDNA) and murine iNOS2 mRNA as quantified by reverse transcriptase PCR with respect to equalized murine GAPDH. The IC₅₀ of miltefosine has been determined from the plot of percentage inhibition against increasing concentrations of miltefosine (0 to 10 μM), based on the treatment of a drug-susceptible strain in vitro (L. donovani strain MHOM/IN/1983/AG83).

To check the involvement of TLR2, the uninfected, HePC-R-infected, and M. indicus pranii + HIP-treated BMDCs (M. indicus pranii [2 × 10⁷] + HIP [50 μg/ml]) were stimulated with a known TLR2 agonist, PAM3Csk4 (10 μg/ml), and the induction of proinflammatory mediators IL-6, IL-12, TNF-α and CXCL10 was measured at the mRNA level by reverse transcriptase PCR (RT-PCR). The MTT assay and RT-PCR were applied to check the resistance in promastigotes and intracellular amastigotes (16, 58, 59). For in vivo experiments, 1-month postinfected mice were treated (subcutaneously [s.c.]), either with PBS (control) or with the indicated doses of M. indicus pranii (10⁸/100 μl) or HIP (100 μg) or M. indicus pranii (10⁸/100 μl) + HIP (D1 = 25, D2 = 50, D3 = 75, and D4 = 100 μg) at day 1, day 3, day 5, day 7, and day 14. For in vivo IL-6 neutralization, the 1-month postinfected mice (infected by drug-resistant L. donovani strain HePC-R) were administered (at 100 μg/mouse intravenously [i.v.]) with LEAF purified anti-mouse IL-6 antibody (MAb; BioLegend) or control rat IgG (R&D Systems) in 100 μl PBS as described earlier (60), along with M. indicus pranii (10⁸/100 μl) + HIP (100 μg) at day 1, day 3, day 5, day 7, and day 14. For the determination of the effect of miltefosine in vivo, 1-month postinfected BALB/c mice (infected both by drug-sensitive L. donovani strain MHOM/IN/1983/AG83 and drug-resistant L. donovani strain HePC-R) were administered orally with increasing doses of miltefosine (1.25, 2.5, 5, and 10 mg/kg body weight) for 5 consecutive days. Mice were sacrificed on day 16 and spleens, livers, and bone marrow cells were collected aseptically for the immune analysis and assessment of parasitemia from the Giemsa-stained stamp smear micrographs by Stauber formula (58). For the estimation of nitric oxide (NO) in infected and uninfected and treated animals, single-cell suspensions were prepared from the murine spleens in complete RPMI 1640 medium containing 1% penicillin-streptomycin (Invitrogen) and 10% FCS (GIBCO), pulsed with Leishmania crude soluble antigen (CSA at 25 μg/ml) and were harvested for 60 h. The cell supernatants were dispersed (100 μl/well) in 96-well plates with an equal volume of Griess reagent, incubated for 15 min at 37°C, and the absorbance was measured at 540 nm by an iMark microplate reader (Bio-Rad, USA) (58).

Reverse transcriptase PCR.

The quantification of different cytokines, chemokines, and TLRs in experimental cells and the expression of resistance-specific markers in L. donovani at the mRNA level in vitro and in vivo were determined by reverse transcriptase PCR as described previously (58). Briefly, total mRNA was extracted by TRIzol reagent. cDNA was synthesized from 2 μg of total RNA by Moloney murine leukemia virus reverse transcriptase (Bangalore Genei) enzyme at 37°C for 1 h, and incubated for 10 min at 70°C. cDNA from each sample was amplified for a total of 35 cycles with Taq DNA polymerase (Bangalore Genei) enzyme under the following conditions: 95°C for 2 min, 94°C for 1 min, 56°C to 62°C for 1 min (as applicable), and 72°C for 1 min, using Eppendorf Master Cycler Pro and the amplified products were run in a 1.2% agarose gel. The primer sequences are given in Table S1.

Flow cytometric analyses.

The in vivo expansion patterns of the different cytokines of Th1/2, regulatory T cells, Th17 cells, the subsets of memory T cells, macrophages/monocytes, and DCs in spleens, together with the status of pre-DCs and common dendritic cell progenitor (CDPs) cells in bone marrow in the different experimental groups of BALB/c mice, were also assessed. Flow cytometric acquisition and analyses were performed in BD FACSVerse using BD FACSuite software. For all the flow cytometric analyses, only viable cells were taken into account. The doublet, clumped, and nonviable cells were excluded by means of the appropriate FSC-A/FSC-H gating strategy and Aqua Live/Dead stain kit (catalog number L34957, Invitrogen) or Green Live/Dead stain kit (catalog number L23101, Invitrogen). Cytofix/cytoperm with the GolgiPlug kit was used for intracellular staining, as per the manufacturer’s instructions (catalog number 555028, BD Biosciences, CA, USA). BD GolgiPlug protein transport inhibitor (containing Brefeldin A) was added ex vivo (1 μg/ml for 6 h) to in vivo-stimulated cells for specifically blocking their intracellular transport processes for staining of intracellular cytokines, unless otherwise specified. To correct the spillover between the emission channels, the compensation matrices were adjusted, using the anti-rat and anti-hamster Ig κ/Negative Control BD CompBeads (catalog number 552845). The appropriate isotypes and fluorescence minus one (FMO) controls were used for characterizing background signals caused by off-target binding of antibodies and for identifying the positive-cell populations for the specific combinations of fluorochrome-conjugated antibodies in all the flow cytometric analyses. Cytokines were quantified from supernatants of Ld-CSA-pulsed splenocytes (25 μg/ml for 48 h) by the BD Cytometric Bead Array Mouse Inflammation kit (catalog number 552364), BD CBA Mouse IL-4 Flex Set (catalog number 558298), BD CBA Mouse IL-5 Flex Set (catalog number 558302), and BD CBA Mouse IL-13 Flex Set (catalog number 558349) and acquired in BD FACSVerse and analyzed in FCAP Array software v 3.0. The specifications of anti-mouse monoclonal antibodies, lineage-specific markers, respective isotypes, and the relevant reagents have been provided in the supplemental material.

Statistical analysis.

For in vivo experiments, data from at least 5 animals per group (uninfected, infected [PBS treated], and M. indicus pranii + HIP treated) were taken in duplicate and were presented in scatterplots as the cumulative data of the each of the experimental groups. The isotype control was randomly pooled from all the experimental groups in a single tube. As each experimental group had five animals, five single tubes of pooled isotype controls were prepared and the data were acquired in duplicate. The flow cytometric contour plots are representative of each group of experimental animals. Results were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett’s or Sidak’s multiple-comparison test using GraphPad Prism v6 software. P < 0.05 reflected statistically significant differences among the different experimental groups for all calculations of percentages and fold changes, which have been provided in Table S2.