Suchandra Bhattacharyya Majumdar

CXC Chemokine–Mediated Protection against Visceral Leishmaniasis: Involvement of the Proinflammatory Response

Visceral leishmaniasis, caused by the protozoan parasite Leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response. In the present study, the comparative potential of CXC chemokines, interferon-gamma-inducible protein-10 (IP-10) and interleukin-8 (IL-8) in restricting Leishmania donovani infection via the release of nitric oxide and proinflammatory cytokines was studied in an in vitro model. Nitric oxide, a crucial mediator for IP-10-mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase 2 (iNOS2) expression and was linked to the mitogen-activated protein kinases (MAPK) signaling pathway. Further, IP-10 was also able to abrogate the survival of Leishmania in an in vivo model of visceral leishmaniasis by restoration of Th1 cytokines and nitric oxide. Thus, this study strongly demonstrates that IP-10, like CC chemokines, is involved in rendering a protective response in visceral leishmaniasis via up-regulation of proinflammatory mediators.

Arabinosylated Lipoarabinomannan—Mediated Protection in Visceral Leishmaniasis through Up-Regulation of Toll-Like Receptor 2 Signaling: An Immunoprophylactic Approach

Visceral leishmaniasis is characterized by severe immunosuppression of the host cell, resulting in loss of the proinflammatory response. Toll-like receptor 2 (TLR2) is involved in myriad disease forms, including visceral leishmaniasis. During Leishmania donovani infection, the parasite modulates TLR2 to suppress interleukin 12 production, indicating the possible involvement of TLR2 in regulation of the immune response against L. donovani infection. Arabinosylated lipoarabinomannan (Ara-LAM) possesses immunomodulatory properties and induces proinflammatory responses via induction of TLR2-mediated signaling. Here, we found that pretreatment of L. donovani-infected macrophages with Ara-LAM caused a significant increase in TLR2 expression along with the activation of TLR2-mediated downstream signaling, facilitating active nuclear translocation of nuclear factor kappaB. These events culminated in up-regulation of the proinflammatory response, which was abrogated by treatment with TLR2-specific small interfering RNA. In vivo experiments were also suggestive of Ara-LAM playing a long-term protective role. This study demonstrates that Ara-LAM confers protection against leishmanial pathogenesis via TLR2 signaling-mediated induction of the proinflammatory response.

Leishmania donovani-Induced Ceramide as the Key Mediator of Akt Dephosphorylation in Murine Macrophages: Role of Protein Kinase Cζ and Phosphatase

ABSTRACT Leishmania donovani is an intracellular protozoan parasite that impairs the host macrophage immune response to render it suitable for its survival and establishment. L. donovani-induced immunosuppression and alteration of host cell signaling is mediated by ceramide, a pleiotropic second messenger playing an important role in regulation of several kinases, including mitogen-activated protein kinase and phosphatases. We observed that the endogenous ceramide generated during leishmanial infection led to the dephosphorylation of protein kinase B (PKB) (Akt) in infected cells. The study of ceramide-mediated Akt phosphorylation revealed that Akt was dephosphorylated at both Thr308 and Ser473 sites in infected cells. Further investigation demonstrated that ceramide was also responsible for the induction of PKCζ, an atypical Ca-independent stress kinase, as well as the ceramide-activated protein phosphatases (e.g., protein phosphatase 2A [PP2A]). We found that Akt dephosphorylation was mediated by ceramide-induced PKCζ-Akt association and PP2A activation. In addition, treatment of L. donovani-infected macrophages with PKCζ-specific inhibitor peptide could restore the translocation of phosphorylated Akt to the cell membrane. This study also revealed that ceramide is involved in the inhibition of proinflammatory cytokine tumor necrosis factor alpha release by infected macrophages. These observations strongly suggest the importance of ceramide in the alteration of normal cellular functions, impairment of the kinase/phosphatase balance, and thereby establishment of leishmaniasis in the hostile macrophage environment.

Tumor-Derived Vascular Pericytes Anergize Th Cells

Immune evasion within the tumor microenvironment supports malignant growth and is also a major obstacle for successful immunotherapy. Multiple cellular components and soluble factors coordinate to disrupt protective immune responses. Although stromal cells are well-known for their parenchymal supportive roles in cancer establishment and progression, we demonstrate for the first time, to our knowledge, that tumor-derived vascular pericytes negatively influence CD4+ T cell activation and proliferation, and promote anergy in recall response to Ag by CD4+CD44+ T cells via regulator of G protein signaling 5– and IL-6–dependent pathways. Our data support a new specific role for tumor-derived pericytes in the immune evasion paradigm within the tumor microenvironment and suggest the targeting of these cell populations in the context of successful immunotherapeutics for the treatment of cancer.

Miltefosine triggers a strong proinflammatory cytokine response during visceral leishmaniasis: role of TLR4 and TLR9.

Visceral leishmaniasis (VL) caused by the protozoan parasite, Leishmania donovani, is associated with irregular fever, weight loss, hepatosplenomegaly and anemia. The therapeutic arsenal against VL is limited and the recent advent of a novel immunomodulatory drug, Miltefosine has shown promising results for effective treatment of VL but its dependence on Toll like receptors (TLR) has not been explored. In this study, we have shown that the non-cytotoxic dose (5 μM) of Miltefosine could render significant protection corresponding to 88% and 95% reduction in intracellular parasite load at 24 h and 48 h in L. donovani infected THP1 cells. This was accompanied by a strong proinflammatory cytokine response in the form of IFN-γ, IL-12 and TNF-α as evident by enzyme linked immunosorbent assay (ELISA) and real time PCR (RT-PCR). This Miltefosine induced proinflammatory cytokine response in infected THP1 cells was also accompanied by simultaneous 10- and 12-fold increase in TLR4 mRNA and TLR9 mRNA. These changes in cytokine response and TLR expression were also studied in peripheral blood mononuclear cells (PBMC) of VL patients treated with Miltefosine by RT-PCR which showed similar results as in THP1 cells. Thereby, suggesting a probable dependence of Miltefosine on TLR4 and TLR9 in triggering a proinflammatory response.

Treatment with IP-10 induces host-protective immune response by regulating the T regulatory cell functioning in Leishmania donovani -infected mice

Visceral leishmaniasis (VL), caused by the protozoan parasite, Leishmaniadonovani, is characterized by an infection in the liver and spleen. The failure of the first-line drugs has led to the development of new strategies for combating VL. Recently, our group has shown that interferon-γ-inducible protein (IP)-10, a CXC chemokine, renders protection against VL. In the present study, we have elucidated the mechanism by which IP-10 renders protection in in vivoL.donovani infection. We observed that IP-10–treated parasitized BALB/c mice showed a strong host-protective T helper cell (Th) 1 immune response along with marked decrease in immunosuppressive cytokines, tumor growth factor (TGF)-β, and interleukin (IL)-10 secreting CD4+ T cells. This IP-10-mediated decrease in immunosuppressive cytokines was correlated with the reduction in the elevated frequency of CD4+CD25+ T regulatory (Treg) cells along with the reduced TFG-β production from these Treg cells in Leishmania-infected mice. This reduction in TGF-β production was due to effective modulation of TGF-β signaling by IP-10, which reduced the immunosuppressive activity of Treg cells. Thus, these findings put forward a detailed mechanistic insight into IP-10-mediated regulation of the Treg cell functioning during experimental VL, which might be helpful in combating Leishmania-induced pathogenesis.

Leishmania-Induced Biphasic Ceramide Generation in Macrophages Is Crucial for Uptake and Survival of the Parasite

The initial macrophage-Leishmania donovani interaction results in the formation of membrane platforms, termed lipid rafts, that help in the entry of the parasite. Therefore, it is imperative that the parasite designs a strategy to modulate its uptake and survival within the macrophages. Herein, we report Leishmania-triggered biphasic ceramide generation. In the first phase, L. donovani promastigotes induce activation of acid sphingomyelinase (ASMase), which catalyzes the formation of ceramide from sphingomyelin. Inhibition of ASMase resulted in reduced uptake and infection with the parasite. In the second phase, de novo synthesis generates ceramide that reduces the cellular cholesterol level and displaces the cholesterol from the membrane, leading to enhanced membrane fluidity, disruption of rafts, and impaired antigen-presentation to the T cells. The results reveal a novel role for ceramide in the perspective of L. donovani infection and help formulate an antileishmanial strategy that can possibly be applied to other intracellular infections as well.

Mycobacterium indicus pranii (Mw) re-establishes host protective immune response in Leishmania donovani infected macrophages: critical role of IL-12.

Leishmania donovani, a protozoan parasite, causes a strong immunosuppression in a susceptible host and inflicts the fatal disease visceral leishmaniasis. Relatively high toxicity, low therapeutic index, and failure in reinstating host-protective anti-leishmanial immune responses have made anti-leishmanial drugs patient non-compliant and an immuno-modulatory treatment a necessity. Therefore, we have tested the anti-leishmanial efficacy of a combination of a novel immunomodulator, Mycobacterium indicus pranii (Mw), and an anti-leishmanial drug, Amphotericin B (AmpB). We observe that Mw alone or with a suboptimal dose of AmpB offers significant protection against L. donovani infection by activating the macrophages. Our experiments examining the anti-leishmanial activity of Mw alone or with AmpB also indicate a p38MAPK and ERK-1/2 regulated pro-inflammatory responses. The Mw-AmpB combination induced nitric oxide production, restored Th1 response, and significantly reduced parasite burden in wild type macrophages but not in IL-12-deficient macrophages indicating a pivotal role for IL-12 in the induction of host-protection by Mw and AmpB treatments. In addition, we observed that Mw alone or in combination with suboptimal dose of AmpB render protection against L. donovani infection in susceptible BALB/c mice. However, these treatments failed to render protection in IL-12-deficient mice in vivo which added further support that IL-12 played a central role in this chemo immunotherapeutic approach. Thus, we demonstrate a novel chemo-immunotherapeutic approach- Mw and AmpB crosstalk eliminating the parasite-induced immunosuppression and inducing collateral host-protective effects.

Antigen-Pulsed CpG-ODN-Activated Dendritic Cells Induce Host-Protective Immune Response by Regulating the T Regulatory Cell Functioning in Leishmania donovani-Infected Mice: Critical Role of CXCL10

Visceral leishmaniasis (VL), caused by Leishmania donovani, is a systemic infection of reticulo-endothelial system. There is currently no protective vaccine against VL and chemotherapy is increasingly limited due to appearance of drug resistance to first line drugs such as antimonials and amphotericin B. In the present study, by using a murine model of leishmaniasis we evaluated the function played by soluble leishmanial antigen (SLA)-pulsed CpG-ODN-stimulated dendritic cells (SLA–CpG–DCs) in restricting the intracellular parasitic growth. We establish that a single dose of SLA–CpG–DC vaccination is sufficient in rendering complete protection against L. donovani infection. In probing the possible mechanism, we observe that SLA–CpG–DCs vaccination results in the significant decrease in Foxp3+GITR+CTLA4+CD4+CD25+ regulatory T cells (Treg) cell population in Leishmania-infected mice. Vaccination with these antigen-stimulated dendritic cells results in the decrease in the secretion of TGF-β by these Treg cells by possible regulation of the SMAD signaling. Moreover, we demonstrate that a CXC chemokine, IFN-γ-inducible protein 10 (IP-10; CXCL10), has a direct role in the regulation of CD4+CD25+ Treg cells in SLA–CpG–DC-vaccinated parasitized mice as Treg cells isolated from IP-10-depleted vaccinated mice showed significantly increased TGF-β production and suppressive activity.

Co-administration of glycyrrhizic acid with the antileishmanial drug sodium antimony gluconate (SAG) cures SAG-resistant visceral leishmaniasis

Since there are very few affordable antileishmanial drugs available, antimonial resistance has crippled antileishmanial therapy, thereby emphasising the need for development of novel therapeutic strategies. This study aimed to evaluate the antileishmanial role of combined therapy with sodium antimony gluconate (SAG) and the triterpenoid glycyrrhizic acid (GA) against infection with SAG-resistant Leishmania (GE1F8R). Combination therapy with GA and SAG successfully limited infection with SAG-resistant Leishmania in a synergistic manner (fractional inhibitory concentration index <1.0). At the same time, mice infected with SAG-resistant Leishmania and co-treated with GA and SAG exhibited a significant reduction in hepatic and splenic parasite burden. In probing the mechanism, it was observed that GA treatment suppressed the expression and efflux activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1), two host ABC transporters responsible for antimony efflux from host cells infected with SAG-resistant parasites. This suppression correlated with greater intracellular antimony retention during SAG therapy both in vitro and in vivo, which was reflected in the reduced parasite load. Furthermore, co-administration of GA and SAG induced a shift in the cytokine balance towards a Th1 phenotype by augmenting pro-inflammatory cytokines (such as IL-12, IFNγ and TNFα) and inducing nitric oxide generation in GE1F8R-infected macrophages as well as GE1F8R-infected mice. This study aims to provide an affordable leishmanicidal alternative to expensive antileishmanial drugs such as miltefosine and amphotericin B. Furthermore, this report explores the role of GA as a resistance modulator in MRP1- and P-gp-overexpressing conditions.