Gaurav Gupta

Quassin alters the immunological patterns of murine macrophages through generation of nitric oxide to exert antileishmanial activity.

OBJECTIVES The aim of this study was to characterize the in vitro antileishmanial activity of quassin, a traditional Chinese herbal medicine. METHODS The cytotoxic effect of quassin was studied in murine peritoneal macrophages at various concentrations using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide method. The role of quassin as an antileishmanial agent was evaluated by microscopic counting of intracellular amastigotes in macrophages stained with Giemsa. To understand the effector mechanism of quassin-treated macrophages against leishmanial parasites, western blot and real-time PCR analysis of inducible nitric oxide (NO) synthase 2 (iNOS2) were done followed by measurement of NO generation by Griess reaction. The effect of quassin on the production of Th1 cytokines such as interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha and Th2 cytokines such as IL-10 and transforming growth factor-beta was measured by ELISA, and the mRNA expression of these cytokines was analysed by real-time PCR. RESULTS Quassin at a dose of 25 microg/mL (64.36 microM) showed less cytotoxicity to the host murine peritoneal macrophages but at the same dose was effective enough to control the intracellular parasitic load compared with higher doses of quassin. Leishmania donovani is known to exert its pathogenic effects mainly by the suppression of NO generation and subversion of the cellular inflammatory responses in the macrophages. Quassin was found to induce a potent host-protective immune response by enhancing NO generation and iNOS2 expression both at a protein and mRNA level and by up-regulating pro-inflammatory cytokines such as TNF-alpha and IL-12 in L. donovani-infected macrophages with concurrent inhibition of anti-inflammatory responses. CONCLUSIONS These findings strongly support the effectiveness of quassin as a potent immunomodulatory tool for controlling the establishment of leishmanial parasite within the host macrophages.

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.

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.

Arabinosylated Lipoarabinomannan Skews Th2 Phenotype towards Th1 during Leishmania Infection by Chromatin Modification: Involvement of MAPK Signaling

The parasitic protozoan Leishmania donovani is the causative organism for visceral leishmaniasis (VL) which persists in the host macrophages by deactivating its signaling machinery resulting in a critical shift from proinflammatory (Th1) to an anti-inflammatory (Th2) response. The severity of this disease is mainly determined by the production of IL-12 and IL-10 which could be reversed by use of effective immunoprophylactics. In this study we have evaluated the potential of Arabinosylated Lipoarabinomannan (Ara-LAM), a cell wall glycolipid isolated from non pathogenic Mycobacterium smegmatis, in regulating the host effector response via effective regulation of mitogen-activated protein kinases (MAPK) signaling cascades in Leishmania donovani infected macrophages isolated from BALB/C mice. Ara-LAM, a Toll-like receptor 2 (TLR2) specific ligand, was found to activate p38 MAPK signaling along with subsequent abrogation of extracellular signal–regulated kinase (ERKs) signaling. The use of pharmacological inhibitors of p38MAPK and ERK signaling showed the importance of these signaling pathways in the regulation of IL-10 and IL-12 in Ara-LAM pretreated parasitized macrophages. Molecular characterization of this regulation of IL-10 and IL-12 was revealed by chromatin immunoprecipitation assay (CHIP) which showed that in Ara-LAM pretreated parasitized murine macrophages there was a significant induction of IL-12 by selective phosphorylation and acetylation of histone H3 residues at its promoter region. While, IL-10 production was attenuated by Ara-LAM pretreatment via abrogation of histone H3 phosphorylation and acetylation at its promoter region. This Ara-LAM mediated antagonistic regulations in the induction of IL-10 and IL-12 genes were further correlated to changes in the transcriptional regulators Signal transducer and activator of transcription 3 (STAT3) and Suppressor of cytokine signaling 3 (SOCS3). These results demonstrate the crucial role played by Ara-LAM in regulating the MAPK signaling pathway along with subsequent changes in host effector response during VL which might provide crucial clues in understanding the Ara-LAM mediated protection during Leishmania induced pathogenesis.

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.

TLR signaling-mediated differential histone modification at IL-10 and IL-12 promoter region leads to functional impairments in tumor-associated macrophages.

Tumor-associated macrophages (TAM) are severely compromised for the induction of proinflammatory mediators following toll-like receptor (TLR) activation. Here, we reported that the defective TLR response in TAM was due to the malfunctioning of the myeloid differentiation primary response gene 88 (MyD88)-dependent signaling cascade in concert with downregulation of tumor necrosis factor receptor-associated factor (TRAF) 6 and interleukin-1 receptor-associated kinase (IRAK) 1. However, the expression of toll-interleukin1 receptor domain-containing adapter-inducing interferon beta (TRIF) and TRAF 3, which act via the TRIF-dependent pathway of TLR signaling, were found to be unaffected in TAM. Although, TRIF-mediated signal inducers, lipopolysaccharide or poly (I:C), induced high level of extracellular signal-regulated kinase (ERK)-1/2 mitogen-activated protein kinase (MAPK) phosphorylation, but they were failed to induce significant p38MAPK phosphorylation in TAM. Consequently, ERK-1/2-dependent histone phosphorylation at the IL-10 promoter elicited enhanced interleukin (IL)-10 production by TAM. Whereas, the lack of transcription favorable histone phosphorylation at the IL-12 promoter was accompanied with a very low amount of IL-12 expression in TAM. Moreover, ERK-1/2 MAPK activation resulted in enhanced IRAK M induction in TAM, a specific inhibitor of MyD88 pathway. Therefore, for the first time, we decipher an unexplored TLR signaling in TAM where ERK-1/2 activation in a MyD88-independent pathway results in transcription favorable histone modification at the IL-10 promoter region to enhance IL-10-mediated immunosuppression. Additionally, by enhancing IRAK M induction, it also polarizes TAM toward a more immunosuppressive form.

Mycobacterium indicus pranii (Mw)-mediated protection against visceral leishmaniasis: involvement of TLR4 signalling

OBJECTIVES The aim of this study was to characterize the antileishmanial activity of heat-killed Mycobacterium indicus pranii (Mw) alone or in combination with a subtoxic dose of amphotericin B [AMB(st)]. METHODS Mw- and Mw + AMB(st)-mediated antileishmanial activity was evaluated by microscopic counting of intracellular amastigotes in Giemsa-stained macrophages and real-time PCR analysis of inducible nitric oxide synthase (iNOS) expression and measurement of nitric oxide generation by Griess reagent. The relationship between Mw and Toll-like receptor 4 (TLR4) signalling was studied by fluorescence-activated cell sorting, western blot and confocal microscopy. The effect of Mw alone or in combination with AMB(st) on the expression and production of interleukin (IL)-12, tumour necrosis factor-α, IL-10 and transforming growth factor-β was analysed by real-time PCR and ELISA, respectively. RESULTS Mw treatment alone or with AMB(st) caused a significant increase in TLR4 expression of L. donovani-infected macrophages along with the activation of TLR4 downstream signalling, facilitating active nuclear translocation of nuclear factor κB (NF-κB). These events culminated in the up-regulation of the proinflammatory response, which was abrogated by treatment with TLR4-specific small-interfering RNA. In addition, this study demonstrates that this chemoimmunotherapeutic strategy confers protection against leishmanial pathogenesis via TLR4-dependent counter-regulation of inducible nitric oxide synthase (iNOS) and arginase1 activity. CONCLUSIONS These results provide a mechanistic understanding of Mw- or Mw + AMB(st)-mediated protection against leishmanial parasites within host macrophages.