Myotubularin-related protein 6 is an ion channel-associated pro-leishmanial phosphatase.

Residing obligatorily as amastigotes within the mammalian macrophages, the parasite Leishmania donovani inflicts the potentially fatal, globally re-emerging disease visceral leishmaniasis (VL) by altering intracellular signaling through kinases and phosphatases. Because the phosphatases that modulate the VL outcome in humans remained unknown, we screened a human phosphatase siRNA-library for anti-leishmanial functions in THP-1, a human macrophage-like cell line. Of the 251 phosphatases, the screen identified the Ca++-activated K+-channel-associated phosphatase myotubularin-related protein-6 (MTMR6) as the only phosphatase whose silencing reduced parasite load and IL-10 production in human macrophages. Virulent, but not avirulent, L. donovani infection increased MTMR6 expression in macrophages. As virulent L. donovani parasites expressed higher lipophosphoglycan, a TLR2-ligand, we tested the effect of TLR2 stimulation or blockade on MTMR6 expression. TLR1/TLR2-ligand Pam3CSK4 enhanced, but TLR2 blockade reduced, MTMR6 expression. L. donovani infection of macrophages ex vivo increased, but miltefosine treatment reduced, MTMR6 expression. Corroboratively, compared to endemic controls, untreated VL patients had higher, but miltefosine-treated VL patients had reduced, MTMR6 expression. The phosphatase siRNA-library screening thus identified MTMR6 as the first TLR2-modulated ion channel-associated phosphatase with significant implications in VL patients and anti-leishmanial functions.

Lupeol and amphotericin B mediate synergistic anti-leishmanial immunomodulatory effects in Leishmania donovani-infected BALB/c mice.

Leishmania donovani, a protozoan parasite, inflicts the disease Visceral leishmaniasis (VL) Worldwide. The only orally bioavailable drug miltefosine is toxic, whereas liposomal amphotericin B (AmpB) is expensive. Lupeol, a triterpenoid from Sterculia villosa bark, was exhibited immunomodulatory and anti-leishmanial activity in experimental VL. Herein, we evaluated synergism between sub-optimum dose of AmpB and lupeol in anti-leishmanial and immunomodulatory effects in L. donovani-infected BALB/c mice. We observed that a combination of sub-optimum dose of lupeol and AmpB significantly reduced the hepatic and splenic parasitic burden accompanied by enhanced nitric oxide production, robust induction of Th1 cytokines (IL-12 and IFN-γ) but suppressed Th2 cytokine (IL-10 and TGF- ß) production. The treatment with the lupeol-AmpB combination enhanced p38mitogen-activated protein kinase (p38MAPK), but reduced extracellular signal-related kinase (ERK-1/2), phosphorylation and up-regulated pro-inflammatory response. The present work thus indicates a lupeol-AmpB-mediated immunotherapeutic approach for eliminating the parasite-induced immunosuppression.

Immunomodulatory effect of Arabinosylated lipoarabinomannan restrict the progression of visceral leishmaniasis through NOD2 inflammatory pathway: Functional regulation of T cell subsets.

NOD like receptors (NLR) are essential pathogen associated molecular pattern receptors of cytoplasmic origin. During several intracellular parasitic infections NLR played vital role for host protective immune response against the pathogen. Amongst various classes of NLR, NOD1 and NOD2 had been extensively studied and were found to be the most active member of the NLR family. Therefore, we wanted to study the role of NOD1/NOD2 during Leishmania donovani infection and the mechanism behind the utilization of this pathway as a therapeutic approach. Using the infected model of macrophage and BALB/c mice the expression of NOD1 and NOD2 were analysed. Our study showed that NOD2 but not NOD1 has been exploited during experimental VL, leading to the imbalance between Th-1/Th-2 cytokines profile. Over-expression of NOD2 and stimulation with its ligand muramyl dipeptide leads to successful clearance of parasite. During in vivo experiments we found that arabinosylated lipoarabinomannan helps in the restoration of NOD2 and with MDP in combination leads to effective clearance of parasite which rescued host protective immunity and comparatively more effective than Mw and MDP combination resulting in increase T cell response. Consequently, our study highlighted the significance of NOD2 during infection the immune-modulations of which can be used as a therapeutic target.

Immunomodulation of dual specificity phosphatase 4 during visceral leishmaniasis.

DUSP4, an inducible protein has a substrate specificity toward ERK1/2, a component of MAP kinase which is enhanced during Leishmania infection. The DUSP4-/- mice show increased susceptibility towards the infection caused by Toxoplasma gondii and Leishmania mexicana. These observations emphatically established the fact that unlike DUSP1, DUSP4 has host protective role. In our study, it has been Leishmania donovani, the causative agent of visceral leishmaniasis (VL) significantly reduced the expression of DUSP4 during infection. In order to find out the host protective role of DUSP4 in macrophages during VL, we silenced DUSP4 prior to infection and the parasite number within macrophage was counted. Under DUSP4 knock-down condition, phosphorylation of p38 MAPK and generation of pro-inflammatory response like IL-12, TNF-α, and iNOS was decreased significantly. Silencing DUSP4 promoted the phosphorylation of ERK1/2 and the generation of anti-inflammatory response like- IL-10, TGF-ß with increased Arginase-1 and Cox-2 activity. Glycyrrhizic Acid (GA), an immunomodulator, already known to suppress L. donovani infection, found to up-regulate DUSP4 expression during L. donovani infection. On the other hand, GA failed to increase Th1 cytokine production and decrease Th2 response during DUSP4 knock-down condition suggesting the key role of DUSP4 while providing protection during L. donovani infection.

Mycobacterium indicus pranii (Mw)-mediated protection against visceral leishmaniasis by reciprocal regulation of host dual-specificity phosphatases.

Leishmania donovani resides within the host macrophages by dampening host defence mechanisms and thereby it modulates the host cell functions for its survival. Multiple host cell factors compete during the interplay between the host and the parasite. Roles for dual-specificity phosphatases (DUSPs) are implicated in various pathological conditions. However, the reciprocity of these DUSPs was unknown in L. donovani infection in a susceptible model. Here, we show that Mycobacterium indicus pranii (Mw), an immunomodulator, reciprocally regulates DUSP1 and DUSP6 through the TLR4 pathway. Association of PKC-ß with DUSP6 increases after Mw treatment resulting in decreased IL-10, phosphorylation of ERK1/2 and Arginase-1, whereas Mw treatment decreases the association between PKC-ε and DUSP1 resulting in increased IL-12, phosphorylation of p38 and inducible nitric oxide synthase expression. Silencing of DUSP1 or over-expression of DUSP6 in L. donovani-infected BALB/c mice decreases the parasite burden by inducing IL-12 and reducing IL-10 production. Therefore, we identify DUSP1 and DUSP6 as therapeutic targets, functions of which could be favourably modulated by Mw during L. donovani infection.

Mycobacterium indicus pranii (MIP) mediated host protective intracellular mechanisms against tuberculosis infection: Involvement of TLR-4 mediated signaling.

Mycobacterium tuberculosis infection inflicts the disease Tuberculosis (TB), which is fatal if left untreated. During M. tuberculosis infection, the pathogen modulates TLR-4 receptor down-stream signaling, indicating the possible involvement of TLR-4 in the regulation of the host immune response. Mycobacterium indicus pranii (MIP) possesses immuno-modulatory properties which induces the pro-inflammatory responses via induction of TLR-4-mediated signaling. Here, we observed the immunomodulatory properties of MIP against tuberculosis infection. We have studied the detailed signaling mechanisms employed by MIP in order to restore the host immune response against the in vitro tuberculosis infection. We observed that in infected macrophages MIP treatment significantly increased the TLR-4 expression as well as activation of its downstream signaling, facilitating the activation of P38 MAP kinase. MIP treatment was able to activate NF-κB via involvement of TLR-4 signaling leading to the enhanced pro-inflammatory cytokine and NO generation in the infected macrophages and generation of protective immune response. Therefore, we may suggest that, TLR4 may represent a novel therapeutic target for the activation of the innate immune response during Tuberculosis infection.

SLA-PGN-primed dendritic cell-based vaccination induces Th17-mediated protective immunity against experimental visceral leishmaniasis: a crucial role of PKCß.

Emergence of drug resistance during visceral leishmaniasis (VL) is a major obstacle imposed during successful therapy. An effective vaccine strategy against this disease is therefore necessary. Our present study exploited the SLA (soluble leishmanial antigen) and PGN (peptidoglycan) stimulated bone marrow-derived dendritic cells (DCs) as a suitable vaccine candidate during experimental VL. SLA-PGN-stimulated DCs showed a significant decrease in hepatic and splenic parasite burden, which were associated with increased production of nitric oxide and pro-inflammatory cytokines such as IL-12, IFN-γ and IL-17. Elevated level of IL-17 was accompanied with the generation of more Th17 cells. Further studies on DC provided the evidence that these SLA-PGN-stimulated DCs played an important role in providing necessary cytokines such as IL-6, IL-23 and TGF-ß for the generation of Th17 cells. Interestingly, inhibition of protein kinase C-ß (PKCß) in DCs led to decreased production of Th17 polarizing cytokines, causing reduction of the Th17 population size. Altogether, our finding highlighted the important role of DC-based PKCß in regulation of the function and generation of Th17 cells.