SHIP1 inhibition via 3-alpha-amino-cholestane enhances protection against Leishmania infection.

Leishmania major and L. donovani cause cutaneous leishmaniasis and visceral leishmaniasis, respectively. Available chemotherapies suffer from toxicity, drug-resistance or high cost of production prompting the need for the discovery of new anti-leishmanials. Here, we test a novel aminosteriodal compound- 3-alpha-amino-cholestane [3AC] - that shows selective inhibition of SHIP1, an inositol-5'-phosphate-specific phosphatase with potent effects on the immune system. We report that 3AC-sensitive SHIP1 expression increases in Leishmania-infected macrophages. Treatment of BALB/c mice, a Leishmania-susceptible host, with 3AC increased anti-leishmanial, but reduced pro-leishmanial, cytokines' production and reduced the parasite load in both L. major and L. donovani infections. These findings implicate SHIPi as a potential novel immunostimulant with anti-leishmanial function.

Maternal diesel particle exposure promotes offspring asthma through NK cell-derived granzyme B.

Mothers living near high-traffic roads before or during pregnancy are more likely to have children with asthma. Mechanisms are unknown. Using a mouse model, here we showed that maternal exposure to diesel exhaust particles (DEP) predisposed offspring to allergic airway disease (AAD, murine counterpart of human asthma) through programming of their NK cells; predisposition to AAD did not develop in DEP pups that lacked NK cells and was induced in normal pups receiving NK cells from WT DEP pups. DEP NK cells expressed GATA3 and cosecreted IL-13 and the killer protease granzyme B in response to allergen challenge. Extracellular granzyme B did not kill, but instead stimulated protease-activated receptor 2 (PAR2) to cooperate with IL-13 in the induction of IL-25 in airway epithelial cells. Through loss-of-function and reconstitution experiments in pups, we showed that NK cells and granzyme B were required for IL-25 induction and activation of the type 2 immune response and that IL-25 mediated NK cell effects on type 2 response and AAD. Finally, experiments using human cord blood and airway epithelial cells suggested that DEP might induce an identical pathway in humans. Collectively, we describe an NK cell-dependent endotype of AAD that emerged in early life as a result of maternal exposure to DEP.

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.

TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner.

Ceramide is one of the important cellular components involved in cancer regulation and exerts its pleiotropic role in the protective immune response without exhibiting any adverse effects during malignant neoplasm. Although, the PKCδ-ceramide axis in cancer cells has been an effective target in reduction of cancer, involvement of PKCδ in inducing nephrotoxicity have become a major questionnaire. In the present study, we have elucidated the mechanism by which cisplatin exploits the ceramide to render cancer cell apoptosis leading to the abrogation of malignancy in a PKCδ independent pathway with lesser toxicity. Our study revealed that cisplatin treatment in PKCδ silenced melanoma cells induces ceramide mediated apoptosis. Moreover, cisplatin induced upregulation of the transcription factor IRF1 leading to the induction of the transcriptional activity of the TNFα promoter was evident from the pharmacological inhibition and RNA interference studies. Increased cellular expression of TNFα resulted in an elevated ceramide generation by stimulating acid-sphingomyelinase and cPLA2. Furthermore, reciprocity in the regulation of sphingosine kinase 1 (Sphk1) and sphingosine kinase 2 (Sphk2) during PKCδ independent ceramide generation was also observed during cisplatin treatment. PKCδ inhibited murine melanoma model showed reduction in nephrotoxicity along with tumor regression by ceramide generation. Altogether, the current study emphasized the unexplored signaling cascade of ceramide generation by cisplatin during PKCδ silenced condition, which is associated with increased TNFα generation. Our findings enlightened the detailed mechanistic insight of ceramide mediated signaling by chemotherapeutic drugs in cancer therapy exploring a new range of targets for cancer treatment strategies.

Mycobacterium indicus pranii (Mw) inhibits invasion by reducing matrix metalloproteinase (MMP-9) via AKT/ERK-1/2 and PKCα signaling: A potential candidate in melanoma cancer therapy.

Invasion and metastasis via induction of matrix metalloproteinases are the main causes of death in melanoma cancer. In this study, we investigated the inhibitory effects of heat killed saprophytic bacterium Mycobacterium indicus pranii (Mw) on B16F10 melanoma cell invasion. Mw reported to be an immunomodulator has antitumor activity however, its effect on cancer cell invasion has not been studied. Highly invasive B16F10 melanoma was found sensitive to Mw which downregulated MMP-9 expression. Mw treatment inhibited nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) transcriptional activity and respective DNA binding to MMP-9 promoter. Moreover, Mw also overcame the promoting effects of PMA on B16F10 cell invasion. Mw decreased PMA-induced transcriptional activation of NF-κB and AP-1 by inhibiting phosphorylation of AKT and ERK-1/2. Furthermore, Mw strongly suppressed PMA-induced membrane localization of protein kinase C α (PKCα) since PKCα inhibition caused a marked decrease in PMA-induced MMP-9 secretion as well as AKT/ERK-1/2 activation. These results suggest that Mw may be a promising anti-invasive agent as it blocks tumor growth and inhibits B16F10 cell invasion by reducing MMP-9 activation through inhibition of PKCα/ AKT/ ERK-1/2 phosphorylation and NF-κB/AP-1 activation.

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.

Immunomodulation of host-protective immune response by regulating Foxp3 expression and Treg function in Leishmania-infected BALB/c mice: critical role of IRF1.

Visceral leishmaniasis (VL), caused by a protozoan parasite Leishmania donovani, is still a threat to mankind due to treatment failure, drug resistance and coinfection with HIV. The limitations of first-line drugs have led to the development of new strategies to combat this dreaded disease. Recently, we have shown the immunomodulatory property of Ara-LAM, a TLR2 ligand, against leishmanial pathogenesis. In this study, we have extended our study to the effect of Ara-LAM on regulatory T cells in a murine model of VL. We observed that Ara-LAM-treated infected BALB/c mice showed a strong host-protective Th1 immune response due to reduced IL-10 and TGF-ß production, along with marked decrease in CD4(+) CD25(+) Foxp3(+) GITR(+) CTLA4(+) regulatory T cell (Treg) generation and activation. The reduction in Foxp3 expression was due to effective modulation of TGF-ß-induced SMAD signaling in Treg cells by Ara-LAM. Moreover, we demonstrated that Ara-LAM-induced IRF1 expression in the Treg cells, which negatively regulated foxp3 gene transcription, resulting in the reduced immunosuppressive activity of Treg cells. Interestingly, irf1 gene knockdown completely abrogated the effect of Ara-LAM on Treg cells. Thus, these findings provide detailed mechanistic insight into Ara-LAM-mediated modulation of Treg cells, which might be helpful in combating VL.

Immunomodulation in host-protective immune response against murine tuberculosis through regulation of the T regulatory cell function.

Tuberculosis, caused by the bacteria Mycobacterium tuberculosis, is characterized by an infection in lung and spleen. In the present study, we have elucidated the mechanism by which Mycobacterium indicus pranii renders protection in in vivo Mycobacterium tuberculosis infection. We observed that Mycobacterium indicus pranii treated infected C57BL/6 mice showed a strong host-protective Th1 immune response along with a marked decrease in immunosuppressive cytokines, TGF-ß, and IL-10-secreting CD4(+) T cells. This Mycobacterium indicus pranii mediated decrease in immunosuppressive cytokines was correlated with the reduction in the elevated frequency of CD4(+)CD25(+) T regulatory cells, along with the reduced TGF-ß production from these T regulatory cells in tuberculosis-infected mice. This reduction in the T regulatory cell population was a result of effective modulation of STAT4-STAT5 transcription factor counter-regulation by Mycobacterium indicus pranii, which in turn, reduced the immunosuppressive activity of T regulatory cells. Thus, these findings put forward a detailed mechanistic insight into Mycobacterium indicus pranii mediated regulation of the T regulatory cell functioning during experimental murine tuberculosis, which might be helpful in combating Mycobacterium-induced pathogenesis.

The host-protective effect of arabinosylated lipoarabinomannan against Leishmania donovani infection is associated with restoration of IFN-γ responsiveness.

Visceral leishmaniasis (VL), which is endemic as a major infectious disease in the tropical and subtropical countries, is caused by a protozoan parasite Leishmania donovani. At present, restricted treatment options and lack of vaccines intensify the problem of controlling VL. Therefore, finding a novel immunoprophylactic or therapeutic principle is a pressing need. Here, we report that arabinosylated lipoarabinomannan (Ara-LAM), a TLR2-ligand isolated from Mycobacterium smegmatis, exhibits a strong immunomodulatory property that conferred protection against L. donovani infection. Although, Ara-LAM modulates TLR2 and MAPK signaling, it is not known whether Ara-LAM involves IFN-γ signaling for effective parasite clearance. Because, it is reported that IFN-γ signaling, a principle mediator of NO generation and macrophage and Tcell activation, is hampered during leishmanial pathogenesis. Ara-LAM increases IFN-γ receptor expression and potentiates IFN-γ receptor signaling through JAK-STAT pathway. Moreover, Ara-LAM reciprocally modulates IRF4 and IRF8 expression and reinstates anti-leishmanial Th1 response that eventuates in significantly reduced parasite load in spleen and liver of L. donovani-infected BALB/c mice. IFN-γRα silencing resulted in the suppression of these host-protective mechanisms affected by Ara-LAM. Thus, Ara-LAM-mediated restoration of IFN-γ responsiveness is a novel immuno-modulatory principle for protection against L. donovani susceptible host.

Immune subversion by Mycobacterium tuberculosis through CCR5 mediated signaling: involvement of IL-10.

Tuberculosis is characterized by severe immunosuppression of the host macrophages, resulting in the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates C-C Chemokine Receptor 5 (CCR5) to enhance IL-10 production, indicating the possible involvement of CCR5 in regulation of the host immune response. Here, we found that Mycobacterium infection significantly increased CCR5 expression in macrophages there by facilitating the activation of its downstream signaling. These events culminated in up-regulation of the immunosuppressive cytokine IL-10 production, which was further associated with the down-regulation of macrophage MHC-II expression along with the up-regulation of CCR5 expression via engagement of STAT-3 in a positive feedback loop. Treatment of macrophages with CCR5 specific siRNA abrogated the IL-10 production and restored MHCII expression. While, in vivo CCR5 silencing was also effective for the restoration of host immune responses against tuberculosis. This study demonstrated that CCR5 played a very critical role for the immune subversion mechanism employed by the pathogen.