Cancer Cells Promote Immune Regulatory Function of Macrophages by Upregulating Scavenger Receptor MARCO Expression.

Expression of macrophage receptor with collagenous structure (MARCO) by tumor-associated macrophages is associated with poor prognosis of multiple types of cancer. In this article, we report that cancer cells (e.g., breast cancer and glioblastoma cell lines) can upregulate surface MARCO expression on human macrophages not only via IL-6-induced STAT3 activation but also via sphingosine-1-phosphate receptor (S1PR)-mediated IL-6 and IL-10 expression followed by STAT3 activation. We further found that MARCO ligation induces activation of the MEK/ERK/p90RSK/CREB signaling cascade, leading to IL-10 expression followed by STAT3-dependent PD-L1 upregulation. Such MARCO-induced macrophage polarization is accompanied by increased expression of PPARG, IRF4, IDO1, CCL17, and CCL22. Ligation of surface MARCO can thus result in decreased T cell responses mainly by reduction of their proliferation. Taken together, cancer cell-induced MARCO expression and its intrinsic regulatory function within macrophages are, to our knowledge, new aspects of cancer immune evasion mechanisms that need to be further studied in the future.

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.

Toll-Like Receptor 2 Targeted Rectification of Impaired CD8⁺ T Cell Functions in Experimental Leishmania donovani Infection Reinstates Host Protection.

Leishmania donovani, a protozoan parasite, causes the disease visceral leishmanisis (VL), characterized by inappropriate CD8+ T-cell activation. Therefore, we examined whether the Toll-like Receptor 2 (TLR2) ligand Ara-LAM, a cell wall glycolipid from non-pathogenic Mycobacterium smegmatis, would restore CD8+ T-cell function during VL. We observed that by efficient upregulation of TLR2 signaling-mediated NF-κB translocation and MAPK signaling in CD8+ T-cells (CD25+CD28+IL-12R+IFN-γR+), Ara-LAM triggered signaling resulted in the activation of T-bet, which in turn, induced transcription favourable histone modification at the IFN-γ, perforin, granzyme-B promoter regions in CD8+ T-cells. Thus, we conclude that Ara-LAM induced efficient activation of effector CD8+ T-cells by upregulating the expression of IFN-γ, perforin and granzyme-B in an NF-κB and MAPK induced T-bet dependent manner in VL.

Glycyrrhizic acid-mediated subdual of myeloid-derived suppressor cells induces antileishmanial immune responses in a susceptible host.

CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs), a heterogeneous population of precursor cells, modulate protective immunity against visceral leishmaniasis by suppressing T cell functions. We observed that CD11b(+) Gr1(+) MDSCs, which initially expanded in soluble leishmanial antigen (SLA)-immunized mice and later diminished, suppressed proliferation of T cells isolated from SLA-immunized mice, but to a lesser extent than the case in naive mice. This lesser suppression of MDSCs accompanied the expression of F4/80 and the production of Cox-2, arginase I, nitric oxide, and PGE2. However, with SLA immunization, there was no difference in the expression of interleukin-2 (IL-2) or gamma interferon (IFN-γ) by T cells, in contrast to the case in nonimmunized mice, in which there is an influence. Glycyrrhizic acid (a triterpenoid compound)-mediated inhibition of Cox-2 in myeloid-derived suppressor cells influenced the capacity of T cells to proliferate and the expression of IL-2 and IFN-γ in Leishmania donovani-infected BALB/c mice. Further characterization confirmed that administration of glycyrrhizic acid to L. donovani-infected BALB/c mice results in an impairment of the generation of MDSCs and a reciprocal organ-specific proliferation of IFN-γ- and IL-10-expressing CD4(+) and CD8(+) T cells. Comprehensive knowledge on the Cox-2-mediated regulation of myeloid-derived suppressor cells might be involved in unlocking a new avenue for therapeutic interventions during visceral leishmaniasis.

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 combination of a novel immunomodulator with a regulatory T cell suppressing antibody (DTA-1) regress advanced stage B16F10 solid tumor by repolarizing tumor associated macrophages in situ.

Tumor associated macrophages and tumor infiltrating regulatory T cells greatly hamper host-protective antitumor responses. Therefore, we utilized a novel immunomodulator, heat-killed Mycobacterium indicus pranii (Mw), to repolarize TAM and an agonistic GITR antibody (DTA-1) to reduce intratumoral regulatory T cell frequency for generation of a host-protective antitumor response. Although, the combination of Mw and DTA-1was found to be effective against advanced stage tumors, however, Mw or DTA-1 failed to do so when administered individually. The presence of high level of regulatory T cells abrogated the only Mw induced antitumor functions, whereas only DTA-1 treatment was found to be ineffective due to its inability to induce TAM repolarization in vivo. The combination therapy was found to be effective since DTA-1 treatment reduced the frequency of regulatory T cells to such an extent where they could not attenuate Mw induced TAM repolarization in vivo. Therefore, the combination therapy involving Mw and DTA-1 may be utilized to the success of advanced stage solid tumor immunotherapies.

Regulation of PKC mediated signaling by calcium during visceral leishmaniasis.

Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca2+ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis.

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.

Overexpressed PKCδ downregulates the expression of PKCα in B16F10 melanoma: induction of apoptosis by PKCδ via ceramide generation.

In the present study, we observed a marked variation in the expression of PKCα and PKCδ isotypes in B16F10 melanoma tumor cells compared to the normal melanocytes. Interestingly, the tumor instructed expression or genetically manipulated overexpression of PKCα isotype resulted in enhanced G1 to S transition. This in turn promoted cellular proliferation by activating PLD1 expression and subsequent AKT phosphorylation, which eventually resulted in suppressed ceramide generation and apoptosis. On the other hand, B16F10 melanoma tumors preferentially blocked the expression of PKCδ isotype, which otherwise could exhibit antagonistic effects on PKCα-PLD1-AKT signaling and rendered B16F10 cells more sensitive to apoptosis via generating ceramide and subsequently triggering caspase pathway. Hence our data suggested a reciprocal PKC signaling operational in B16F10 melanoma cells, which regulates ceramide generation and provide important clues to target melanoma cancer by manipulating the PKCδ-ceramide axis.

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.

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.