IFNγ insufficiency during mouse intra-vaginal Chlamydia trachomatis infection exacerbates alternative activation in macrophages with compromised CD40 functions.

Chlamydia trachomatis (C.tr), an obligate intracellular pathogen, causes asymptomatic genital infections in women and is a leading cause of preventable blindness. We have developed in vivo mouse models of acute and chronic C. trachomatis genital infection to explore the significance of macrophage-directed response in mediating immune activation/suppression. Our findings reveal that during chronic and repeated C. trachomatis infections, Th1 response is abated while Treg response is enhanced. Additionally, an increase in exhaustion (PD1, CTLA4) and anergic (Klrg3, Tim3) T cell markers is observed during chronic infection. We have also observed that M2 macrophages with low CD40 expression promote Th2 and Treg differentiation leading to sustained C. trachomatis genital infection. Macrophages infected with C. trachomatis or treated with supernatant of infected epithelial cells drive them to an M2 phenotype. C. trachomatis infection prevents the increase in CD40 expression as observed in western blots and flow cytometric analysis. Insufficient IFNγ, as observed during chronic infection, leads to incomplete clearance of bacteria and poor immune activation. C. trachomatis decapacitates IFNγ responsiveness in macrophages via hampering IFNγRI and IFNγRII expression which can be correlated with poor expression of MHC-II, CD40, iNOS and NO release even following IFNγ supplementation. M2 macrophages during C. trachomatis infection express low CD40 rendering immunosuppressive, Th2 and Treg differentiation which could not be reverted even by IFNγ supplementation. The alternative macrophages also harbour high bacterial load and are poor responders to IFNγ, thus promoting immunosuppression. In summary, C. trachomatis modulates the innate immune cells, attenuating the anti-chlamydial functions of T cells in a manner that involves decreased CD40 expression on macrophages.

Host Lipid Manipulation by Intracellular Bacteria: Moonlighting for Immune Evasion.

Lipids are complex organic molecules that fulfill energy demands and sometimes act as signaling molecules. They are mostly found in membranes, thus playing an important role in membrane trafficking and protecting the cell from external dangers. Based on the composition of the lipids, their fluidity and charge, their interaction with embedded proteins vary greatly. Bacteria can hijack host lipids to satisfy their energy needs or to conceal themselves from host cells. Intracellular bacteria continuously exploit host, from their entry into host cells utilizing host lipid machinery to exiting through the cells. This acquisition of lipids from host cells helps in their disguise mechanism. The current review explores various mechanisms employed by the intracellular bacteria to manipulate and acquire host lipids. It discusses their role in manipulating host membranes and the subsequence impact on the host cells. Modulating these lipids in macrophages not only serve the purpose of the pathogen but also modulates the macrophage energy metabolism and functional state. Additionally, we have explored the intricate pathogenic relationship and the potential prospects of using this knowledge in lipid-based therapeutics to disrupt pathogen dominance.

Chlamydia trachomatis infection co-operatively enhances HPV E6-E7 oncogenes mediated tumorigenesis and immunosuppression.

Chlamydia trachomatis and human papilloma virus (HPV) are the two most common sexually transmitted infections among women. HPV infection can increase the risk of cervical cancer and infertility while C. trachomatis induces pelvic inflammatory disease. Here, we elucidate the molecular conundrum of the co-infection of HPV and C. trachomatis infection and their outcome with respect to cervical cancer. HPV infection was mimicked by overexpression of HPV 16 E6-E7 or using human cervical cell lines SiHa and C33a (with and without HPV 16 respectively). HPV transfected co-infection increased cell proliferation and resistance to H202 and TNFα-induced cell death compared to individual infections. These changes are brought by alteration in the cell cycle proteins (CDK2, CDK6 and Bcl2) and thus increasing the stemness of the epithelial cells as observed by increased colony forming units and CD133 expression. The co-infection also induces change in the mRNA levels of cells which are involved in mesenchymal phenotype. C. trachomatis in presence of E6-E7 overexpression caused cervical epithelial neoplasm in mice with increased Ki67 expression and decreased P53 levels. Stem cell marker, CD133 expression also increased in the cervical tissues of both infected and co-infected group of mice. The cells obtained from the cervix were able to grow continuously in ex vivo cultures. All these results indicate the co-existence of the C. trachomatis and HPV 16 might increase the risk of cervical cancer.

Design, synthesis, characterization and anticancer activity evaluation of deoxycholic acid-chalcone conjugates.

A series of deoxycholic acid-chalcone amides were synthesised and tested against the human lung cancer cell line, A549 and the cervical cancer cell line, SiHa. Among the synthesised deoxycholic acid-chalcone conjugates, some conjugates showed encouraging results as anticancer agents with good in vitro activity. More precisely, deoxycholic acid-chalcone conjugates 4b (IC50: 0.51 µM) and 4e (IC50: 0.84 µM) having 2­nitrophenyl and 3,4,5­trimethoxyphenyl groups exhibited a good activity against human cancer cell-line SiHa and while 4d (IC50: 0.25 µM) and 4b (IC50: 1.71 µM) showed better activity against A549 lung cancer cell line with respect to deoxycholic acid and chalcones. The anticancer activity of the bile acid conjugated chalcones was more than the activity of chalcone and deoxycholic acid alone. The results indicate that a bile acid conjugate strategy may be beneficial in improving the biological activity of chalcone derivatives. The enhanced activity of certain compounds may be due to their increased bioavailability.

Saga of monokines in shaping tumour-immune microenvironment: Origin to execution.

Cellular communication mediated by cytokines is an important mechanism dictating immune responses, their cross talk and final immune output. Cytokines play a major role in dictating the immune outcome to cancer by regulating the events of development, differentiation and activation of innate immune cells. Cytokines are pleiotropic in nature, hence understanding their role individually or as member of network cytokines is critical to delineate their role in tumour immunity. Tumour systemically manipulates the immune system to evade and escape immune recognition for their uncontrollable growth and metastasis. The developing tumour comprise a large and diverse set of myeloid cells which are vulnerable to manipulation by the tumour-microenvironment. The innate immune cells of the monocytic lineage skew the fate of the adaptive immune cells and thus dictating cancer elimination or progression. Targeting cells at tumour cite is preposterous owing to their tight network, poor reach and abundance of immunosuppressive mechanisms. Monocytic lineage-derived cytokines (monokines) play crucial role in tumour regression or progression by either directly killing the tumour cells with TNFα or promoting its growth by TGFß. In addition, the monokines like IL-12, IL-1ß, IL-6, IL-10 and TGFß direct the adaptive immune cells to secrete anti-tumour cytokines, TNFα, IFNγ, perforin and granzyme or pro-tumour cytokines, IL-10 and TGFß. In this review, we elucidate the roles of monokines in dictating the fate of tumour by regulating responses at various stages of generation, differentiation and activation of immune cells along with the extensive cross talk. We have attempted to delineate the synergy and antagonism of major monokines among themselves or with tumour-derived or adaptive immune cytokines. The review provides an update on the possibilities of placing monokines to potential practical use as cytokine therapy against cancer.

Insights into inflammasome regulation: cellular, molecular, and pathogenic control of inflammasome activation.

Maintenance of immune homeostasis is an intricate process wherein inflammasomes play a pivotal role by contributing to innate and adaptive immune responses. Inflammasomes are ensembles of adaptor proteins that can trigger a signal following innate sensing of pathogens or non-pathogens eventuating in the inductions of IL-1ß and IL-18. These inflammatory cytokines substantially influence the antigen-presenting cell's costimulatory functions and T helper cell differentiation, contributing to adaptive immunity. As acute and chronic disease conditions may accompany parallel tissue damage, we analyze the critical role of extracellular factors such as cytokines, amyloids, cholesterol crystals, etc., intracellular metabolites, and signaling molecules regulating inflammasome activation/inhibition. We develop an operative framework for inflammasome function and regulation by host cell factors and pathogens. While inflammasomes influence the innate and adaptive immune components' interplay modulating the anti-pathogen adaptive immune response, pathogens may target inflammasome inhibition as a survival strategy. As trapped between health and diseases, inflammasomes serve as promising therapeutic targets and their modus operandi serves as a scientific rationale for devising better therapeutic strategies.

Berberine mediates tumor cell death by skewing tumor-associated immunosuppressive macrophages to inflammatory macrophages.

BACKGROUND: Berberine is a plant-derived alkaloid with potent anti-cancer activities. Berberine may redirect the tumor-promoting immunosuppressive M2 macrophages, to tumoricidal activated M1 macrophages. But such an anti-tumor function remains to be demonstrated. HYPOTHESIS: Polarization of macrophages to an immunosuppressive phenotype within the tumor microenvironment promotes tumor growth and contributes to resistance to chemotherapy. We examined if berberine would target macrophage polarization to reinstate anti-tumor immune response. STUDY DESIGN: Using a B16F10 mouse melanoma model, we assessed berberine-induced re-polarization of immunosuppressive M2 macrophages to anti-tumor M1 macrophages and subsequent T-cell activation within the immunosuppressive tumor microenvironment. METHODS: The B16F10 culture supernatant along with tumor antigen was used as tumor mimicking conditioned medium (CM). The bone marrow-derived macrophages were cultured in CM for 5 days. The CM-induced skewing of macrophages to M2-like phenotype was confirmed by flow cytometry and ELISA. The T-cells were co-cultured with macrophages to decipher the effect of berberine on T-cell differentiation. In vivo efficacy of berberine was analyzed using melanoma model of solid tumor. RESULTS: Berberine inhibited rIL-6-induced STAT-3 phosphorylation and IL-10 release from B16F10 cells. It enhanced tumor antigen-induced IL-1ß, IL-12 and TNFα, but suppressed IL-6 and TGF-ß release. Berberine significantly prevented the tumor antigen-mediated IL-10-enhanced IL-6 and TGF-ß expression. The CM skewed the bone marrow-derived macrophages to CD206-high but MHC-II-low M2-like tumor-associated macrophages. Berberine partially prevented the generation of these macrophages and was associated with reduced C/EBPß and Egr2 mRNA expression and lowered IL-10 and TGF-ß production. Berberine significantly reduced Arginase-1 expression in CM-treated M1 and M2-like macrophages. Berberine increased MHC-II and CD40 expression on the macrophages augmenting the CTL activity and the number of IFNγ-producing CD4+ T-cells. Berberine significantly lowered tumor volume, weight and enhanced the frequency of M1-like macrophages in mice. CONCLUSION: These data indicate that berberine interferes with pro-tumor macrophage polarization and IL-10 and TGF-ß release but restores Tcell anti-tumor cytotoxicity in the tumor microenvironment.

Caspase-1 inhibition by YVAD generates tregs pivoting IL-17 to IL-22 response in ß-glucan induced airway inflammation.

BACKGROUND: During Aspergillus fumigatus mediated lung inflammation, NLRP3 inflammasome is rapidly activated that aggravates IL-1ß production contributing to lung inflammation. Previously, we have shown the protective role of SYK-1 inhibition in inhibiting inflammasome activation during lung inflammation. In the current manuscript, we explored the protective role of direct caspase-1 inhibition during ß-glucan-induced lung inflammation. METHODS: We have mimicked the lung inflammation by administering intranasal ß-glucan in mice model. YVAD was used for caspase-1 inhibition. RESULTS: We have shown that caspase-1 inhibition by YVAD did not alter inflammasome independent inflammatory cytokines, while it significantly reduced inflammasome activation and IL-1ß secretion. Caspase-1 inhibited bone marrow derived dendritic cells (BMDCs), co-cultured with T cells showed decreased T-cell proliferation and direct them to secrete high TGF-ß and IL-10 compared to the T cells co-cultured with ß-glucan primed dendritic cells. Caspase-1 inhibition in BMDCs also induced IL-22 secretion from CD4+T cells. Caspase-1 inhibition in intranasal ß-glucan administered mice showed decreased tissue damage, immune cell infiltration and IgA secretion compared to control mice. Further, splenocytes challenged with ß-glucan show high IL-10 secretion and increased FOXp3 and Ahr indicating an increase in regulatory T cells on caspase-1 inhibition. CONCLUSION: Caspase-1 inhibition can thus be an attractive target to prevent inflammation mediated tissue damage during Aspergillus fumigatus mouse model and can be explored as an attractive therapeutic strategy.HIGHLIGHTSCaspase-1 inhibition protects lung damage from inflammation during ß-glucan exposureCaspase-1 inhibition in dendritic cells decreases IL-1ß production resulting in decreased pathogenic Th17Caspase-1 inhibition promotes regulatory T cells thereby inhibiting lung inflammation.

microRNAs (miR 9, 124, 155 and 224) transdifferentiate mouse macrophages to neurons.

Development is an irreversible process of differentiating the undifferentiated cells to functional cells. Brain development involves generation of cells with varied phenotype and functions, which is limited during adulthood, stress, damage/degeneration. Cellular reprogramming makes differentiation reversible process with reprogramming somatic/stem cells to alternative fate with/without stem cells. Exogenously expressed transcription factors or small molecule inhibitors have driven reprogramming of stem/somatic cells to neurons providing alternative approach for pre-clinical/clinical testing and therapeutics. Here in, we report a novel approach of microRNA (miR)- induced trans-differentiation of macrophages (CD11b high) to induced neuronal cells (iNCs) (neuronal markershigh- Nestin, Nurr1, Map2, NSE, Tubb3 and Mash1) without exogenous use of transcription factors. miR 9, 124, 155 and 224 successfully transdifferentiated macrophages to neurons with transient stem cell-like phenotype. We report trans differentiation efficacy 18% and 21% with miR 124 and miR 155. in silico(String 10.0, miR gator, mESAdb, TargetScan 7.0) and experimental analysis indicate that the reprogramming involves alteration of pluripotencygenes like Oct4, Sox2, Klf4, Nanog and pluripotency miR, miR 302. iNCs also shifted to G0 phase indicating manipulation of cell cycle by these miRs. Further, CD133+ intermediate cells obtained during current protocol could be differentiated to iNCs using miRs. The syanpsin+ neurons were functionally active and displayed intracellular Ca+2 evoke on activation. miRs could also transdifferentiate bone marrow-derived macrophages and peripheral blood mononuclear cells to neuronal cells. The current protocol could be employed for direct in vivo reprogramming of macrophages to neurons without teratoma formation for transplantation and clinical studies.

Spleen tyrosine kinase inhibition ameliorates airway inflammation through modulation of NLRP3 inflammosome and Th17/Treg axis.

Repeated exposure to the fungal pathogen Aspergillus fumigates triggers spleen tyrosine kinase (SYK) signalling through dectin-1 activation, which is associated with deleterious airway inflammation. ß-Glucan-induced dectin-1 signalling activates the NLRP3 inflammasome, which in turn rapidly produces IL-1ß, a master regulator of inflammation. IL-1ß expression results in Th17/Treg imbalance, pulmonary inflammation, and bystander tissue injury. This study reports that 3,4 methylenedioxy-ß-nitrostyrene (MNS), a potent SYK inhibitor, markedly decreased the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokines in vitro. Furthermore, SYK inhibition markedly decreased ß-glucan-induced IL-1ß expression, suggesting that SYK is indispensable for NLRP3 inflammasome activation. Decreased IL-1ß expression correlated with reduced Th17 response and enhanced immunosuppressive Treg response. Notably, SYK inhibition ameliorated inflammation caused by repeated intranasal ß-glucan challenge in BALB/C mice. SYK inhibition also restored the Th17/Treg balance via decreased Th17 and increased Treg responses, as evidenced by decreased IL-17 and ror-γ levels. Additionally, inhibition of SYK increased IL-10 secreting CD4+FOXP3+ T cells that accompanied reduced T cell proliferation. Decreased IgA in the Bronchoalveolar lavage (BAL) fluid and serum also indicated the immunosuppressive potential of SYK inhibition. Histopathology data revealed that repeated ß-glucan challenge caused substantial pulmonary damage, as indicated by septal thickening and interstitial lymphocytic, neutrophil and granulocyte recruitment. These processes were effectively prevented by SYK inhibition, resulting in lung protection. Collectively, our findings suggest that SYK inhibition ameliorates dectin-1- mediated detrimental pulmonary inflammation and subsequent tissue damage. Therefore, SYK can be a new target gene in the therapeutic approach against fungal induced airway inflammation.