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Singh V.,Pennsylvania State University | Yeoh B.S.,Pennsylvania State University | Chassaing B.,Georgia State University | Zhang B.,Georgia State University | And 6 more authors.
CMGH Cellular and Molecular Gastroenterology and Hepatology | Year: 2016

Background & Aims: Lipocalin 2 (Lcn2) is a multifunctional innate immune protein whose expression closely correlates with the extent of intestinal inflammation. However, whether Lcn2 plays a role in the pathogenesis of gut inflammation is unknown. Herein, we investigated the extent to which Lcn2 regulates inflammation and gut bacterial dysbiosis in mouse models of IBD. Methods: Lcn2 expression was monitored in murine colitis models and upon microbiota ablation/restoration. Wild-type (WT) and Lcn2 knockout (Lcn2KO) mice were analyzed for gut bacterial load, composition by 16S ribosomal RNA gene pyrosequencing, and their colitogenic potential by co-housing with interleukin (Il)10KO mice. Acute (dextran sodium sulfate) and chronic (IL10R neutralization and T-cell adoptive transfer) colitis were induced in WT and Lcn2KO mice with or without antibiotics. Results: Lcn2 expression was dramatically induced on inflammation and was dependent on the presence of a gut microbiota and MyD88 signaling. Use of bone marrow-chimeric mice showed that nonimmune cells are the major contributors of circulating Lcn2. Lcn2KO mice showed increased levels of entA-expressing gut bacteria burden, and, moreover, a broadly distinct bacterial community relative to WT littermates. Lcn2KO mice developed highly colitogenic T cells and showed exacerbated colitis on exposure to DSS or neutralization of IL10. Such exacerbated colitis could be prevented by antibiotic treatment. Moreover, exposure to the microbiota of Lcn2KO mice, via cohousing, resulted in severe colitis in Il10KO mice. Conclusions: Lcn2 is a bacterially induced, MyD88-dependent protein that plays an important role in gut homeostasis and a pivotal role on challenge. Hence, therapeutic manipulation of Lcn2 levels may provide a strategy to help manage diseases driven by alteration of the gut microbiota. © 2016 The Authors.

Tiwari R.L.,Industrial Research Central Drug Research Institute | Singh V.,Industrial Research Central Drug Research Institute | Singh A.,Industrial Research Central Drug Research Institute | Rana M.,Industrial Research Central Drug Research Institute | And 4 more authors.
Journal of Lipid Research | Year: 2014

This study examined the role of interleukin (IL)-1 receptor-associated kinase (IRAK) and protein kinase C (PKC) in oxidized LDL (Ox-LDL)-induced monocyte IL-1 β production. In THP1 cells, Ox-LDL induced time-dependent secretory IL-1 β and IRAK1 activity; IRAK4, IRAK3, and CD36 protein expression; PKC β -JNK1 phosphorylation; and AP-1 activation . IRAK1/4 siRNA and inhibitor (INH)- attenuated Ox-LDL induced secreted IL-1 β and pro-IL-1 β mRNA and pro-IL-1 β and mature IL-1 β protein expression, respectively. Diphenyleneiodonium chloride (NADPH oxidase INH) and N -acetylcysteine (free radical scavenger) attenuated Ox-LDL-induced reactive oxygen species generation, caspase-1 activity, and pro-IL-1 β and mature IL-1 β expression. Ox-LDL-induced secretory IL-1 β production was abrogated in the presence of JNK INH II, Tanshinone IIa, Ro-31-8220, Go6976, Rottlerin, and PKC β siRNA. PKC β siRNA attenuated the Ox-LDL-induced increase in IRAK1 kinase activity, JNK1 phosphorylation, and AP-1 activation. In THP1 macrophages, CD36, toll-like receptor (TLR)2, TLR4, TLR6, and PKC β siRNA prevented Ox-LDLinduced PKC β and IRAK1 activation and IL-1 β production. Enhanced Ox-LDL and IL-1 β in systemic inflammatory response syndrome (SIRS) patient plasma demonstrated positive correlation with each other and with disease severity scores. Ox-LDL-containing plasma induced PKC β and IRAK1 phosphorylation and IL-1 β production in a CD36-, TLR2-, TLR4-, and TLR6-dependent manner in primary human monocytes . Results suggest involvement of CD36, TLR2, TLR4, TLR6, and the PKC β -IRAK1-JNK1- AP-1 axis in Ox-LDL-induced IL-1 β production. -Tiwari, R. L., V. Singh, A. Singh, M. Rana, A. Verma, N. Kothari, M. Kohli, J. Bogra, M. Dikshit, and M. K. Barthwal. PKC β - IRAK1 axis regulates oxidized LDL-induced IL-1 β production in monocytes. J. Lipid Res. 2014. 55: 1226 - 1244 . © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Singh N.,Industrial Research Central Drug Research Institute | Yadav M.,Industrial Research Central Drug Research Institute | Singh A.K.,Industrial Research Central Drug Research Institute | Kumar H.,Industrial Research Central Drug Research Institute | And 11 more authors.
Molecular Endocrinology | Year: 2014

The synthetic nuclear bile acid receptor (farnesoid X receptor [FXR]) agonist GW4064 is extensively used as a specific pharmacological tool to illustrate FXR functions. We noticed that GW4064 activated empty luciferase reporters in FXR-deficient HEK-293T cells.Wepostulated that this activity of GW4064 might be routed through as yet unknown cellular targets and undertook an unbiased exploratory approach to identify these targets. Investigations revealed that GW4064 activated cAMP and nuclear factor for activated T-cell response elements (CRE and NFAT-RE, respectively) present on these empty reporters. Whereas GW4064-induced NFAT-RE activation involved rapid intracellular Ca2+ accumulation and NFAT nuclear translocation, CRE activation involved soluble adenylyl cyclase-dependent cAMP accumulation and Ca2+-calcineurin-dependent nuclear translocation of transducers of regulated CRE-binding protein 2. Use of dominant negative heterotrimeric G-protein minigenes revealed that GW4064 caused activation of Gαi/o and Gq/11 G proteins. Sequential pharmacological inhibitor- based screening and radioligand-binding studies revealed that GW4064 interacted with multiple G protein-coupled receptors. Functional studies demonstrated that GW4064 robustly activated H1 and H4and inhibitedH2histamine receptor signaling events.Wealso found that MCF-7 breast cancer cells, reported to undergo GW4064-induced apoptosis in an FXR-dependent manner, did not express FXR, and the GW4064-mediated apoptosis, also apparent in HEK-293T cells, could be blocked by selective histamine receptor regulators. Taken together, our results demonstrate identification of histamine receptors as alternate targets for GW4064, which not only necessitates cautious interpretation of the biological functions attributed to FXR using GW4064 as a pharmacological tool but also provides a basis for the rational designing of new pharmacophores for histamine receptor modulation. © 2014 by the Endocrine Society.

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