Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Monoclonal/therapeutic use , COVID-19 Drug Treatment , Receptors, Interleukin-6/antagonists & inhibitors , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Female , Humans , Male , Meta-Analysis as Topic , Randomized Controlled Trials as TopicABSTRACT
Post translational modifications, ubiquitination and its reversal by deubiquitination play an important role in regulating innate immune system. USP12 is a poorly studied deubiquitinase reported to regulate T-cell receptor signalling however the functional role of USP12 in macrophages, the principal architects of inflammation, is unknown. Thus, in this study we probed the involvement of USP12 in macrophage mediated inflammatory responses using bacterial endotoxin, LPS, as the model system. Here, we observed that the expression of USP12 was altered in time dependent manner in LPS stimulated RAW 264.7 macrophages at both mRNA and protein levels as revealed by qPCR and western blot analysis, respectively. Further analysis showed that LPS reduced the levels of Sp1 which enhanced the transcriptional levels of USP12. We observed that siRNA mediated ablation of USP12 expression in mouse macrophages suppressed the induction of LPS-induced iNOS and IL-6 expression but failed to alter IFN-ß synthesis, oxidative stress and phagocytic ability of macrophages. Mechanistic analysis suggest that USP12 may be required for the activation of NFκB pathway as knockdown of USP12 reduced the inhibitory phosphorylation of IκBα, a well characterized inhibitor of NFκB nuclear translocation. Further, USP12 was observed to be required for LPS elicited phosphorylation of ERK1/2 and p38. Collectively, our data suggest that USP12 may be a key mediator of LPS stimulated macrophage responses.
Subject(s)
Endopeptidases/metabolism , Lipopolysaccharides/toxicity , Macrophages/drug effects , Macrophages/metabolism , NF-KappaB Inhibitor alpha/antagonists & inhibitors , Animals , Endopeptidases/deficiency , Endopeptidases/genetics , Gene Expression , Gene Knockdown Techniques , Inflammation/metabolism , Interferon-gamma/pharmacology , Macrophage Activation/drug effects , Macrophage Activation/immunology , Macrophage Activation/physiology , Macrophages/immunology , Mice , Nitric Oxide Synthase Type II/metabolism , Phosphorylation , RAW 264.7 Cells , RNA, Small Interfering/genetics , Ubiquitin ThiolesteraseABSTRACT
Chronic inflammatory diseases such as insulin resistance, Type 2 diabetes, neurodegenerative diseases etc., are shown to be caused due to imbalanced activation states of macrophages. MicroRNAs which are transcriptional/post-transcriptional regulators of gene expression drive several pathophysiological processes including macrophage polarization. However the functional role of microRNAs in regulating inflammation induced insulin resistance is ill defined. In our current study we observed that the expression of miR-712 was reduced in macrophages exposed to LPS and IFN-γ. Ectopic expression of miR-712 in RAW 264.7 mouse macrophages impaired the expression of iNOS protein and secretion of pro-inflammatory cytokines such as TNF-α, IL-6 and IFN-ß which in turn led to improved insulin stimulated glucose uptake in co-cultured L6 myoblasts. Mechanistically, we identified that miR-712 targets the 3'UTR of a potent inflammatory gene LRRK2 and dampens the phosphorylation of p38 and ERK1/2 kinases. Taken together, our data underscore the regulatory role of miR-712 in restoring insulin stimulated glucose uptake by myoblasts through down-regulating macrophage mediated inflammatory responses.
