1712-P: DOC2b Enrichment Mitigates Cytokine-Induced CXCL10 Expression Via Attenuating IKK-NFkB and STAT-1 Signaling in Beta Cells

Introduction & Objective: The reduction of beta-cell dysfunction, and demise hold promise for therapeutic intervention for type 1 diabetes. Previously, we have reported the beneficial role of SNARE-regulatory protein DOC2b in preventing cytokine-induced beta-cell stress and apoptosis. However, the underlying mechanism remains unknown. We hypothesize that DOC2b enrichment mitigates cytokine-induced beta-cell apoptosis via attenuating CXCL10 signaling. Methods: To test the hypothesis, we have used RNA-seq, qPCR, proteomics, biochemical studies, and immuno-confocal microscopy using DOC2b-enriched or -depleted primary islets (human and mouse) and clonal beta-cells challenged with or without proinflammatory cytokines. Results: Human islet RNAseq analysis revealed a significant decline in NFkB signaling pathway in islets with DOC2b enrichment vs control. Our proteomics and co-immunoprecipitation studies identified IKK, NFkB p65 and STAT-1 as binding partners of DOC2b in clonal beta-cells. Western blot studies demonstrated that DOC2b enrichment corresponds with a significant reduction in total and activated levels of IKK, NFkB p65 and STAT-1 proteins in cytokine-stressed human islets. Immunofluorescent confocal microscopy showed DOC2b enriched cytokine-stressed clonal beta-cells to harbor reduced levels of NFkB p65 in the nucleus. In beta-cells, cytokine-induced chemokine ligand CXCL10 expression is known to be regulated via upstream NFkB p65 and STAT-1 mediated signaling. Intriguingly, qPCR and western blot studies revealed marked attenuation of cytokine-induced CXCL10 levels in DOC2b-enriched human islets and clonal beta-cells; by contrast, CXCL10 is elevated in DOC2b-depleted beta cells and mouse islets. Conclusion: These data support a model in which DOC2b protects beta-cells from cytokine stress and apoptosis via attenuation of IKK-NFkB and STAT-1 signaling, thereby reducing CXCL10 expression. Disclosure D. Chatterjee Bhowmick: None. M. Ahn: None. S. Bhattacharya: None. D.C. Thurmond: Consultant; Truebindings. Funding JDRF Postdoctoral fellowship (3-PDF-2020-934-A-N); Diabetes Research Connection grant National Institutes of Health (DK067912, DK112917, and DK102233)