Research Projects

Diabetes is associated with increased inflammation and chance of infection. Neutrophils which are specialized type of phagocytic cells use neutrophil extracellular traps (NETs) to trap pathogens and protect against infection. NETs are produced when the neutrophils expel their chromatin into the cytoplasm forming web-like structures to entrap pathogens. In addition to infection, NET formation (NETosis) is also observed in response to ‘sterile’ stimuli such as cytokines, immune complexes, and autoantibodies. In both cases, NETosis is initiated by the activation of its key mediator, protein-arginine deiminase type 4 (PAD4). PAD4 along with other mediators such as myeloperoxidase and neutrophil elastase promote chromatin decondensation prior to expulsion to the cytoplasm. Chromatin decondensation also depends on histone post-translational modifications such as histone citrullination, catalyzed by PAD4. Uncontrolled NETosis has been implicated in the pathogenesis of cancer, autoimmune, and inflammatory diseases including diabetes. However, the detailed underlying molecular mechanisms of NETosis and the role of NETs in both T1DM and T2DM is largely unknown. Our overarching hypothesis is that diabetic conditions augment NETosis via distinct mechanisms in T1DM and T2DM. We will test this hypothesis via three specific aims. In specific Aim1 we will study NOX-dependent and NOX-independent pathways of NETosis involved in T1DM and T2DM by performing NETosis assays followed by gene expression, western blot and immunofluorescence analysis using NETosis markers. In specific Aim2, we will study PAD4 dependent and independent mechanisms of chromatin decondensation in diabetes induced NETosis. We will use PAD4 knockout mice, PAD4 catalytically inactive mutant, PAD4 depleted neutrophils to understand PAD4 role as well as to identify PAD4 independent mechanisms. To investigate the role of PAD4 in initiating NETosis epigenetic program, RNA-seq and ATAC-seq will be performed in PAD4-depleted HL-60 cells and PAD4 knockout mice. Further, we will employ PAD4-immunoprecipitation followed by mass-spectrometry to identify the PAD4 interactome. In specific Aim3 we will determine whether neddylation, a type of post-translational modification has any roles in NETosis. We will use neddylation-specific antibodies, mass-spectrometry, and other biochemical assays. The proposed study will identify distinct signalling cascades that activate PAD4-mediated enhanced NETosis in T1DM and T2DM. Moreover, it will discover additional key players involved in NETosis apart from PAD4 including chromatin remodellers and post-translational modifications such as neddylation. Successful completion of the proposal will identify mechanisms of enhanced NETosis in diabetes, role of PAD4 and novel factors that can be targeted in addition to PAD4 to prevent excessive NETosis. These studies could lead to therapeutic strategies to tackle onset of diabetes-associated complications in diabetic patients.