30-OR: Sirtuin 1 Dysfunction Underlies the Pathological Upregulation of NETosis in Diabetes

Activated neutrophils can release their chromatin, forming neutrophil extracellular traps (NETs). Being highly procoagulant and pro-inflammatory, NETs are implicated in conditions that are prevalent in diabetes, such as thrombosis, stroke, and delayed wound healing. While diabetes is found to prime neutrophils for NET formation (NETosis), a knowledge gap persists for years as to its underlying mechanism. Here, we report an important role of Sirtuin 1 (SIRT1) in the modulation of NETosis. We found that Ex-527 (a SIRT1 inhibitor), and SIRT1 knockdown using siRNA, predisposed blood neutrophils isolated from healthy mice and humans, as well as HL-60-derived neutrophils (dHL-60 cells), to NETosis. While neutrophils from mice and humans with diabetes and dHL-60 cells primed in high glucose naturally generated more NETs when compared to their healthy or basal glucose-cultured counterparts, Ex-527 failed to further exacerbate NETosis in these cells, implicating that SIRT1 is dysfunctional in hyperglycemia and diabetes, leading to maximal NETosis even without Ex-527. Cell-based assays reflected that SIRT1 acts upstream of peptidylarginine deiminase 4 (PAD4, an enzyme critical for NETosis), and suppresses NETosis via PAD4 inhibition in healthy neutrophils and basal glucose-cultured dHL-60 cells, but not neutrophils isolated from mice and humans with diabetes, nor dHL-60 cells primed in high glucose. Remarkably, acute treatment using resveratrol (a polyphenol known to activate SIRT1) normalized NETosis in neutrophils from mice and humans with diabetes, alongside a concomitant reduction in PAD4 activity. To conclude, SIRT1 serves as an endogenous suppressor of NETosis in health and becomes defective in hyperglycemia and diabetes, resulting in excessive NETosis. Reactivation of SIRT1 using resveratrol effectively abrogated NETosis by inhibiting PAD4 activity. Revitalizing SIRT1 could be a novel therapeutic strategy in alleviating NET-mediated inflammation and diabetic complications. Disclosure L.Wang: None. R.Dalan: None. S.Wong: None. Funding Nanyang Assistant Professorship; Singapore Ministry of Education Academic Research Fund Tier 1 (RG25/20); Nanyang President's Graduate Scholarship