LB 02.3 - Sirtuin 1 as an endogenous inhibitor of NETosis

Wednesday, July 13, 2022

10:45 AM – 11:00 AM

Location: ICC Auditorium

Presentation Format: In-Person

Co-Author(s)

LW

Liang De Wang

PhD Candidate
Lee Kong Chian School of Medicine, Nanyang Technological University Singapore
Singapore, Singapore
CY

Chung Yin Daryl Yuen

Research Assistant
Lee Kong Chian School of Medicine, Nanyang Technological University Singapore
Singapore, Singapore

Presenting Author(s)

Siu Ling Wong, PhD

Nanyang Assistant Professor
Nanyang Technological University Singapore
Singapore, Singapore

Background: Neutrophil extracellular traps (NETs) are implicated in thrombosis, stroke, and delayed wound healing, conditions that are prevalent in diabetes. While diabetes is known to predispose neutrophils to NETosis, a knowledge gap persists for years as to how diabetes upregulates NETosis.

Aims: The study sought to dissect the dysregulated pathways that exacerbates NETosis in diabetes.

Methods: NETosis propensity was examined in blood neutrophils freshly isolated from humans and mice, as well as differentiated HL-60 cells (dHL-60 cells), by immunofluorescence microscopy. The role of Sirtuin 1 (SIRT1) was examined by targeted knockdown via siRNA transfection in dHL-60 cells.

Results: Screening of anti-aging compounds showed that pharmacological inhibition of SIRT1 using Ex-527 increased NETosis in neutrophils isolated from healthy humans and mice. Excessive NETosis was also observed in dHL-60 cells transfected with siRNA targeting SIRT1, confirming SIRT1 as an intrinsic inhibitor of NETosis. Interestingly, Ex-527 gave no further increase in NETosis in neutrophils isolated from diabetic mice, suggesting that SIRT1 is defective in diabetes, leading to a full-blown NETosis level which is not exacerbated by exogenous inhibition using Ex-527. We next examined the relationship between SIRT1 and peptidylarginine deiminase 4 (PAD4), an enzyme critical for NETosis. Ex-527 increased the levels of H4Cit^high neutrophils isolated from healthy subjects and mice, as well as dHL-60 cells cultured in basal glucose, when challenged. Notably, the effect was abolished in neutrophils isolated from diabetic mice and dHL-60 cells that had been primed in high glucose, indicating that SIRT1 acts upstream of PAD4 and limits NETosis via PAD4 inhibition in healthy neutrophils and dHL-60 cells, but not those under diabetic or hyperglycemic conditions.

Conclusion(s):

SIRT1, the endogenous inhibitor of NETosis, is dysfunctional in diabetes, thus causing excessive NETosis. Strategies that can revitalize SIRT1 activity are promising in suppressing NETosis and hence curtail NET-mediated diabetic complications.