Stimulus-Induced Histone H3 Citrullination and Neutrophil Extracellular Trap Formation Are Impaired in Patients with Proliferative Diabetic Retinopathy

Background: Proliferative diabetic retinopathy (PDR) is the most advanced stage of diabetic retinopathy (DR). Although spontaneous release of Neutrophil Extracellular Traps (NETs) has been reported in DR patients, the capacity of neutrophils from PDR patients to undergo inducible NET formation remains unexplored. This study aimed to evaluate inducible NET release and the underlying regulatory mechanisms in PDR. Methods: We performed a case–control study including 9 nondiabetic controls, 17 patients with type 2 diabetes (T2D) without DR, and 15 patients with PDR. Peripheral neutrophils were isolated and stimulated with ionomycin to assess NET release and histone H3 citrullination (H3Cit). Peptidylarginine deiminase 4 (PADI4)promoter methylation and mRNA expression were analyzed, together with the surface expression of the activation markers CD11b and CD66b. An ex vivo high-glucose model was used to examine the effect of persistent hyperglycemia on NET induction. Results: Compared with those from controls and T2D patients, neutrophils from PDR patients exhibited significantly reduced inducible NET release and impaired H3Cit. The NET index (stimulated/nonstimulated ratio) was markedly lower in the PDR group. Despite increased PADI4 mRNA expression in PDR neutrophils, promoter methylation, as well as that of cell activation markers, remained unchanged. Notably, persistent hyperglycemia in healthy neutrophils reproduced the impaired NET release and reduced H3Cit observed in PDR. Conclusions: Neutrophils from PDR patients display selective defects in inducible NET release and histone citrullination, which are likely driven by chronic hyperglycemia and metabolic stress rather than epigenetic silencing of PADI4. These findings are consistent with impaired inducible neutrophil responsiveness in PDR and suggest that altered regulation of PAD4-dependent histone citrullination may contribute to this phenotype, warranting further mechanistic investigation.