Citrullination of H3 and Neutrophil Extracellular Traps are reduced upon activation in Proliferative Diabetic Retinopathy patients

Background Proliferative diabetic retinopathy (PDR) is the most advanced stage of diabetic retinopathy (DR). Although spontaneous NET release has been reported in DR, the capacity of neutrophils from PDR patients to undergo inducible NET formation remains unexplored. This study aimed to evaluate inducible NET formation and underlying regulatory mechanisms in PDR. Methods We performed a case–control study including 9 non-diabetic 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 formation and histone H3 citrullination (H3Cit). PADI4 promoter methylation and mRNA expression were analyzed, together with the surface expression of activation markers CD11b and CD66b. An ex vivo high-glucose model was performed to examine the impact of persistent hyperglycemia on NET induction. Results Neutrophils from PDR exhibited significantly reduced inducible NET formation and impaired H3 citrullination compared with controls and T2D patients. The NETs Index (stimulated/non-stimulated ratio) was markedly lower in the PDR group. Despite increased PADI4 mRNA expression in PDR neutrophils, promoter methylation remained unchanged, as well as cell activation markers. Notably, persistent hyperglycemia in healthy neutrophils reproduced the impaired NET formation and reduced H3 citrullination observed in PDR. Conclusions Neutrophils from PDR patients display a selective defect in inducible NET formation and histone citrullination, likely driven by chronic hyperglycemia and metabolic stress rather than epigenetic silencing of PADI4. These findings support the concept of neutrophil functional exhaustion in PDR and identify PAD4 as a potential therapeutic target in advanced diabetic retinopathy