Actin remodeling governs neutrophil activation: Implications for retinal capillary degeneration in diabetic retinopathy.

Abstract

Purpose : Vascular inflammation plays a causal role in the degeneration of retinal capillaries in early diabetic retinopathy (DR). We have recently reported that, in addition to proinflammatory cytokines (e.g. TNF-α) and high glucose (HG), lysyl oxidase (LOX) also contributes greatly to retinal vascular inflammation in diabetes. Since neutrophil activation is a hallmark of inflammation and known to cause retinal capillary degeneration in diabetes, here we asked whether and how the aforementioned proinflammatory factors promote neutrophil activation, with a focus on the role of actin dynamics that is implicated in cell shape regulation upon activation.

Methods : Neutrophil-like differentiated HL-60 (dHL-60) cells or primary mouse neutrophils were treated with proinflammatory DR stressors LOX (75 ng/ml), TNF-α (10 ng/ml), and HG (20 mM) for 6h. For proof-of-concept, some dHL-60 cells were treated with Jasplakinolide (JASP; 1 uM; 6h) or cytochalasin D (CytoD; 2.5 uM; 6h) to stabilize or depolymerize F-actin. Activation was assessed by measuring superoxide (using dihydroethidium dye, DHE; 5 mM), neutrophil elastase release (EnzChek Elastase Assay Kit), and retinal endothelial cell (REC) cytotoxicity (Annexin-PI labeling). Temporal F-actin dynamics was visualized by TRITC-Phalloidin staining and epifluorescence imaging from 5 min through 6h.

Results : LOX and TNF-α activated dHL-60 cells and mouse neutrophils, as judged by increased superoxide and neutrophil elastase release and greater cytotoxicity towards RECs. In contrast, HG had no effect. LOX and TNF-a caused rapid (within 15-30 min) and transient F-actin depolymerization, followed by increase in actin intensity and polarization. In contrast, HG had no effect on F-actin. Crucially, preventing the transient actin depolymerization (using JASP) blocked the LOX-induced neutrophil activation while inducing actin depolymerization using CytoD was sufficient to activate neutrophils. Finally, this transient actin depolymerization is required for the membrane assembly of p47 (for NADPH activation) and CD63 (for neutrophil release).

Conclusions : Rapid and transient F-actin depolymerization is necessary for neutrophil activation by proinflammatory stressors LOX and TNF-α. These findings implicate F-actin regulation in neutrophils as a potential therapeutic target for inflammatory conditions marked by LOX and TNF-α upregulation such as DR.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.