Lysyl oxidase promotes actin-dependent neutrophil activation and cytotoxicity in diabetes: Implications for diabetic retinopathy

Abstract

Activated neutrophils contribute to retinal endothelial cell (EC) death and capillary degeneration associated with early diabetic retinopathy (DR). However, the factors and mechanisms driving neutrophil activation and cytotoxicity in diabetes remain insufficiently understood. Here we show that lysyl oxidase (LOX), a collagen crosslinking and matrix stiffening enzyme that increases retinal EC susceptibility to activated neutrophils, simultaneously activates neutrophils in its alternate soluble form. Specifically, treatment of diabetic mice with LOX inhibitor β-aminopropionitrile (BAPN) prevented the diabetes-induced increase in neutrophil activation (extracellular release of neutrophil elastase and superoxide) and cytotoxicity towards co-cultured mouse retinal ECs. Mouse neutrophils and differentiated (neutrophil-like) human HL-60 cells treated with recombinant LOX alone exhibited similar activation and cytotoxicity. Mechanistically, this LOX-induced neutrophil activation was associated with biphasic F-actin remodeling, with the initial and rapid (<15 min) F-actin depolymerization followed by a significant increase in F-actin polymerization and polarization. Preventing the initial F-actin depolymerization blocked LOX-induced neutrophil activation and cytotoxicity towards co-cultured retinal ECs. Finally, we show that this biphasic F-actin remodeling is essential for LOX-induced membrane clustering of azurophilic granule marker CD63 and NADPH organizer p47 that are associated with extracellular release of neutrophil elastase and superoxide, respectively. By revealing a causal and previously unrecognized link between LOX and actin-dependent neutrophil activation in diabetes, these findings provide fresh mechanistic insights into the proinflammatory role of LOX in early DR that goes beyond its canonical matrix-stiffening effects.

Keywords: Actin; Diabetes; Leukocytes; Lysyl oxidase; Neutrophil elastase; Retinal endothelial cells.

Figure 1:. LOX mediates diabetes-induced neutrophil activation and cytotoxicity towards retinal ECs.

(A) RT-qPCR analysis of mouse whole retina indicates that LOX inhibitor BAPN prevents the (n=6 mice/group; 10 wk diabetes) diabetes-induced increase (by 1.5-fold; p<0.01) in LOX mRNA level. (B) Flow cytometry-based analysis of FITC-Annexin V-labeled mRECs following 16h co-culture with neutrophils isolated from nondiabetic (ND) mice or diabetic (D) mice ± LOX inhibitor BAPN (D+BAPN; 3 mg/kg BW) (n=6 mice/group; 10 wk diabetes) revealed that the significant increase in mREC apoptosis caused by neutrophils from D mice is prevented by LOX inhibition. (C) Representative Western blot bands and cumulative densitometric analysis of mouse neutrophils (n=6 mice/group; 10 wk diabetes) indicate that the diabetes-induced ~2-fold increase (p<0.05) in neutrophil elastase (NE) protein expression is prevented by LOX inhibition (using BAPN). (D) fMLP (10 nM) stimulation of neutrophils isolated from ND, D, or D+BAPN mice (n=6 mice/group; 10 wk diabetes) and subsequent EnzChek^™ elastase assay revealed that the diabetes-induced ~40% increase (p<0.05) in extracellular NE activity is prevented by LOX inhibition. (E) fMLP (10 nM) stimulation of neutrophils isolated from ND or 4-wk D (four weeks’ duration of diabetes) mice (n=7 mice/group) and subsequent EnzChek^™ elastase assay indicate a lack of change in extracellular NE activity in 4-wk D mice. (F) Flow cytometry-based analysis of FITC-Annexin V-labeled mRECs following co-culture with mouse neutrophils isolated from ND or 4-wk D mice (n=7 mice/group) revealed no increase in neutrophil-mediated mREC apoptosis in shorter-term diabetes. (G) RT-qPCR analysis of mouse whole retina indicates that LOX mRNA levels do not increase in 4-wk D mice (n=7 mice/group). Bar graphs indicate mean ± SD.

Figure 2.. LOX induces neutrophil activation and CD63 aggregation.

(A) Neutrophils isolated from ND mice were either left untreated (Unt.) or treated with 75 ng/mL (6h) recombinant mouse LOX before brief stimulation with fMLP (10 nM) and supernatant collection. Fluorometric analysis of the supernatant/EnzChek^™ elastase substrate mix revealed a 40% increase (p<0.05) in extracellular NE activity in LOX-treated neutrophils. (B) Flow cytometry quantification of the total number of FITC Annexin V-labeled mRECs following 16h of co-culture with LOX-treated (75 ng/mL; 6h) ND neutrophils revealed a 1.5-fold increase (p<0.001) in mREC apoptosis caused by LOX-treated neutrophils. (C) Human neutrophil-like dHL-60 cells were either left untreated (Unt.) or treated with recombinant human LOX (75 ng/mL) ± NE inhibitor sivelestat (50 μM) for 6h before brief stimulation with fMLP (10 nM) and supernatant collection. Fluorometric analysis of the supernatant/EnzChek^™ elastase substrate mix revealed that the LOX-induced ~1.6-fold increase (p<0.001) in extracellular NE activity is blocked by sivelestat. (D) Flow cytometry-based analysis of FITC-Annexin V-labeled HRECs following 16h co-culture with LOX-pretreated or untreated (Unt.) dHL-60 cells in the absence or presence of NE inhibitor sivelestat (50 μM) revealed that LOX-treated dHL-60 cells cause a significant increase in HREC apoptosis, which is blocked in the presence of sivelestat. (E) Untreated (Unt.) or LOX-treated (75 ng/mL; 6h) dHL-60 cells were labelled with anti-CD63 (to visualize NE-containing granules; green) and Phalloidin-594 (to visualize F-actin; red). Representative confocal images and subsequent quantitative analysis, performed as depicted in the schematic, demonstrate that LOX causes a significant increase in co-localized CD63 aggregation and F-actin polarization (indicated by arrowheads in fluorescence images). Column scatter plots indicate mean and distribution from ≥80 cells. (F) AFM stiffness measurement of untreated (Unt.) or LOX-treated (75 ng/mL; 6h) dHL-60 cells (n=30) revealed that LOX induces significant softening (by ~70%, p<0.001) of dHL-60 cells. Bar graphs indicate mean ± SEM. Scale bar, 5 μm.

Figure 3:. LOX induces rapid and transient actin remodelling in neutrophils.

(A) dHL-60 cells were either left untreated (Unt.) or treated with LOX (75 ng/mL) for the indicated durations prior to labelling with Phalloidin-594 to visualize F-actin. Representative fluorescence images and subsequent quantitative analysis revealed that LOX causes a rapid and transient decrease in F-actin intensity followed by a significant increase at 360 min (6h). Line graph indicates mean ± SD from ≥200 cells; intensity was normalized with respect to untreated (Unt.) cells. (B) Untreated (Unt.) or LOX-treated (6h) dHL-60 cells were labelled with Phalloidin-594 (red) and anti-CD63 (to visualize NE-containing granules; green). Representative confocal images and subsequent quantitative analysis demonstrate that LOX-induced CD63 aggregation is spatiotemporally associated with F-actin polarization (arrowheads) at 360 min (6h). Plots indicate mean and distribution from ≥90 cells. (C) dHL-60 cells were treated with 75 ng/mL LOX either continuously for 360 min (6h) or transiently for 10 min prior to labelling with Phalloidin-594 at the indicated time points. Representative fluorescence images and subsequent quantitative analysis revealed that removal of LOX after 10 min does not inhibit F-actin repolymerization seen at 6h. Plots indicate mean and distribution from ≥110 cells; F-actin intensity was normalized with respect to value at 0 min. Bar graphs indicate mean ± SEM. Scale bar, 5 μm.

Figure 4:. Transient actin depolymerization is necessary for LOX-induced neutrophil activation.

(A, B) dHL-60 cells were treated with 75 ng/mL LOX ± actin stabilizing drug JASP (1 μM) for the indicated durations prior to labelling with Phalloidin-594 to visualize F-actin. Representative fluorescence images (A) and subsequent quantitative analysis (B) revealed that the LOX-induced F-actin remodeling is blocked by JASP. Line graph in (B) indicates mean ± SD from ≥80 cells; intensity was normalized with respect to untreated (Unt.) dHL-60 cells. Plots indicate mean and distribution from ≥65 cells. $p<0.001 for Unt Vs LOX treated cells; #p=ns for unt vs LOX+JASP treated cells. (C) AFM stiffness measurement of dHL-60 cells (n=40) that were either left untreated (Unt.) or treated with 75 ng/mL LOX ± actin stabilizing drug JASP (1 μM) for 6h revealed that the LOX-induced softening of dHL-60 cells is significantly inhibited (by 40%; p<0.05) by JASP. (D) Untreated (Unt.) or LOX ± JASP-treated (6h) dHL-60 cells were labelled with Phalloidin-594 (red) and anti-CD63 (to visualize NE-containing granules; green). Representative confocal images and subsequent quantitative analysis revealed that JASP treatment concurrently blocks LOX-induced F-actin polarization and CD63 aggregation (arrowheads). Plots indicate mean and distribution from ≥70 cells. (E) Untreated (Unt.) or LOX ± JASP-treated (6h) dHL-60 cells were briefly stimulated with fMLP (10 nM) prior to supernatant collection. Fluorometric analysis of the supernatant/EnzChek^™ elastase substrate mix revealed that the LOX-induced increase in extracellular NE activity is significantly inhibited (by ~70%; p<0.05) by JASP. Unt: Untreated. (F) HRECs were co-cultured with [LOX±JASP]-pretreated or untreated (Unt.) dHL-60 cells for 16h prior to FITC Annexin V/PI labeling and flow cytometry. Quantification of the total number of Annexin V-positive HRECs (plotted as % of Unt.) revealed that the increase in HREC apoptosis caused by LOX-pretreated dHL-60 cells is significantly inhibited (by 70%; p<0.001) by JASP. Bar graphs indicate mean ± SEM. Scale bar, 5 μm.

Figure 5:. Transient actin depolymerization is sufficient to cause neutrophil activation and cytotoxicity.

(A, B) dHL-60 cells were either left untreated (Unt.) or treated with actin depolymerization drug cytoD (2.5 μM) for the indicated durations prior to labelling with Phalloidin-594 to visualize F-actin. Representative fluorescence images and subsequent quantitative analysis revealed that cytoD causes a transient decrease in F-actin intensity followed by a significant increase in F-actin polymerization and polarization at 360 min (6h). Line graph indicates mean ± SD from ≥200 cells; intensity was normalized with respect to untreated (Unt.) cells. Plots indicate mean and distribution from ≥70 cells. (C) Untreated (Unt.) or cytoD-treated (6h) dHL-60 cells were labelled with Phalloidin-594 (red) and anti-CD63 (to visualize NE-containing granules; green). Representative confocal images and subsequent quantitative analysis revealed that cytoD treatment leads to a significant increase in co-localized F-actin polarization and CD63 aggregation (arrowheads) at 360 min (6h). Plots indicate mean and distribution from ≥65 cells. (D) Untreated (Unt.) or cytoD-treated (6h) dHL-60 cells were briefly stimulated with fMLP (10 nM) prior to supernatant collection. Fluorometric analysis of the supernatant/EnzChek^™ elastase substrate mix revealed that cytoD-treated dHL-60 cells exhibit a significant increase (by 30%; p<0.01) in extracellular NE activity when compared with untreated (Unt.) cells. (E) HRECs were co-cultured with cytoD-pretreated or untreated (Unt.) dHL-60 cells for 16h prior to FITC Annexin V/PI labeling and flow cytometry. Quantification of the total number of Annexin V-positive HRECs (plotted as % of Unt.) revealed that cytoD-treated dHL-60 cells cause a significant increase (by ~40%; p<0.05) in HREC apoptosis. Bar graphs indicate mean ± SEM. Scale bar, 5 μm.

Figure 6:. LOX inhibition blocks diabetes-induced F-actin remodeling and CD63 aggregation in neutrophils.

Mouse bone marrow neutrophils isolated from nondiabetic (ND), diabetic (D), or D mice treated with BAPN (D+BAPN; 3 mg/kg BW) (n≥90 cells/group; 10 wk diabetes) were labelled with Phalloidin-594 (to visualize F-actin; red) and anti-CD63 (to visualize NE-containing granules; green). (A) Representative confocal images and (B) subsequent quantitative analysis revealed that LOX inhibition using BAPN prevented the diabetes-induced concurrent increase in F-actin intensity, F-actin polarization, and CD63 aggregation. Arrowheads indicate F-actin polarization or CD63 aggregation. Plots indicate mean and distribution from ≥90 cells. Scale bar, 5 μm. (C) AFM stiffness measurement of bone marrow neutrophils (n≥15 cells) isolated from nondiabetic (ND), diabetic (D), or D mice treated with BAPN (D+BAPN; 3 mg/kg BW) (n=6 mice/group; 10 wk diabetes) revealed that the diabetes-induced reduction in neutrophil stiffness (by 41%; p<0.01) is significantly inhibited (by 48%; p<0.05) by LOX inhibitor BAPN.

Figure 7:. Schematic illustration of LOX-induced regulation of neutrophil actin dynamics, activation, and cytotoxicity in diabetes.

Based on our current findings, we propose that LOX is a mechanical determinant of neutrophil activation wherein it alters actin cytoskeleton and cell stiffness to increase extracellular NE and superoxide release that, together, exert cytotoxic effects on retinal ECs. This newly identified mechanism of neutrophil activation begins with LOX-induced rapid (within ~10 min) F-actin depolymerization. At this early time, the membrane distribution of CD63 and p47^Phox, which reflects the cell’s ability to release extracellular NE and superoxide, respectively, remains uniform and unchanged. However, over time (6h), as F-actin recovers through repolymerization, it simultaneously becomes polarized. Interestingly, this late-phase F-actin remodeling causes redistribution (aggregation) of CD63 and p47^Phox that predictably leads to increased extracellular release of NE and superoxide, key cytotoxic mediators of retinal EC death in diabetes.