Neutrophil extracellular traps and peptidylarginine deiminase 4-mediated inflammasome activation link diabetes to cardiorenal injury and heart failure

Abstract

Background and aims: Diabetes is associated with increased risk of cardiovascular and renal disease. This study investigated the role of peptidylarginine deiminase 4 (PAD4), neutrophil extracellular traps (NETs), and inflammasome activation in diabetic cardiomyopathy (DCM) and kidney disease (DKD).

Methods: Endomyocardial biopsies (EMB) from heart failure (HF) patients (n = 20) with or without diabetes were stained for NETs. Wild-type (WT) and PAD4⁻/⁻ mice were subjected to streptozotocin (STZ)-induced diabetes and cardiac function, blood glucose, body weight, and exercise tolerance were assessed longitudinally. NETosis and ASC specks were evaluated in mouse and human neutrophils. Cardiac and renal fibrosis was assessed by Sirius Red/Fast Green staining. Confocal microscopy, ELISA, and flow cytometry were used to quantify NETs, IL-1β, von Willebrand factor (VWF), cytokine transforming growth factor beta-1 (TGF-β1), and neutrophil infiltration.

Results: Myocardial NET burden was increased in HF patients with diabetes. High glucose triggered inflammasome activation in human neutrophils. After STZ, PAD4⁻/⁻ and WT mice developed hyperglycaemia and weight loss, yet only WT neutrophils showed increased NETosis and ASC speck formation. Only diabetic WT mice exhibited elevated IL-1β and VWF levels, impaired cardiac function, reduced exercise tolerance, and pulmonary oedema; PAD4⁻/⁻ mice were protected. Wild-type diabetic hearts and kidneys showed greater fibrosis, neutrophil infiltration, NETs, and TGF-β1 levels. Kidney injury in WT mice was reflected by albuminuria and renal fibrosis, whereas PAD4⁻/⁻ mice preserved renal function.

Conclusions: Diabetes promotes neutrophil inflammasome activation and NETosis, driving cardiac and renal inflammation and fibrosis. Peptidylarginine deiminase 4 deficiency prevents heart failure and preserves kidney function in experimental diabetes.

Keywords: Chronic kidney disease; Diabetes; Heart failure; Inflammasome; Neutrophil extracellular traps (NETs); PAD4.

Figure 1

Myocardial neutrophil extracellular trap burden is increased in diabetic heart failure, and high glucose promotes neutrophil inflammasome activation and NETosis. (A) Representative confocal microscopy images (63×) of endomyocardial biopsy sections from patients with heart failure without and with diabetes, immunostained for myeloperoxidase (MPO), citrullinated histone 3 (H3Cit), and nuclei (DAPI). (B) Quantification of myocardial neutrophil infiltration (MPO⁺ cells mm⁻²) and NET burden (% H3Cit⁺ neutrophils) in heart failure patients without (n = 10) and with diabetes (n = 10). Data are presented as median (IQR) and compared using the Mann–Whitney U test. (C) Correlation between left ventricular ejection fraction and myocardial neutrophil extracellular trap burden across all patients. Correlation was assessed using Spearman’s rank correlation, and the regression line was fitted by simple linear regression using ordinary least squares; dashed curves indicate the 95% confidence band of the fit (Spearman r = −.57, 95% CI −.81 to −.15, p = .009). (D) Representative confocal microscopy images (63×) of peripheral blood neutrophils from healthy donors (n = 7) incubated for 2 h with physiological (5.5 mM) or high (22 mM) glucose, with or without ionomycin (8 μM), immunostained for MPO, ASC, and nuclei (DAPI). (E) Quantification of ASC speck formation (% of cells) and neutrophil extracellular trap formation (% H3Cit⁺ cells) under the indicated conditions. Data are shown as mean ± SD and analysed by repeated-measures two-way ANOVA [factors: glucose (5.5 mM vs 22 mM) and stimulation (±8 μM ionomycin)]. Individual donor trajectories can be found in Supplementary data online, Figure S2B. Exact P values are shown in the figure. ASC, apoptosis-associated speck-like protein containing a CARD; DAPI, 4′,6-diamidino-2-phenylindole; H3Cit, citrullinated histone 3; HF, heart failure; LVEF, left ventricular ejection fraction; MPO, myeloperoxidase; NET, neutrophil extracellular trap

Figure 2

Neutrophil inflammasome activation and NETosis are peptidylarginine deiminase 4–dependent in streptozotocin-induced type 1 diabetes. (A) Timeline of experimental procedures: streptozotocin (50 mg/kg/day, i.p.) was injected for 5 consecutive days in wild-type and PAD4⁻/⁻ mice to induce type 1 diabetes mellitus. Blood glucose, echocardiography, and exercise tolerance were monitored every 2 weeks; analyses were performed 8 weeks post-induction. (B) Fed plasma glucose levels over time in WT + STZ (n = 10), PAD4⁻/⁻+ STZ (n = 10), and WT controls (n = 9). (C) Body weight (left) and relative body weight gain (right) over time in the same groups. Corresponding area under the curve analyses for glucose and body weight trajectories are provided in Supplementary data online, Figure S3. (D) Representative fluorescence microscopy images of neutrophils from streptozotocin-treated mice, unstimulated or treated with ionomycin (8 μM, 4 h); DNA (DAPI), ASC; red arrows indicate ASC speck oligomerization (representative of n = 5 experiments). (E) Quantification of ASC speck⁺ neutrophils (%) from WT + STZ, PAD4⁻/⁻+ STZ, and WT controls ± ionomycin (n = 5). (F) IL-1β in supernatants of neutrophils from WT + STZ and PAD4⁻/⁻+ STZ mice ± ionomycin (n = 5). (G) Plasma IL-1β and (H) plasma von Willebrand factor concentrations at 8 weeks post-streptozotocin (n = 8). (I) Neutrophil-to-lymphocyte ratio at 8 weeks in WT + STZ (n = 9), PAD4⁻/⁻+ STZ (n = 9), and WT controls (n = 8). Panels (B) and (C), (E) and (F), and (I) show data as mean ± SD and were analysed by ordinary one-way ANOVA; panel (G) shows median (IQR) and was analysed using the Mann–Whitney U test; panel (H) shows mean ± SD and was analysed using an unpaired t-test. Exact P values are shown in the figure. ASC, apoptosis-associated speck-like protein containing a CARD; DAPI, 4′,6-diamidino-2-phenylindole; IL-1β, interleukin-1β; NLR, neutrophil-to-lymphocyte ratio; PAD4, peptidylarginine deiminase 4; STZ, streptozotocin; T1D, type 1 diabetes mellitus; VWF, von Willebrand factor; WT, wild type

Figure 3

Peptidylarginine deiminase 4 deficiency preserves cardiac function and limits adverse remodelling in streptozotocin-induced type 1 diabetes. (A) Representative M-mode images at end-systole and end-diastole 8 weeks after streptozotocin in wild-type and PAD4⁻/⁻ mice. (B and C) Systolic function over time: (B) ejection fraction and (C) fractional shortening at baseline, 2, 4, 6, and 8 weeks post-streptozotocin (n = 9–10 per genotype). (D) Representative pulsed-wave Doppler and tissue Doppler traces across the mitral valve at 8 weeks; E-wave, A-wave, and e′ indicated. (E and F) Diastolic function over time: (E) E/e′ ratio and (F) isovolumetric relaxation time (n = 9–10). (G) Representative left ventricular sections stained with Sirius Red/Fast Green at Week 8 (scale bar, 50 μm); arrows indicate fibrosis. (H) Quantification of fibrosis (% left ventricular area) at Week 8 (n = 6 per group). (I) Exercise capacity: distance run on a forced treadmill at baseline and 4 and 8 weeks (n = 9–10). (J) Plasma troponin-I at Week 8 (n = 8–9). (K) Lung wet/dry weight ratio at Week 8 in WT controls (n = 8) and streptozotocin-treated WT (n = 13) and PAD4⁻/⁻ mice (n = 12). (L) Heart weight/body weight at Week 8 in the same groups (n = 6–9). Area under the curve analyses of integrated echocardiographic parameters (B and C, E and F) and exercise capacity (I) are provided in Supplementary data online, Figures S6 and S7. Panels (B) and (C), (E) and (F), and (I) show data as mean ± SD; per-time-point group differences were analysed using unpaired t-test or Mann–Whitney U test. Panel (H) shows mean ± SD and was analysed using an unpaired t-test; panel (J) shows median (IQR) and was analysed by Mann–Whitney U test; panels (K) and (L) show mean ± SD and were analysed using ordinary one-way ANOVA. Exact P values are shown in the figure. Dia, diastole; EF, ejection fraction; E/e′, ratio of early mitral inflow velocity to early diastolic mitral annular velocity; FS, fractional shortening; IVRT, isovolumetric relaxation time; LV, left ventricle; PAD4, peptidylarginine deiminase 4; STZ, streptozotocin; Sys, systole; WT, wild type

Figure 4

Peptidylarginine deiminase 4 deficiency reduces cardiac fibrosis, neutrophil infiltration, neutrophil extracellular trap burden, and inflammatory cytokines in streptozotocin-induced type 1 diabetes. (A) Representative immunofluorescence microscopy images of LV sections stained for collagen type I, vasculature (CD31), and nuclei (DAPI) at Week 8 post-streptozotocin (scale bar, 50 μm). (B) Quantification of collagen type I deposition (% left ventricular area) and (C) capillary density (% CD31⁺ of left ventricular area) in WT + STZ and PAD4⁻/⁻+ STZ mice (n = 6 per group). (D) Flow cytometry gating strategy for digested heart tissue: CD45⁺/CD11b⁺ double-positive events defined myeloid cells; Ly6G⁺ within this population defined neutrophils. (E) Quantification of cardiac neutrophils (Ly6G⁺ of CD45⁺/CD11b⁺) and (F) cardiac myeloid cells (CD11b⁺ of CD45⁺) at Week 8 post-streptozotocin (n = 6). (G) Representative immunofluorescence images (60×) of left ventricular sections from WT + STZ and PAD4⁻/⁻+STZ mice stained for Ly6G, H3Cit, and nuclei (DAPI); extracellular H3Cit adjacent to Ly6G⁺ cells was classified as neutrophil extracellular traps. (H) Quantification of total extracellular H3Cit⁺ area fraction (% left ventricular section) (n = 6). (I) IL-1β concentrations and (J) TGF-β1 concentrations in heart tissue at Week 8 post-streptozotocin, determined by ELISA in WT controls (n = 6), WT + STZ (n = 9), and PAD4⁻/⁻+ STZ (n = 6). Panels (B) and (C) show data as mean ± SD and were analysed using unpaired t-tests; panels (E), (F), (H), and (J) show data as median (IQR) and were analysed using the Mann–Whitney U test; panel (I) shows data as mean ± SD and was analysed using ordinary one-way ANOVA. Exact P values are shown in the figure. CD, cluster of differentiation; CD31, platelet endothelial cell adhesion molecule-1; DAPI, 4′,6-diamidino-2-phenylindole; H3Cit, citrullinated histone 3; IL-1β, interleukin-1β; LV, left ventricle; Ly6G, lymphocyte antigen 6 complex locus G6D; NET, neutrophil extracellular trap; PAD4, peptidylarginine deiminase 4; STZ, streptozotocin; TGF-β1, transforming growth factor-β1; WT, wild type

Figure 5

Peptidylarginine deiminase 4 deficiency reduces renal injury, fibrosis, inflammation, and neutrophil extracellular trap burden in streptozotocin-induced type 1 diabetes. (A) Urinary albumin concentrations (μg/mL) at Week 8 post-streptozotocin, measured by ELISA, in WT + STZ (n = 9) and PAD4⁻/⁻+ STZ mice (n = 8). (B) Kidney weight/body weight (mg/g) at Week 8 in WT controls (n = 6), WT + STZ (n = 9), and PAD4⁻/⁻+ STZ mice (n = 8). (C) Representative kidney sections stained with Sirius Red/Fast Green at Week 8 (scale bar, 50 μm); arrows indicate fibrotic tissue. (D) Quantification of renal fibrosis (% Sirius Red⁺ area) (n = 5 per group). (E) Representative kidney sections immunostained for CD68, TGF-β, and nuclei (DAPI) at Week 8 (scale bar, 40 μm). (F) Quantification of CD68⁺ cells (mm⁻²) in kidney sections (n = 5). (G) TGF-β1 concentrations in kidney tissue at Week 8, measured by ELISA. (H) Representative immunofluorescence microscopy images (60×) of kidney sections immunostained for Ly6G, H3Cit, and nuclei (DAPI); extracellular H3Cit deposits were considered neutrophil extracellular traps. (I) Quantification of total extracellular H3Cit⁺ area fraction (% of kidney section) (n = 5). Panels (A) and (I) show data as median (IQR) and were analysed using the Mann–Whitney U test; panel (B) shows data as mean ± SD and was analysed using Welch’s ANOVA (unequal variances); panels (D), (F), and (G) show data as mean ± SD and were analysed using unpaired t-tests. Exact P values are shown in the figure. CD, cluster of differentiation; CD68, cluster of differentiation 68; DAPI, 4′,6-diamidino-2-phenylindole; H3Cit, citrullinated histone 3; IL-1β, interleukin-1β; Ly6G, lymphocyte antigen 6 complex locus G6D; NET, neutrophil extracellular trap; PAD4, peptidylarginine deiminase 4; STZ, streptozotocin; TGF-β1, transforming growth factor-β1; WT, wild type