Neutrophil extracellular traps induce endothelial dysfunction in systemic lupus erythematosus through the activation of matrix metalloproteinase-2

Abstract

Rationale: The structural and functional integrity of the endothelium is crucial in maintaining vascular homeostasis and preventing atherosclerosis. Patients with systemic lupus erythematosus (SLE) have an increased risk of developing endothelial dysfunction and premature cardiovascular disease. Neutrophil extracellular trap (NET) formation is increased in SLE and has been proposed to contribute to endothelial damage, but the mechanism remains unclear.

Objective: To determine the mechanism by which enhanced NET formation by low-density granulocytes (LDGs) in SLE contributes to endothelial damage and disrupts the endothelium.

Results: The putative role of NET-externalised matrix metalloproteinases (MMPs) in altering the functional integrity of the endothelium was examined. MMP-9 externalised by lupus LDGs during NET formation specifically impaired murine aortic endothelium-dependent vasorelaxation and induced endothelial cell apoptosis. Endothelial dysfunction correlated with the activation of endothelial MMP-2 by MMP-9 present in NETs, while inhibition of MMP-2 activation restored endothelium-dependent function and decreased NET-induced vascular cytotoxicity. Moreover, immunogenic complexes composed of MMP-9 and anti-MMP-9 were identified in SLE sera. These complexes, as well as anti-MMP-9 autoantibodies, induced NETosis and enhanced MMP-9 activity.

Conclusions: These observations implicate activation of endothelial MMP-2 by MMP-9 contained in NETs as an important player in endothelial dysfunction, and MMP-9 as a novel self-antigen in SLE. These results further support that aberrant NET formation plays pathogenic roles in SLE.

Figure 1. Active MMP-9 and MMP-25 decorate NETs and this is enhanced in lupus LDGs

A. MMP-9 and human cationic antimicrobial protein (h-CAP18; LL-37 precursor) were quantified by immunoblot in purified NETs from LPS-stimulated controls (Ctrl-N) and spontaneously formed NETs by lupus neutrophils (SLE-N) and lupus LDGs (LDG) (n=2-3/group). B. Densitometry of MMP-9 in NETs is displayed mean arbitrary units (AUs) ± SEM; n=3 per group; p<0.05); C. Zymogram displaying MMP-9 and MMP-25 activities in purified NETs, when compared to neutrophil lysate. D. and E Representative images where red represents MMP-9 or MMP-25; green represents hCAP-18, mitochondria (Mito) or elastase, and blue is Hoechst. Scale bars, 10 μm.

Figure 2. NETs activate endothelial MMP-2 and promote endothelial damage

A. HUVECs were incubated for 3 h in the absence (-) or presence of control neutrophil (Ctrl) or lupus LDGs NETs. Images display morphological changes induced by NETs. B-C. Localization of NET proteins in HUVECs’ plasma membrane. D. Zymography analysis of MMP activity in control and lupus NETs before and after 3 h incubation with HUVECs; arrows indicate inactive and active MMP-2, MMP-9 and a complex between MMP-9 and neutrophil gelatinase associated lipocalin (NGAL). E. Elevated levels of active MMP-9 in LDGs-NET is associated with activation of endothelial MMP-2 following incubation with NETs. Results are presented as AUs and represent 3 experiments. F. NETs impair endothelium-dependent vasorelaxation in response to acetylcholine (Ach). Comparisons are made using NETs isolated from the same number of LPS-stimulated control neutrophils (Ctrl NETs per volume) and spontaneously-generated in lupus LDGs (LDG NETs), or equal concentrations of control NETs (Ctrl NETs per μg) and LDG NETs. Results represent mean ± SEM % relaxation of 3 independent experiments; *p< 0.05.

Figure 3. MMP-9 inhibition abrogates endothelial dysfunction induced by NETs

A. HUVECs and HAECs were incubated for 3 h in the absence or presence of NETs (+), MMP-9-depleted (MMP-9 dep)-NETs, histone H2A-depleted (H2A dep)-NETs, NETs plus neutralizing anti-MMP-9 (MMP-9 Ab) or NETs plus MMP inhibitor (MMP inh). Images display HUVEC morphological changes induced by NETs. B. Apoptotic ECs were quantified by FACS. Results are adjusted to background levels (no NETs) and represent mean ± SEM of six independent experiments. C. Zymography analysis performed on supernatants after 3 h incubation. MMP-2 (representative image, bottom) was quantified by densitometry. Results are presented active MMP-2/pro-MMP-2 ratio and expressed as median AUs ± SEM. D Improvement in aortic endothelium-dependent vasorelaxation after incubation with lupus LDG NETs with and without neutralizing anti-MMP-9 (MMP-9 Ab) or MMP inhibitor (MMP inh). Results represent mean ± SEM of two experiments. *p< 0.05, **p< 0.005, ***p<0.001, #### p< 0.0001 for Ctrl vs lupus LDG comparison; acetylcholine (Ach).

Figure 4. Active MMP-9 induces endothelial damage through MMP-2 activation

A. Representative images display EC morphological changes induced by MMP-9. B. EC death was quantified by FACS. C. MMP-2 activity was quantified on the supernatants after 3 h of incubation with MMP-9.

Figure 5. Anti-MMP-9 Abs and ICs containing MMP-9 stimulate NETosis

A. MMP-9 activity was quantified in control and SLE sera by zymography. Results represent median AUs ± SEM of three independent experiments. B. MMP-9 activity was quantified in IgG fractions purified from healthy control sera (n=2) or SLE sera with high anti-dsDNA titers (n= 3). Results are expressed as mean AU ± SEM. C. Purified SLE IgG enhances NETosis in control neutrophils through ROS and NOX-dependent processes. PMA and LPS are positive controls. Diphenyleneiodonium chloride (DPI); N-Acetylcysteine (NAC). Results are expressed as relative fluorescent units (RFUs) of DNA fluorescence using SYTOX Green and represent mean ± SEM of three independent experiments. D. Quantification of anti-MMP-9 Abs in SLE (n= 20), RA (n=12) or healthy control (n= 10) sera. Results are expressed as optical density (OD) index (OD in patient serum/ mean OD in control sera). E. Recombinant LL-37 and MMP-9 were resolved in a gradient gel. Membrane was incubated with SLE sera (n=5) and signal detected by chemiluminescence. F. Anti-MMP-9 isolated from SLE sera and MMP-9 ICs enhances NETosis in control neutrophils. This is blocked by FcγR inhibition. Results represent mean ± SEM of three independent experiments. NETs were detected as in 5C. G. Anti-MMP-9 Abs bind to MMP-9 externalized in NETs. H. MMP-9 activity was measured in NETs in presence or absence of anti-MMP-9 autoAb using FITC-coupled gelatin. Results represent mean RFUs± SEM of three independent experiments. Scale bars, 10 μm. *p< 0.05, **p< 0.005, ***p<0.001

Figure 6. Proposed model of the role of NET-bound MMP-9 in endothelial dysfunction

ICs containing MMP-9 and anti-MMP-9 enhance NETosis in LDGs. In turn, LDG-NETs are a source of active MMP-9 that activates pro-MMP-2 in ECs, triggering endothelial dysfunction. LDG-NETs also serve as a source of MMP-9 autoantigen and promote further propagation of a vicious cycle.