Ligation of signal inhibitory receptor on leukocytes-1 suppresses the release of neutrophil extracellular traps in systemic lupus erythematosus

Abstract

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of systemic Lupus erythematosus (SLE), since netting neutrophils release potentially immunogenic autoantigens including histones, LL37, human neutrophil peptide (HNP), and self-DNA. In turn, these NETs activate plasmacytoid dendritic cells resulting in aggravation of inflammation and disease. How suppression of NET formation can be targeted for treatment has not been reported yet. Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) is a surface molecule exclusively expressed on phagocytes. We recently identified SIRL-1 as a negative regulator of human neutrophil function. Here, we determine whether ligation of SIRL-1 prevents the pathogenic release of NETs in SLE. Peripheral blood neutrophils from SLE patients with mild to moderate disease activity and healthy donors were freshly isolated. NET release was assessed spontaneously or after exposure to anti-neutrophil antibodies or plasma obtained from SLE patients. The formation of NETs was determined by microscopic evaluation using DNA dyes and immunostaining of NET components, as well as by live cell imaging. We show that SLE neutrophils spontaneously release NETs. NET formation is enhanced by stimulation with antibodies against LL37. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and MEK-ERK signaling prevents NET release in response to these antibodies. Signaling via the inhibitory receptor SIRL-1 was induced by ligation with anti-SIRL-1 specific antibodies. Both spontaneous and anti-neutrophil antibody-induced NET formation is suppressed by engagement of SIRL-1. Furthermore, NET release by healthy neutrophils exposed to SLE plasma is inhibited by SIRL-1 ligation. Thus, SIRL-1 engagement can dampen spontaneous and anti-neutrophil antibody-induced NET formation in SLE, likely by suppressing NAPDH oxidase and MEK-ERK activity. Together, these findings reveal a regulatory role for SIRL-1 in NET formation, potentially providing a novel therapeutic target to break the pathogenic loop in SLE.

Figure 1. Localization of MPO and NE on extracellular DNA upon NET release in response to anti-neutrophil antibodies.

A) Fluorescence imaging of healthy neutrophils cultured with 10 µg/ml control IgG, 10 µg/ml anti-LL37 antibody, 10 µg/ml anti-HNP antibody or 25 ng/ml PMA as positive control and stained for total DNA with Hoechst 33342 (left panels) and extracellular DNA (Sytox Green, right panels) after 3 h of incubation. Scale bars, 50 µm. B) Quantification of NET release by healthy neutrophils using fluorescence microscopy. The density of extracellular DNA (stained with Sytox Green) over the image area after 3 h of incubation with control IgG (n=4), anti-LL37 antibody (n=4), anti-HNP antibody (n=3) or PMA (n=3) is shown as mean±SD. **p<0.01; ***p<0.001, ANOVA (adjusted for Dunnett’s test). C) Immunostaining for NET components (green, myeloperoxidase (MPO); red, neutrophil elastase (NE); blue, DNA). The experiment was repeated three times with neutrophils from independent donors, with similar results. Scale bars, 10 µm. Figures show details of larger fields of view, that were used to quantify NET NE and NET MPO density. Original magnification 20x. D) Quantification of NET release using fluorescently conjugated NET-specific antibodies (either anti-MPO or anti-NE antibodies). The MPO- and NE-stained areas are shown as mean±SD (n=3). *p=0.0498; ***p=0.0006, Student’s t test.

Figure 2. NADPH oxidase activity and MAPK signaling are required for NET formation in response to antibodies against LL37.

Healthy neutrophils were pretreated for 30 min with or without the NADPH oxidase inhibitor DPI (10 µM) or U0126 (50 µM), a specific mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor, before incubation with anti-LL37 antibodies for 3 h. DMSO was used as vehicle control. A) One out of three independent experiments is shown. NET release is determined by staining for DNA and visualized by fluorescence microscopy. Scale bars, 50 µm. B) The amount of NET-DNA is quantified as in Figure 1B. Mean±SD of three independent donors is indicated. **p<0.01, ANOVA (adjusted for Dunnett’s test).

Figure 3. Ligation of SIRL-1 suppresses both spontaneous and anti-LL37-triggered NET release by SLE neutrophils.

A) Healthy (HD) or SLE neutrophils were incubated with anti-LL37 antibodies with or without pretreatment with anti-SIRL-1 mAb. NET-DNA release was quantified after 3 h in multiple experiments by fluorescence microscopy to reflect the area covered by extracellular DNA (as described in Figure 1B). HD, mean±SEM of four independent donors is shown. **p=0.0014, paired Student’s t test. SLE, mean±SEM is shown (medium, n=11; anti-LL37, n=13). *p=0.0117; **p=0.0052, paired Student’s t test. B) Representative images of anti-LL37-stimulated SLE neutrophils with or without SIRL-1 ligation stained for extracellular DNA (Sytox Green, left panels) and total DNA (Hoechst 33342, right panels). Scale bars, 50 µm. C) Correlation of spontaneous and anti-LL37 antibody-induced NET formation by neutrophils from individuals with SLE. r²=0.4413; p=0.0258, Pearson’s correlation test. D) Flow cytometry analysis of SIRL-1 surface expression on freshly purified neutrophils from healthy donors and SLE patients. Representative histograms (grey filled, isotype-matched antibody; white dotted, anti-SIRL-1 mAb) are shown. E) Results from multiple independent donors (HD; n=6, SLE; n=7) are given. Average mean fluorescence intensity of each group±SD is indicated, Student’s t test.

Figure 4. Engagement of SIRL-1 prevents NETosis in response to SLE plasma.

A) NET release from healthy neutrophils exposed to SLE plasma visualized as extracellular DNA (Sytox Green) by live cell imaging (Video S1) (neutrophils are blue; NETs are green). Individual frames are shown. Scale bar, 10 µm. B) Representative images of netting healthy neutrophils in response to SLE plasma with or without ligation of SIRL-1. Scale bars, 50 µm. C) Results from multiple independent donors are given. Neutrophils from 4 healthy donors were cultured for 3 h with 20% plasma from either nonautologous healthy controls (n=4) or individuals with SLE (n=5 or 6) in the presence or absence of anti-SIRL-1 mAb. NET formation was analyzed by fluorescence microscopy and the amount of NET-DNA quantified. Average density of each pooled group±SEM is shown. ***p<0.0001, paired Student’s t test.