Fig 2: AAV neutrophils are primed to respond to sCD95L.Cytosolic ROS production ([ROS]cyt) was measured with the fluorescent probe H2DCFDA, Ratio values (R) were normalized to pre-stimulated values (R0) to yield R/R0 values (relative [ROS]). Sera from healthy donors and AAV patients were diluted (1/1000) in the extracellular recording medium (HBSS). Data are representative of three independently performed experiments. A left panel: Relative cytosolic ROS production in PNN (polynuclear neutrophils) from HDs after stimulation with sera from AAV patients (N = 4, red) or healthy donors (N = 4, black). For each condition, data represent mean ± SD of n = 41 cells. Right panel: Representation of areas under the curve (AUC) of the ROS production. Data represent mean ± SD; * p < 0.05 using two-tailed Mann-Whitney test. B Relative cytosolic ROS in PNN from HD after stimulation with sera from AAV patients before (M0) or after treatment (M6). For each condition, data represent mean ± SD of n = 41 cells. Data are representative of three independently performed experiments. C HD PNN were pre-treated with or without DB550 (1 µM) and then bathed with sera from AAV patients. To block CD95L/CD95 interaction, the neutralizing anti-CD95L mAb NOK-1 or isotypic control (1 µg/ml) was pre-incubated in sera for 10 min prior to incubation with HD neutrophils and relative cytosolic ROS production was measured. Inset: histograms represent mean ± SD of areas under the curve (AUC) of the cytosolic ROS response. ** p < 0.01, *** p < 0.001, **** p < 0.0001 using unpaired and two-tailed Mann-Whitney test. D Cytosolic ROS in PNN isolated from HD or AAV patients exposed to sCD95L (100 ng/ml). For each condition, data represent mean ± SD of n = 41 cells. Data are representative of three independently performed experiments. E HD neutrophils were pre-treated in the presence or absence (control) of the pro-inflammatory cytokines TNF (5 ng/ml), IL-1β (10 ng/ml), IL-6 (10 ng/ml) or C5a (10 ng/mL) for 15 min and then stimulated with sCD95L (100 ng/ml). The cytosolic ROS was assessed. For each condition, data represent mean ± SD of n = 41 cells. Data are representative of three independently performed experiments.

Fig 3: DB550 treatment alleviates clinical symptoms in two AAV-like mouse models.A Anti-MPO-induced ANCA vasculitis model. Mice (AAV-like, Ctrl) received anti-GBM serum (40 mg/kg, day 0) followed by anti-MPO (30 mg/kg) and LPS (0.5 mg/kg, day 5). Untreated controls received non-immune serum, isotype IgG, and PBS. AAV-like mice were treated or not with DB550 (10 mg/kg, three times weekly) (n = 5/group). B Serum soluble CD95L levels were quantified by ELISA at day 12 (mean ± SD; *p < 0.05, two-tailed Mann-Whitney). C Localization of CD95L (red) and CD31 (green) in kidney sections of AAV and control mice; nuclei stained with DAPI (blue). Scale bar, 50 µm. D Membrane-bound (48 kDa) and soluble (26 kDa) CD95L in kidney lysates were analyzed by immunoblotting. E Kidney histology assessed by Masson’s trichrome staining. Scale bar, 50 µm. F Glomerular injury (% crescents) was scored (mean ± SD; **p < 0.01, two-tailed Mann-Whitney; n = 5/group). G Renal function assessed by urine protein/creatinine ratio and serum urea (mean ± SD; **p < 0.01, two-tailed Mann-Whitney; n = 5/group). H ANCA pulmonary vasculitis (AAPV) model. Mice received intratracheal LPS (0.5 mg/kg) and fMLP (0.5 mg/kg), followed by anti-MPO (50 mg/kg). Controls received PBS and isotype IgG. AAPV mice were treated or not with DB550 (10 mg/kg, at D-1 and D3) (n = 3-4/group). I Soluble CD95L levels in bronchoalveolar lavage (BAL) were measured by ELISA at day 12 (mean ± SD; ns for not significant, two tailed Mann-Whitney). J Membrane-bound and soluble CD95L in lung lysates were analyzed by immunoblotting. K Left: Lung histology by hematoxylin/eosin staining (scale bar, 2 mm). Right: Lung hemorrhage scores (mean ± SD; *p < 0.05, two tailed Mann-Whitney; n = 3-4/group). L Neutrophil counts in BAL (mean ± SD; ns or *p < 0.05, two-tailed Mann-Whitney; n = 3-4/group). All data are representative of at least three independent experiments unless stated otherwise.

Fig 4: AAV neutrophils are primed to respond to sCD95L.Cytosolic calcium concentration ([Ca2+]cyt) was assessed using Cal-520-AM. Ratio values (R) were normalized to pre-stimulated values (R0) to yield R/R0 values (relative [Ca2+]). Data represent the mean ± SD (n = 41 cells). Data are representative of three independently performed experiments. Sera were injected, and calcium responses were monitored in neutrophils isolated from healthy donors and AAV patients. A Left panel: Relative Cytosolic Ca2+ concentration in neutrophils isolated from HD and exposed to sera of AAV patients or healthy donors. For each condition, data represent mean ± SD of n = 41 cells. Right panel: histograms of areas under the curve (AUC) of the cytosolic Ca2+ concentration measured in the left panel. Data represent mean ± SD; * p < 0,05 using two-tailed Mann-Whitney test. B Cytosolic Ca2+ concentration was assessed in HD neutrophils stimulated with sera from AAV patients. Briefly, neutrophils were pre-incubated in the presence or absence of DB550 (1 µM) for 30 min and then incubated with sera of AAV patients. To block CD95L/CD95 interaction, the neutralizing anti-CD95L mAb NOK-1 or isotypic control (1 µg/ml) was pre-incubated in the indicated sera for 10 min prior to incubation with HD neutrophils. For each condition, data represent mean ± SD of n = 41 cells. Insets: Cytosolic Ca2+ concentration AUC of indicated conditions. Data represent mean ± SD; *** p < 0.001, **** p < 0.0001 using unpaired and two-tailed Mann-Whitney test. C Neutrophils isolated from AAV patients or healthy subjects were stimulated with sCD95L (100 ng/mL) and cytosolic Ca2+ concentration was monitored. For each condition, data represent mean ± SD of n = 41 cells. Data are representative of three independently performed experiments. D Cytosolic Ca2+ concentration was assessed in PNN from HD, which were pre-incubated pre-treated in the presence or absence (control) of the pro-inflammatory cytokines TNF (5 ng/ml), IL-1β (10 ng/ml), IL-6 (10 ng/ml) or C5a (10 ng/mL) for 15 min and then stimulated with sCD95L (100 ng/ml). The cytosolic Ca2+ concentration was assessed. For each condition, data represent mean ± SD of n = 41 cells. Data are representative of three independently performed experiments.