Fig 2: Induction of CXCL1 in the DRG is not dependent of neutrophil accumulation in the DRG. (a) Time course of mechanical allodynia in neutrophil-depleted mice after MOG35–55 immunization. Mice were treated with IgG2a (500 µg, intraperitoneally) or anti-Ly6G (500 µg, intraperitoneally) before MOG35–55 immunization. n = 8 mice per group, two-way repeated measures ANOVA, F(1,98) antibody treatment = 217.8, ***P < 0.001. (b) Images show the immunofluorescence of MPO (green) and Nissl (red) in the lumbar 5 (L5) DRG of neutrophil-depleted mice on day 5 after MOG35–55 immunization. Arrowheads indicate MPO-positive cells. The inset indicates an enlarged image. Scale bar = 50 µm. (c) Columns represent statistical data of neutrophil density in the L5 DRG of neutrophil-depleted mice on day 5 after MOG35–55 immunization. n = 4 mice per group, unpaired t-test. **P = 0.0072. (d) Relative neutrophil elastase (NE) activity in the L5 DRG of neutrophil-depleted mice on day 5 after MOG35–55 immunization. n = 4 mice per group, unpaired t-test. ***P = 0.0003. (e) Immunoblot shows protein levels of CXCL1 in the DRG 5 days after MOG35–55 immunization in neutrophil-depleted mice. Columns represent statistical data of CXCL1 protein normalized to ß-actin. n = 4 mice per group, unpaired t-test, n.s. P = 0.8278. n.s.: not significant. All values are the mean ± SEM.

Fig 3: tACPA prevents NET-mediated tissue damage and disease progression in chronic CIA mice. a A schematic overview of the CIA mouse model of IA. To induce chronic IA, mice were injected twice (days 0 and 21) with CII. Therapeutic treatment started after onset of the disease (between days 21 and 28), when the MAS was =0.75, and included four injections (4-day intervals) with tapered dosing regimens of cIgG (50/50/50/50 mg/kg) or hz-tACPA (30/30/30/10, 50/50/50/15 or 50/10/10/10 mg/kg). The mice were terminated 14 days after the start of treatment. b The MAS of CIA mice was evaluated for 14 days (n = 10 mice per group). c Representative immunofluorescence and H&E images of NET release in joints of right hind paws showing citH3 (red), DAPI (blue), MPO (yellow), and Ly6G (green). Scale bars: 100 µm. d Quantification of NETs (colocalization of citH3 and MPO) in the tibiotarsal, proximal intertarsal, distal intertarsal, and tarsometatarsal joints of the right hind paws of mice (n = 10). e Significant correlation of macroscopic score (paw swelling) and NETs per joint. f Bone damage to the right and left hind knees and ankles was analyzed by X-ray at day 14 after the first antibody injection (n = 10). H&E and SO staining of joints from right and left ankles showing g inflammatory cell influx, h bone erosion, i cartilage erosion, j cartilage PG depletion, and k chondrocyte death at day 14 after the first antibody injection (n = 16–20 mouse ankles). The results are presented as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using two-way ANOVA with Dunnett’s multiple comparisons test (b; cIgG was used to calculate significant differences), unpaired two-tailed Student’s t test (d), two-tailed Mann–Whitney statistical test (f–k), or Spearman r test (e)

Fig 4: Type I IFN activates neutrophils to promote bacterial growth.Ifnarfl/fl MRP8-Cre negative (Creneg; circles) or positive (Crepos; squares) mice were infected and treated with Ctrl Ab (closed symbols) or aGM-CSF (open symbols) as in Fig. 1a. a Viable bacterial loads determined in the lungs at day 21 post-infection. **P < 0.0022. Data representative of two biological experiments. Represented is the mean ± SEM; each dot represents an individual mouse: n = 4–6 mice/group/experiment. b Representative images of lung sections stained with citrullinated histone H3 (citH3; red), MPO (green), and DAPI (blue). NETs are visualized by colocalization of citH3 and DAPI staining (merged images). Scale bars, 50 µm. Data representative of two biological experiments (n = 3–4 mice/group/experiment). c Percentage of NET area in the lung normalized to MPO positive signal. Data pooled from two biological experiments. Represented is the mean ± SD; each dot represents an individual mouse: n = 7–8 mice/group. Source data are provided as a Source Data file. Statistical analysis was performed using two-tailed Mann–Whitney test.

Fig 5: IFNAR signalling is required for severity of lung NETosis in C3HeB/FeJ mice.a Schematic of experimental design: C3HeB/FeJ mice were treated by intraperitoneal (i.p.) injection of 0.5 mg of anti-IFNAR (aIFNAR; open circles) or isotype control (labelled as Ctrl Ab; closed circles) mAbs the day prior low dose HN878 aerosol infection, followed by i.p. injections of 0.5 mg of aIFNAR or Ctrl mAbs, three times per weekly until harvest on day 26 post-infection. b Viable bacterial loads were determined. **P = 0.0079. c Representative hematoxylin and eosin (H&E) staining of infected lung sections (n = 3 mice/group). Scale bars from top to bottom: 1 mm; 200 µm; 50 µm, 10 µm. d Relative lung lesion burden from H&E stained sections (Supplementary Data 1). 0 = no lesions, 1 = focal lesion, 2 = multiple focal lesions, 3 = one or more focal severe lesions, 4 = multiple focal lesions that are extensive and coalesce, 5 = extensive lesions that occupy the majority of the lung lobe. *P = 0.0152. e Relative necrosis (left) and intra-alveolar necrotic debris (right) scores from H&E stained lung sections (Supplementary Data 1). 0 = not present, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. **P = 0.0023. f Representative Ziehl–Neelsen staining of infected lung sections. Scale bars, 20 µm. g Representative images of lung sections labelled with citrullinated histone H3 (citH3; red), MPO (green), and DAPI (blue). NETs are visualized by colocalization of citH3 and DAPI staining (merged images). Scale bars, 50 µm. h Percentage of NET area in the lung normalized to MPO positive signal. **P < 0.0012. b, c, f, g Data representative of two biological replicate experiments. d, e, h Data pooled from two biological experiments. Represented is the mean ± SD; each dot represents an individual mouse: n = 5 mice/group (b) or n = 6–7 mice/group (d, e, h). Source data are provided as a Source Data file. Statistical analysis was performed using the two-tailed Mann–Whitney test.