Fig. 2: NET formation drives CAIA severity in Micl−/− mice.
From: Recognition and control of neutrophil extracellular trap formation by MICL
a, ROS generation by MSU, PMA, zymosan or A. fumigatus hyphae-stimulated bone marrow neutrophils, depicted as relative light units (RLU), over time. Data are a representative example of n = 4 independent experiments and mean ± s.d. performed in triplicate. Area under curve was analysed using unpaired two-tailed Student’s t-test. WT versus Micl−/− MSU P < 0.0001, A. fumigatus P = 0.0056 and not significant (NS). b, Sytox green fluorescent images of MSU-induced NETs and NET formation percentage by thioglycollate-elicited neutrophils (3 fields of view per condition) 4 h post-stimulation with PMA or MSU, or medium alone (–). Data are represented as mean ± s.d. (n = 3 independent experiments performed in triplicate). Scale bars, 500 µm. Statistical significance was determined by two-way ANOVA with Bonferroni’s multiple comparisons test. WT versus Micl−/− MSU P = 0.0304. c, Representative confocal immunofluorescence microscopy of NETs in CAIA WT and Micl−/− day 11 synovial sections (as per Extended Data Fig. 1a; n = 1 experiment with 3 mice per group). GR-1 (purple), DAPI (blue), citrullinated histone 3 (cit-H3; yellow) and DNA/H1 (green) are shown. NETs are defined as GR-1+cit-H3+DNA/H1+-stained cells. Scale bars, 100 µm. d, Schematic of the PAD4 inhibitor CAIA treatment regime. e,f, Image stream quantification of NET-positive cells (e) and neutrophils isolated from arthritic ankle joints (f) at day 11 during PAD4 inhibitor (BB-CL-amidine) treatment. Pooled data are from 2 independent experiments with n = 7 biologically independent mice per group, represented as mean ± s.d. Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparisons test. WT versus Micl−/− P = 0.0063 and Micl−/− versus Micl−/− + PAD4 inhibitor P = 0.0295 (e). g, Severity scoring of WT and Micl−/− mice during CAIA treated with vehicle or the PAD4 inhibitor (GSK484). The black arrow indicates the start of treatment. Data shown are a representative example of n = 2 experiments with 4 mice per group, represented as mean ± s.d. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. Days 8–10 Micl−/− versus Micl−/− + GSK484 P < 0.0001. *P < 0.05, **P < 0.01 and ****P < 0.0001. The diagram in panel d was created using BioRender (https://biorender.com).
