Figure 9. RIPK3 deficiency, but not MLKL loss, decreases innate K/B × N arthritis chronicity.

(a–e) WT, c1^LysMcrec2^−/− and c1^LysMcrex^−/−c2^−/− BM chimeras were injected with 100 μl K/B × N pathogenic serum, and (a,b) clinical severity of disease (0–3 per limb) was measured; data are represented as mean±s.e.m., (a) n=4–5 mice per group, *P<0.05, (b) n≥11 mice per group (WT versus c1^LysMcrex^−/−c2^−/−, P=0.0196; WT versus c1^LysMcrec2^−/−, P=0.083). P values were calculated using the Mann–Whitney two-sample rank test. (c) MPO activity was measured in limbs of mice at day 2 of model. Data show individual mouse MPO average radiance, and a representative bioluminescent image of MPO levels in arthritic mice. Mean±s.e.m., *P<0.05, Mann–Whitney two-sample rank test. (d,e) Serum (d) and ankle joint secretions (e) from mice at day 5 of K/B × N arthritis were analyzed for cytokine levels by ELISA. Symbols represent individual mice, mean±s.e.m. *P<0.05, **P<0.01, Student’s two-tailed t-test. (f–i) WT (n=5–6/ experiment), IL-1 R^−/− (n=5), IL-1α^−/− (n =5), Myd88^−/− (n=4) and Trif^−/− (n=5–6 per experiment) mice were injected with 100 μl K/B × N serum and (f,h) clinical severity and (g,i) MPO activity (average radiance of individual mice) measured. Mean±s.e.m. *P<0.05, **P<0.01. (WT versus Trif^−/−, total clinical course (f) 0.03, (h) NS, not significant; resolution phase (h) P=0.0079, (f) 0.0173). P values were calculated using the Mann–Whitney two-sample rank test. (j,k) WT mice were treated with 200 μg antibodies to IL-1β (B122, n=4 mice) or control polyclonal hamster antibody (n=6 mice) on days −1, 0, 2, 4, 6, 8 and 10 after injection of 100 μl K/B × N pathogenic serum. Mice were evaluated (j) daily for clinical severity, and (k) MPO activity (average radiance) was measured in limbs on days 7 and 12. Mean±s.e.m. **P<0.01, Mann–Whitney two-sample rank test. One of two experiments. (l,m) WT, Ripk3^−/− and Ripk3^−/−Caspase-8^−/− mice (n≥6 mice per group) were injected with 100 μl K/B × N serum. (l) Clinical severity, (m) MPO activity (average radiance) in limbs of individual mice. Mean±s.e.m. Representative of at least one of two independent experiments. (WT versus Ripk3^−/−, clinical course NS, resolution phase P=0.008; WT versus Ripk3^−/−Caspase-8^−/− clinical course NS, resolution phase P=0.0075). *P<0.05, **P<0.01, Mann–Whitney two-sample rank test. (n,o) WT and Mlkl^−/− mice (n=6 mice per group) were injected with 100 μl K/B × N serum. (n) Clinical severity and (o) MPO activity in limbs of individual mice are shown. Mean±s.e.m. (p–s) WT, Ripk3^−/− and Ripk3^−/−Caspase-8^−/− mice (n=5–6 mice per group) were injected with 100 μl K/B × N serum and during the resolution phase of disease (day 10) analyzed for (p) clinical severity, (q) MPO activity in limbs, and IL-1β levels measured in (r) serum and (s) ankle joint secretions. Data symbols are individual mice and different symbols within each group indicate separate experiments. Mean±s.e.m. *P<0.05, **P<0.01. P values were calculated using the Mann–Whitney two-sample rank test (p,q) or the Student’s two-tailed t-test (r,s).