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. Author manuscript; available in PMC: 2022 Feb 27.
Published in final edited form as: Sci Immunol. 2019 Apr 19;4(34):eaaw4341. doi: 10.1126/sciimmunol.aaw4341

Fig. 4. Nod2 and Cybb regulate neutrophil recruitment and killing to control Mucispirillum accumulation.

Fig. 4.

(A) Total number of colonic neutrophils isolated from ~4-week-old Tac-DKO NI (n = 6), Tac-DKO I (n = 6), and Tac-WT (n = 4). Bars show mean; data are from two independent experiments. *P < 0.05; **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. (B) Total number of luminal neutrophils isolated from ~4-week-old Tac-DKO NI (n = 4), Tac-DKO I (n = 5), and Tac-WT (n = 4). Bars show median; data are from two independent experiments. *P < 0.05 by Kruskal-Wallis test followed by Dunn’s post test. (C)Presence of Mucispirillum in luminal neutrophils isolated from ~5-week-old Tac-DKO NI (n = 13), Jax-DKO (n = 7), Tac-WT (n = 7), Jax-WT (n = 6), Tac-Nod2−/− (n = 6), and Tac-Cybb−/− (n = 7). Bars indicate median; data are from five independent experiments. (D) Presence of Mucispirillum in LP neutrophils (~5-week-old mice; Tac-DKO NI, n = 17; Jax-DKO, n = 7; Tac-WT, n = 8; Jax-WT, n = 10; Tac-Nod2−/−, n = 8; Tac-Cybb−/−, n = 6). Bars show median; data are from four independent experiments. (E and F) Presence of Firmicutes (E) and A. muciniphila (F) in LP neutrophils (~5-week-old mice; Tac-DKO NI, n = 17; Jax-DKO, n = 7; Tac-WT, n = 8; Jax-WT, n = 10; Tac-Nod2−/−, n = 8; and Tac-Cybb−/−, n = 6). Bars indicate median; data are from four independent experiments. (C to F) Results were normalized to GAPDH expression. Kruskal-Wallis test followed by Dunn’s post test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (G) Representative chemiluminescence curves of ROS production frombone marrow–derived neutrophils isolated from Tac-WT (n = 3), Tac-Cybb−/− (n = 3), Tac-Nod2−/− (n = 3), and Tac-DKO (n = 3) mice activated by PMA. The relative light units (RLU) were monitored at 98-s intervals and shown up to 14.42 min. Results are mean ± SEM; data are from three independent experiments. **P < 0.01; ****P < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparisons test. (H) Total number of LP neutrophils (~5-week-old mice; age-matched Tac-DKO NI, n = 6; Jax-DKO, n = 6; Tac-WT, n = 7; Jax-WT, n = 7; Tac-Nod2−/−, n = 7; and Tac-Cybb−/−, n = 6). Bars show median; data are from three independent experiments. (I) Absolute number of luminal neutrophils isolated from ~5-week-old, age-matched Tac-DKO NI (n = 15), Jax-DKO (n = 7), Tac-WT (n = 7), Jax-WT (n = 6), Tac-Nod2−/− (n = 6), and Tac-Cybb−/− (n = 7). Bars indicate median; data are from five independent experiments. (H and I) Kruskal-Wallis test followed by Dunn’s post test. *P < 0.05; **P < 0.01. (J and K) Gene expression in colonic tissues harvested from fostered mice. Results were normalized to GAPDH. Results are mean ± SEM of at least six individual mice; data are from two independent experiments. *P < 0.05; **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test.