Figure 6. Analysis of proinflammatory mediators in LIP-trained BM.

(A-E) Mice were subjected, or not (control) to LIP. After 14 days, indicated cytokines were analysed in BM extracellular fluid (A) and serum (B) (n=4–10 mice/group). (C) After 21 days, relative mRNA expression (normalized to Gapdh) of indicated cytokines was analysed in the gingiva (n=8–10 mice/group). (D,E) After 14 days, BM cells were harvested and the indicated cell types were FACS sorted (Gating strategies of LSK, CMP and GMP in Figures 1 and S1; gating strategies for pre-neutrophils, CD11b⁺CD115⁻Gr-1⁺cKit⁺CXCR4⁺; immature neutrophils, CD11b⁺CD115⁻Gr-1⁺cKit⁻CXCR4⁻Ly6G⁺CXCR2⁻; mature neutrophils, CD11b⁺CD115⁻Gr-1⁺cKit⁻CXCR4⁻Ly6G⁺CXCR2⁺; monocytes, CD11b⁺Ly6G⁻Ly6C⁺) and examined for Csf3r, Il1b and Il1r1 expression (D). Mature neutrophils isolated from the BM of LIP-trained or untrained controls were stimulated with recombinant mouse G-CSF for 24h and IL-1β was measured in culture supernatants (n=6 mice/group) (E). Data are means±SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, NS, not significant vs. control (A-C, E) or mature neutrophils (D); two-tailed Student’s t-test (A-C), except for IL-12p70 in panel A (two-tailed Mann-Whitney U test); one-way ANOVA with Dunnett’s multiple-comparisons test (D,E).