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Commensal microbiota in vivo regulation of pro-inflammatory cytokines. 11 to 12 week-old male SPF & GF mice were euthanized; tissues harvested for (a–d) gene expression assays, (e) flow cytometry assays, and (g,h) ELISA assays. (a–d) RNA was isolated from tissues and qRT-PCR analysis was performed in (a) bone marrow (n = 4/gp), (b) ileum (n = 6/gp), (c) liver (n = 6/gp), (d) spleen (n = 4/gp) to assess Tnf, Il6, Csf1, Ccl2, Cxcl1 mRNA. Relative quantification of mRNA was performed via the comparative CT method (ΔΔCT); Gapdh was utilized as an internal control gene; data expressed as fold difference relative to SPF. (e) Mesenteric lymph node (MLN) cells and liver lymph node (LLN) cells were isolated and stained for flow cytometric analysis (n = 4/gp) to assess the frequency of CD11b+LY6G-F4/80+LY6Chi (inflammatory monocyte) cells. Cell percentages are expressed relative to total gated monocyte cells. (f) Schematic of newly identified/proposed Gut-Liver-Bone Axis. (g,h) Serum was isolated from whole blood (n = 9–10/gp); ELISA analysis of (g) TNF levels and (h) CSF1 levels. Data are reported as mean ± SEM. *p < 0.05 vs. SPF; **p < 0.01 vs. SPF; ***p < 0.001 vs. SPF.