Microbiota depletion affects metabolic activity of CD11b ⁺ CD45 ^hi myeloid cells in the brain, and TMAVA administration does not affect T cell populations in the periphery. (a and b) Targeted mass spectrometry analysis of polar metabolites in FACS-sorted CD11b⁺ CD45^int cells from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle, on D+14 after allo-HCT. Quantification of intracellular levels of (a) acetylcarnitine and (b) palmitoylcarnitine. Experiments were performed two times and results (mean ± SEM) pooled. n = 7 per group. Each data point represents a biological replicate. P values were calculated using either two-sided Student’s unpaired t test. *P < 0.05, ns = 0.059. (c and d) Flow cytometry–based SCENITH analysis of CD11b⁺ CD45^hi cells in the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle. Quantification of (c) FAO and AAO capacity of CD45^hi CD11b⁺ cells, and (d) GD of CD11b⁺ CD45^hi cells. The experiment was performed once. n = 6 or 7 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test. *P < 0.05. (e) GVHD-related mortality of SPF BALB/c (H2-k^d+) recipients undergoing allo-HCT and treated with antibiotics (all animals) and either TMAVA or vehicle (water). The experiment was performed once. n = 5 per group. Each data point represents a biological replicate. P values were calculated using the Mantel–Cox test. The Bonferroni-corrected threshold was set as 0.017. (f) Clinical scoring of aGVHD symptoms in SPF BALB/c (H2-k^d+) recipients undergoing allo-HCT and treated with antibiotics (all animals) and either TMAVA or vehicle (water). The experiment was performed once. n = 5 per group. Each data point represents a biological replicate. P values were calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons test. Bonferroni-corrected threshold was set as 0.017. (g–i) Histological scoring of GVHD severity in colon, small intestine, and liver samples stained for H&E on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics (all animals) and either TMAVA or vehicle (water). Quantification of GVHD histological scores in the (g) colon, (h) liver, and (i) small intestine. The experiment was performed once. n = 6 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test. (j–x) Flow cytometry analysis of the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics (all animals) and either TMAVA or vehicle (water). Quantification of (j) percentage of CD11b⁺ CD45^int cells, and absolute counts of (k) CD11b⁺ CD45^int cells, (l) CD3⁺ CD4⁺ cells and (m) CD3⁺ CD8⁺ cells. Quantification of IFN-γ in (n) CD3⁺ CD4⁺ cells and (o) CD3⁺ CD8⁺ cells. Absolute counts of (p) CD3⁺ CD4⁺ IFN-γ⁺ cells, and (q) CD3⁺ CD8⁺ IFN-γ⁺ cells, (r) percentage of Ly6G⁺ cells, (s) absolute counts of Ly6G⁺ cells, (t) percentage of B220⁺ CD19⁺ cells, (u) absolute counts of CD19⁺ B220⁺ cells, (v) percentage of CD206⁺ cells, (w) absolute counts of CD206⁺ cells, and (x) E-cadherin on CD31⁺ CD45⁻ cells. The experiment was performed once (k and n–t), two times (l, m, and u–x), or three times (j). n = 5–15 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test.