Microbiota depletion increases microglial activation in aGVHD. (a–c) Flow cytometry analysis of the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). (a and b) Quantification of the percentage of CD11b⁺ CD45^int cells (a) along with (b) representative flow cytometry plots of each group. (c) Quantification of absolute numbers of CD11b⁺ CD45^int cells. Experiments were performed three (c) or nine times (a and b) and results (mean ± SEM) pooled. n = 12–43 per group. Each data point represents a biological replicate. P values were calculated using either two-sided Student’s unpaired t test or the Mann–Whitney U test. *P < 0.05, ****P < 0.0001. (d–g) Histology of brain samples stained for Iba-1⁺ cells and Iba-1⁺ Ki67⁺ cells analyzed on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). Quantification of the absolute number (per mm²) of (d) Iba-1⁺ and (f) Iba-1⁺ Ki67⁺ cells in the cerebral cortex, along with representative images of (e) Iba-1⁺ cells (scale bars, 1,000 μm) and (g) Iba-1⁺ Ki67⁺ cells (scale bars, 50 μm) in each group. Experiments were performed two (f and g) or three times (d and e) and results (mean ± SEM) pooled. n = 8–10 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test. **P < 0.01. (h) Gene set enrichment analysis on microarray analysis shows differentially regulated cell proliferation–related pathways from FACS-sorted CD11b⁺ CD45^int cells isolated from the brains of SPF BALB/c (H2-k^d+) recipients treated with antibiotics (n = 2) or vehicle (n = 2) and analyzed on D+14 after allo-HCT. The experiment was performed once. (i–n) Flow cytometry analysis of the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). Quantification of (i) CD11c, (k) P2RY12, and (m) TLR4 on CD11b⁺ CD45^int cells along with representative histograms of (j) CD11c, (l) P2RY12, and (n) TLR4 in each group. Experiments were performed three (k and l) or six times (i, j, m, and n) and results (mean ± SEM) pooled. n = 10–22 per group. Each data point represents a biological replicate. P values were calculated using either two-sided Student’s unpaired t test or the Mann–Whitney U test. *P < 0.05, **P < 0.01. (o–p) Flow cytometry analysis of the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics and transplanted on D0 with different T cell numbers. (o and p) Quantification of MyD88 in CD11b⁺ CD45^int cells (o) along with (p) representative histograms of each group. Experiments were performed two times and results (mean ± SEM) pooled. n = 5–8 per group. Each data point represents a biological replicate. P values were calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, ***P < 0.001. (q) Flow cytometry analysis of the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics. Correlation analysis using simple linear regression between TLR4 (MFI) on CD11b⁺ CD45^int cells and percentage of CD3⁺ CD45⁺ cells in the brain. Experiments were performed two times and results pooled.