Antibiotic-mediated microbiota depletion in aGVHD exacerbates systemic inflammation. (a–n) Analysis of cecum, colon, stool, and bone marrow cells on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). (a) Schematic depiction of treatment and allo-HCT. (b–l) Quantification of cecum length (b) and (c) representative images of each group. Quantification of (d) SYTO 9⁺ bacteria normalized to stool weight and (e) percentage of SYTO 9⁺ bacteria using flow cytometry, along with (f) representative flow cytometry plots of each group on D−1 (after 14 days of antibiotics/vehicle treatment). Quantification of (g) SYTO 9⁺ bacteria normalized to stool weight and (h) percentage of SYTO 9⁺ bacteria using flow cytometry, along with (i) representative flow cytometry plots of each group on D+13 (after 27 days of antibiotics/vehicle treatment). Quantification of (j) acetate, (k) butyrate, and (l) propionate in colon using targeted mass spectrometry analysis of polar metabolites. (m) Quantification of percentage of body weight of mice at different time points compared with the weight at start of antibiotic treatment (D−14). (n) Quantification of percentage of H2-k^b+ cells in the bone marrow using flow cytometry. The experiment was performed once (b, c, m, and n) or two times (d–l) and results (mean ± SEM) pooled. n = 5–11 per group. Each data point represents a biological replicate. P values were calculated using either two-sided Student’s unpaired t test, the Mann–Whitney U test, or two-way ANOVA with Šidák’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (o–q) Histological scoring of GVHD severity in colon, liver, and small intestine samples stained for H&E on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). Quantification of GVHD histological scores in (o) colon, (p) liver, and (q) small intestine. Experiments were performed two times and results (mean ± SEM) pooled. n = 9 or 10 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test. ****P < 0.0001. (r and s) Analysis of the spleen on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). Quantification of (r) CD80 and (s) CD40 on H2-k^b+ F4/80⁺ cells in the spleen using flow cytometry. The experiment was performed once (s) or two times (r) and results (mean ± SEM) pooled. n = 5–10 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.