Neutrophil granulocytes selectively form clusters in the ileum after TBI. (A-B) Leukocytes were isolated from duodenum, ileum, and colon from untreated C57BL/6 mice, as well as after TBI, and analyzed by flow cytometry. Neutrophils are defined as viable CD45⁺CD11b⁺Ly6G⁺ cells. (A) Representative plots of neutrophil numbers from untreated samples and 48 hours after TBI. (B) Pooled data from 2 separate experiments showing the percentages of CD11b⁺Ly6G⁺ cells of all leukocytes (CD45⁺) at different points after TBI (n = 6-8). (C) C57BL/6 mice underwent allo-HCT (BALB/c into C57BL/6; 5 × 10⁶ BM + 10⁶ CD4⁺/CD8⁺ T cells). Leukocytes were isolated at different points after allo-HCT, and the percentages of CD11b⁺Ly6G⁺ cells of all leukocytes (CD45⁺) are shown (n = 4). (D) Three-dimensional light sheet fluorescence microscopy of ileum 48 hours after TBI showed cluster formation of neutrophils (Ly6G, red; CD11b, green; tissue autofluorescence, blue). (E) Immunofluorescence staining of ileal cross-section 48 hours after TBI showed localization of neutrophils adjacent to intestinal crypts (Collagen IV, green; Ly6G, red; scale bar, 50 µm). (F) Bacterial load in lamina propria of duodenum, ileum, and colon of untreated C57BL/6 mice and C57BL/6 mice 48 hours after TBI was analyzed by quantitative reverse transcription-polymerase chain reaction. 16S rDNA content was normalized to mouse genomic DNA (glyceraldehyde-3-phosphate dehydrogenase). Pooled data from 2 individual experiments are shown (n = 6).