(A) Fecal microbiota from Clec4n^–/– mice were cultured on MRS medium supplemented with neomycin for 3 days. Single colonies were isolated and identified by PCR amplification and sequencing of bacterial 16S rDNA. (B–F) L.j. WXY strain was isolated from mouse feces. WT mice were treated with antibiotics (ABX), followed by oral transfer of the indicated bacterial strains twice over 7 days. Whole fecal microbiota from normal WT mice were then transferred back for 3 days, followed by DSS treatment for 7 days and sacrifice on day 10. Body weight loss (B), DAI (C), colon length measurement (D), distal colon histology (E), and cLP neutrophil and Treg frequencies (F) (B–D, and F, PBS, n = 7; L.j. WXY, n = 7; Lactobacillus mixture, n = 8; E. coli, n = 7; E, n = 3 /group). (G) Correlation between fecal Lactobacillus abundance and colonic S100a8/S100a9 mRNA expression in C57BL/6J mice under steady-state conditions (n = 18). (H) L.j. WXY was cultured anaerobically at 37°C with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each). Bacterial growth was quantified spectrophotometrically at 6 and 24 hours (n = 6 technical replicates/group). (I) Clec4n^–/– mice received intrarectal administration of recombinant S100A8 + S100A9 for 5 hours, followed by quantification of fecal L.j. abundance by qPCR (n = 10). (J and K) WT and Clec4n^–/– recipient mice were lethally irradiated and reconstituted with BM cells from WT or Clec4n^–/– donors. After 30 days, colonic expression of S100a8 and S100a9 was assessed by qPCR (J), and fecal Lactobacillus abundance was measured by qPCR on days 10, 20, and 30 after transfer (K) (WT→WT, n = 8; WT→Clec4n^–/–, n = 7; Clec4n^–/–→WT, n = 8; Clec4n^–/–→Clec4n^–/–, n = 8.). (L–N) WT and Clec4n^–/– mice were treated intraperitoneally with anti-Ly6G neutralizing Ab or isotype control IgG (100 μg/mouse) every other day for 5 doses. Two days after the final injection, colonic tissues and feces were collected. S100A8 and S100A9 protein levels in colon lysates were measured by ELISA (L), fecal L.j. abundance was determined by qPCR (M), and colonic Il6 and Tnf expression was assessed by qPCR (N) (control [con] IgG, n = 3–4; anti-Ly6G, n = 3–4). (O–S) WT mice were orally administered culture supernatant (sup.) from L.j. WXY or heat-killed (hk) bacteria daily for 3 days before and throughout 7 days of DSS treatment (n = 10 total administrations). Body weight loss (O), DAI (P), colon length measurement at sacrifice on day 9 (Q), distal colon histology (R), and cLP Treg frequencies (S) (PBS n = 8; WXY-sup. n = 8; WXY-hk n = 9). (T) CD11b⁺ and CD11c⁺ cells isolated from WT cLP were stimulated in vitro with L.j. WXY, Lactobacillus mixture, or E. coli. After 12 hours, Il10 and Tgfb1 mRNA expression was quantified by qPCR (n = 4 technical replicates/group). (U) CD62L⁺ naive CD4⁺ T cells isolated from WT spleen and lymph nodes were polarized toward Treg differentiation in the presence of L.j. WXY culture supernatant. After 6 days, Il10 and Tgfb1 expression was measured by qPCR (n = 9 technical replicates from 3 biological replicates/group). (V and W) ABX-treated WT mice received oral L.j. WXY or PBS, followed by fecal microbiota transplantation from normal WT mice and subsequent DSS treatment. Cecal contents were collected on day 10 for untargeted metabolomic analysis by liquid chromatography. Histogram (V) shows the top 20 enriched metabolites, and volcano plot (W) highlights significantly altered metabolites in WXY-treated mice. (X) Targeted metabolomic analysis of l-glutamic acid levels in cecal contents from 8-week-old WT and Clec4n^–/– mice under physiological conditions (n = 3/group). (Y) l-Glutamic acid concentrations in culture supernatants of L.j. WXY, Lactobacillus mixture, or E. coli measured by targeted metabolomics (n = 3 replicates/group). Data in B–F, H–K, and O–S are pooled from 2 independent experiments. Data in B–F, J, L–U, X, and Y are presented as mean ± SD. Statistical analysis: 1-way ANOVA with Bonferroni’s multiple-comparison test (B, C, K, O, and P); 1-way ANOVA with Tukey’s multiple-comparison test (D–F, J, L–N, Q–T, and Y); Spearman’s correlation test (G); 2-way ANOVA test with repeated measures (H); paired Student’s t test (I); or 2-tailed unpaired Student’s t test (U and X).