Fig 2: Transfer of ROR?tS182A CD4+ T cells promote exacerbated wasting in Rag1-/- mice(A) Weight changes of Rag1-/- mice receiving ROR?tWT (n = 6) or ROR?tS182A (n = 6) naive CD4+ T cells. Each dot represents the result from 1 mouse. Each bar represents the sample mean. **p < 0.01 and ***p < 0.001 (multiple t test).(B) RNA expression level of Il17a and Il10 in total cLP cell lysate from Rag1-/- mice in (A). Each dot represents the result from 1 mouse. Each bar represents the sample mean. *p < 0.05; n.s., not significant (t test).(C) Right: Activated ROR?tWT (transduced with GFP expressing pMIG construct) or ROR?tS182A (transduced with Thy1.1 expressing MSCV construct) CD4+ T cells were mixed in a 1:1 ratio and injected into Rag1-/- mice. Left: Summarized proportion of IL-17A+ and IL-10+ colonic Th17 and ROR?t+ Treg cells from Rag-/- recipients 33 days post-transfer (n = 5). Each line represents results from same Rag1-/- recipient. Each bar represents the mean from 5 Rag1-/- recipients. *p < 0.05; n.s., not significant (multiple t test).(D) Representative flow cytometry analysis of IL-17A and IL-10 in colonic Th17 and ROR?t+ Treg cells from (C).

Fig 3: Common and distinct ROR?tS182-dependent gene programs in colonic Th17 and ROR?t+ Treg cells(A) Number of ROR?tS182-dependent genes (DEG, p < 0.05) in colonic Th17 (cluster 0 and 3) and ROR?t+ Treg cells (cluster 2d) from steady-state or DSS-challenged mice.(B) Venn diagram showing overlap and subset-specific ROR?tS182-regulated genes in Th17 and ROR?t+ Treg cells from (A).(C) Percentage of cells expressing ROR?tS182-dependent genes (gray dots, p < 0.05) in colonic ROR?t+ Treg cells (subset 2d) from DSS-challenged ROR?tWT and ROR?tS182A mice. Select genes were labeled and highlighted in blue.(D) Violin plot of Il10 expression in ROR?t+ Treg cells (subset 2d) from control or DSS-challenged ROR?tWT and ROR?tS182A mice.(E) Proportion and cell number of IL-10-producing colonic Th17, ROR?t+ Treg, and conventional Treg cells from DSS-challenged ROR?tWT and ROR?tS182A mice. Each dot represents the result from 1 mouse. Each bar represents the sample mean. *p < 0.05; ***p < 0.001; n.s., not significant (paired multiple t test).(F) Representative flow cytometry analysis of IL-17A and IL-10 in colonic ROR?t+ Treg and conventional Treg cells from DSS-challenged mice harvested on day 10.

Fig 4: Murine fecal metabolites suppress activated immune cells and are enriched with hypoxanthine/inosine and glutamate/glutamine.A Schematic illustration of in vitro fecal supernatant–immune cell cultures and stool metabolite analysis. mRNA expression for Il10 (B), Foxp3 (C), and Tnfa (D) as a ratio to Gapdh in RRV-primed hepatic MNCs cultured in the presence of fecal supernatants from neonatal mice of water- or butyrate-fed mothers (mean ± SD, two-tailed unpaired Student’s t test, n = 4 per group; *p < 0.05, **p < 0.01, ***p < 0.001). E NMDS and ANOSIM of metabolites in fecal supernatants of neonatal mice from water- or butyrate-fed mothers at 14 days of age. F Volcano plot illustrating fecal metabolites with p values and fold changes between neonatal mice from water- and butyrate-fed mothers (inset depicts metabolites of lower distance). The replicate values were determined using biologically distinct samples and p values calculated using unpaired Student’s t test with two-tailed distribution. G Fecal metabolites ordered by Euclidean distance measured from the volcano plot. Source data for this figure are provided as a Source data file.

Fig 5: Camptothecin impedes the inflammatory response in stimulated primary microglia ACytotoxicity of CPT on stimulated microglia was determined by measuring the release of LDH in the supernatants (n = 4 technical replicates).BSupernatants were collected after 24 h LPS/IFN? stimulation and measured for nitrite, as an index of nitric oxide (NO) production (n = 4 technical replicates).C, DImmunostaining of iNOS (red) and Phalloidin (green) in microglia (n = 3 technical replicates with more than 5 random fields per well) (bar = 50 µm).EThe kinetics of mRNA expression of Nos2, Tnfa, Il1b, Il6, and Il10 after 1, 3, 6, and 24 h of LPS/IFN? stimulation (n = 3–6 technical replicates).FCytokine release (TNF, IL-6, MCP-1, and IL-10) to the supernatants were measured by cytometric beads assay after 24 h LPS/IFN? stimulation (n = 2 or 5 technical replicates).G, HThe production of TNF-a within microglia was determined by flow cytometry (n = 3 to 5 technical replicates). Data information: Data shown are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = no statistical significance. Statistical analyses were performed using one-way ANOVA with Dunnett’s Multiple Comparison Test for (A), (B), and (D), and the comparisons between the LPS/IFN?and LPS/IFN?+CPT group in (E-H) were performed using the student’s unpaired two-tailed t-test.