Fig 2: Clinical Relevance of S100A8 by RNF5 in Samples from IBD Patients(A) Representative images of IHC staining for RNF5 and S100A8 in colon sections from healthy control and IBD patients (un-inflamed and inflamed area). Scale bars, 50 µm.(B) Quantification of RNF5 and S100A8 IHC scores of IECs in colon sections from healthy control (n = 17) and IBD patients (un-inflamed and inflamed area) (n = 19).(C) Scatterplots between the IHC score (IECs) of RNF5 and S100A8 in colitis from inflamed area of IBD patients (n = 19).(D) Number of RNF5- or S100A8-positive cells (403) in stroma of colon sections from healthy control (n = 17) and IBD patients (un-inflamed and inflamed area) (n = 19).(E) Scatterplots between number of RNF5- and S100A8-positive cells in stroma of colitis from inflamed area of IBD patients (n = 19).(F) RNF5 IHC score (IECs) and S100A8 IHC score (IECs) in the pathological severity (PS)-low (n = 4) or PS-high (n = 12) colitis from inflamed area of IBD patients.(G) Number of RNF5- or S100A8-positive cells (stroma) in the PS-low (n = 4) or PS-high (n = 12) colitis from inflamed area of IBD patients.Graphs show mean ± SEM. *p < 0.01, **p < 0.001, and ***p < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test (B and D). Pearson’s correlation (C and E). n.s., not significant (p > 0.05). *p < 0.05 and ***p < 0.001 by two-tailed t test (F and G).

Fig 3: 27-OH-induced RAGE increase in neurons. A Western blots from rat primary neurons treated with 27-OH (1 µM, 24 h) showing protein levels of RAGE (Vehiclemean = 1.0, SEM = 0.08, n = 8 wells; 27-OHmean = 1.34, SEM = 0.09, n = 8 wells. Unpaired t test P = 0.01). B Western blots from rat primary cultures treated with S100A8 (12.5 ug/ml, 24 h) showing protein levels of RAGE (Vehiclemean = 1.0, SEM = 0.08, n = 12 wells; 27-OHmean = 1.39, SEM = 0.13, n = 12 wells. Unpaired t test P = 0.02). C Western blots from rat primary neurons treated with 27-OH (1 µM, 24 h) showing protein levels of RXR? (Vehiclemean = 1.0, SEM = 0.08, n = 8 wells; 27-OHmean = 1.34, SEM = 0.09, n = 8 wells. Unpaired t test P = 0.053) and D mRNA levels of RXR? were diminished in the RXR?-knockdown primary neurons treated with 27-OH (Controlmean = 0.96, SEM = 0.1247, n = 4 wells; 27-OHmean = 2.007, SEM = 0.26, n = 6 wells; iRXRgmean = 0.12, SEM = 0.056, n = 4 wells; iRXRg + 27-OHmean = 0.58, SEM = 0.04, n = 4 wells. ANOVA P < 0.0001. Tukey’s multiple comparisons (**) P = 0.005; (***) P = 0.002), and a similar effect was found on RAGE mRNA expression (Controlmean = 1.057, SEM = 0.033, n = 4 wells; 27-OHmean = 1.54, SEM = 0.101, n = 4 wells; iRXRgmean = 0.29, SEM = 0.035, n = 4 wells; iRXRg + 27-OHmean = 0.59, SEM = 0.35, n = 4 wells. ANOVA P = 0.0003. Tukey’s multiple comparisons (**) P = 0.0045)

Fig 4: Neutralizing Antibodies to S100A8 Attenuate Acute Colitis in DSS-Treated Rnf5-/- Mice(A) Body weight of WT and Rnf5-/- mice administered 2.5% DSS and treated with control rabbit IgG or rabbit anti-S100A8-neutralizing antibody (200 µg, i.p.) on days –1, 1, and 3, relative to the start of DSS treatment on day 0 (n = 6 mice/group), as shown by red arrows (on day 9: ****p < 0.0001; Rnf5-/- + control IgG versus Rnf5-/- + anti-S100A8, ****p < 0.0001; WT + control IgG versus Rnf5-/- +control IgG, n.s.; WT + anti-S100A8 versus Rnf5-/- +anti-S100A8).(B) Disease activity index (see STAR Methods) of anti-S100A8- or control rabbit IgG-treated WT or Rnf5-/- mice after 2.5% DSS treatment as described for (A) (n = 5) (on day 9: *p < 0.05; Rnf5-/- + control IgG versus Rnf5-/- + anti-S100A8, *p < 0.05; WT + control IgG versus Rnf5-/- +control IgG, n.s.; WT + anti-S100A8 versus Rnf5-/- + anti-S100A8).(C) Survival of anti-S100A8- or control IgG-treated WT and Rnf5-/- mice after 2.5% DSS treatment (n = 6 mice/group) by Kaplan-Meier method.(D) Representative images (left) of colon and cecum and quantification (right) of colon length from anti-S100A8- or control rabbit IgG-treated WT or Rnf5-/- mice as described for (A) on day 9 after 2.5% DSS treatment (n = 5).(E) Representative images of H&E-stained and anti-CD4-stained colon sections from WT and Rnf5-/- mice treated as described for (A). Sections are from mice sacrificed on day 9. Scale bars, 50 µm.(F) Quantification of histological score and CD4+ cells (per 203 field) from WT and Rnf5-/- mice treated with anti-S100A8 antibody or control rabbit IgG after 2.5% DSS treatment for (E) (n = 3 mice/group, three fields each).(G) Body weight of WT and Rnf5-/- mice administered 2.5% DSS for 5 days and treated with control rabbit IgG or rabbit anti-S100A8-neutralizing antibody (200 µg, i.p.) on days 5, 7, and 9 (n = 6 mice/group), as shown by red arrows (on day 9: **p < 0.01; Rnf5-/- + control IgG versus Rnf5-/- + anti-S100A8, **p < 0.01; WT + control IgG versus Rnf5-/- +control IgG, n.s.; WT + anti-S100A8 versus Rnf5-/- +anti-S100A8).All data are representative of two or three independent experiments. Graphs show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test (A, B, and G) or one-way ANOVA (D, F). Survival by log rank test (C).

Fig 5: 27-OH increases S100A8 and RAGE expressions administered acutely in the brain. Intracerebroventricularly injected 27-OH (ICV27-OH) (10 µM) into the lateral ventricle of wild-type mice showing (A) mRNA levels of S100A8 in aCSF controls (mean = 1.22, SEM = 0.23, n = 4 animals, P = 0.018) and on 27-OH mice (mean = 4.30, SEM = 1.43, n = 4 animals). Protein levels by western blot of S100A8 (B) (WTmean = 100, SEM = 3.81, n = 4 animals; CYP27Tgmean = 110.3, SEM = 10.3, n = 4 animals, P = 0.025) and RAGE (C) (WTmean = 100, SEM = 34.97, n = 4 animals; CYP27Tgmean = 123.8, SEM = 5.94, n = 4 animals, P = 0.021) of ICV27-OH