Fig 2: IMQ dermatitis mice did not increase intestinal permeability but altered immune cell composition in the intestine. (A) Analysis for intestinal permeability of mice induced psoriasis-like dermatitis (on day 17 of IMQ or vehicle treatment), by using 4 kilodaltons (left) or 70 kilodaltons (right) FITC-dextran. Plasma FITC-dextran concentrations were determined by fluorescence intensity, after 1, 2 and 4 hours from oral administration of FITC-dextran (n = 4, in each group). Seventy kilodaltons FITC-dextran was administered intravenously as a positive control. (B) Quantitative PCR analysis for ZO-1 and occludin in colonic epithelial cells after topical IMQ treatment (on day 17) analyzed by real-time PCR. (C) Western blot analysis for occludin expression in colonic epithelial cells after topical IMQ treatment (on day 17). Western blot (left) and relative protein levels (right) of occludin. (D) Immunofluorescence staining was performed in control and IMQ mice. ZO-1 (top, green), occludin (middle, red), claudin (bottom, green) stained with 4',6-diamidino-2-phenylindole (DAPI; blue) in colon tissue. Four to 5 sections from each mice were obtained and the average positive area of each protein was compared with the DAPI-positive area measured (n = 4–5, each dot represents each mouse). (F) Quantitative PCR analysis for Reg III? in colonic epithelial cells after topical IMQ treatment (on day 17) analyzed by real-time PCR. (G) Representative plots of flow cytometry analysis for macrophages (CD11b+CD64+ cells) in CD45+CD3-CD19- B220-NK1.1- colonic lamina propria after IMQ treatment (on day 17) (n = 6 in each group). Percentage (left) and absolute number (right) of macrophages in colonic lamina propria. (H and I) Percentage and absolute number of CD206+ (left) or CD80+ (right) macrophages. (I) Quantitative PCR analysis of IL10, Tnf-a, Il6, IL12b, Arg-1, and Nos2 in LP CD11b+s (n = 3–4 in each group). (J) CD11b+ cells were sorted by magnetic activated cell sorting from colonic lamina propria mononuclear cells on day 17 of IMQ treatment (LP CD11b+s). LP CD11b+s were cultured with lipopolysaccharide (LPS) or peptidoglycan (PGN) for 24 hours in vitro, and concentrations of cytokines in the supernatant were measured by cytometric bead array. Unstimulated control was cultured with medium (Med). (K) Western blot analysis for NLRP3 in peritoneal cavity cells, which are composed mainly of macrophages. Western blot (left) and relative protein levels (right) of NLRP3 (n = 7–8, pooled from 2 independent experiments, in each group). (L) Percentage of ILC3 in CD45+CD3-CD19- cells and (M) pDCs in CD45+CD11b-CD11c+ cells in colonic lamina propria (ILC3, CD45+CD3-CD19-Ror?t+ cells; pDC, CD45+CD11b-CD11c+B220+PDCA-1+ cells. Each symbol represents an individual mouse (n = 4–8). Statistical analyses were performed with the Student t test. *P < .05, **P < .01, ***P < .001. Error bars represent the SEM of samples within a group.

Fig 3: Fecal microbiome transplantation of IMQ mice exacerbated DSS colitis. (A) GF mice were transplanted with a fecal microbiome derived from control mice (cont gnoto mice) or IMQ-treated mice (IMQ gnoto mice). Three weeks after transplantation, immune cells were analyzed by flow cytometry. (B) Percentage (left) and absolute number (right) of CD11b+CD64+ cells in CD45+CD3-CD19-B220- NK1.1- cells from colonic lamina propria. (C) Percentage (left) and absolute number (right) of B cells in CD45+ colonic lamina propria. (D) Percentage of IgA+ (left), IgD+ (middle), and IgM+ (right) B cells in the total B cells from colonic lamina propria (n = 6–7 in each group). (E) Experimental protocol. Cont gnoto mice and IMQ gnoto mice (shown in panel A) were further induced with acute colitis by 3% (wt/vol) DSS. After administration of DSS for 7 days, the severity of colitis was assessed. (F) Representative photograph of colon on day 7 of DSS colitis. (G) Body weight on day 7 of DSS colitis. (H) Colon length on day 7 of DSS colitis. (I) Partial DAI score on day 7 of DSS colitis. Partial DAI score was calculated as the sum of scores for perianal bleeding (0–4) and diarrhea (0–4) (part of the DAI score except for the body weight loss component). (J) Representative H&E staining of distal colon section on day 7 of DSS colitis. Scale bar: 100 µm. (K) Histologic score of colitis on day 7 of DSS (n = 3–5 in each group). (L) Experimental protocol. Mice treated with IMQ or vehicle subsequently were induced with colitis by 2% (wt/vol) DSS for 7 days. Feces derived from control mice, or saline, was administered daily by oral gavage, from the day before starting DSS to the end of the experiment. (M) Body weight changes during administration of DSS and (N) the percentage of the initial value (day 0 of DSS) of body weight on day 7 of DSS colitis. (O) DAI score on day 7 of DSS colitis (n = 6 in each group). (P) Experimental protocol. Mice treated with IMQ or vehicle were subsequently induced with colitis by 2% (wt/vol) DSS for 7 days. A mixture of L johnsonii and L reuteri, or saline, was administered daily by oral gavage, from the day before starting DSS to the end of the experiment. (Q) Body weight changes during administration of DSS and (R) the percentage of the initial value (day 0 of DSS) of body weight on day 7 of DSS colitis. (S) DAI score on day 7 of DSS colitis (n = 8 in each group). Each symbol represents an individual mouse. Statistical analyses were performed with the Student t test. *P < .05, **P < .01, ***P < .001. Error bars represent the SEM of samples within a group.

Fig 4: A gut microbiome in IMQ mice is essential to induce severe DSS colitis. (A) Mice were administered a mixture of ampicillin, neomycin, vancomycin, and metronidazole by oral gavage 3 times a week until the end of the experiment. IMQ treatment was started 1 week after starting Abx treatment. Colonic lamina propria mononuclear cells after IMQ treatment (on day 17) were analyzed by flow cytometry. (B and C) Flow cytometry analysis, the ratio and cell number of CD11b+CD64+ cells in CD45+CD3-CD19- B220-NK1.1- cells from colon lamina propria mononuclear cells. (D and E) Flow cytometry analysis, the ratio and cell number of CD19+ cells in CD45+ cells from colon lamina propria mononuclear cells. (F–H) Percentage in the total B cells (left) and absolute number (right) of (F) IgA+ B cells, (G) IgD+ B cells, and (H) IgM+ B cells in CD45+ cells from colon lamina propria mononuclear cells. (I) Mice were administered Abx (a mixture of ampicillin, neomycin, vancomycin, and metronidazole) or distilled water by oral gavage 3 times a week until the end of the experiment. Topical IMQ treatment was started 1 week after starting Abx or distilled water. A total of 2% (wt/vol) DSS subsequently was administered to all mice for 7 days after topical treatment of IMQ or vehicle. (J) Psoriasis Area and Severity Index (PASI) score on day 16 of IMQ or vehicle treatment. (K) Body weight changes during administration of DSS. (L) Body weight percentages on day 7 of DSS colitis. (M) Representative photograph of colon on day 7 of DSS colitis. (N) Colon length on day 7 of DSS colitis. (O) DAI score on day 7 of DSS colitis. (P) Representative H&E staining of distal colon section on day 7 of DSS colitis. Scale bar: 100 µm. (Q) Histologic score for colitis on day 7 of DSS colitis (water-cont-DSS and water-IMQ-DSS, n = 11; Abx-cont-DSS, n = 9; Abx-IMQ-DSS, n = 7). Each symbol represents an individual mouse. Statistical analyses were performed with the Student t test or 1-way analysis of variance followed by the Tukey multiple comparison test. *P < .05, **P < .01, ***P < .001. Error bars represent the SEM of samples within a group. Data are pooled from 2 independent experiments.

Fig 5: Psoriasis-like dermatitis exacerbates DSS colitis. (A) Experimental protocol for induction of psoriasis-like dermatitis by IMQ. (B) Skin appearance of psoriasis-like dermatitis on day 14 of IMQ or vehicle treatment. (C) Clinical score for psoriasis-like dermatitis (psoriasis area severity index [PASI] score) at the end of IMQ treatment (on day 17). (D) Representative H&E staining of colon sections at the end of IMQ treatment (on day 17). Scale bar: 100 µm. (E) Experimental protocol. Mice treated topically with IMQ or vehicle as shown in panel A, were subsequently induced with acute colitis by 2% (wt/vol) DSS for 7 days (n = 8 in each group). (F) Body weight changes during administration of DSS. (G) Representative photograph of colon on day 7 of DSS colitis. (H) Colon length on day 7 of DSS colitis. (I) DAI score on day 7 of DSS colitis. (J) Representative H&E staining of distal colon section on day 7 of DSS colitis. Scale bar: 100 µm. (K) Histologic score for colitis on day 7 of DSS colitis. (L) Representative plots of flow cytometry analysis for colonic lamina propria macrophages (CD45+ lineage-CD11b+CD64+ cells). The plots are gated on CD45+CD3-CD19-B220-NK1.1- cells. (M) Percentage (left) and absolute number (right) of colonic lamina propria macrophages. (N and O) Representative plots of flow cytometry analysis for CD80+ macrophages in colonic lamina propria. Percentage of CD80+ cells in macrophages. (P) Representative plots of flow cytometry analysis for CD45+ colonic lamina propria lymphocytes. (Q and R) Percentage and absolute number of B cells (left) and T cells (right) in CD45+ cells. (S) Absolute number of IgA+ B cells (left), IgD+ B cells (middle), and IgM+ B cells (right). (T) Representative plots of flow cytometry analysis for IgA+ plasma cells in colonic lamina propria (left) and absolute number (right). The plots are gated on CD45+CD3-CD4-CD8-NK1.1- cells. (U) Percentage of IgA-coated fecal bacteria analyzed by flow cytometry. Each symbol represents an individual mouse (n = 3–8). Statistical analyses were performed with the Student t test. *P < .05, **P < .01, ***P < .001, NS; not significant. Error bars represent the SEM of samples within a group.