(A) WT and Myd88^−/− mice received 2% DSS alone or 2% DSS+Abx (ampicillin + vancomycin + neomycin sulfate + metronidazole) in their drinking water for the duration of the experiment. Mice were monitored for 60 days after the initiation of DSS treatment. The results shown are representative of nine experiments, each of which included at least four mice per group. (B) Myd88^−/− mice received 2% DSS+Abx for 30 days and were subsequently switched to drinking water containing DSS only. The results shown are representative of three experiments, each of which included at least four mice per group. (C) Myd88^−/− mice (four mice per group) were left untreated (open bars), treated with Abx (black bars), treated with DSS (red bars) or treated with DSS+Abx (blue bars) in their drinking water, as described in Figure 1A. The bacterial loads in the liver and lungs were analyzed on day 10 post-treatment by plating tissue lysates on blood agar. Similar numbers of bacteria were detected under aerobic (shown here) and anaerobic conditions (not shown). The results shown are representative of four experiments, each of which included at least four mice per group. (D) Serum levels of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were analyzed in WT and Myd88^−/− mice on day 10 post-treatment. The results shown are representative of three experiments. * P< 0.01, ** P< 0.001

(A) WT mice (depicted in black), complete Myd88^−/− mice (depicted in red) and mice with a cell type-specific deletion of MyD88 in epithelial cells (Villin-Cre ×Myd88 ^flox/flox, depicted in purple), dendritic cells (CD11c-Cre ×Myd88 ^flox/flox, depicted in blue), epithelial cells and DCs (Villin-Cre × CD11c-Cre ×Myd88 ^flox/flox, depicted in gray), monocytes and macrophages (LysM-Cre × Myd88^flox/flox, depicted in orange), T cells (Lck-Cre ×Myd88 ^flox/flox, depicted in yellow) or B cells (CD19-Cre ×Myd88 ^flox/flox, depicted in green) were treated with 2% DSS in their drinking water and monitored for survival for 30 days. (B) The histological changes in the colon were analyzed on day 10 post-DSS treatment. The results are representative of six experiments, each of which included between four and six mice per group.

The IgM levels in the stools of naïve and DSS-treated WT, Myd88^−/− and B cell-Myd88^−/− mice were measured by (A) ELISA or (B) flow cytometry. The experiment shown is representative of three experiments, each of which included at least four mice per group. The black bars and histograms depict WT mice, and the red and blue bars and histograms depict B cell-specific and complete Myd88^−/− mice, respectively. (C) IgM secretion by purified WT and Myd88^−/− B cells stimulated with commensal bacterial extract (10 μg/ml) for 72 hours. IgM levels in the cell culture supernatant were measured by ELISA. The results shown are representative of three experiments. *** P< 0.0001. (D) Complement deposition in the colons of WT, Myd88^−/− and B cell-Myd88^−/− mice treated with DSS was visualized by anti-C3 staining. (E) The percentages of commensal bacteria covered with C3 (green curves) were analyzed by flow cytometry. Black curves represent isotype controls. The experiment shown is representative of three experiments, each of which included at least four mice per group. (F) WT, Myd88^−/−, and B-Myd88^−/− mice were left untreated (naïve), treated with 2% DSS, or treated with 2% DSS+Abx in their drinking water. Serum levels of C3 were analyzed on day 10 post-treatment. The results shown are representative of three experiments. (G) The survival and (H) the dissemination of commensal bacteria in the livers and lungs of WT, Myd88−/−, and C3^−/− mice were analyzed on day 10 post-DSS or DSS+Abx treatment. The experiment shown is representative of four experiments, each of which included at least four mice per group.

(A) WT and Myd88^−/− mice were left untreated (top panel), treated with 2% DSS (middle panel) or treated with 2% DSS+Abx (ampicillin + vancomycin + neomycin sulfate + metronidazole, bottom panel) in their drinking water. The histological changes in the colon were analyzed on day 10 post-treatment based on the following additive scoring system: crypt integrity, 0 = normal, 1 = irregular crypts, 2 = mild crypt loss; 3 = severe crypt loss, 4 = complete crypt loss with intact epithelial cell layer, 5 = complete loss of crypts and surface epithelium (<10 crypt width), and 6 = complete loss of crypts and surface epithelium (>10 crypt width); infiltration of inflammatory cells into the mucosa, 0 = normal, 1 = mild; 2 = modest, and 3 = severe; infiltration into the submucosa, 0 = normal, 1 = mild; 2 = modest, and 3 = severe; and infiltration into the muscle, 0 = normal, 1 = mild; 2 = modest, and 3 = severe. The scores for each parameter were added, resulting in a total scoring range of 0 to 17. Antibiotic treatment alone had no detectable effects on the architecture of WT or Myd88^−/− colons (not shown). (B) IL-12p40, TNF, and IL-1β secretion from the colons isolated from DSS- or DSS+Abx-treated WT and Myd88^−/− mice on day 10 after the initiation of treatment. The results shown are representative of three experiments, each of which included at least four mice per group. A green asterisk indicates the position of the gut lumen in the histological images. * P< 0.01.

(A) Mice with a cell -specific deletion of MyD88 in epithelial cells, DCs, monocytes/macrophages, T cells and B cells and control animals were treated with 2% DSS in their drinking water. Aerobic (left panel) and anaerobic (right panel) bacteria were recovered from the livers of the mice on day 10 post-DSS treatment. The colonies were analyzed by plating the bacteria on blood agar. * P< 0.01(B) The dissemination of bacteria in the livers of WT mice and of mice lacking MyD88 in all tissues was analyzed compared to the cell type-specific Myd88-deficient mice on day 10 post-DSS treatment by in situ hybridization. Yellow arrows point to the detected bacteria. Histopathological analysis of livers from the same mice after H&E staining is shown below the in situ hybridization images. The results shown are representative of at least four experiments including three or four mice per group, and the images shown are representative of multiple sections examined. (C) Bacterial DNA was isolated from the livers of Myd88^−/− (left) and B cell-Myd88^−/− mice (right), and the relative frequencies of the major phylogenetic groups were analyzed by 16SRNA sequencing (blue: Firmicutes, green: Bacteroidetes, red: Proteobacteria, yellow: Deferribacteres, and black: Actinobacteria).