Wild type (WT) and Clec7a^−/− littermates were treated with 2.5% DSS for 7 days and kept on water for 4 additional days. Colitis progress and severity were assessed by measuring body weight during treatment (A) and histology (B, C), and TNF-α production in the colon (D) on day 11. (E, F) WT and Clec7a^−/− mice were given an antibiotic cocktail including fluconazole for 3 weeks, transplanted as indicated (red) with fecal microflora from WT or Clec7a^−/− mice and treated with DSS as in (A). Disease severity was accessed by histology score (E) and by cytokine production by anti-CD3/anti-CD28 stimulated LI-LP and MLN T cells (F). Each symbol represents a different mouse. One of four independent experiments is shown. Error bars, s.d., * P < 0.05, ** P < 0.01.

(A) WT and Clec7a^−/− mice were given fluconazole in their drinking water for total of 14 days (starting 2 days prior the induction of DSS colitis), and body weight was measured. Weight loss is shown in (A) (p<0.05). Histology score (B), the percentage of IL-17 and IFN-γ producing CD4⁺ T cells in LI-LP (C), and IL-17 and IFN-γ production in MLNs (D) were determined 4 days after the 7 days of DSS treatment. Each symbol represents a different mouse. One of three independent experiments with similar results is shown. Error bars, s.d., * P < 0.05, ** P < 0.01. (E) Specific CLEC7A haplotypes associate with medically refractory ulcerative colitis (MRUC). Haplotypes were formed from rs2078178 and rs16910631 using PHASE v2.3. Haplotypes listed as “Other Combinations” were those that could not be reliably determined (posterior p<0.95). (F) The CLEC7A “AG/AG” haplotype associates with severity of disease as indicated by earlier progression to colectomy. Haplotypes were tested for association with time to surgery by fitting the MRUC/non-MRUC and time to surgery with a Cox proportional hazards model.

(A) Prevalence of fungi in mucosa isolated from ileum, caecum, proximal (prox) and distal (dist) colon of C57BL/6J mice. ITS1-2 rDNA level was analyzed by qPCR and normalized to β-actin DNA. (B) Visualization of commensal fungi in the intestine. Colon sections were stained with a soluble Dectin-1 probe (sDEC-1) and counterstained with DAPI. The DAPI signal has been amplified in lower panels (B) to show that DAPI-stained bacteria and fungi are in close proximity to each other. (C) Intestinal fungi are recognized by Dectin-1. Fecal pellets were homogenized and labeled with sDEC-1 in presence (gray histogram) or absence (black histogram) of laminarin (a soluble β-glucan) to block specific binding. Binding was assessed by flow cytometry (left panels). Dectin-1-binding fungi were sorted (right panels) and visualized by confocal microscopy. (D) ASCA generation after DSS colitis. Mice were exposed twice to 2.5% DSS-supplemented water for 7 days each separated by two weeks of recovery. Serum samples were collected before DSS treatment (day 0) and 2 weeks after the last DSS cycle (42 days total) and ASCA IgM and IgG were measured by ELISA. Each symbol represents a mouse, all error bars indicate the s.d. *P < 0.05; unpaired t test. All data are representative of at least two independent experiments with similar results.

(A) DNA was isolated from murine feces and mycobiome analysis was performed using Roche 454 and Illumina GA sequencing of ITS1-2 rDNA. The taxonomic distribution of the most abundant fungal genera is shown (large pie chart), and species breakdown for major groups are provided (small pie charts). (B) Quantitative analysis of the major intestinal fungal genera in wild type and Clec7a^−/− mice before and after treatment with DSS. Illumina GA data were analyzed and presented as relative percentage of dominant fungal genera (n=16 mice). (C) Fungal invasion of colonic tissue in Clec7a^−/− mice during colitis. Colon sections from WT and Clec7a^−/− mice before and after colitis were stained with the sDEC-1 probe and counterstained with DAPI. (D) Intestinally conditioned dendritic cells were incubated with live C. tropicalis and killing was assessed after 6 and 18 hours. (E) Histology score of WT and Clec7a^−/− mice supplemented or not with four doses of C. tropicalis or S. fibuligera every other day, and then treated with 2.5% DSS for 7 days and kept on water for 4 additional days. Data are representative of at least two independent experiments with similar results. Error bars, s.d., * P < 0.05, ** P < 0.01.