The C-Type Lectin Receptor SIGNR3 Binds to Fungi Present in Commensal Microbiota and Influences Immune Regulation in Experimental Colitis

Abstract

Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern-recognition receptors (PRRs) sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs) are PRRs recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 (SIGNR3) in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homolog of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze whether this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium-induced colitis model was employed. SIGNR3(-/-) mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3(-/-) mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis.

Keywords: C-type lectin receptor; SIGNR3; carbohydrate recognition; colitis; fungi; host innate immunity; microbiota.

Figures

Figure 1

Binding of SIGNR3 to fungi present in commensal microbiota. (A) ELISA analysis of binding of CLR-hFc fusion proteins to heat inactivated intestinal microbiota demonstrated significant binding of SIGNR3-hFc. Binding of SIGNR3-hFc to zymosan, and MGL1-hFc to colon microbiota were used as positive controls. If indicated, incubation and washing was performed in 25 mM EDTA buffer to analyze Ca2+-dependency of the interaction. Results are representative of three independent experiments. (B) Representative dot plots of SytoRed stained gut microbes (APC+) incubated with hFc, SIGNR3-hFc, or DCAR-hFc. Detection with a PE-labeled goat anti-hFc antibody demonstrates binding of SIGNR3-hFc to gut microbes. (C) Corresponding statistical analysis of flow cytometry data representative from three independent experiments. (D) The interaction of SIGNR3-hFc and microbes was inhibited by preincubation with S. cerevisiae-derived mannan in the concentrations of 0.01–10 mg/ml. Data are representative of two independent experiments. (E) Staining with a FITC-labeled anti-C. albicans antibody reported to cross-react with other fungi revealed about 7% of the microbes were fungi. Staining of cells with fusion proteins demonstrated that SIGNR3-hFc recognizes>60% of the commensal fungi in the colon. (F) Statistical analysis of commensal fungi detected by SIGNR3- and DCAR-hFc as determined by flow cytometry. Shown are data and gating representative for three independent experiments. (A–F) Data are presented as mean + SEM. The p-values were determined using unpaired Student’s t-test. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

Wild-type and SIGNR3−/− mice were fed 3% DSS supplemented to the drinking water for seven consecutive days (n = 31 for SIGNR3−/− mice, n = 30 for wild-type mice). (A) Weight was recorded daily. On day 7, disease activity index (B), stool score (C), and colon length (D) were measured. SIGNR3−/− mice displayed an increased susceptibility to DSS colitis as indicated by a more severe weight loss and exacerbated colitis symptoms. A summary of four independent experiments is shown. Data are expressed as mean + SEM. The p-values were determined using a mixed linear model (A) and unpaired Student’s t-test (B–D). Significance is indicated by asterisks (*), *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

After 7 days of DSS treatment, mice were sacrificed and paraffin sections of the colon of each mouse were prepared and stained with hematoxylin and eosin (H&E). A histopathological evaluation of each section was performed in a blinded manner. (A) H&E stained sections from colon of C57BL/6 wild-type, and SIGNR3−/− mice at day 7, and untreated control mice. (B) Each colon was divided into three segments of identical length (oral, middle, rectal) and each segment of the colon was analyzed separately. The degree of infiltration of inflammatory cells and mucosal erosion/ulceration was graded from none (score 0) to mild (score 1), moderate (score 3), or severe (score 4) (n = 5). Data are expressed as mean + SEM. The p-values were determined using unpaired Student’s t-test. Arrows show examples of mucosal ulceration. Significance is indicated by asterisks (*), *p < 0.05.

Figure 4

Cytokines in colon homogenates from wild-type and SIGNR3−/− mice fed with 3% DSS were analyzed by cytometric bead array. The levels of TNF-α (A), IL-6 (B), and IL-1β (C) were measured (n = 8 for DSS-treated SIGNR3−/− and wild-type mice, n = 5 for untreated wild-type mice). Data are expressed as mean + SEM. The p-values were determined using unpaired Student’s t-test. Significance is indicated by asterisks (*), *p < 0.05.