Impaired Mucosal Immunity to Commensal Fungi Influence Colitis

Abstract

Interactions between commensal microbiota and the immune system are critical for establishing a proper balance between immune host defense mechanisms and tissue health. Changes in the composition of gut bacterial communities have been associated with intestinal inflammation and obesity. Recent studies have begun to note that a fraction of mucosa-associated microorganisms are not bacterial. Mucosal fungal infections are relatively common in Crohn’s Disease patients, and antibodies against fungal antigens (ASCA) are a widely accepted clinical marker for disease severity. What fungi populate the intestine and how immunity to them might play a role in inflammatory disease is currently unknown. Fungi are sensed by number of innate immune receptors among which Dectin-1 has emerged as the main innate immune receptor for recognition, phagocytosis, and killing of fungi by myeloid phagocytes. Commensal fungi were visualized and quantified by staining with Dectin-1 probe followed by microscopy and FACS analysis, and additionally detected in fecal samples by qPCR for fungal 18S rDNA. To define the mouse intestinal fungal microbiome (the mycobiome), we isolated DNA from murine feces, amplified the internal transcribed spacer region (ITS1-2) of fungal rDNA, and performed high-throughput sequencing. To induce colitis wild type and Dectin-1-/- mice were treated with DSS. In some cases mice were supplemented with C. tropicalis - a common commensal fungus we found in murine gut and subjected to DSS. To determine whether the altered fungal burden during colitis contributes to disease severity, we suppressed fungal growth with fluconazole, a specific antifungal drug. We found that mice lacking Dectin-1, recognizing fungal cell wall β-glucan, are more susceptible to experimental colitis characterized by increased infiltration of Th17 and Th1 cells in the colon. Interestingly this pathology was driven by intestinal fungus, and antifungal therapy ameliorated colitis severity in knockout mice. Deep sequencing analysis of the fungal mycobiome revealed fungal species that are overrepresented in the gut during experimental colitis. When Dectin-1-/- mice were supplemented with Candida tropicalis, a specific commensal fungus found in the intestine during colitis, they experienced more severe intestinal inflammation and augmented Th17 mucosal responses in absence of Dectin-1. Consistently, intestinal dendritic cells (DCs) from Dectin-1-/- mice, but not WT DCs, showed reduced ability to kill fungi. Therefore the data suggest that an inability of Dectin-1-/- mice to mount effective immune responses to specific intestinal fungi creates conditions that promote inflammation. Since the mouse model suggested that Dectin-1 is involved in contributing to the severity of colonic disease, we focused on the severity of ulcerative colitis (UC), the form of IBD that always affects the colon. In particular we focused on severe UC, termed medically refractory UC (MRUC), consisting of patients requiring colectomy as a result of lack of response to medication. We found that a specific variant of the gene for Dectin-1 is strongly associated with a severe form of ulcerative colitis requiring colectomy. Together our findings reveal a novel eukaryotic fungal community in the gut and show that altered interactions between the fungal microflora and the host intestinal phagocytes can profoundly influence intestinal pathology.