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Intelectins are carbohydrate-binding proteins implicated in innate immunity and highly conserved across chordate evolution, including both ascidians and humans. Human intelectin-1 (ITLN1) is highly abundant within the intestinal mucosa and binds microbial but not host glycans. Genome-wide association studies identified SNPs in ITLN1 that are linked to susceptibility for Crohn’s disease. Moreover, ITLN1 has been implicated in the pathophysiology of obesity and associated metabolic disease. To gain insight on biological activities of human ITLN1 in vivo, we developed a C57BL/6 mouse model genetically targeting the gene encoding the functional mouse ortholog. In wild-type C57BL/6 mice, both mRNA and protein analysis showed high expression of Itln1 in the small intestine, but manifold lower levels in colon and other extraintestinal tissues. Whereas intestinal expression of human ITLN1 localizes to goblet cells, our data confirm that mouse Itln1 is expressed in Paneth cells. Compared to wild-type littermate controls, mice homozygous for the Itln1 hypomorphic trapping allele had reduced expression levels of Itln1 expression (~10,000-fold). The knockout mice exhibited increased susceptibility in an acute model of experimentally induced colitis with 2% w/v dextran sulfate sodium (DSS). In a model of chronic colitis using a lower dose of DSS (1.5% w/v), which enabled a detailed view of disease activity across a protracted period, no differences were observed in body weight, fecal texture, hemoccult scores, food/water intake, or colon length at necropsy, but there was a statistically significant genotype over time effect for the combined fecal scores of disease activity. In model of diet-induced obesity, using two western-style diets, which varied in amounts of sugar (as sucrose) and saturated fat (as lard), mice with Itln1 expression ablated showed no increased susceptibility, in terms of weight gain, food intake, plasma markers of obesity compared to wildtype littermates. While the mouse genetic knockout model for Itln1 holds promise for elucidating physiological function(s) for mammalian intelectins, results reported here suggest that Itln1, a Paneth cell product in C57BL/6 mice, likely plays a minor role in the pathophysiology of chemically induced colitis or diet-induced obesity.

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INTRODUCTION: An intricate balance with our intestinal microbes is pivotal to health. A key interface of host-microbial interactions occurs in the mucus covering the intestinal epithelial surface. In the colon, the mucus layer serves as a barrier inhibiting direct epithelial contact with the dense population of microbes. Defects in this system are a hallmark of colitis. The mucus layer is structurally dependent on the polymeric mucin MUC2, and it is synthesized by goblet cells (GCs), specialized secretory cells classically viewed as a homogenous cell type. Studies identifying divergent functional features in GC subpopulations, including differential mucus biosynthesis rate and responses to bacteria, suggests GC populations may in fact be heterogeneous. RATIONALE: In the present study, we characterized intestinal GC expression diversity, and defined how a specific GC subtype, localized in the intercrypt surface epithelium, functionally contributes to the formation of the mucus barrier. RESULTS: Using mCherry-MUC2 transgenic mice to sort and isolate GCs, we generated transcriptomic and proteomic profiles to characterize the GC expression landscape in both the small intestine and colon. Single cell transcriptomic analysis revealed several unique GC clusters in each tissue regions, which segregated into two separate trajectories. One trajectory had enriched expression of known GC-specific genes (e.g., Clca1, Fcgbp), and was designated as canonical GCs. Conversely, the other trajectory was enriched for expression of genes typically associated with enterocytes (e.g., Dmbt1, Gsdmc4), which we designated as non-canonical GCs. In the colon, the most differentiated GCs are the high mucus turnover cells localized to the surface epithelium between crypts and were thus designated as intercrypt GCs (icGCs). These cells had distinct expression profiles from crypt-resident GCs, thus we investigated their role in forming the mucus barrier. We exploited the lectin binding features of mucus to resolve the three-dimensional organization of mucus in live tissue explants. Results demonstrated that icGCs secreted distinct mucus that filled the spatial regions between mucus plumes secreted from crypt openings. The intercrypt mucus was impenetrable to bacteria-sized beads; however, it was more penetrable to smaller molecules compared to crypt plume mucus. Penetrable surface mucus may be important for absorption of ions, and other compounds, while denser mucus within the crypt compartment contributes to the shielding of the stem cell niche. Both mucus subtypes appear to be important for the overall protective function of mucus, as a barrier impenetrable to bacteria was formed by the mixed net-like organization of intercrypt and crypt plume mucus. A mouse model with dysfunctional icGCs lacking normal intercrypt mucus exhibited an inadequate mucus barrier and was more susceptible to both chemically-induced and spontaneous (age-dependent) colitis, thus arguing for the indispensable role of icGCs in maintaining a functional mucus barrier. Furthermore, biopsies from patients with ulcerative colitis, also in remission, exhibited increased GC shedding and reduced icGC numbers. These characteristics was associated with structural defects in the mucus barrier, including gaps in the intercrypt mucus that exposed areas of the surface epithelium. CONCLUSION: The current study identifies GCs as a heterogeneous population of cells with diverse functional features that indicate a dynamic cellular system. GCs at different locations along the crypt-surface axis contribute to a functional mucus barrier that protects the epithelium from microorganisms. The icGCs possess a unique role in mucus organization, where their malfunction is associated with colitis in both mice and humans.