Mucin-type O-linked oligosaccharides (O-glycans) are primary components of the intestinal mucins thatform the mucus gel layer overlying the gut epithelium. Impaired expression of intestinal O-glycans has been observed in patients with ulcerative colitis (UC), but its role in the etiology of this disease is unknown. Here, we report that mice with intestinal epithelial cell-specific deficiency of core 1-derived O-glycans, the predominant form of O-glycans, developed spontaneous colitis that resembled human UC, including massive myeloid infiltrates and crypt abscesses. The colitis manifested in these mice was also characterized by TNF-producing myeloid infiltrates in colon mucosa in the absence of lymphocytes, supporting an essential role for myeloid cells in colitis initiation. Furthermore, induced deletion of intestinal core 1-derived O-glycans caused spontaneous colitis in adult mice. These data indicate a causal role for the loss of core 1-derived O-glycans in colitis. Finally, we detected a biosynthetic intermediate typically exposed in the absence of core 1 O-glycan, Tn antigen, in the colon epithelium of a subset of UC patients. Somatic mutations in the X-linked gene that encodes core 1 β1,3-galactosyltransferase-specific chaperone 1 (C1GALT1C1, also known as Cosmc), which is essential for core 1 O-glycosylation, were found in Tn-positive epithelia. These data suggest what we believe to be a new molecular mechanism for the pathogenesis of UC.

Altered intestinal O-glycan expression has been observed in patients with ulcerative colitis and colorectal cancer, but the role of this alteration in the etiology of these diseases is unknown. O-glycans in mucin core proteins are the predominant components of the intestinal mucus, which comprises part of the intestinal mucosal barrier. Core 3–derived O-glycans, which are one of the major types of O-glycans, are primarily expressed in the colon. To investigate the biological function of core 3–derived O-glycans, we engineered mice lacking core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT), an enzyme predicted to be important in the synthesis of core 3–derived O-glycans. Disruption of the C3GnT gene eliminated core 3–derived O-glycans. C3GnT-deficient mice displayed a discrete, colon-specific reduction in Muc2 protein and increased permeability of the intestinal barrier. Moreover, these mice were highly susceptible to experimental triggers of colitis and colorectal adenocarcinoma. These data reveal a requirement for core 3–derived O-glycans in resistance to colonic disease.

Circulating lymphocytes continuously enter lymph nodes (LNs) for immune surveillance through specialised blood vessels named high endothelial venules (HEVs)1–5, a process that increases dramatically during immune responses. How HEVs permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here, we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α)6–8 in maintaining HEV barrier function. Mice with postnatal deletion of PDPN lost HEV integrity and exhibited spontaneous bleeding in mucosal LNs, and bleeding in the draining peripheral LN after immunisation. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells (FRCs)7, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2)9,10. Mice lacking FRC PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (VE-cad), which is essential for overall vascular integrity11,12, on HEVs. Infusion of wild-type (WT) platelets restored HEV integrity in CLEC-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate (S1P)13,14 from platelets, which promoted expression of VE-cad on HEVs ex vivo. Furthermore, draining peripheral LNs of immunised mice lacking S1P had impaired HEV integrity similar to PDPN- and CLEC-2-deficient mice. These data demonstrate that local S1P release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.

Mucin-type O-glycans (O-glycans

Introduction Leukocyte recruitment during inflammation is a multistep process involving tethering and rolling of leukocytes on vascular surfaces, followed by firm adhesion and transmigration into the affected tissues (1). Interactions of selectins with glycoconjugate ligands primarily mediate the initial tethering and rolling adhesion of leukocytes (2, 3). L-selectin, expressed on leukocytes, binds to ligands on other leukocytes and on activated endothelial cells. P-selectin, expressed on activated platelets and endothelial cells, and E-selectin, expressed on activated endothelial cells, bind to ligands on leukocytes. Studies with selectin-deficient mice and blocking mAb's indicate that the selectins have both unique and overlapping functions (4, 5). In the microcirculation of the cremaster muscle, P-selectin mediates most tethering and rolling of leukocytes early after trauma-induced inflammation (6), whereas L-selectin supports significant tethering and rolling 6-8 hours after exposure to TNF-α (7). E-selectin supports slow rolling of leukocytes on venules 2-4 hours after stimulation with TNF-α (8). Selectin ligands require α2,3-sialylation and α1,3fucosylation on capping structures such as sialyl Lewis x (sLe x), which bind to the C-type lectin domains of the selectins (9). In addition, sulfation is a critical requirement of ligands that bind to P-and L-selectin. A subset of glycoproteins binds with high affinity or avidity to the selectins (2). Of these, only P-selectin glycoprotein ligand-1 (PSGL-1) has a clearly documented function in mediating selectin-dependent cell adhesion under flow (3, 10). Human and murine PSGL-1 are extended homodimeric sialomucins that are expressed on most leukocytes. PSGL-1 binds to all three selectins in vitro. P-and L-selectin bind to the N-terminal region of PSGL-1 (11-13), whereas E-selectin binds to one or more additional sites on PSGL-1 (14). Human PSGL-1 binds to P-selectin with specific stereochemical requirements that include optimal orientation of three tyrosine sulfates, amino acids, and a core-2 O-glycan capped with sLe x (15-17). PSGL-1 does not require tyrosine sulfation to bind to E-selectin, but expression of sLe x on core-2 O-glycans enhances binding (18). mAb's against the N-terminal region of PSGL-1 markedly inhibit rolling of human and murine leukocytes on P-selectin in vitro (11, 12), rolling of human neutrophils on P-selectin in rat venules in vivo (19), and rolling of murine leukocytes on P-selectin in murine venules in vivo (12, 20). These data demonstrate that