Objective The inner mucus layer in mouse colon normally separates bacteria from the epithelium. Do humans have a similar inner mucus layer and are defects in this mucus layer a common denominator for spontaneous colitis in mice models and ulcerative colitis? Methods and Results The colon mucus layer of mice deficient in the Muc2 mucin, Core 1 O-glycans, Tlr5, IL10 and Slc9a3 (Nhe3) together with dextran sulfate (DSS) treated mice was immunostained for Muc2 and the bacterial localization in the mucus was analyzed. All murine colitis models revealed bacteria in contact with the epithelium. Additional analysis of the less inflamed IL10−/− mice revealed a thicker mucus layer than WT, but the properties were different as the inner mucus layer could be penetrated both by bacteria in vivo and by fluorescent beads the size of bacteria ex vivo. Clear separation between bacteria or fluorescent beads and the epithelium mediated by the inner mucus layer was also evident in normal human sigmoid colon biopsies. In contrast, mucus on colon biopsies of ulcerative colitis (UC) patients with acute inflammation had a highly penetrable mucus. Most UC patients in remission had similar to controls an impenetrable mucus layer. Conclusions Normal human sigmoid colon has an inner mucus layer impenetrable to bacteria. The colon mucus in animal models that spontaneously develop colitis and in UC patients with active disease allows bacteria to penetrate and reach the epithelium. Thus colon mucus properties can be modulated and suggest a novel model of UC pathophysiology.

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The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately 1 percent of the world population, are not well understood [1][2][3] . Using a mouse model of epilepsy, we show that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1) and leukocyte integrins α4β1 and αLβ2. Inhibition of leukocytevascular interactions either with blocking antibodies, or in mice genetically deficient in functional PSGL-1, dramatically reduced seizures. Treatment with blocking antibodies following acute seizures prevented the development of epilepsy. Neutrophil depletion also inhibited acute seizure induction and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability, was induced by acute seizure activity but was prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Consistent with potential leukocyte involvement in the human, leukocytes were more abundant in brains of epileptics than of controls. Our results Correspondence should be addressed to: P.F.F (E-mail: paolo.fabene@univr.it) or G.C. (E-mail: gabriela.constantin@univr.it). Author contribution G.N.M., D.B., A.C., L.Z., F.S. performed epilepsy experiments, telemetry and open field behavior. M.M., B.R., L.O., S.B., S.A., performed intravital microscopy, in vivo staining for adhesion molecules, adhesion assays and contributed to the obtainment of behavioral data. A.O. provided the human samples. F.M., A.C. and F.O. performed immunohistochemistry on human and animal samples. P.M., E.N. and A.S provided MRI expertise. J.W.H., L.X., J.B.L., R.P.M provided vital reagents and mice. E.C.B contributed experimental suggestions, reagents and assistance with writing. P.F.F and G.C. designed the study, analyzed the data and wrote the paper NIH Public Access suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy.Experimental data from animal models as well as human evidence indicate that seizures can lead to neuronal damage and cognitive impairement 2, 3 . However, the molecular mechanisms leading to seizures and epilepsy are not well understood. Recent data suggests that inflammation may play a role in the pathogenesis of epilepsy 4, 5 . For instance, elevation in inflammatory cytokines are seen in the central nervous system (CNS) and plasma in experimental models of seizures and in clinical cases of epilepsy 4, 5 . Moreover, CNS inflammation is associated with breakdown in the blood-brain barrier (BBB), and BBB leakage has been implicated both in the induction of seizures and in the progression to epilepsy 6-9 . Leukocyte recruitment is a hallmark of and a point of therapeutic intervention in tissue inflammation 10,11 , but a role for leukocyte-endothelia...

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here, we demonstrate that podoplanin (PDPN) regulated actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor, CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, resulting in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, resulting in an expanded reticular network and enhanced immunity.

Normal bone marrow (BM) contains T cells whose function and origin are poorly understood. We observed that CD8+ T cells in BM consist chiefly of CCR7+ L-selectin+ central memory cells (TCMs). Adoptively transferred TCMs accumulated more efficiently in the BM than naive and effector T cells. Intravital microscopy (IVM) showed that TCMs roll efficiently in BM microvessels via L-, P-, and E-selectin, whereas firm arrest required the VCAM-1/alpha4beta1 pathway. alpha4beta1 integrin activation did not depend on pertussis toxin (PTX)-sensitive Galphai proteins but was reduced by anti-CXCL12. In contrast, TCM diapedesis did not require CXCL12 but was blocked by PTX. After extravasation, TCMs displayed agile movement within BM cavities, remained viable, and mounted potent antigen-specific recall responses for at least two months. Thus, the BM functions as a major reservoir for TCMs by providing specific recruitment signals that act in sequence to mediate the constitutive recruitment of TCMs from the blood.

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.

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.

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.