Furthermore, the crypt denuded mucosal surface was heavily infiltrated by leukocytes in EPCR?/? mice (Fig

Furthermore, the crypt denuded mucosal surface was heavily infiltrated by leukocytes in EPCR?/? mice (Fig.?3A). dextran sulfate sodium (DSS)-induced colitis, manifested by increased weight loss, macrophage infiltration, and inflammatory cytokines in the colon tissue. DSS treatment of EPCR?/? mice resulted in increased bleeding, bodyweight loss, anemia, fibrin deposition, and loss of colon epithelial and goblet cells. Administration of coagulant factor VIIa significantly attenuated the DSS-induced colon length shortening, rectal bleeding, bodyweight Ephb3 loss, and disease activity index in the wild-type mice but not EPCR?/? mice. In Mefloquine HCl summary, our data provide direct evidence that EPCR plays a crucial role in regulating the inflammation in the colon during colitis. erythrocyte membrane protein 1 (pfEMP1), and T cell receptor (TCR) present on a subset of V2? T cells19. These observations suggest that EPCR may influence various cellular functions in pathophysiology by coupling with different ligands19. Recent studies demonstrate that this protein C-EPCR pathway governs the intestinal inflammation during IBD20C22. EPCR and TM were expressed around the mucosal endothelium, but their expression was decreased in IBD, which in turn caused impairment of protein C activation20,23. The downregulation of the protein C pathway was shown to correlate to the active disease during colitis clinically20. The APC treatment was shown to suppress mucosal barrier permeability and inflammation during experimental colitis20,21. Consistent with the above data, PC?/?/PC (low-PC) mice, expressing only 3% of protein C, were prone to severe experimental colitis21. Although the above studies demonstrate the importance of the EPCR-protein C pathway in the pathogenesis of IBD, owing to the presence of diverse ligands to EPCR, the use of protein C-deficient mice alone does not provide a full picture of the pathological significance of EPCR during colitis. In this context studies employing EPCR?/? mice may provide more relevant and direct information on the consequences of the loss of EPCR in IBD in the progression of the disease. In the present study, we confirm that EPCR is usually expressed in epithelial and leukocytes surrounding the crypts in the colon mucosa, and the loss of EPCR expression in experimental colitis is usually associated with an increased disease index. EPCR-deficient mice are highly susceptible to DSS-induced colitis. FVIIa treatment reduced the severity of the DSS-induced experimental colitis in wild-type mice but not ECR?/? mice. Materials and methods Materials Dextran sulfate sodium was purchased from Affymetrix (ThermoFisher Scientific, Waltham, MA, USA). ELISA kits for IL-1, MCP-1, and IL-6, and CD11c (N418)-PE conjugated antibodies were obtained from eBioscience (ThermoFisher Scientific). Rat CD21/CD35 AF-594 conjugated antibody was purchased from BioLegend, CA. The antibodies against mouse EPCR were described elsewhere24. The antibodies against fibrin and F4/80 were from EMD Millipore (Burlington, MA, USA). Hemoccult Sensa fecal occult blood test kit was from Modomed (Grand Rapids, MI, USA). rhFVIIa was a gift from the late Dr. Walter Kisiel, School of Medicine, The University of New Mexico, Albuquerque, NM, USA. Mice The generation of EPCR?/? mice (Procr?/?) was described previously25. Wild-type C57BL/6 mice were generated from the in-house breeding program or obtained from Jackson Laboratory (Bar Harbor, ME). DSS induced colitis in the mice All animal studies reported herein were approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center at Tyler, TX, USA. Animal Mefloquine HCl husbandry and experiments were conducted according to the animal welfare guidelines outlined in the Guide for the Care and Use of Laboratory Animals. Colitis Mefloquine HCl was induced in mice by giving 2.5% DSS (w/v) dissolved in the sterile drinking water ad libitum for 10?days. Control mice received sterile drinking water with no DSS. The food and water intake of the mice were monitored daily throughout the experimental period (10?days). The severity of the disease was assessed based on the clinical symptoms of colitis, such as body weight loss, stool consistency, and blood in the stools. A fecal occult blood test kit was used to measure fecal blood according to the manufacturers instructions. All three parameters were weighted equally. The scoring as followed, bodyweight loss, 0C4 (0?=? ?1%, 1?=?1C5%, 2?=?5C10%, 3?=?10C15%, and 4?=? ?15%), stool consistency, 0C4 (0, normal; 2, soft; 4, diarrhea), and stool blood, 0C4 (0, no blood; 2, low to moderate levels; 4, high levels). Disease activity index (DAI) was calculated using the sum of the values of body weight loss, diarrhea, and stool blood, with a maximum score of 12 for severe colitis26,27. After 10?days of colitis, an aliquot of blood was collected for the measurement of hemoglobin in peripheral blood..