engleski

Challenging of immuno-compromised Tff2 knock-out mice with Yersinia enterocolitica infection

Introduction: Trefoil factor family 2 (TFF2) protein is expressed predominantly in the mucous cells of gastric mucosa contributing to its protection as an effector molecule. It’s not so prominent presence in immune cells seems to be more relevant for protection of gastric mucosa due to its role as a regulator of immune response. To estimate contribution of TFF2 in defence of intestinal mucosa to bacterial infection we exposed Tff2 deficient mice to Y.enterocolitica infection and monitored several relevant parameters. Materials and Methods: Specific pathogen free, six to eight weeks Tff2-/- and Tff2+/+ mice were orally infected with Y.enterocolitica-WA314. Animal survival was monitored twice a day over a period of 15 days. The group of animals was sacrificed on 3rd day post infection. To determine the concentration of the bacteria per organ (CFU/organ), serial dilutions of homogenized Ileal peyer’s patches were plated out on Yersinia selective media (LB agar) and incubated at 27°C for 48 hours. Different regions of GI tract were collected for RNA isolation to estimate expression of various genes involved in mucosal immune response : Crbp1, Gus, Timp, Psmb5, Bag2, Tap1, Lysosyme P, Tff3, Mmp7, Cathepsin C, Crip. Various organs (stomach, duodenum, jejunum, ileum, caecum, colon, peyer’s patches, spleen, liver, kidney and lung) were collected to monitor histopathological changes. Presence of macrophages in intestinal mucosa was investigated by immunohistochemistry using Mac3 antibody. Results and conclusions: 1. Survival curve showed significantly lower survival ratio for Tff2 deficient mice. All Tff2-/- mice died on day 7 upon infection while 80 % of Tff2+/+ mice survived at that stage. 2. Bacterial titer indicated a higher bacterial load in the illeal Peyer's patches of Tff2-/- mice as compared to Tff2+/+ mice. 3. Payer’s patches Tff2-/- mice had more macrophages (stained with Mac 3 antibody) compared to wild type mice. 4. Histopathological analysis showed a sever structural impairment of the spleen in Tff2-/- mice as compared to Tff2+/+ mice. 5. Ileal genes involved in immune response of mucosa that previously showed modulated expression in comparison of healthy Tff2-/- and Tff2-/- animals, had the same level of expression in Y.enterocolitica challenged animals. These new data show that Tff2 has an important role in immune system by protecting the organism from consequences of infection with Y.enterocolitica-WA314.

TFF2 knock out mice ; immune response ; Yersinia enterocolitica infection

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