Exploring the interplay of barrier function and leukocyte recruitment in intestinal inflammation by targeting fucosyltransferase VII and trefoil factor 3

Abstract

Intestinal mucosal integrity is dependent on epithelial function and a regulated immune response to injury. Fucosyltransferase VII (Fuc-TVII) is an essential enzyme required for the expression of the functional ligand for E- and P-selectin. Trefoil factor 3 (TFF3) is involved in both protecting the intestinal epithelium against injury as well as aiding in wound repair following injury. The aim of the present study was to assess the interplay between barrier function and leukocyte recruitment in intestinal inflammation. More specifically, we aimed to examine how targeted disruption of Fuc-TVII either in wild-type or TFF3(-/-) mice would alter their susceptibility to colonic injury. TFF3 and Fuc-TVII double-knockout mice (TFF3/Fuc-TVII(-/-) mice) were generated by mating TFF3(-/-) and Fuc-TVII(-/-) mice. Colitis was induced by administration of dextran sodium sulfate (DSS) (2.5% wt/vol) in the drinking water. Changes in baseline body weight, diarrhea, and fecal blood were assessed daily. Upon euthanasia, extents of colonic inflammation were assessed macroscopically, microscopically, and through quantification of myeloperoxidase (MPO) activity. Colonic lymphocyte subpopulations were assessed at 6 days after administration of DSS by flow cytometry and immunohistochemistry. No baseline intestinal inflammation was found in TFF3/Fuc-TVII(-/-), TFF3(-/-), Fuc-TVII(-/-), or wild-type mice. Loss of Fuc-TVII resulted in a reduction in disease severity whereas TFF3(-/-) mice were markedly more susceptible to DSS-induced colitis. Remarkably, the loss of Fuc-TVII in TFF3(-/-) mice markedly decreased the severity of DSS-induced colitis as evidenced by reduced weight loss, diarrhea, decreased colonic MPO levels and improved survival. Furthermore, the loss of TFF3 resulted in increased severity of spontaneous colitis in IL-2/beta-microglobulin-deficient mice. These studies highlight the importance of the interplay between factors involved in the innate immune response, mucosal barrier function, and genes involved in regulating leukocyte recruitment and other aspects of the immune response.

Fig. 1.

Characterization of TFF3/Fuc-TVII^−/− mice. A: deletion of the TFF3 and Fuc-TVII genes was confirmed by Southern blot and PCR analysis. Loss of TFF3 protein expression was confirmed by Western blotting of colonic mucosal tissue. Loss of fucosyltransferase activity was confirmed in the stomach, duodenum, jejunum, ileum, and colon (***P < 0.001 compared with wild-type). Some fucosyltransferase activity was noted in the colon and likely represents a cross-reaction with other fucosyltransferase species. B: fucosylated glycan expression in the colon of the wild-type (A and B) and TFF3/Fuc-TVII^−/− mice (C and D). UEA1 lectin binding of the fucosylated glycans was very high in the wild-type mice (A), and the expression was compromised in the mutant mice (C). B and D: Nomarski image of the same section of the colon, demonstrating normal morphology of the tissue. Wt, wild-type.

Fig. 2.

Impact of the loss of TFF3 and/or Fuc-TVII in dextran sodium sulfate (DSS)-induced colitis. A: body weight throughout the 9 days of DSS exposure expressed as percent of basal body weight. Significantly different from wild-type: *P < 0.05; **P < 0.01. B: diarrhea assessed throughout the 9 days of DSS exposure scored on a 0–3 scale. Significantly different from wild-type: *P < 0.05. Significantly different from TFF^−/−: ‥P < 0.01; …P < 0.001. C: fecal blood loss during the 9 days of DSS exposure, (see materials and methods for further details). Significantly different from wild-type: *P < 0.05; **P < 0.01; ***P < 0.001. Significantly different from TFF^−/−: ·P < 0.05; ‥P < 0.01; …P < 0.001. D: colonic myeloperoxidase (MPO) levels assessed from tissue isolated prior to DSS (untreated) after 3 or 9 days of DSS exposure (*P < 0.05 compared with wild-type; **P < 0.05 compared with TFF3^−/− and wild-type mice; N = 19–37). E: histological scoring of DSS-induced colitis at DSS day 3; inflammation score is 0–4 scale (see materials and methods), and percent ulceration is the percentage of tissue with disruption of the epithelium. F: colonic sections from wild-type (A), TFF3^−/− (B), Fuc-TVII^−/− (C), and TFF3/Fuc-TVII^−/− (D) after 9 days of DSS exposure, stained with hematoxylin and eosin. Parametric ANOVA was performed in A and D, and nonparametric ANOVA was used for B, C, and E, both followed by posttests (see materials and methods).

Fig. 3.

Analysis of immune cell infiltrate in the colon. Flow cytometry of lamina propria cells populations at day 8 of DSS treatment indicating the CD4-to-CD8 ratio (A) and natural killer (NK) cell populations (B) (*P < 0.05). C–D: immunohistochemical staining of macrophages stained with F4/80 antibody in frozen sections from DSS day 6. In C, A is from a TFF3^−/− mouse, and B is from a TFF3/Fuc-Fuc-TVII^−/− mouse. The loss of Fuc-Fuc-TVII in the setting of TFF3 deficiency markedly reduced the number of macrophages that infiltrated the colonic lamina propria (**P < 0.01 TFF3^−/− vs. Fuc-Fuc-TVII^−/−). hpf, High-powered field.

Fig. 4.

Impact of the loss of TFF3 and/or Fuc-TVII on survival in DSS-induced colitis. The Kaplan-Meier method and survival comparisons were performed by using the log-rank or Mantel-Haenszel test. Note that survival was assessed as outlined in materials and methods and animals were removed (euthanized) from the study if they appeared in distress or lost more than 25% of basal body weight as per our institutional animal care and use committee-approved animal care protocol.

Fig. 5.

Impact of the loss of TFF3 in an immune-mediated model of spontaneous colitis. IL-2/β2m^−/− mice were mated with TFF3^−/− mice. IL-2/β2m/TFF3^−/− line was confirmed as described previously (41, 42) and above. Ten animals from both IL-2/β2m/TFF3^−/− and IL-2/β2m^−/− were followed to 25–30 wk prior to euthanasia. A: colonic tissues levels of MPO activity (*P < 0.05). B: bowel wall thickness and inflammation and damage scores in mice euthanized at 25–30 wk of age (**P < 0.01). C: histology of IL-2/β2m^−/− (A) and IL-2/β2m/TFF3^−/− (B); both animals were euthanized at week 25.