Bovine colostrum increases pore-forming claudin-2 protein expression but paradoxically not ion permeability possibly by a change of the intestinal cytokine milieu

Abstract

An impaired intestinal barrier function is involved in the pathogenesis of inflammatory bowel disease (IBD). Several nutritional factors are supposed to be effective in IBD treatment but scientific data about the effects on the intestinal integrity remain scarce. Bovine colostrum was shown to exert beneficial effects in DSS-induced murine colitis, and the present study was undertaken to explore the underlying molecular mechanisms. Western blot revealed increased claudin-2 expression in the distal ileum of healthy mice after feeding with colostrum for 14 days, whereas other tight junction proteins (claudin-3, 4, 10, 15) remained unchanged. The colostrum-induced claudin-2 induction was confirmed in differentiated Caco-2 cells after culture with colostrum for 48 h. Paradoxically, the elevation of claudin-2, which forms a cation-selective pore, was neither accompanied by increased ion permeability nor impaired barrier function. In an in situ perfusion model, 1 h exposure of the colonic mucosa to colostrum induced significantly increased mRNA levels of barrier-strengthening cytokine transforming growth factor-β, while interleukine-2, interleukine-6, interleukine-10, interleukine-13, and tumor-necrosis factor-α remained unchanged. Thus, modulation of the intestinal transforming growth factor-β expression might have compensated the claudin-2 increase and contributed to the observed barrier strengthening effects of colostrum in vivo and in vitro.

Figure 1. Bovine colostrum increases claudin-2 expression in the distal ileum of healthy mice.

(A) Representative western blot of elevated claudin-2 protein expression in whole tissue extracts from the distal ileum of mice after 14 days feeding with bovine colostrum (BC) and untreated controls. β-actin was used as internal control for equal protein loading. (B) Increased relative expression of claudin-2 protein upon BC-feeding compared to untreated controls. *p<0.05 vs. untreated, n = 5–6.

Figure 2. Changes of claudin-2 protein expression in intestinal epithelial cells.

(A) Western blot analysis revealed elevated claudin-2 expression in 14 days postconfluent differentiated Caco-2 cells after 48 h incubation with bovine colostrum (BC) or fetal bovine serum (FBS) compared to complete media (CM). β-actin was used as internal control for equal protein loading. (B) Relative expression of claudin-2 protein in Caco-2 cells was increased upon 48 h pre-incubation with BC compared to CM and after stimulation with IL-6 (50 ng/ml) for 24 h. *p<0.05 vs. CM, n = 5–6. (C) Immunofluorescence of Caco-2 cells after incubation with BC confirmed elevation of claudin-2 (red) by BC and IL-6 (24 h, 50 ng/ml) compared to CM after 48 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Representative images of 3 individual experiments. Magnification: ×600.

Figure 3. Increased claudin-2 expression is not paralled by decreased transepithelial electrical resistance.

Apical stimulation of filter-grown differentiated 14 days postconfluent Caco-2 cells with IL-6 (50 ng/ml) decreased transepithelial electrical resistance (TER) after 24 h compared to unstimulated controls (complete media, CM). Incubation with bovine colostrum (BC) or fetal bovine serum (FBS) increased TER. TER in the presence of CM at the beginning of the incubation was set as 100%. *p<0.05, n = 3–4.

Figure 4. Bovine colostrum does not impair integrity of Caco-2 monolayers.

(A) Permeability of filter-grown differentiated 14 days postconfluent Caco-2 cells for 4 kDa fluorescein isothiocyanate-dextrane and IL-8 cytokine secretion (B) were not affected by 48 h incubation with bovine colostrum (BC) or fetal bovine serum (FBS) compared to complete media (CM, control), n = 4 per assay and per group.

Figure 5. BC influences intestinal cytokine milieu.

RNA-transcript levels of TGF-β but not IL- 6, 2, 13, 10, and TNF-α were significantly elevated in mouse proximal colon exposed to BC compared to Hank’s balanced salt solution control group (HBSS) *p<0.05, n = 4–6.