Influence of orally fed a select mixture of Bacillus probiotics on intestinal T-cell migration in weaned MUC4 resistant pigs following Escherichia coli challenge

Abstract

Efficient strategies for treating enteritis caused by F4(+) enterotoxigenic Escherichia coli (ETEC)/verocytotoxigenic Escherichia coli (VTEC)/enteropathogenic E. coli (EPEC) in mucin 4 resistant (MUC4 RR; supposed to be F4ab/ac receptor-negative [F4ab/acR(-)]) pigs remain elusive. A low (3.9 × 10(8) CFU/day) or high (7.8 × 10(8) CFU/day) dose of Bacillus licheniformis and Bacillus subtilis spore mixture (BLS-mix) was orally administered to MUC4 RR piglets for 1 week before F4(+) ETEC/VTEC/EPEC challenge. Orally fed BLS-mix upregulated the expression of TLR4, NOD2, iNOS, IL-8, and IL-22 mRNAs in the small intestine of pigs challenged with E. coli. Expression of chemokine CCL28 and its receptor CCR10 mRNAs was upregulated in the jejunum of pigs pretreated with high-dose BLS-mix. Low-dose BLS-mix pretreatment induced an increase in the proportion of peripheral blood CD4(-)CD8(-) T-cell subpopulations and high-dose BLS-mix induced the expansion of CD4(-)CD8(-) T cells in the inflamed intestine. Immunostaining revealed that considerable IL-7Rα-expressing cells accumulated at the lamina propria of the inflamed intestines after E. coli challenge, even in pigs pretreated with either low- or high-dose BLS-mix, although Western blot analysis of IL-7Rα expression in the intestinal mucosa did not show any change. Our data indicate that oral administration of the probiotic BLS-mix partially ameliorates E. coli-induced enteritis through facilitating upregulation of intestinal IL-22 and IκBα expression, and preventing loss of intestinal epithelial barrier integrity via elevating ZO-1 expression. However, IL-22 also elicits an inflammatory response in inflamed intestines as a result of infection with enteropathogenic bacteria.

Figure 1

Effect of orally fed BLS-mix on TLR4- and NOD-mediated inflammatory responses in the small intestine. The relative expression of mRNAs for genes encoding A TLR4, B NOD1, C NOD2, E iNOS, and F IL-8 in both jejunal and ileal tissues collected from the indicated pigs 1 week after F4⁺ ETEC/VTEC/EPEC challenge was analyzed using quantitative real-time PCR. D Western blot analysis of IκBα expression in jejunal tissues collected from the indicated pigs 1 week after F4⁺ ETEC/VTEC/EPEC challenge. Representative IκBα expression in jejunal tissues collected from 2 pigs of each group is shown (left panel). Results are presented as the ratio of IκBα band intensity to the intensity of the β-actin band (right panel). Data are expressed in arbitrary units (A.U.) as the mean ± SEM for each tissue (n = 8 per group). *P < 0.05; **P < 0.01.

Figure 2

Effect of orally fed BLS-mix on peripheral blood lymphocytes. Peripheral blood samples were collected from the indicated pigs at 0, 24, and 144 h after F4⁺ ETEC/VTEC/EPEC challenge. A FSC-A/SSC-A dot plot of peripheral blood lymphocytes, cells with no gating. B CD3 dot plot, cells were gated on lymphocytes. C CD4/CD8 dot plot, cells were gated on CD3⁺. Flow cytometry analysis of the percentages of D CD4⁺CD8⁻, E CD4⁻CD8⁺, F CD4⁺CD8⁺, G CD4⁻CD8⁻ cells among CD3⁺ T cells. Data are presented as the mean ± SEM for each time point (n = 8 per group). *P < 0.05; ***P < 0.001.

Figure 3

Orally fed BLS-mix upregulates the expression of IL-22 and chemokine mRNAs in the small intestine. The expression of mRNAs for the A IL-22, B IL-23p19, C CCL25, D CCL28, E CCR9, and F CCR10 genes in both jejunal and ileal tissues collected from the indicated pigs 1 week after F4⁺ ETEC/VTEC/EPEC challenge was analyzed using quantitative real-time PCR. Data are presented as the mean ± SEM for each tissue (n = 8 per group). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Presence of IL-7Rα–expressing cells in the ileal mucosa. A Representative photomicrographs of immunostaining of IL-7Rα and negative control of irrelevant rabbit serum in ileal tissues collected from the indicated pigs 1 week after F4⁺ ETEC/VTEC/EPEC challenge. IL-7Rα–positive cells were predominantly localized in the surface epithelium and the lamina propria, as well as Peyer’s patches, with sporadic positive staining. Scale bars, 100 μm. B Representative Western blot results for IL-7Rα in ileal tissues collected from 2 pigs of each group are shown (upper panel). Results are presented as the ratio of the IL-7Rα band intensity to the intensity of the GAPDH band (lower panel). Data are expressed in arbitrary units (A.U.) as the mean ± SEM for each tissue (n = 8 per group).

Figure 5

Effect of orally fed BLS-mix on intestinal lymphocytes. A Representative dot plots showed the gating strategy for gut T-cell subpopulations. Flow cytometry analysis of the percentage of B CD4⁺CD8⁻, C CD4⁻CD8⁺, D CD4⁺CD8⁺, E CD4⁻CD8⁻ cells among intestinal CD3⁺ T cells. Peyer’s patch lymphocytes (PPLs), intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs) were collected from jejunal and ileal tissues from the indicated pigs 1 week after F4⁺ ETEC/VTEC/EPEC challenge. Data are presented as the mean ± SEM for each tissue (n = 8 per group). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Effect of orally fed BLS-mix on PKC-α, ZO-1, and occludin expression. Western blot analysis of PKC-α, zonula occludens-1 (ZO-1), and occludin expression in jejunal tissues collected from pigs 1 week after F4⁺ ETEC/VTEC/EPEC challenge. A Representative Western blot results for PKC-α in jejunal tissues collected from 1 pig of each group are shown. B Representative Western blot results for ZO-1 and occludin in jejunal tissues collected from 2 pigs of each group are shown. C Results are presented as the ratio of the intensity of the ZO-1 or occludin band to the intensity of the β-actin band. Data are expressed in arbitrary units (A.U.) as the mean ± SEM for each tissue (n = 8 per group). *P < 0.05; **P < 0.01.