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. 2010 Jul 1;161(1):187-96.
doi: 10.1111/j.1365-2249.2010.04140.x. Epub 2010 Mar 16.

An imbalance in mucosal cytokine profile causes transient intestinal inflammation following an animal's first exposure to faecal bacteria and antigens

B C Sydora  1 , S M MacFarlane, M Lupicki, A L Dmytrash, L A Dieleman, R N Fedorak
Affiliations

An imbalance in mucosal cytokine profile causes transient intestinal inflammation following an animal's first exposure to faecal bacteria and antigens

B C Sydora et al. Clin Exp Immunol. .

Abstract

Intestinal microflora play a critical role in the initiation and perpetuation of chronic inflammatory bowel diseases. In genetically susceptible hosts, bacterial colonization results in rapid-onset chronic intestinal inflammation. Nevertheless, the intestinal and systemic immune response to faecal bacteria and antigen exposure into a sterile intestinal lumen of a post-weaned animal with a mature immune system are not understood clearly. This study examined the effects of faecal bacteria and antigen exposure on the intestinal mucosal and systemic immune system in healthy axenic mice. Axenic wild-type mice were inoculated orally with a crude faecal slurry solution derived from conventionally raised mice and were analysed prior to and then at days 3, 7, 14 and 28 post-treatment. Ingestion of faecal slurry resulted in a transient, early onset of proinflammatory interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-17 response that was maximal at day 3. In contrast, the transient release of the anti-inflammatory cytokines IL-10 and IL-4 occurred later and was maximal at day 7. Both responses subsided by day 14. This early cytokine imbalance was associated with a brief rise in colonic and caecal histopathological injury score at day 7. The bacterial antigen-specific systemic response was found to follow the intestinal immune response with a maximal release of both pro- and anti-inflammatory cytokines at day 7. Thus, first exposure of healthy axenic wild-type mice to normal faecal flora and antigens results in an early proinflammatory cytokine response and transient colonic inflammation that then resolves in conjunction with a subsequent anti-inflammatory cytokine profile.

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Figures

Fig. 1
Fig. 1
Kinetics of cytokine release from colonic and caecal mucosal explant cultures derived from axenic 129/SvEv wild-type mice following ingestion of faecal slurry. Spontaneous release of interferon (IFN)-γ, interleukin (IL)-17, IL-4, IL-10 and granulocyte-colony stimulating factor (G-CSF) was assessed in supernatants after 24 h using standard enzyme-linked immunosorbent assay (ELISA) techniques. Production of proinflammatory cytokines (IFN-γ, IL-17) as well as production of G-CSF preceded the secretion of anti-inflammatory cytokines (IL-14, IL-10). Bars represent mean values ± standard error of the mean for four to six mice per group; *P > 0·05.
Fig. 2
Fig. 2
Histological injury assessment in Swiss Webster mice prior to and on days 3, 7 and 14 post-faecal slurry ingestion. Injury score (a) and histological inflammation (b) in both colon and caecum is maximal at day 7 and declines thereafter. Values represent mean values ± standard error of the mean for five mice per group. Fig. 2b shows haematoxylin and eosin staining of a histology preparation from a representative mouse for each time-point.
Fig. 3
Fig. 3
Epithelial barrier integrity as measured by colonic transmural mannitol flux in Ussing chambers pre- and post-faecal slurry ingestion. Bars represent mean values ± standard error of the mean for three Swiss Webster mice per group. All mice were assessed in duplicate.
Fig. 4
Fig. 4
Systemic immune response prior to and on days 3, 7 and 14 following faecal slurry ingestion as measured by cytokine release in 24-h antigen-stimulated cultures of spleen cells from 129/SvEv mice. At day 7 a temporary maximal increase in cytokine production [interferon-γ, interleukin (IL)-17 and IL-10, but not IL-4] was observed in response to (a) sterile lysates prepared from caecal matter of specific pathogen-free mice containing several endogenous intestinal bacteria to (b) sterile bacterial antigens prepared from pure cultures of Bacteroides vulgatus, Enterococcus cloacae and Lactobacillus reuteri and to (c) cross-linked anti-CD3 monoclonal antibodies. The results from stimulation with the three bacterial antigens are combined in one bar. Values represent mean values ± standard error of the mean for five to 10 mice per group; *P > 0·05.
Fig. 4
Fig. 4
Systemic immune response prior to and on days 3, 7 and 14 following faecal slurry ingestion as measured by cytokine release in 24-h antigen-stimulated cultures of spleen cells from 129/SvEv mice. At day 7 a temporary maximal increase in cytokine production [interferon-γ, interleukin (IL)-17 and IL-10, but not IL-4] was observed in response to (a) sterile lysates prepared from caecal matter of specific pathogen-free mice containing several endogenous intestinal bacteria to (b) sterile bacterial antigens prepared from pure cultures of Bacteroides vulgatus, Enterococcus cloacae and Lactobacillus reuteri and to (c) cross-linked anti-CD3 monoclonal antibodies. The results from stimulation with the three bacterial antigens are combined in one bar. Values represent mean values ± standard error of the mean for five to 10 mice per group; *P > 0·05.
Fig. 5
Fig. 5
Flow cytometric assessment and enumeration of spleen cells pre- and post-faecal slurry ingestion. No significant increase in the population of CD25-positive cells was noted (a). At day 7 CD11b-positive leucocytes increased significantly in the spleen cell population (b). The total number of spleen cells remained similar at various time-points post-feeding (c). Values represent mean values ± standard error of the mean (s.e.m.) for three to four mice per group (a) and mean values ± s.e.m. for five to 10 mice per group (b,c); *P > 0·05. Results for CD25 staining were derived from one experiment with Swiss Webster mice. Results for CD11b staining as well as spleen cell enumeration data were derived from experiments with 129/SvEv mice; however, data from Swiss Webster mice generated similar results.
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