Roles of chloroform-resistant variants in mouse models of experimental colitis

Abstract

We have recently reported that the intestinal microflora, especially chloroform-resistant variants (CRV), plays an important role in the maturation of murine intestinal mucosal T cell developments. In this study, to elucidate the role of CRV in the development of colitis, we compared the susceptibility to the colitis of the specific-pathogen-free (SPF) flora-associated and CRV-associated ex-GF Balb:c mice by means of intra-rectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS). The association of SPF flora or CRV with GF Balb/c induced phenotypic maturation in large intestinal (LI)-intestinal intraepithelial lymphocytes (IELs) and LI-lamina propria (LP) T cells. In LI-IELs, the CD4/CD8 ratio decreased with contamination by SPF flora or CRV with GF Balb/c mice. Among LP cells, the number of αβ T cell receptor (TCR) T cells increased. The percentage of the CD4 + CD45RB high phenotype in CD4 + αβ TCR LI-LP T cells, which is a critical marker molecule of virgin T cells, decreased in both SPF flora and CRV-associated Balb/c mice compared to GF Balb/c mice. These results suggested that the CRV flora converted the LI-IEL and LI-LP T cell characteristics from the GF to the conventional type. After the induction of colitis by means of an intra-rectal enema of TNBS in 50% ethanol, the body weight decreased in the group of Balb/c mice contaminated with the fecal flora of SPF mice but not that of CRV flora mice. Moreover, the colo-rectal tissue weight increased in the SPF flora-associated Balb/c mice but not in CRV-associated ones. Flow-cytometric analysis indicated that the percentage of LI-LP αβ TCR T cells increased in both the SPF flora and CRV-associated Balb/c mice. However, CD4 + CD45RB high LI-LP αβ TCR T cells only decreased in the SPF flora-associated Balb/c mice. Semi-quantitative RT-PCR analysis indicated that mRNA in both IL-12 and IFN- γ in LI-LP cells was higher in SPF flora-associated Balb/c mice than in CRV-associated ones. Taken together, these results suggested that the CRV flora is important of the maturation for LI-IELs and LI-LP T cells from GF to the conventional type. However, this flora is not critical for the development of colitis. Keywords: Clostridia, colitis, cytokine, inflammatory bowel disease, intestinal flora, intestinal intraepithelial lymphocyte, lamina propria, TNBS.