장내 세균총의 변화가 DSS 장염 감수성에 미치는 영향

Abstract

Background/Aims: The role of intestinal microbiota in maintaining the host's homeostasis cannot be more emphasized. It is well known that inflammatory bowel disease (IBD) is caused by excessive immunologic reaction towards certain intestinal microbiota in genetically susceptible host. Intestinal microbiota plays an essential role in the induction of colitis in experimental animal model. In addition, some epidemiologic studies have proved that the changes in intestinal microbiota due to the use of antibiotics can affect the development of the disease. IL-17A is an inflammatory mediator secreted by Th17 cells, related to various autoimmune diseases such as IBD and rheumatoid arthritis. However, whether IL-17A plays a pathogenic or protective role in the development of chronic colitis is not yet clear. In the present study, we have evaluated whether the change in the composition of intestinal microbiota due to the administration of antibiotics causes any effect in the manifestation of DSS (dextran sodium sulfate)-induced colitis during the sensitive stage of intestinal microbiota establishment in wild type (WT) C57B/6 and IL-17A deficient mouse. Method: WT C57BL/6 mice (n=16) and IL-17A KO mice (n=16) were divided into four groups. The four groups were categorized as follows; group 1, control group with no administration of antibiotics or DSS; group 2, DSS administered from 5th week for 5 days; group 3, antibiotics (vancomycin, ampicillin, neomycin, metronidazole) administered from 3rd week for 2 weeks; group 4, antibiotics administered from 2nd week for 3 weeks followed by 5 days of DSS administration. Weight change and any clinical changes such as hematochezia were monitored during the experiment. Mice were sacrificed 7 days after DSS administration, and the morphologic changes as well as the length of colon and cecum were examined. The degree of inflammation was evaluated through microscopic evaluation of the cecal and distal colonic specimen after H&E staining, while the change in cytokine (IFN-γ, IL-12p40, IL-6) levels were analyzed through ELISA (Enzyme linked immunosorbent assay) using the supernatant of colonic tissue culture. Fecal material was aseptically collected from each mouse before and after the antibiotics administration. After DNA extraction, PCR amplification was carried out using 16S rRNA primer. Finally, Illumina sequencing was performed to analyze the change in the composition of intestinal microbiota. Result: A temporary decrease in weight was documented in the antibiotics administered groups compared to the control group in both WT and IL-17A KO mice. Cecal diameter was greatly increased after antibiotics administration in both the WT and IL-17KO mice, which resembled findings seen in germ-free state. The decrease in weight and colon length after DSS administration were less in the IL-17KO mice compared to WT mice, while the histologic scoring and the increase in inflammatory cytokine were also lower in the IL-17KO mice. Both WT and IL-17A KO mice demonstrated a decrease in the degree of colitis induced by DSS after antibiotics administration, and the decrease in colitis induced by antibiotics was more prominent in the WT mouse. A distinct difference in the intestinal composition during the weaning stage between the WT and IL-17A KO mouse has been documented. In both groups, the level of Firmicutes decreased while Proteobacteria increased in the phylum level, and Lachnospiraceae and Lactobacillaceae decreased while Enterobacteriaceae increased in the family level after antibiotics administration. Conclusion: In this mouse model, the alteration in intestinal microbiota induced by antibiotics during the infant stage changes the susceptibility to DSS-induced colitis. Deficiency in IL-17A ameliorates the severity of DSS-induced colitis. The change in intestinal microbiota induced by antibiotics administration during the infant stage may possibly cause a temporary growth delay of the host. The composition of intestinal microbiota during weaning stage depends on the genotype of the host, while the administration of antibiotics results in decreased intestinal microbiota diversity and induces a distinct change in the composition of intestinal microbiota.