Functional analysis of the Interleukin-10 (IL-10) network by induction of colitis in conditional IL-10 and IL-10 receptor knock-out mice

Elucidation of the cellular interleukin-10 (IL-10) network is an important issue for understanding how IL-10 regulates inflammatory responses. The comprehension of such regulatory mechanisms might help find a way to limit the immune response in a chronic inflammation such as Inflammatory Bowel Disease (IBD). The generation and analysis of conditional knock-out mice is a way to demonstrate the cell specific function of a gene in the context of an entire organism. The work in this thesis involves the establishment of 3 conditional IL-10 receptor (IL-10R) mouse strains and the development of a colitis model in order to analyse these strains and conditional IL-10 knock-out mouse strains in addition, for their susceptibility to colitis. In the first part, 3 conditional IL-10R knock-out mouse strains, specific for B-cells, T-cells and macrophages and neutrophils, were bred and the efficiency and the specificity of the deletion was confirmed by Southern blot analysis of sorted cell populations. Furthermore, a mouse strain carrying the ubiquitous IL-10R knock-out (IL-10-/-) was established. The evaluation of a colitis model was the next step conducted, because IL-10 complete knock-out mice maintained at the Helmholtz Centre for Infection Research were found to be devoid of signs of colitis. Induction of colitis using dextran sulfate sodium (DSS) was found to be a suitable model and gave the following results: IL-10-/- mice developed a severe acute suppurative colitis, whereas wildtype mice exhibited only a mild colitis. The cytokine profile in IL-10-/- mice was characterised by proinflammatory cytokines and chemokines. These results demonstrate the induction of an innate immune response by DSS and its inhibition by IL-10. In order to elucidate the role of the intestinal bacteria in DSS induced colitis, germfree mice were exposed to DSS. Surprisingly, germfree wildtype as well as IL-10-/- mice were even more susceptible to DSS than IL-10-/- mice harbouring intestinal flora. This implies that intestinal bacteria are protective against DSS induced damage and that the protective effect is mediated by IL-10. Hence, DSS causes colitis through a toxic effect to the intestinal epithelial cells. The analysis of the mouse strains carrying a cell type specific deletion of the IL-10R in B-cells, T-cells and macrophages and neutrophils in the DSS colitis model did not reveal significant differences to wildtype controls. The mixed genetic background of the animals might be the cause for the large deviation observed. A backcross of the three conditional IL-10R mouse strains to C57BL/6 might reveal differences that were masked by the background effect in this thesis. The exposure of T-cell and macrophage and neutrophil specific conditional IL-10 knock-out mice to DSS did not reveal significant differences, either. Mice carrying the specific deletion of IL-10 in T-cells though, exhibited a tendency towards an enhanced susceptibility to DSS. In order to elucidate the role of T-cell derived IL-10 in a Th1 or Th2 dominated immune response upon DSS exposure, double knock-out mice carrying the deletion of IL-10 in T-cells and the complete knock-out of IL-4 or IL-12 in addition, were analysed in the DSS colitis model. Unexpectedly, the lack of IL-12 led to an increased susceptibility to DSS induced colitis. The lack of T-cell derived IL-10 in combination with the lack of IL-4 increased the susceptibility to DSS induced colitis as well. Hence, a T helpercell immune response seems to down-regulate the innate immune response to DSS induced damage of the epithelium, independent of a Th1 or Th2 polarisation. In conclusion the data obtained in this study indicate that DSS causes damage to the intestinal epithelium due to a toxic effect, thereby inducing an innate immune response. This innate immune response is inhibited by IL-10. The main IL-10 producing cells are most probably T-cells that are induced by intestinal bacteria. The most important target cell of IL-10 was not found in this study, the cytokine profile of an innate immune response though, suggests a crucial role of macrophages and neutrophils or intestinal epithelial cells. The analysis of the recently available mouse strain carrying the Cre-transgene for intestinal epithelial cells would be of interest in the DSS colitis model. Furthermore, the role of dendritic cells in this model remains to be determined.