Studies on the intestinal immunity of Tribolium castaneum to use the larvae as a model for inflammatory bowel diseases

Inflammatory bowel diseases (IBD) are exuberating infections of the intestines. Most studies on IBD are based on experiments with mouse models. IBD symptoms are induced in the animals in various ways, but the most common is oral administration of DSS via the drinking water, which results in chronic or acute colitis depending on the concentration. But this treatment also means a significant burden for the mice, which thereby suffer from rectal prolapse, diarrhea cramps and pain. Larvae of T. castaneum can almost be considered as gut on legs, since the intestines fill most of their body. The midgut is covered with a mucosal layer, the peritrophic matrix (PM) consisting of chitin and glycosylated PM proteins. Similar to the mucus found in vertebrate intestines, the PM serves as protection of the epithelium against bacteria and mechanical influences. IBD often go along with impaired mucus protection and the associated loss of the barrier function, which increases contact of bacteria and the epithelium, triggering immune responses. Further, the microbiome of the larvae includes bacterial phyla, which are also predominantly found in the human microbiome. Additionally, the innate immune responses of insects and mammals share similar and partially conserved mechanisms of pathogen recognition and defense. For this purpose, insects may be a suitable supplementary model for IBD research to reduce experiments based on mice. In the present thesis, the effects of DSS on the larvae of Tribolium castaneum, were analyzed. Further, the pyrimidine uracil, which is known to induce the production of reactive oxygen (ROS) in a DUOX dependent manner that is an enzyme belonging to the NADPH oxidase family, was used to similarly induce inflammatory phenotypes within the gut. Both chemicals had significant influence on intestinal health, including reduced growth, increased mortality and impaired development as well as midgut thickening, epithelial swelling and loss of epithelial barrier function, which may facilitate the entry of midgut bacteria, triggering innate immune responses that in return also affect the gut microbiome. In line with that, parts of the immunity like AMPs and DUOX showed increased expression and it was found that both treatments resulted in dysbiosis that resemble the microbial changes in the gut of IBD patients. The mis-regulation of AMPs and the associated intensified contact between bacteria and the intestinal epithelium appears to play a decisive role, particularly in the pathogenesis of Crohn's disease. In the present study, the influence of missing AMPs on the intestinal microbiome of T. castaneum larvae was investigated, whereby the AMPs TcATTA1, TcCEC2, TcCOL1 and TcDEF1 were downregulated with help of RNA interference (RNAi). The absence of the investigated AMPs (except TcCOL1) led to significantly altered compositions of the microbiome. In particular, TcATTA1 appears to affect gram-negative β/γ-Proteobacteria and the Family of Enterobacteriaceae, while TcDEF1 and TcCEC2 may regulate the class of gram-positive Bacilli, which otherwise repress bacteria such as Actinobacteria and Enterobacteriaceae. In addition, the inducibility of these AMPs by gram-positive and gram-negative pathogenic stimuli was investigated, whereby the influence of the IMD-pathway and the Toll- pathway was also considered by RNAi mediated knockdowns of either TcIMD or TcTOLL1. The IMD-pathway is known to induce immune reactions such as the release of AMPs in response to gram-negative bacteria, while the Toll-pathway recognizes gram-positive bacteria and fungi. However, no completely clear induction of the analyzed AMPs by a specific pathogen or by a specific immune signaling pathway could be identified. Rather, the signaling pathways also appear to be cross-activatable. Possibly, some of the phenotypic changes due to DSS or uracil treatment were caused by increased ROS. To test this assumption, an RNAi-mediated knockdown of TcDUOX expression was performed. But it was found that this knockdown enhanced the effects of DSS and uracil on mortality and also did not improve the observed reduced growth and delayed development, which may indicate that DUOX is an important defense mechanism. In insects, the activation of DUOX expression as well as DUOX activity are not fully understood so far. It was suggested that DUOX expression can be also performed via the IMD-pathway under a high pathogenic load, but not by commensal microorganism. Supporting this hypothesis, in the present thesis it was found that oral challenge of larvae with the gram-negative entomopathogenic Photorhabdus luminescens increases significantly TcDUOX-expression in the gut, which was significantly impaired in TcIMD knockdown larvae. But challenge with pathogenic, gram-positive Bacillus thuringiensis also significantly increased TcDUOX expression even if the larvae have a knockdown of TcIMD. It was previously described that B. thuringiensis, unable to produce uracil, were no longer able to stimulate DUOX expression. As mentioned before, uracil, which is probably only released by pathogenic bacteria, was described as a main stimulus to induce both, the DUOX expression as well as the DUOX activity pathway, but uracil recognition is still elusive. It was proposed, that a G-protein coupled receptor (GPCR) is involved in uracil dependent DUOX pathway activation. So far, no pyrimidinergic receptor has been described in insects, particularly none that binds uracil nucleotides or sugar derivatives. To identify potential candidates for insect pyrimidine receptors, a human P2Y4 receptor was used as a template to screen the Tribolium castaneum reference proteome. Four promising receptor candidates were identified, of which two showed significantly increased expression in the gut of uracil treated larvae. Therefore, knockdowns of two candidate genes (TcGPCR41, TcGPCR52) were performed by RNAi to analyze their influence on uracil-induced TcDUOX expression, HOCl formation and development in control larvae and larvae that were challenged with B. thuringiensis. Larvae, which were injected with dsTcGPCR41 showed impaired uracil-induced TcDUOX expression and HOCl formation. Furthermore, the development of challenged larvae was affected in a manner like that observed in a TcDUOX knockdown. These findings suggest, that this receptor may play a role in the uracil-dependent activation of the DUOX-pathways.