Multi-Omic Approaches Reveal Novel Targets and Mechanisms in Intestinal Inflammation and Gut-Liver Axis

Due to rapid advances in sequencing technology, multi-omic approaches integrating RNA sequencing, shotgun metagenomics, 16S rRNA sequencing, and metabolomics have emerged as appealing options for the study of complex disorders in the gut-liver axis due to their ability to generate large amounts of high-throughput data in a relatively short period of time. These approaches are especially attractive in the gut-liver axis as we are learning more about the roles of the gut microbiome in the development of such diseases such as inflammatory bowel diseases, alcoholic liver disease, and non-alcoholic liver disease. Inflammatory bowel diseases (IBD) and alcoholic liver disease (ALD) are complex disorders which present several challenges to public health. Some common factors in both diseases include the involvement of intestinal functions such as impairment of intestinal barrier function, and gut microbial dysbiosis. We investigated the pathophysiology of both diseases through the integration of biochemical approaches with transgenic animal models, the use of metabolomics, and bioinformatic analysis. The downregulation of SLC6A4, otherwise known as the serotonin transporter (SERT), has been implicated in IBD patients and in pre-clinical models of IBD. While SERT is emerging as a novel therapeutic target for gut disorders, its underlying mechanisms remain unclear and were the scope of the first aim of this work as presented in Chapter II. In order to investigate the mechanisms of SERT downregulation in intestinal inflammation, we utilized SERT KO mice as well as a newly generated model for intestinal epithelial cell specific SERT overexpression (SOEΔIEC). We performed serum and fecal metabolomics in SERT KO mice subjected to chronic DSS colitis and found diminished intestinal barrier function in SERT KO mice during inflammatory insult, basal metabolic profiles resembling those of WT mice with chronic colitis, and changes in microbial metabolites that corresponded to disease severity. Further, we found in a proof of concept that IEC SERT overexpression conferred protection against intestinal inflammation. Regarding ALD, recent studies have begun to explore the roles of the aryl hydrocarbon receptor (AhR), an environmental sensing transcription factor, in the pathogenesis of alcohol mediated liver injury due to the roles of AhR in the maintenance of intestinal barrier function. It is also worth noting that our group has recently uncovered that serotonin, or 5-HT is an indirect activator of AhR. In Chapter III, our preliminary studies have shown that a loss of intestinal epithelial cell AhR is protective towards the development of alcohol induced liver injury in female mice, with significant clinical implications given that women have a higher susceptibility to the development of ALD. In parallel, we have also utilized metabolomic approaches in the serum of a cohort of ALD patients with and without cirrhosis at the University of Illinois Hospital (UIH). Our studies have corroborated previous metabolomic studies in patients with ALD while contributing novel findings such as increases in the microbial metabolite methionine sulfoxide and decreases in guanosine. Furthermore, we were able to construct multiple linear regression models containing patient demographic, clinical, and metabolite data that were predictive of fibrosis stage. Finally, we were able to construct receiver operator curves for the measured metabolites and also found several candidate biomarkers with potential for the diagnosis of cirrhosis which is often diagnosed once patients have uncompensated cirrhosis with severe clinical complications. This work demonstrates the utility of utilizing multiple approaches for the investigation of complex diseases in the gut-liver axis. This work has shown the benefits of integrating novel, cell-specific, inducible transgenic mouse models, with broad scale metabolomics and bioinformatics. With these tools, we were able to gain mechanistic insight into how SERT deficiency leads to increased intestinal inflammation, discover a potential new treatment strategy for IBD in the form of intestinal epithelial SERT overexpression, find a protective role for the loss of intestinal epithelial cell AhR in the development of alcohol mediated liver injury in females, and contribute to the relatively few metabolomic studies of patients with alcoholic cirrhosis with the identification of new potential biomarkers for identifying those with severe disease.