Bridging the Bench to the Bedside in IBD:
Understanding the molecular basis for intestinal macrophage tolerance:
IBD results from an inappropriately directed inflammatory response to the enteric microbiota in a genetically susceptible host. Of the pro-inflammatory genes that are induced through interactions with microbes, macrophage specific genes play a central role in driving intestinal inflammation in IBD. In contrast to macrophages from other organs, intestinal macrophages are in a default state of hypo- responsiveness. Intestinal macrophages efficiently eradicate luminal microbes that have breached the intestinal epithelial barrier without mounting a potent inflammatory response. The importance of the functional adaptations of macrophages to perform their role in this unique environment is best illustrated by failure of these homeostatic mechanisms during the development of IBD. Our overall objective is to identify variations in chromatin and active gene regulatory elements in intestinal macrophages that drive intestinal macrophage tolerance in response to the enteric microbiota. To address this objective we combine the power of gnotobiotic rodents, validated models of experimental colitis and whole genome analysis using high throughput sequencing (seq) technology targeting the murine and human genome in macrophages and other immune cell populations in the intestine.
Characterizing non-coding DNA variants in IBD:
Converging human genetic (GWAS) and functional findings have linked many single nucleotide polymorphisms (SNPs) within genes of the innate immune system to IBD. However, which of these SNPs is causal with respect to IBD pathogenesis is unknown. Most IBD-associated SNPs fall into non-coding regions of the genome that are completely unannotated with respect to function. The overall objective of this project is to identify variations in chromatin and active gene regulatory elements in colon tissue obtained from patients with IBD. We use chromatin accessibility to catalog regulatory elements in colon tissue from healthy persons and patients with IBD and elucidate the functional significance of these regions in maintaining intestinal immune homeostasis. We determine chromatin status throughout the genome in colon tissue harvested from patients with IBD by FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) and DNase I hypersensitive (HS), and messenger RNA analysis for gene expression, combined with Illumina high-throughput sequencing. We further adapt and refine these techniques for human cellular analysis through their application to human intestinal and peripheral macrophages.
MicroRNAs and IBD:
MicroRNAs (miRNAs) are a group of short noncoding RNAs that can regulate gene expression at the posttranscriptional level. MicroRNAs have emerged as key regulators of a wide variety of biological processes. Using whole genome smallRNA-sequencing we have identified a number of miRNAs as regulators of severity of Inflammatory Bowel Diseases’ (IBD) disease progression. IBD includes Crohn’s disease (CD) and ulcerative colitis (UC). We have demonstrated that specific miRNAs can serve as markers of distinct disease behaviors in CD. In addition to the potential prognostic utility of miRNAs in adult CD, we are identifying the clinical utility and functions of these microRNAs in pediatric patients with IBD. We are elucidating the role of microRNAs in different intestinal cell types, specifically immune cells and intestinal epithelial cells (IECs) including intestinal stem cells and enterocytes (goblet and paneth cells and enterocytes). We utilize functional assays like intestinal barrier integrity as well as an ex vivo human mini-gut system called enteroids. Finally, using a statistical simulation strategy, we are exploring candidate miRNA drivers of the gene expression profiles associated with CD using RNA-sequencing data generated in the same set of samples and experiments.
Linking the Enteric Microbiota to Clinical Crohn’s Disease Phenotype:
Crohn’s disease is a chronic gastrointestinal disease that has a heterogeneous clinical presentation. In the presence of Crohn’s disease, the microbiota of the intestinal mucosa—enteric microbiota—undergoes abnormal shifts in its composition and studies have shown that the enteric microbiota plays a key role in the CD. We hypothesize that there are distinct microbial signatures (1) that characterize behavioral phenotypic differences in CD and (2) that are associated with post-operative CD recurrence. A better understanding of the abnormal alterations in the enteric microbiota is necessary for a more comprehensive understanding of phenotypic behavioral differences seen in CD and the role they play in post-operative CD recurrence as well as improved diagnostic capabilities.
