Prostaglandins and calprotectin are genetically and functionally linked to the Inflammatory Bowel Diseases

Abstract

BackgroundGenome wide association studies (GWAS) have identified and validated more than 200 genomic loci associated with the inflammatory bowel disease (IBD), although for most the causal gene remains unknown. Given the importance of myeloid cells in IBD pathogenesis, the current study aimed to uncover the role of genes within IBD genetic loci that are endogenously expressed in this cell lineage. MethodsThe open reading frames (ORF) of 43 genes from IBD-associated loci were expressed via lentiviral transfer in the THP-1 model of human monocytes and the impact of each of these on the cells transcriptome was analyzed using a novel RNA sequencing-based approach. We used a combination of genetic and pharmacologic approaches to validate our findings in the THP-1 line with further validation in human induced pluripotent stem cell (hiPSC)-derived-monocytes. ResultsThis functional genomics screen provided evidence that genes in four IBD GWAS loci (PTGIR, ZBTB40, SLC39A11 and NFKB1) are involved in controlling S100A8 and S100A9 genes expression, which encode the two subunits of calprotectin (CP). We demonstrated that increasing PTGIR expression and/or stimulating PTGIR signaling resulted in increased CP expression in THP-1. This was further validated in hiPSC-derived monocytes. Conversely, knocking-down PTGIR endogenous expression and/or inhibiting PTGIR signaling led to decreased CP expression. These analyses were extended to the known IBD gene PTGER4, whereby its specific agonist also led to increased CP expression. Furthermore, we demonstrated that the PTGIR and PTGER4 mediated control of CP expression was dependent on signaling via adenylate cyclase and STAT3. Finally, we demonstrated that LPS-mediated increases in CP expression could be potentiated by agonists of PTGIR and PTGER4, or diminished by their antagonists, with an opposite effect on TNF-alpha expression. ConclusionOur results support a causal role for the PTGIR, ZBTB40, SLC39A11 and NFKB1 genes in IBD, with all four genes regulating the expression of CP in myeloid cells, as well as an important role for the prostacyclin/prostaglandin biogenesis and signaling pathways in IBD susceptibility and pathogenesis. Author SummaryCrohns Disease and Ulcerative colitis are the two main types of inflammatory bowel diseases (IBD). These are debilitating chronic inflammatory diseases of the digestive tract. IBD pathogenesis is complex and involves multiple different cell types within the intestinal mucosa. While over 200 regions of the genome have been associated with susceptibility to IBD, for most the causal gene remains to be identified. In the current study we have focused on genes from IBD loci that are endogenously expressed in monocytes or macrophages, given the importance of these cells in IBD pathogenesis. Specifically, we modulated the expression of 43 genes from within validated IBD loci, in a human monocyte/macrophage cell line, and determined the impact of this increased expression on the rest of the transcriptome. We found evidence that four of these genes (PTGIR, ZBTB40, SLC39A11 and NFKB1) control the expression of calprotectin, which is a proinflammatory molecule that is used as a marker of intestinal mucosal inflammation. We then elucidated how prostaglandin signaling via prostaglandin receptors PTGIR and PTGER4, another IBD gene, regulates calprotectin expression. This work provides evidence that all five genes are causal and provide the first link between calprotectin and disease susceptibility.