Defining cell-type specific Stat3 enhancers, their role in IBD development, and monocyte differentiation into colonic and ileal macrophages

Introduction

Macrophages are long-lived, tissue-resident phagocytes that, in most organs, are established prenatally. In the gut and other barrier tissues, however, macrophages display continuous replenishment by blood monocytes. To investigate what governs this steady state differentiation of monocytes into intestinal macrophages we employed a combination of cell ablation and precursor engraftment. We have identified factors associated with gradual adaption of monocytes to tissue residency in the ileum and colon. In the colon, monocyte-derived gut macrophages must sense IL-10 to maintain intestinal homeostasis. Mice with a macrophage-specific Il10ra deficiency develop severe colitis, as do children harboring IL10RA loss-of-function (LOF) mutations (Zigmond et al., 2014, Glocker et al. 2009). Mechanistically, IL-10Ra deficient macrophages produce IL-23, which activates T_H17 cells to secrete IL-22, which is sensed by epithelial cells exacerbating intestinal inflammation (Bernshtein et al., 2019). Interestingly, all the above cytokine responses require STAT3 for signaling and indeed Stat3B loci-associated single nucleotide polymorphisms (SNPs) were identified by GWAS as inflammatory bowel disorders (IBD) risk factors. In contrast, patients carrying these LOF mutations do not display IBD, but suffer from an autosomal dominant Hyper-IgE syndrome (AD-HIES). To understand, the underlying logic for this observation, we are in the process to define enhancer elements driving Stat3 expression in man and mice.

Material and methods

STAT3 regulatory elements and SNPs were retrieved from the GeneHancer (Fishilevich et al 2007) and UCSC genome browser. CRISPR/Cas9 technology is being used to generate mutant human cell lines and knock-out mice. To study monocyte differentiation into macrophages, monocytes were transferred into macrophage-depleted mice. Engrafted macrophages were then sorted and subjected to transcriptome profiling on various time points after transfer.

Results and discussion

Three out of 4 IBD-associated STAT3 SNPs were found to be located in regulatory elements, one of which (rs6503695) is predicted to be solely active in macrophages. Investigating monocyte differentiation, we identified a total of 634 and 539 genes, including transcription factors, involved in adaption to the colonic and ileal microenvironments, respectively.

Conclusion

Understanding cell-type specific Stat3 enhancers, particularly in myeloid cells, and differentiation of monocytes into gut macrophages might reveal critical pathways in the development of IBD. This will potentially allow the development of novel therapeutics.