Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

Abstract The stromal cell compartment plays a central role in maintaining tissue homeostasis by coordinating with the immune system throughout the inception, amplification, and resolution of inflammation(Henderson et al. 2020). Chronic inflammation can impede the phased regulation of tissue restitution, resulting in the scarring complication of fibrosis. In inflammatory bowel disease (IBD), stromal fibroblasts have been implicated in treatment-refractory disease and fibrosis; however, their mechanisms of activation remain undefined. Through integrative single-cell and spatial profiling of intestinal tissues from IBD patients, we uncovered a pathological cell nexus centered on inflammation-associated fibroblasts (IAFs). These IAFs were induced by pro-inflammatory macrophages (FCN1+, IL1B+) and, in turn, produced the pro-fibrotic cytokine IL-11. Mechanistic dissection of the IAF activation program was achieved through genome-wide CRISPR knockout and activation screens, identifying the transcription factor GLIS3 as a key regulator of a gene-regulatory network governing expression of inflammatory and fibrotic genes. We further demonstrate that the magnitude of the GLIS3 gene expression program in intestinal biopsies stratifies patients with ulcerative colitis (UC) by disease severity, and fibroblast-specific deletion of Glis3 in mice alleviates pathological features of chronic colitis. Taken together, our findings unveil a critical immune–stromal cell circuit that functions as a central node in the inflammation–fibrosis cycle. Vladislav Pokatayev1,2,3,*, Alok Jaiswal2,3,*, Angela R. Shih4, Åsa Segerstolpe5, Bihua Li2,3, Elizabeth A. Creasey1,2, Yanhua Zhao3, Crystal Lin5, Shane Murphy5, Chih-Hung Chou3, Daniel B. Graham1,2,3,5,6,#, Ramnik J. Xavier1,2,3,5,6,# 1Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA 2Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA 3Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA 4Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA 5Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA 6Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, MA 02114, USA * These authors contributed equally # Correspondence: dgraham@broadinstitute.org (D.B.G.), xavier@molbio.mgh.harvard.edu (R.J.X.)