Fibrin(ogen) Promotes Immune Cell Infiltration, Dysbiosis and ROS Production in Experimental Colitis
Authors: Sharma Balkrishan , Kudira Ramesh , Rosenfeldt Leah A. , Gourley Benjamin E , Cantrell Rachel , Watanabe Miki , Edwards Olivia , Romick-Rosendale Lindsey E , Haslam David , Flick Matthew J. , Palumbo Joseph S. 
Published: November 2021

Abstract
Elevated stool fibrinogen has recently been shown to predict disease course in ulcerative colitis (UC), suggesting that fibrin(ogen) in the colitis microenvironment promotes UC pathogenesis. This conclusion is consistent with previous studies from our laboratory showing that fibrin(ogen)/leukocyte interactions mediated by the integrin receptor α Mβ 2 promote experimental colitis and colitis-associated cancer, but the mechanisms are not well defined. To delineate the mechanism coupling fibrinogen to colitis pathogenesis, we induced colitis in Fib WT and mice carrying a mutant fibrinogen lacking the α Mβ 2 binding motif (Fibg 390-396A) with Dextran Sulfate Sodium (DSS). We performed flow cytometric, protein analyses of colons and fecal microbiome and metabolomics analyses after DSS exposure. Five days after DSS challenge, a timepoint prior to significant epithelial damage, we observed significantly diminished infiltration of natural killer cells, T cells, dendritic cells, macrophages and neutrophils in colons harvested from DSS-challenged Fibg 390-396A mice relative to Fib WT mice. We also observed significantly diminished proinflammatory cytokine production by NK cells, macrophages and dendritic cells isolated from DSS-challenged Fibg 390-396A colons. It is well-established that the microbiome composition is a major determinant of colitis in humans and mice. One mechanism by which microbiome contents alter the course of colitis is by the elaboration of certain fecal metabolites. NMR-based fecal metabolomics analyses demonstrated no significant changes in short-chain fatty acids (acetate, propionate, and butyrate) and various amino acids (valine, proline, alanine) however we found significantly less uracil in fecal from Fibg 390-296A relative to Fib WT mice following DSS exposure. Uracil is a key ligand for Duox2 (dual NADPH oxidase), a gut epithelial specific enzyme, that drives reactive oxygen species production by gut epithelial in response to dysbiosis. Notably, Duox2 is the highest induced gene in inflammatory bowel disease (IBD) and has been identified as a risk gene. Our molecular analyses showed no difference in colonic Duox2 expression between genotypes at baseline, but Duox2 expression significantly increased in Fib WT relative to Fibg 390-296A after just 5 days of DSS exposure. Further western blot analyses revealed that Duox2 expression only in the colonic epithelial cells and not in the lamina propria cells. To determine if fibrinogen cause dysbiosis in DSS-induced colitis model, we performed shotgun sequencing on fecal samples from Fib WT and Fibg 390-396A mice at baseline and after 7 days of DSS challenge. Interesting shotgun sequencing analyses revealed major fibrin genotype dependent significant differences in the microbiomes of Fib WT and Fibg 390-396A mice at baseline as well as following DSS challenge. Our studies are the first to show that fibrin(ogen) is a major determinant of the gut-microbiome in the context of experimental colitis. Altogether, these studies demonstrate that fibrin(ogen) in the colitis microenvironment promotes the infiltration and activation of multiple leukocyte subsets that drive colitis pathogenesis. These results also suggest that fibrin(ogen) promotes colitis-associated dysbiosis and Duox2 expression, leading to ROS production that contributes to colitis pathogenesis and possibley tumorigenesis. Overall, these studies suggest that fibrin(ogen)-α Mβ 2 interactions represent an attractive therapeutic target for IBD without incurring the potential bleeding risks associated with anticoagulants or other modalities targeting fibrin deposition.

Disclosures
No relevant conflicts of interest to declare.