The TNF superfamily factor TWEAK promotes colonic inflammatory fibroblast differentiation and fibroblast-monocyte interaction

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Background and Aims: Colonic fibroblasts have emerged as key players in inflammatory bowel disease (IBD) for their potential to acquire a pro-inflammatory phenotype, but the factors driving this process and how these fibroblasts interact with the immune system is incompletely understood. The TNF superfamily factor 12 (TNFSF12/TWEAK) has gained interest as a potential mediator of chronic inflammation and tissue remodelling in IBD. Here we explore its role as a driver of inflammatory fibroblast differentiation and as a modulator of fibroblast-monocyte interaction. Methods: Human primary colonic fibroblasts were stimulated with TWEAK or vehicle for 24 hours. fibroblasts were co-cultured with THP1 cells or primary monocytes for an additional 24 hours to investigate their interaction. RNA sequencing, immunoblotting, ELISA and flow cytometry were used for analysis. Results: TWEAK up-regulated the expression of >600 and down-regulated the expression of >500 genes in primary colonic fibroblasts (pAdj<0.05, fold>1.5). The up-regulated transcriptome was characterised by a pro-inflammatory programme including numerous cytokines, chemokines and immune receptors, which resembled an inflammatory stroma subpopulation previously identified in in ulcerative colitis (29% transcriptional alignment). The shared transcriptome included a marked signature of non-canonical NF-κB genes. At protein level, TWEAK inflicted changes in the expression of stromal markers (PDPN, PDGFRα); stimulated the secretion of IL-6, CCL2 and CXCL10; And enhanced the expression of surface adhesion molecules ICAM-1 and VCAM-1. Co-culture of THP1 cells with fibroblasts induced their adhesion and promoted a CD14high/ICAM-1high phenotype in THP1 cells, both of which were further enhanced when fibroblasts were pre-stimulated with TWEAK, while TWEAK did not directly affect adhesion or differentiation of THP1 cultured alone. Moreover, treatment of fibroblasts with a small molecule inhibitor of non-canonical NF-κB partially abrogated the fibroblast-mediated increase in CD14 expression on THP1 cells. In primary monocytes extracted from whole blood of 7 human donors, co-culture with TWEAK-treated fibroblasts promoted differentiation to a CD16intermediate, TREM-1high phenotype. Moreover, media from TWEAK-treated fibroblast-monocyte co-cultures displayed elevated levels of CXCL1 and IL-8 compared to vehicle-treated co-cultures, or to either cell type cultured alone, pointing towards reciprocal pro-inflammatory crosstalk. Conclusions: Our results indicate that TWEAK can promote pro-inflammatory polarisation in colonic fibroblasts and positions TWEAK as a mediator in the pro-inflammatory interaction between colonic fibroblasts and monocytes. This work is supported by funding from Science Foundation Ireland and The Irish Research Council under the Pathway program (21/PATH-S/9621), Enterprise Ireland, and the Conway Institute Director’s Strategic Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.