Complementary genetic screens identify novel regulators of fibroblast pathogenicity

Abstract

Rheumatoid arthritis (RA), a disease affecting 1.5 million people in the US, is a chronic autoimmune condition in which the immune system attacks the joints, leading to inflammation, tissue remodeling, and, ultimately, bone erosion and joint deformity. While several therapies have transformed RA treatment over the past two decades, approximately 50% of patients fail to respond to TNF inhibitors, and up to 25% remain refractory to all available therapies. These multidrug-resistant patients represent a significant unmet clinical need, underscoring the importance of discovering new therapeutic targets. Synovial fibroblasts, a key stromal cell type in the joint synovium, adopt pathological behaviors in RA, including inflammatory cytokine production, tissue invasion, and immune cell modulation. However, the molecular pathways governing these behaviors remain incompletely understood.To address this gap, we developed a gene editing toolbox that enables both loss-of- function and gain-of-function genetic perturbations in primary RA patient-derived synovial fibroblasts. Using these tools, we conducted complementary arrayed CRISPR deletion and open reading frame (ORF) overexpression screens to identify regulators of fibroblast inflammatory and invasive programs. We modeled synovial fibroblast activation in vitro using a panel of RA- relevant cytokines (TNFα, IL-17, IL-1β, IFNγ), and assessed phenotypic responses via multiplexed ELISA and bulk RNA-seq. Our screens uncovered both known and previously unrecognized regulators of fibroblast effector functions. Notably, we identified NFIL3, ITGB5, MAFF, SOX5, and ETV7 as regulators of distinct pathological features, ranging from inflammatory gene expression to matrix remodeling behavior. Together, this work establishes a robust experimental and analytical pipeline for medium- throughput functional interrogation of primary human fibroblasts. The insights gained not only deepen our understanding of fibroblast biology in RA but also offer a resource of candidate regulators and pathways that could be leveraged to develop next-generation therapies. As fibroblasts contribute to disease processes across tissues, including cancer, fibrosis, and other autoimmune diseases, the tools and discoveries presented here have broad relevance for tackling fibroblast-driven pathology in RA and beyond.