Synchronous fibrosis in multiple organs is the defining hallmark of systemic sclerosis (SSc), but its pathogenesis remains poorly understood, and there is an urgent need to discover “druggable” targets. The pattern recognition receptor Toll-like receptor 4 (TLR4), a vital mediator of innate immunity, is expressed on both immune and stromal cells, and can be activated by endogenous ligands called “damage-associated molecular patterns” (DAMPs). Published and preliminary work from our laboratories show that TLR4 and its endogenous ligand tenascin-C are likely to play important roles in multi-organ fibrosis in SSc. Notably, tenascin-C itself elicits core fibrotic responses including ECM production and matrix stiffening in fibroblasts and 3D human skin organoid models. TLR4 activity is regulated by the ubiquitin-editing enzyme A20, which is a major risk gene for SSc. However, the mechanism linking SSc-associated A20 variants and pathogenesis are unknown. We demonstrated that A20 expression is reduced in SSc skin biopsies. Surprisingly, we found that A20 inhibited core fibrotic responses, and mice that are haploinsufficient for A20 showed markedly aggravated non-inflammatory skin fibrosis. We hypothesize that persistence of fibrosis in SSc could be explained by activated TLR4 signaling that is triggered by tenascin-C and other DAMPs, and chronically sustained by impaired A20 function. The cell types with increased profibrotic TLR4 pathway activity, and the ensemble of profibrotic DAMPs and specific domains, remain unknown. Moreover, the clinical correlates of reduced A20 in SSc, cell type-specific regulation and anti-fibrotic activity of A20, and the mechanisms linking reduced A20 and SSc pathogenesis, have never been investigated. To address these critical gaps, Aim 1 will determine cell type- and stimulus-specific roles and mechanisms of DAMP-TLR4 signaling in two separate models of experimentally-induced multi-organ fibrosis; map key tenascin-C domains and identify additional DAMPs as potential SSc biomarkers and therapeutic targets; Aim 2 will define the clinical correlates of A20 expression in a longitudinal cohort of SSc patients; and define distinct functions of A20 in the fibrotic process using novel A20-deficient and A20 humanized BAC transgenic mice. Aim 3 will determine the cellular sources and function of TLR4 signaling pathway activity in skin and lung from SSc patients and controls. Employing human disease samples from the established Northwestern and Yale Scleroderma Programs, combined with in vitro and in vivo disease models and state-of-the-art technologies including comprehensive matrisome analysis and unbiased single cell RNA sequencing of multiple tissue, our investigative team is poised to generate notable advances in understanding TLR4 signaling in SSc. The information in turn will guide discovery of novel biomarkers and therapies.
|Effective start/end date||6/13/19 → 4/30/24|
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (1R01AR074997-01)
Toll-Like Receptor 4
RNA Sequence Analysis
Pattern Recognition Receptors