Fibronectin-EDA accumulates via reduced ubiquitination downstream of Toll-like receptor 9 activation in SSc-ILD fibroblasts

Abstract

Accumulation of excessive extracellular matrix (ECM) components from lung fibroblasts is a feature of Systemic Sclerosis-associated Interstitial Lung Disease (SSc-ILD), and there is increasing evidence that innate immune signaling pathways contribute to these processes. Toll-Like Receptors (TLRs) are innate immune sensors activated by danger signals derived from pathogens or host molecular patterns. Several Damage-Associated Molecular Pattern (DAMP) molecules are elevated in SSc-ILD plasma, including ligands that activate TLR9, an innate immune sensor recently implicated in driving profibrotic responses in fibroblasts. Fibronectin and the isoform Fibronectin-Extra Domain A (FN-EDA) is prominent in pathologic extracellular matrix accumulation, but mechanisms promoting FN-EDA accumulation are only partially understood. Here, we show that TLR9 activation increases FN-EDA accumulation in MRC5 and SSc-ILD fibroblasts, but that this effect is independent of changes in FN-EDA gene transcription. Rather, we describe a novel mechanism where TLR9 activation inhibits FN-EDA turnover via reduced FN-EDA ubiquitination. TLR9 ligand ODN2006 reduces ubiquitinated FN-EDA destined for lysosomal degradation, an effect abrogated with TLR9 knockdown or inhibition. Taken together, these results provide rationale for 1) disrupting the TLR9 signaling axis or FN-EDA degradation pathways to reduce FN-EDA accumulation in SSc-ILD fibroblasts. More broadly, enhancing intracellular degradation of ECM components through TLR9 inhibition or enhanced ECM turnover could be a novel strategy to attenuate pathogenic ECM accumulation in SSc-ILD.

Use of a pulmosphere model to evaluate drug antifibrotic responses in interstitial lung diseases

“…Fibronectin and Collagen are important ECM proteins which are expressed in non-IPF ILD’s [ 12 – 14 ]. Fibronectin levels are elevated in profibrotic monocytes, macrophages, and fibroblasts in scleroderma associated ILD [ 15 , 16 ]. We analyzed VATS biopsy tissues of the control subjects and compared them to 5 ILD patients with IHC for fibronectin-EDA.…”