PMID- 35997276 OWN - NLM STAT- MEDLINE DCOM- 20221006 LR - 20231002 IS - 1522-1504 (Electronic) IS - 1040-0605 (Print) IS - 1040-0605 (Linking) VI - 323 IP - 4 DP - 2022 Oct 1 TI - Fibronectin-EDA accumulates via reduced ubiquitination downstream of Toll-like receptor 9 activation in SSc-ILD fibroblasts. PG - L484-L494 LID - 10.1152/ajplung.00019.2022 [doi] AB - 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) are prominent in pathological 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 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. FAU - Tuncer, Ferhan AU - Tuncer F AUID- ORCID: 0000-0003-4225-8627 AD - Aging Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Bulik, Melissa AU - Bulik M AD - Division of Rheumatology, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Villandre, John AU - Villandre J AD - Aging Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. AD - Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Lear, Travis AU - Lear T AUID- ORCID: 0000-0001-9156-0844 AD - Aging Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. AD - Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Chen, Yanwen AU - Chen Y AD - Aging Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Tuncer, Beyza AU - Tuncer B AD - Aging Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Kass, Daniel J AU - Kass DJ AD - Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. AD - Simmons Center for Interstitial Lung Disease, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Valenzi, Eleanor AU - Valenzi E AD - Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. AD - Simmons Center for Interstitial Lung Disease, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Morse, Christina AU - Morse C AD - Division of Rheumatology, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Sembrat, John 2nd AU - Sembrat J 2nd AUID- ORCID: 0000-0003-4108-803X AD - Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Lafyatis, Robert AU - Lafyatis R AD - Simmons Center for Interstitial Lung Disease, University of Pittsburgh, Pittsburgh, Pennsylvania. AD - Division of Rheumatology, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Chen, Bill AU - Chen B AD - Aging Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. AD - Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. FAU - Evankovich, John AU - Evankovich J AUID- ORCID: 0000-0003-2070-0883 AD - Aging Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. AD - Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. LA - eng SI - figshare/10.6084/m9.figshare.20383605.v1 GR - K08 HL144820/HL/NHLBI NIH HHS/United States GR - P50 AR060780/AR/NIAMS NIH HHS/United States GR - R35 HL139860/HL/NHLBI NIH HHS/United States GR - K99 AG078342/AG/NIA NIH HHS/United States GR - T32 HL110849/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20220823 PL - United States TA - Am J Physiol Lung Cell Mol Physiol JT - American journal of physiology. Lung cellular and molecular physiology JID - 100901229 RN - 0 (Fibronectins) RN - 0 (Ligands) RN - 0 (Protein Isoforms) RN - 0 (Toll-Like Receptor 9) SB - IM MH - Fibroblasts/metabolism MH - *Fibronectins/metabolism MH - Humans MH - Ligands MH - *Lung Diseases, Interstitial/metabolism MH - Protein Isoforms/metabolism MH - Toll-Like Receptor 9/genetics MH - Ubiquitination PMC - PMC9550569 OTO - NOTNLM OT - Toll-like receptor 9 OT - fibronectin OT - systemic sclerosis COIS- R.L. has grants from: Corbus, Formation, Moderna, Regeneron, Astra Zeneca, Pfizer, and Kiniksa, and consulting with Pfizer, Bristol Myers Squibb, Boehringer-Ingleheim, Formation, Sanofi, Boehringer-Mannheim, Merck and Genentech/Roche, Biogen. None of the other authors has any conflicts of interest, financial or otherwise, to disclose. EDAT- 2022/08/24 06:00 MHDA- 2022/10/07 06:00 PMCR- 2023/10/01 CRDT- 2022/08/23 08:03 PHST- 2022/08/24 06:00 [pubmed] PHST- 2022/10/07 06:00 [medline] PHST- 2022/08/23 08:03 [entrez] PHST- 2023/10/01 00:00 [pmc-release] AID - L-00019-2022 [pii] AID - 10.1152/ajplung.00019.2022 [doi] PST - ppublish SO - Am J Physiol Lung Cell Mol Physiol. 2022 Oct 1;323(4):L484-L494. doi: 10.1152/ajplung.00019.2022. Epub 2022 Aug 23.