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Anti-carbamylated protein antibodies drive AEC II toward a profibrotic phenotype by interacting with carbamylated TLR5

This study looked at the role of anti-carbamylated protein (anti-CarP) antibodies in contributing to lung fibrosis in CTD-associated interstitial lung disease (ILD) in an autoantigen-dependent manner. ELISA was used to test serum samples, including 89 from the CTD-ILD group and 170 from the non-CTD-...

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Published in:Rheumatology (Oxford, England) England), 2024-10, Vol.63 (10), p.2874-2886
Main Authors: Xu, Wei, Huang, Minghua, Dong, Rongrong, Yan, Suyan, An, Yan, Liu, Baocheng, Ma, Zhenzhen, Mu, Kun, Yang, Qingrui
Format: Article
Language:English
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Summary:This study looked at the role of anti-carbamylated protein (anti-CarP) antibodies in contributing to lung fibrosis in CTD-associated interstitial lung disease (ILD) in an autoantigen-dependent manner. ELISA was used to test serum samples, including 89 from the CTD-ILD group and 170 from the non-CTD-ILD group, for anti-CarP levels. Male C57BL/6 mice were used for the pulmonary fibrosis model and anti-CarP treatment in vivo (n = 5) and patient serum-derived or commercialized anti-CarP was used for cell treatment. We identified the carbamylated membrane protein via immunofluorescence (IF) and co-immunoprecipitation followed by mass spectrometry (MS) analysis. Quantitative RT-PCR, IF and western blot were performed to explore the antigen-dependent role of anti-CarP. A native electrophoretic mobility shift assay and MS analysis were used to verify direct interaction and carbamylation sites. A significantly higher serum anti-CarP level was observed in CTD with ILD than without ILD. In vivo, intrapulmonary delivery of anti-CarP induces epithelial-mesenchymal transition (EMT) and microfibrotic foci. Carbamylation was enriched in type II alveolar epithelial cells (AEC II). A novel carbamylated membrane receptor, specifically recognized by anti-CarP, was identified as toll-like receptor 5 (TLR5). We found anti-CarP induces the nuclear translocation of NF-κB and downstream events, including EMT and expression of inflammatory cytokines in AEC II, which were reversed by TLR5 blocking or TLR5 knockdown. Moreover, up to 12 lysine carbamylation sites were found in TLR5 ectodomain, allowing the interaction of anti-CarP with carbamylated TLR5. Overall, we found anti-CarP drives aberrant AEC II activation by interacting with carbamylated TLR5 to promote ILD progression.
ISSN:1462-0324
1462-0332
1462-0332
DOI:10.1093/rheumatology/keae111