TNC-targeted CAR-macrophage therapy alleviates liver fibrosis in mice

Abstract Background Tenascin-C (TNC) is an extracellular matrix (ECM) protein involved in tissue damage and fibrosis. Chimeric antigen receptor (CAR) cell therapy is a novel therapeutic approach that has attracted increasing attention in recent years. Here, we engineered CAR-macrophages targeting TNC (TNC-CAR-Ms) and explored the underlying mechanism through which TNC-CAR-Ms treat liver fibrosis. Methods The role of TNC in liver fibrosis was studied in established Tnc knockout (KO) and littermate control mice. A TNC-targeted single-chain variable fragment (scFv) was designed to generate TNC-CAR-Ms and evaluate their biological function. The phagocytosis and killing effects of TNC-CAR-Ms were tested in vitro, while the antifibrotic efficacy and safety of TNC-CAR-Ms were evaluated in vivo. The underlying mechanism through which TNC-CAR-Ms treat liver fibrosis was investigated by Western blotting, flow cytometry, and RNA sequencing. Results TNC expression was significantly upregulated in the liver and activated hepatic stellate cells (HSCs) in carbon tetrachloride (CCl4)-treated mice. Animal studies showed that Tnc KO protects mice from CCl4-induced liver damage and fibrosis. Upon demonstrating their ability to engulf and kill activated HSCs, we intravenously administered TNC-CAR-Ms to fibrotic mice and found that TNC-CAR-Ms significantly reduced liver fibrosis. Mechanistically, TNC-CAR-Ms specifically migrated to liver tissues, potently reduced TNC expression, and decreased the activity of the Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) and integrin/focal adhesion kinase (FAK) signaling pathway. In addition, TNC-CAR-Ms significantly modified the hepatic immune microenvironment, characterized mainly by an increase in the numbers of M2-polarized macrophages and CD8+ T cells in the liver. Finally, in CCl4-treated mice, the depletion of CD8+ T cells with an anti-CD8α antibody significantly impaired the antifibrotic effect of TNC-CAR-Ms. Conclusions Our proof-of-concept study demonstrates the therapeutic potential of TNC-CAR-Ms in alleviating liver fibrosis and may inform the development of future therapeutic strategies for the treatment of a range of liver diseases with a fibrotic phenotype.