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.

Keywords: Chimeric antigen receptor (CAR); Hepatic stellate cells (HSCs); Liver fibrosis; Macrophage; Tenascin-C (TNC).

Fig. 1

TNC deficiency protects mice from CCl₄-induced hepatic fibrosis. a Schematic of the CCl₄-induced liver fibrosis models. Wild-type (WT) and Tnc gene knockout (Tnc KO) mice were intraperitoneally injected with 0.6 ml/kg CCl₄ twice weekly for 6 weeks (n = 6). Olive oil was used as a control treatment. The 12 × refers to the repeated injections of CCl₄ for 12 times. P56 and P98 indicate the mice’s age in postnatal days (day 56 and day 98, respectively). b Serum ALT and AST levels. After the final CCl₄ injection, blood samples were collected, and serum was obtained by centrifugation. c Liver tissues from WT, WT + CCl₄, Tnc KO, and Tnc KO + CCl₄ mice were sectioned and subjected to H&E staining, Sirius Red staining, and α-SMA IHC staining. Quantification of Sirius Red staining and α-SMA IHC staining was performed using ImageJ software. Scale bar = 200 μm. d Measurement of the hydroxyproline content in liver tissues from mice in the WT, WT + CCl₄, Tnc KO, and Tnc KO + CCl₄ groups. e Measurement of serum albumin levels in WT, WT + CCl₄, Tnc KO and Tnc KO + CCl₄ mice. f ELISA of IL-1β protein levels in the serum of mice from the WT, WT + CCl₄, Tnc KO, and Tnc KO + CCl₄ groups (n = 6). g RT-qPCR analysis of the mRNA expression levels of proinflammatory genes (Tnfα, Il1β, and Cxcl1) and profibrogenic genes (Acta2, Col1a1, and Col2a1) in liver tissues from WT, WT + CCl₄, Tnc KO, and Tnc KO + CCl₄ mice. Data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns non-significant. ALT alanine aminotransferase, AST aspartate aminotransferase, H&E hematoxylin and eosin, α-SMA α-smooth muscle actin, IL-1β interleukin-1β, Tnfα tumor necrosis factor-α, Cxcl1 chemokine (C-X-C motif) ligand 1, Acta2 actin alpha 2, Col1a1 collagen type I alpha 1 chain, Col2a1 collagen type II alpha 1 chain, IHC immunohistochemistry, ELISA enzyme-linked immunosorbent assay, RT-qPCR reverse transcription quantitative polymerase chain reaction, TNC tenascin-C

Fig. 2

Generation and functional validation of CAR-Ms targeting TNC in vitro. a Schematic diagram of the TNC-CAR vector sequence. b Microscopy images of TNC-CAR-transfected BMDMs and non-transfected cells, where the TNC-CAR-transfected cells exhibited green fluorescence under a fluorescence microscope because they expressed the EGFP. Scale bar = 100 μm. c TNC scFv expression in TNC-CAR-Ms and non-transfected (UTD) control cells was detected using flow cytometry. TNC-CAR-transfected cells were incubated with biotinylated protein L antibodies, followed by staining with fluorescent dye-labeled secondary antibodies. d RT-qPCR analysis of the expression levels of macrophage polarization-related genes, including the M1 markers Nos2, Il1β, Tnfα, and the M2 marker Arg1, Il10, in Mock-CAR-Ms, TNC-CAR-Ms, and UTD macrophages. e Illustration of the HSC and CAR-Ms coculture system for the killing assay. f In vitro killing assay. TGF-β-stimulated HSCs (stably expressing luciferase) were cocultured with Mock-CAR-Ms and TNC-CAR-Ms. After coculture for 12, 24, and 36 h, cell viability and the fluorescence intensity were assessed to evaluate the cytotoxicity of TNC-CAR-Ms and Mock-CAR-Ms toward HSCs. ^*P < 0.05, ^**P < 0.01, vs. Mock-CAR-Ms. g Illustration of the HSC and CAR-Ms coculture system for the phagocytosis assay. h Fluorescence microscopy images of CAR-Ms and HSCs. The CAR-Ms appeared round and nonfluorescent before they were cocultured with EGFP-labeled fusiform HSCs. The merged signal refers to the combination of white light and green fluorescence (EGFP-labeled HSCs). Scale bar = 100 µm. i Detection of phagocytic activity (F4/80⁺EGFP⁺ cells) in the coculture system using flow cytometry. Data are presented as mean ± SD. ^**P < 0.01. EGFP enhanced green fluorescent protein, T2A thosea asigna virus 2A peptide, scFv single-chain fragment variable, CD8 cluster of differentiation 8, CD3 cluster of differentiation 3, P2A porcine teschovirus-1 2A peptide, PuroR puromycin resistance gene, BMDMs bone marrow-derived macrophages, PE phycoerythrin, Nos2 nitric oxide synthase 2, Il1β interleukin-1β, Tnfα tumor necrosis factor-α, Arg1 arginase 1, Il10 interleukin-10, HSCs hepatic stellate cells, TGF-β transforming growth factor-β, RT-qPCR reverse transcription quantitative polymerase chain reaction, TNC tenascin-C, CAR-Ms chimeric antigen receptor-macrophages

Fig. 3

TNC-CAR-Ms exerted antifibrotic effects in vivo. a Experimental diagram of CAR-Ms therapy in liver fibrosis mouse models. The CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, and CCl₄ + TNC-CAR-Ms groups were treated with PBS solution, Mock-CAR-Ms, or TNC-CAR-Ms (i.v., 2 × 10⁶ cells/mouse), respectively, 24 h after the 8th CCl₄ injection. The control group of WT mice was injected with olive oil (n = 6). The 12 × refers to the repeated injections of CCl4 for 12 times. P56, P84, and P98 indicate the mice’s age in postnatal days (day 56, day 84, and day 98, respectively). b Serum ALT and AST levels in mice from the WT control, CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, and CCl₄ + TNC-CAR-Ms groups. c Representative images of liver tissues from mice in the WT control, CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, and CCl₄ + TNC-CAR-Ms groups. d Liver tissues were embedded, sectioned, and subjected to H&E staining, Sirius Red staining, and α-SMA IHC staining. Quantification of Sirius Red staining and α-SMA IHC staining was performed using ImageJ software. Scale bar = 200 μm (H&E staining and Sirius Red staining) and 50 μm (α-SMA IHC staining). e Experimental diagram of CAR-Ms therapy in cirrhosis models. The CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, and CCl₄ + TNC-CAR-Ms groups were treated with PBS solution, Mock-CAR-Ms, or TNC-CAR-Ms (i.v., 2 × 10⁶ cells/mouse), respectively, 24 h after the 20th CCl₄ injection. The control group of WT mice was injected with an equivalent dose of olive oil (n = 6). The 24 × refers to the repeated injections of CCl₄ for 24 times. P56, P126, and P140 indicate the mice’s age in postnatal days (day 56, day 126, and day 140, respectively). f Serum ALT levels in the WT control, CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, and CCl₄ + TNC-CAR-Ms groups. g Liver tissues were embedded, sectioned, and subjected to H&E staining, Sirius Red staining, and α-SMA IHC staining. Quantification of Sirius Red staining and α-SMA IHC staining was performed using ImageJ software. Scale bar = 200 μm. Data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns non-significant. ALT alanine aminotransferase, AST aspartate aminotransferase, H&E hematoxylin and eosin, α-SMA α-smooth muscle actin, IHC immunohistochemistry, TNC tenascin-C, CAR-Ms chimeric antigen receptor-macrophages

Fig. 4

TNC-CAR-Ms migrate to the liver and reduce TNC expression. a Assessment of the biological distribution and transport of TNC-CAR-Ms in vivo. Three days after the TNC-CAR-Ms infusion, the mice were anesthetized, euthanized, and tissues were dissected. The distribution of luciferase-expressing TNC-CAR-Ms in brain, heart, liver, spleen, lung, and kidney tissues was monitored with a Clinx-IVScope 8000 imaging system. The distribution of TNC-CAR-Ms in the major organs of mice was quantitatively and statistically analyzed. b TNC protein expression in liver tissues from CCl₄ + PBS and CCl₄ + TNC-CAR-Ms mice was detected by Western blotting and quantitatively analyzed using Quantity One software. c Images of immunofluorescence (IF) staining for the TNC protein in liver tissues from WT, CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, and CCl₄ + TNC-CAR-Ms mice. Scale bar = 200 μm (upper) and 50 μm (lower). d Images of IF staining for the TNC and α-SMA proteins in liver tissues from CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, and CCl₄ + TNC-CAR-Ms mice. DAPI was used for nuclear staining. Scale bar = 200 μm (5 ×) and 50 μm (20 ×). Data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. GAPDH glyceraldehyde-3-phosphate dehydrogenase, α-SMA α-smooth muscle actin, DAPI 4',6-diamidino-2-phenylindole, TNC tenascin-C, CAR-Ms chimeric antigen receptor-macrophages

Fig. 5

RNA sequencing analysis of liver tissues from Mock-CAR-Ms and TNC-CAR-Ms mice. a Heatmap showing the results for differentially expressed genes (DEGs) identified by RNA sequencing in the Mock-CAR-Ms and TNC-CAR-Ms groups. b Volcano plot of DEGs identified by RNA sequencing in the Mock-CAR-Ms and TNC-CAR-Ms groups, where upregulated genes are shown in red and downregulated genes are shown in blue. c The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the RNA sequencing data revealed the most significant biological processes that were changed by TNC-CAR-Ms compared with Mock-CAR-Ms. The bubble size represents the number of genes, and the color represents the P-value of the difference. d Levels of the α-SMA, integrin αV, p-FAK, p-NF-κB, IκBα, and TLR4 proteins in liver tissues from the CCl₄ + Mock-CAR-Ms and CCl₄ + TNC-CAR-Ms mice were detected by Western blotting and quantitatively analyzed using Quantity One software. Data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns non-significant. Integrin αV integrin alpha V, p-FAK phosphorylated focal adhesion kinase, α-SMA α-smooth muscle actin, TLR4 Toll-like receptor 4, p-NF-κB/p65 phosphorylated nuclear factor kappa B-p65 subunit, IκBα inhibitor of kappa B alpha, GAPDH glyceraldehyde-3-phosphate dehydrogenase, TNC tenascin-C, CAR-Ms chimeric antigen receptor-macrophages

Fig. 6

M2-polarized TNC-CAR-Ms enhanced fibrosis regression in mice. a The relative proportions of F4/80⁺CD80⁺ cells in liver tissues from WT, CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, and CCl₄ + TNC-CAR-Ms mice were detected by flow cytometry and quantitatively compared (n = 3). b RT-qPCR analysis of the expression levels of M2 polarization-related genes, including Mrc1 (CD206), Il10, and Arg1, in TNC-CAR-Ms and IL-4-stimulated TNC-CAR-Ms. c The relative proportions of CD206⁺ cells among TNC-CAR-Ms and TNC-CAR-Ms + IL-4 cells were determined by flow cytometry and quantitatively compared. d Detection of phagocytic activity (F4/80⁺EGFP⁺ cells) in the coculture system using flow cytometry. e Experimental illustration of the effects of M2-polarized TNC-CAR-Ms on the liver fibrosis model. The CCl₄-treated mice received PBS solution, Mock-CAR-Ms, TNC-CAR-Ms or IL4-stimulated TNC-CAR-Ms (i.v., 2 × 10⁶ cells/mouse) 24 h after the 8th CCl₄ injection (n = 6). 12 × refers to the repeated injections of CCl₄ for 12 times. P56, P84, and P98 indicate the mice’s age in postnatal days (day 56, day 84, and day 98, respectively). f Liver tissues from mice in the CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, CCl₄ + TNC-CAR-Ms, and CCl₄ + TNC-CAR-Ms + IL-4 cell infusion groups were fixed with paraformaldehyde, embedded, sectioned, and subjected to H&E staining, Sirius Red staining, and α-SMA IHC staining. Quantification of Sirius Red staining and α-SMA IHC staining was performed using ImageJ software. Scale bar = 200 μm (H&E staining and Sirius Red staining) and 50 μm (α-SMA IHC staining). g RT-qPCR analysis of the expression of fibrinolytic genes, including Mmp12 and Mmp13, in liver tissues from the CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, CCl₄ + TNC-CAR-Ms, and CCl₄ + TNC-CAR-Ms + IL-4 cell infusion groups. h TNC protein expression in activated HSCs after TNC-CAR-Ms and TNC-CAR-Ms + IL-4 treatment was detected by Western blotting and quantitatively analyzed using Quantity One software. Data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns non-significant. CD80 cluster of differentiation 80, APC allophycocyanin, PE phycoerythrin, Mrc1 mannose receptor C-type 1, Il10 interleukin-10, Arg1 arginase 1, IL-4 interleukin-4, CD206 cluster of differentiation 206, EGFP enhanced green fluorescent protein, Mmp12 matrix metallopeptidase 12, Mmp13 matrix metallopeptidase 13, GAPDH glyceraldehyde-3-phosphate dehydrogenase, HSCs hepatic stellate cells, RT-qPCR reverse transcription quantitative polymerase chain reaction, α-SMA α-smooth muscle actin, IHC immunohistochemistry, TNC tenascin-C, CAR-Ms chimeric antigen receptor-macrophages

Fig. 7

TNC-CAR-Ms exerted antifibrotic effects in a CD8⁺ T cell-dependent manner. a The relative proportions of CD3⁺CD4⁺ and CD3⁺CD8⁺ cells in liver tissues from WT, CCl₄ + PBS, CCl₄ + Mock-CAR-Ms, and CCl₄ + TNC-CAR-Ms mice were determined by flow cytometry and quantitatively compared (n = 3). b Experimental illustration of the effect of CD8α antibody blockade on the antifibrotic effect of TNC-CAR-Ms on mice. The CCl₄-treated mice received monoclonal CD8α antibody (anti-CD8α) or isotype control IgG antibody (anti-Iso) treatment every 3 d (i.p., 1 mg/kg), starting 1 d before the PBS and TNC-CAR-M (i.v., 2 × 10⁶ cells/mouse) infusions for 2 weeks (n = 6). The 12 × refers to the repeated injections of CCl₄ for 12 times. P56 and P98 indicate the mice’s age in postnatal days (day 56 and day 98, respectively). c The relative proportions of CD3⁺CD8⁺ cells in the spleens of CCl₄ + anti-Iso + PBS, CCl₄ + anti-CD8α + PBS, CCl₄ + anti-Iso + TNC-CAR-Ms, and CCl₄ + anti-CD8α + TNC-CAR-Ms mice were determined by flow cytometry and quantitatively compared (n = 3). d Liver tissues from CCl₄ + anti-Iso + PBS, CCl₄ + anti-CD8α + PBS, CCl₄ + anti-Iso + TNC-CAR-Ms, and CCl₄ + anti-CD8α + TNC-CAR-M mice were fixed with paraformaldehyde, embedded, sectioned, and subjected to H&E staining, Sirius Red staining, and α-SMA IHC staining. Quantification of Sirius Red staining and α-SMA IHC staining was performed using ImageJ software. Scale bar = 200 μm (H&E staining and Sirius Red staining) and 50 μm (α-SMA IHC staining). e The relative proportions of CD3⁺CD8⁺ and CD69⁺ cells from the cocultured cell system, which consisted of CD3⁺ cells, HSCs, and CAR-Ms, were detected by flow cytometry and quantitatively compared (n = 3). Data are presented as mean ± SD. ^**P < 0.01, ^***P < 0.001, ns non-significant. CD3 cluster of differentiation 3, CD4 cluster of differentiation 4, CD8 cluster of differentiation 8, Iso isotype control, APC allophycocyanin, PE phycoerythrin, H&E hematoxylin and eosin, α-SMA α-smooth muscle actin, HSCs hepatic stellate cells, CD69 cluster of differentiation 69, SSC-A side scatter area, IHC immunohistochemistry, TNC tenascin-C, CAR-Ms chimeric antigen receptor-macrophages

Fig. 8

Diagram of the proposed mechanism of TNC-engineered CAR-Ms. At the cellular level, on one hand, the TNC-CAR-Ms harboring TNC scFv specifically recognize the TNC protein on the surface of HSCs to engulf activated HSCs, thereby inhibiting HSC activation and ECM deposition. On the other hand, TNC-CAR-Ms can also indirectly inhibit HSC activation and ECM deposition by recruiting CD8⁺ T cells and M2-polarized macrophages from the liver immune microenvironment. At the molecular level, TNC-CAR-Ms treatment can effectively prevent the activity of the TLR4/NF-κB inflammatory signaling pathway to reduce the expression of inflammatory factors IL-1β and TNF-α, and simultaneously inhibit Integrin-mediated FAK activation to prevent the fibrotic process. scFv single-chain fragment variable, CD8 cluster of differentiation 8, HSC hepatic stellate cell, ECM extracellular matrix, TLR4 Toll-like receptor 4, NF-κB nuclear factor kappa-B, FAK focal adhesion kinase, IL-1β interleukin-1β, TNF-α tumor necrosis factor-α, TNC tenascin-C, CAR-Ms chimeric antigen receptor-macrophages