Tenascin-C aggravates autoimmune myocarditis via dendritic cell activation and Th17 cell differentiation

Abstract

Background: Tenascin-C (TN-C), an extracellular matrix glycoprotein, appears at several important steps of cardiac development in the embryo, but is sparse in the normal adult heart. TN-C re-expresses under pathological conditions including myocarditis, and is closely associated with tissue injury and inflammation in both experimental and clinical settings. However, the pathophysiological role of TN-C in the development of myocarditis is not clear. We examined how TN-C affects the initiation of experimental autoimmune myocarditis, immunologically.

Methods and results: A model of experimental autoimmune myocarditis was established in BALB/c mice by immunization with murine α-myosin heavy chains. We found that TN-C knockout mice were protected from severe myocarditis compared to wild-type mice. TN-C induced synthesis of proinflammatory cytokines, including interleukin (IL)-6, in dendritic cells via activation of a Toll-like receptor 4, which led to T-helper (Th)17 cell differentiation and exacerbated the myocardial inflammation. In the transfer experiment, dendritic cells loaded with cardiac myosin peptide acquired the functional capacity to induce myocarditis when stimulated with TN-C; however, TN-C-stimulated dendritic cells generated from Toll-like receptor 4 knockout mice did not induce myocarditis in recipients.

Conclusions: Our results demonstrated that TN-C aggravates autoimmune myocarditis by driving the dendritic cell activation and Th17 differentiation via Toll-like receptor 4. The blockade of Toll-like receptor 4-mediated signaling to inhibit the proinflammatory effects of TN-C could be a promising therapeutic strategy against autoimmune myocarditis.

Keywords: TLR4; Th17; dendritic cell; myocarditis; tenascin‐C.

Figure 1.

Tenascin‐C (TN‐C) expression in cardiac myosin‐induced autoimmune myocarditis. BALB/c mice were immunized twice, on days 0 and 7, with 100 μg of cardiac myosin epitope peptide (MyHC‐α). A, Representative histology of myocarditis on days 5, 14, and 25, respectively. Hematoxylin and eosin staining (i) and immunostaining for TN‐C (ii). Scale bars=50 μm. B, The expression of TN‐C in hearts obtained from immunized mice at the indicated time points. Immunization on days 0 and 7 are indicated with red arrows. TN‐C mRNA expression was evaluated by quantitative reverse transcription–polymerase chain reaction. The results are reported as the fold change in the gene expression relative to the expression on day 0. The TN‐C protein levels were measured by an ELISA. n=4 per group at each time point. Error bars represent the mean±SEM. *P<0.05 vs day 0. CFA indicates complete Freund's adjuvant.

Figure 2.

Tenascin‐C (TN‐C) deficiency inhibits inflammation in the heart. Wild‐type (WT) and TN‐C knockout (TNKO) mice were immunized with cardiac myosin peptide on days 0 and 7. A, Representative hematoxylin and eosin–stained sections of hearts on day 14 from WT and TNKO mice. Scale bar=100 μm. B, Severity of myocarditis in the heart sections. C, Heart‐to‐body‐weight ratios (HW/BW) in WT and TNKO mice before and after experimental autoimmune myocarditis (EAM) induction (day 14). D, Circulating troponin I (TnI) concentration of WT and TNKO mice before and after EAM induction (day 14). E through G, Inflammatory cells infiltrating the heart were isolated and analyzed by flow cytometry of WT and TNKO mice before and after EAM induction (on day 14). E, Absolute number of CD45⁺ cells, frequency of CD45⁺ cells within live cells, and representative plots are shown. F, Absolute number of CD4⁺ cells, frequency of CD4⁺ cells within CD45⁺ cells, and representative plots are shown. Representative plots (gated on CD4⁺ T cells) and the frequency of both the interferon (IFN)‐γ⁺ (Th1), interleukin (IL)‐17⁺ (Th17), and Foxp3⁺ (regulatory T‐cell) cells among all CD4⁺ cells (G) are shown. n=5 to 8 per group (B through G). H, Splenocytes in naïve WT and TNKO mice were isolated and analyzed by a flow cytometric analysis. Representative plots (gated on CD4⁺ T cells) and the frequency of the IFN‐γ⁺ (Th1), IL‐17⁺ (Th17), and Foxp3⁺ cells among all CD4⁺ cells is shown. n=4 per group. The bar graphs show the group mean±SEM. The results of 1 of 2 representative experiments are shown. *P<0.01, **P<0.05. Foxp indicates Forkhead box protein; ND, not detected; SSC, side scatter.

Figure 3.

Effects of tenascin‐C (TN‐C) deficiency on the hemodynamic parameters in experimental autoimmune myocarditis (EAM) mice. A, Heart rate; (B) Left ventricular (LV) systolic pressure (LVSP); (C) LV end‐diastolic pressure (LVEDP); (D) Maximal rate of the increase in the LV pressure (+dP/dt); and (E), Maximal rate of the decrease in the LV pressure (−dP/dt). Naïve or EAM wild‐type (WT) and TN‐C knockout (TNKO) mice (day 14) were analyzed. n=5 to 7 per group. Bar graphs show the group mean±SEM. *P<0.05.

Figure 4.

Tenascin‐C (TN‐C) deficiency affected the cytokine milieu in the heart. Cytokine and chemokine secretion in homogenized hearts obtained from naïve and experimental autoimmune myocarditis (EAM) (on day 14) wild‐type (WT) and TN‐C knockout (TNKO) mice was assessed by an ELISA. n=4 to 5 per group. The bar graphs show the group mean±SEM. The results of 1 of 2 representative experiments are shown. *P<0.05, **P<0.01. IL indicates interleukin; IP, IFN‐γ‐induced protein; KC, keratinocyte chemoattractant; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; ND, not detected; RANTES, regulated on activation, normal T‐cell expressed and secreted; TGF, transforming growth factor; TNF, tumor necrosis factor.

Figure 5.

Tenascin‐C (TN‐C) stimulated production of proinflammatory cytokines and chemokines by bone marrow (BM)–derived dendritic cells (DCs) and differentiated naïve CD4⁺ cells into Th17 cells. A, DCs generated from BM (BMDCs) were cultured in the presence or absence of lipopolysaccharide (LPS) 1 μg/mL for 72 hours. TN‐C secretions from BMDCs and the TN‐C concentration in the medium were measured by an ELISA. B, BMDCs were cultured in the presence of 10 μg/mL of TN‐C for 48 hours, and the supernatants were subjected to multiplex immunoassay panels for the production of proinflammatory cytokines, chemokines, and growth factors. C, BMDCs were cultured in the presence of the indicated dose of TN‐C for 48 hours. TN‐C‐dose‐dependent IL‐6 secretions from BMDCs were measured by an ELISA. D, CD62^high naïve CD4⁺ T cells were cultured with DCs, which were obtained from the spleen, in the presence of anti‐CD3 mAb (1 μg/mL), TGF‐β (2 ng/mL), and TN‐C (10 μg/mL) for 72 hours. In some wells, anti‐IL‐6 Ab (10 μg/mL) was added. The secretion of IL‐17 in the supernatants was analyzed by an ELISA. The values are expressed as means±SEM of triplicate culture wells. The results of 1 of 2 representative experiments are shown. *P<0.05, **P<0.01 (compared to no TN‐C stimulation). GM‐CSF indicates granulocyte/macrophage colony‐stimulating factor; IFN, interferon; IL, interleukin; IP, IFN‐γ‐induced protein; KC, keratinocyte chemoattractant; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NS, not significant; RANTES, regulated on activation, normal T‐cell expressed and secreted; TGF, transforming growth factor; TNF, tumor necrosis factor.

Figure 6.

Blocking of toll‐like receptor (TLR) 4‐mediated tenascin‐C (TN‐C) signaling reduced the IL‐6 secretion and Th17 generation. A, Bone marrow‐derived dendritic cells (BMDCs) generated from TLR4 knockout mice were cultured in the presence of 10 μg/mL of TN‐C for 48 hours. The supernatants were subjected to an ELISA analysis for the production of proinflammatory cytokines and chemokines. B, BMDCs generated from wild‐type mice were cultured in the presence of 10 μg/mL of TN‐C for 48 hours. In some wells, TLR4 inhibitor TAK242 (0.1 μmol/L) was added. The supernatants were subjected to an ELISA analysis for the production of proinflammatory cytokines and chemokines. C, CD62^high naïve CD4⁺ T cells were cocultured with BMDCs generated from TLR4 knockout mice in the presence of anti‐CD3 mAb (1 μg/mL), TGF‐β (2 ng/mL), and full TN‐C (10 μg/mL) for 72 hours. The secretion of IL‐17 in the supernatants was analyzed by an ELISA. D, CD62^high naïve CD4⁺ T cells were co‐cultured with DCs, which were obtained from the spleen, in the presence of anti‐CD3 mAb (1 μg/mL), TGF‐β (2 ng/mL), and TN‐C (10 μg/mL) for 72 hours. In some wells, TLR4 inhibitor TAK242 (0.1 μmol/L) was added. The secretion of IL‐17 in the supernatants was analyzed by an ELISA. The values are expressed as means±SEM of triplicate culture wells. The results of 1 of 2 representative experiments are shown. *P<0.05, **P<0.01. IFN indicates interferon; IL, interleukin; IP, IFN‐γ‐induced protein; KC, keratinocyte chemoattractant; MIP, macrophage inflammatory protein; NS, not significant; RANTES, regulated on activation, normal T‐cell expressed and secreted; TGF, transforming growth factor.

Figure 7.

Tenascin‐C (TN‐C) deficiency does not affect Toll‐like receptor (TLR) 4 expression and NF‐ĸB signaling. A, Western blot of TLR4 expression in naïve wild‐type (WT) and TN‐C knockout (TNKO) DCs left untreated or 15 minutes after stimulation with TN‐C (10 μg/mL). B, Western blot of phosphorylation of NF‐ĸB p65 at Ser 536 and NF‐ĸB p65 in naïve WT and TNKO DCs 15 minutes after stimulation with TN‐C (10 μg/mL) or TNF‐α (20 ng/mL). DCs indicates dendritic cells; TNF, tumor necrosis factor.

Figure 8.

Transfer of myosin‐specific bone marrow–derived dendritic cells (BMDCs) generated from Toll‐like receptor 4 knockout (TLR4‐KO) mice reduced the myocardial inflammation. BMDCs generated from wild‐type (WT) or TLR4KO mice were pulsed overnight with 10 μg/mL MyHC‐α peptide and stimulated for another 4 hours with 10 μg/mL TN‐C and 5 μg/mL anti‐CD40L. Recipient mice received 5×10⁵ pulsed and activated WT‐BMDCs or TLR4KO‐BMDCs i.p. on days 0, 2, and 4 and were killed 10 days after the first injection. A, Representative histology (hematoxylin and eosin staining) of the myocarditis on day 10 after the DC transfer. Left image, WT‐BMDCs transferred myocarditis, right image, TLR4KO‐BMDCs transferred myocarditis. Scale bars=100 μm. B, Severity of myocarditis on day 10 after DC transfer. C, Heart‐to‐body‐weight ratios (HW/BW) on day 10 after DC transfer. D, IL‐6 secretion in the homogenized hearts 10 days after BMDC transfer was assessed by an ELISA. The bar graphs show the group mean±SEM. n=7 per group. *P<0.01. IL indicates interleukin; MyHC, myosin H‐chain peptide; ND, not detected; TN‐C, tenascin‐C.

Figure 9.

Schematic illustration showing how tenascin‐C (TN‐C)‐stimulated dendritic cells (DCs) induce myocarditis. TN‐C aggravates myocardial inflammation by stimulation of myosin‐loaded DCs via Toll‐like receptor (TLR)‐4‐mediated signaling. DCs stimulated by TN‐C produce cytokines and growth factors (IL‐6, IL‐1, and GM‐CSF) that contribute to the generation of cardiac myosin epitope peptide (MyHC)‐α‐specific Th17 cells. Chemokines secreted by TN‐C‐stimulated DCs help to accumulate inflammatory cells into the inflamed heart. GM‐CSF indicates granulocyte/macrophage colony‐stimulating factor; IL, interleukin; IP, IFN‐γ‐induced protein; KC, keratinocyte chemoattractant; MHC, major histocompatibility complex; MIP, macrophage inflammatory protein; RANTES, regulated on activation, normal T‐cell expressed and secreted; TCR, T‐cell receptor.