A novel role of endothelin-1 in linking Toll-like receptor 7-mediated inflammation to fibrosis in congenital heart block

Abstract

Autoimmune associated congenital heart block (CHB) may result from pathogenic cross-talk between inflammatory and profibrosing pathways. Incubation of macrophages with immune complexes (IC) composed of Ro60, a target of the pathologic maternal autoantibodies necessary for CHB, hY3 ssRNA, and affinity-purified anti-Ro60 antibody induces the Toll-like receptor 7 (TLR7)-dependent generation of supernatants that provoke a fibrosing phenotype in human fetal cardiac fibroblasts. We show herein that these cells are a major source of TGFβ and that endothelin-1 (ET-1) is one of the key components responsible for the profibrosing effects generated by stimulated macrophages. Supernatants from macrophages incubated with IC induced the fibroblast secretion of TGFβ, which was inhibited by treating the macrophages with an antagonist of TLR7. Under the same conditions, the induced fibroblast secretion of TGFβ was decreased by inhibitors of the ET-1 receptors ETa or ETb or by an anti-ET-1 antibody but not by an isotype control. Exogenous ET-1 induced a profibrosing phenotype, whereas fibroblasts transfected with either ETa or ETb siRNA were unresponsive to the profibrosing effects of the IC-generated macrophage supernatants. Immunohistochemistry of the hearts from two fetuses dying with CHB revealed the presence of ET-1-producing mononuclear cells in the septal region in areas of calcification and fibrosis. In conclusion, these data support a novel role of ET-1 in linking TLR7 inflammatory signaling to subsequent fibrosis and provide new insight in considering therapeutics for CHB.

Figures

FIGURE 1.

Macrophage supernatants generated following TLR7 ligation induce TGFβ secretion by fibroblasts dependent on ET-1, ETa, and ETb. A, additions to cultured human fetal cardiac fibroblasts (5 × 104 cells) included supernatants obtained from macrophages transfected with hY3 in the presence and absence of IRS661 (32 ng/ml) or treated with IC composed of affinity-purified anti-Ro60 antibody, native Ro60, and hY3, in the presence and absence of IRS661. After 16 h, TGFβ levels (pg/ml) were determined from fibroblast supernatants by ELISA. Statistical significances (p) are indicated (n = 4 for each condition). B, human fetal cardiac fibroblasts (5 × 104 cells) were incubated with supernatants generated from macrophages transfected with hY3 or treated with IC as described in A, in the absence or presence of BQ-123 (100 nm), BQ-788 (100 nm), anti-ET-1 antibody (10 μg/ml), or normal rabbit IgG (isotype control, 10 μg/ml). After 16 h, TGFβ levels (pg/ml) were determined from the fibroblast supernatants by ELISA. Statistical significances (p) are indicated (n = 4 for each condition). Error bars, S.E.

FIGURE 2.

Exogenous ET-1 induces a profibrosing phenotype in cultured human fetal cardiac fibroblasts. A and B, TGFβ (pg/ml) (A) and collagen (ng/ml) (B) were measured in the supernatants generated from human fetal cardiac fibroblasts incubated with ET-1 (100 nm) alone or in the presence of BQ-123 (100 nm), BQ-788 (100 nm), anti-ET-1 antibody (10 μg/ml), or normal rabbit IgG (isotype control; 10 μg/ml). ET-1 was also incubated with anti-TGFβ antibody (10 μg/ml) in the collagen assay. TGFβ was measured 16 h postincubation. Soluble collagen was measured after 16 h by the soluble collagen binding assay (C). In parallel, human fetal cardiac fibroblasts (5 × 104 cells) cultured under identical conditions were fixed, permeabilized, and incubated with anti-α-SMA antibody, anti-Col1A antibody (to assess collagen), or normal mouse IgG (isotype control) for immunofluorescence analysis; red color indicates the presence of α-SMA (left column) or collagen (middle column). Images (×20 magnification) are representative of three experiments. Bar, 40 μm. Error bars, S.E.

FIGURE 3.

Supernatants from IC-stimulated macrophages do not induce a profibrosing phenotype in ETa or ETb siRNA-transfected fibroblasts. A and B, human fetal cardiac fibroblasts (5 × 104 cells) were transfected with siRNA targeting either ETa or ETb or control scrambled siRNA. After 72 h, expression levels of ETa and ETb were verified by qRT-PCR. siRNA-treated and control cells were incubated with supernatants from untreated or IC-incubated macrophages (as described in the legend to Fig. 1). After 16 h, collagen released (ng/ml) into the fibroblast supernatants was measured (A), and α-SMA production was analyzed by immunofluorescence (B). Images (×40 magnification) are representative of three experiments. Error bars, S.E.

FIGURE 4.

TLR7 ligation increases macrophage EDN1 gene mRNA expression and protein expression and secretion of ET-1. A and B, macrophages (5 × 105 cells) were transfected with hY3 or incubated with IC (as described in the legend to Fig. 1) for 16 h in the absence or presence of IRS661. Transfection with hY3 A/U was evaluated as a negative control. The mRNA expression of EDN1 relative to GAPDH was measured by qRT-PCR after isolation of total mRNA (A). Macrophages (5 × 105 cells) were transfected with hY3 for 16 h in the absence or presence of IRS661 (B). The next day cells were fixed, stained with mouse monoclonal ET-1 antibody, and analyzed by fluorescence microscopy (original magnification, ×40). Results are representative of three experiments. Supernatants were evaluated for ET-1 (pg/ml) by ELISA (C). Error bars, S.E.

FIGURE 5.

ET-1 expression in fetal heart. Sections from the septal region of a 40-week fetus (CHB1) dying with CHB, a 29-week fetus dying with CHB (CHB2), and a 24-week non-CHB fetus (control) were stained with anti-ET-1/2/3 antibody or normal rabbit IgG (isotype control for anti-ET-1/2/3 antibody). Stains were visualized using anti-rabbit IgG peroxidase (brown) and counterstained with hematoxylin. Locations of mononuclear cells in CHB hearts and endothelial cells (lining the vessel wall) are indicated by white and black arrows, respectively. Multinucleated giant cells are indicated by red arrows. Anti-ET-1/2/3 strongly stained mononuclear cells in both CHB hearts. Anti-ET-1/2/3 antibody also stained endothelial cells in both CHB hearts and the non-CHB heart. Bar, 100 μm.

FIGURE 6.

Schematic representation of the proposed role of ET-1 as one of the profibrosing factors linking TLR7 inflammatory signaling to fibrosis. Binding and uptake of IC composed of the maternal anti-Ro60 antibody, hY3 ssRNA, and the antigen Ro60 by FcγR present on macrophages induces TLR7-dependent release of ET-1 as one of the key profibrotic factors of the inflammatory signaling cascade. ET-1 subsequently induces the resident cardiac fibroblasts to release TGFβ and promote its transdifferentiation and subsequent scarring.