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 TGF␤ 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 TGF␤, which was inhibited by treating the macrophages with an antagonist of TLR7. Under the same conditions, the induced fibroblast secretion of TGF␤ 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.Cardiac conduction defects detected before or at birth, in the absence of structural abnormalities, are strongly associated with maternal autoantibodies to SSA/Ro and/or SSB/La ribonucleoproteins, independent of whether the mother has systemic lupus erythematosus or Sjögren's syndrome or is asymptomatic (1, 2). The mechanism by which maternal anti-SSA/ Ro-SSB/La antibodies initiate and perpetuate inflammation with consequent scarring of the atrioventricular node (the signature lesion of CHB) 2 and endocardium (3) has not been fully defined. One proposed pathologic cascade centers apoptosis of the fetal cardiocytes as the cellular event that accounts for the surface expression of the otherwise intracellular target antigens (4 -6), facilitating subsequent opsonization by circulating maternal autoantibodies. Experimental data suggest that hY3 ssRNA associated with the SSA/Ro protein bound by affinitypurified anti-Ro60 antibody (AP60) gains access to the macrophage endosome via Fc␥R uptake with subsequent ligation of the Toll-like receptor 7 (TLR7). In clearing the opsonized apoptotic cardiomyocytes, infiltrating macrophages secrete factors that promote a scarring phenotype of the resident cardiac fibroblasts, as evidenced by their transdifferentiation to myofibroblasts and production of collagen (7,8).Immunohistochemical studies (5) of hearts from several fetuses dying with CHB demonstrate abundant TGF␤ in the septal region. TGF␤ probably contributes to s...

Endothelin-1 overexpression exacerbate experimental allergic encephalomyelitis

“…As mentioned, plasma levels of ET-1 are elevated in several autoimmune diseases (Miyasaka et al, 1992), ET-1 stimulates release of proinflammatory cytokines in human monocytes/macrophages (Cunningham et al, 1997) and ET-1 mediated macrophage-dependent inflammation leads to vascular injury (Javeshghani et al, 2013). In addition, ET-1 plays an important role in linking toll-like receptor 7 (TLR7) mediated inflammation to fibrosis in autoimmune associated congenital heart block by inducing cardiac fibroblast to secrete transforming growth factor-beta (TGF-β) which leads to cardiac fibrosis (Alvarez et al, 2011). In this study, pathogenic maternal autoantibodies (anti-Ro antibody) formed immune complex (IC) with autoantigen (Ro), and incubation of macrophages with IC induced TLR7-dependent generation of ET-1 which induced TGF-β secretion from fibroblasts, leading to fibrosis (Alvarez et al, 2011).…”

“…In addition, ET-1 plays an important role in linking toll-like receptor 7 (TLR7) mediated inflammation to fibrosis in autoimmune associated congenital heart block by inducing cardiac fibroblast to secrete transforming growth factor-beta (TGF-β) which leads to cardiac fibrosis (Alvarez et al, 2011). In this study, pathogenic maternal autoantibodies (anti-Ro antibody) formed immune complex (IC) with autoantigen (Ro), and incubation of macrophages with IC induced TLR7-dependent generation of ET-1 which induced TGF-β secretion from fibroblasts, leading to fibrosis (Alvarez et al, 2011). Importantly, intrathecal administration of ET-1 receptor antagonist ameliorated EAE severity in EAE rats (Shin et al, 2001).…”

Toll-like receptor-mediated, enhanced production of profibrotic TIMP-1 in monocytes from patients with systemic sclerosis: role of serum factors

“…This IC uptake leads to TLR7/8 activation with subsequent release of proinflamatory and profibrotic factors by infiltrating macrophages in cardiac fibrosis, or pDCs in SSc 13 21 34. Furthermore, the presence of ICs recognising self-RNA is responsible for disease manifestations found in patients and animal models of other autoimmune diseases, including of SLE and Sjögren's syndrome 35 36.…”

Complement Receptor 3 Influences Toll-like Receptor 7/8-Dependent Inflammation

“…The potential of SSA/Ro-associated hY3 RNA to perpetuate an inflammatory step via a TLR7/8 pathway has been experimentally substantiated by the transdifferentiation of human fetal cardiac fibroblasts to a scarring phenotype following exposure to supernatants generated by incubation of macrophages with surrogate immune complexes comprised of SSA/Ro, hY3, and affinity-purified anti-SSA/Ro antibody (4,21). Consistent with the observation that chloroquine, an inhibitor of endosomal acidification, decreased the RNA-induced TLR signaling, two retrospective studies suggest that use of hydroxychloroquine may reduce the frequency of congenital heart block in anti-SSA/Ro-exposed mothers (23,24).…”

“…Monocytes (CD14-positive cells) were isolated using anti-CD14 microbeads (Miltenyi Biotech) and cultured in Teflon beakers (RPMI 1640/10% FBS) for 7 days in the presence of 10 ng/ml GM-CSF to obtain macrophages (4,21). For in vitro assays, monocyte-derived macrophages (4 ϫ 10 5 /ml) were plated in growth medium and incubated 37°C for 48 h. Neutrophils were obtained from a healthy donor using an isolate from dextran sedimentation and Ficoll-Hypaque separation.…”