Abstract
Activation of TLR by ssRNA after FcgammaR-mediated phagocytosis of immune complexes (IC) may be relevant in autoimmune-associated congenital heart block (CHB) where the obligate factor is a maternal anti-SSA/Ro Ab and the fetal factors, protein/RNA on an apoptotic cardiocyte and infiltrating macrophages. This study addressed the hypothesis that Ro60-associated ssRNAs link macrophage activation to fibrosis via TLR engagement. Both macrophage transfection with noncoding ssRNA that bind Ro60 and an IC generated by incubation of Ro60-ssRNA with an IgG fraction from a CHB mother or affinity purified anti-Ro60 significantly increased TNF-alpha secretion, an effect not observed using control RNAs or normal IgG. Dependence on TLR was supported by the significant inhibition of TNF-alpha release by IRS661 and chloroquine. The requirement for FcgammaRIIIa-mediated delivery was provided by inhibition with an anti-CD16a Ab. Fibrosis markers were noticeably increased in fetal cardiac fibroblasts after incubation with supernatants generated from macrophages transfected with ssRNA or incubated with the IC. Supernatants generated from macrophages with ssRNA in the presence of IRS661 or chloroquine did not cause fibrosis. In a CHB heart, but not a healthy heart, TLR7 immunostaining was localized to a region near the atrioventricular groove at a site enriched in mononuclear cells and fibrosis. These data support a novel injury model in CHB, whereby endogenous ligand, Ro60-associated ssRNA, forges a nexus between TLR ligation and fibrosis instigated by binding of anti-Ro Abs to the target protein likely accessible via apoptosis.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Antibodies, Antinuclear / biosynthesis
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Antibodies, Antinuclear / genetics
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Antibodies, Antinuclear / metabolism*
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Autoantigens / genetics
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Autoantigens / immunology
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Autoantigens / metabolism*
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Binding Sites, Antibody / genetics
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Cells, Cultured
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Coculture Techniques
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Female
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Fetal Diseases / immunology*
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Fetal Diseases / metabolism
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Fetal Diseases / pathology
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Fibroblasts / immunology
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Fibroblasts / metabolism
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Fibroblasts / pathology
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Fibrosis
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Heart Block / congenital
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Heart Block / immunology*
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Heart Block / pathology
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Humans
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Inflammation Mediators / immunology
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Inflammation Mediators / metabolism*
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Ligands
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Maternal-Fetal Exchange / genetics
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Maternal-Fetal Exchange / immunology
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Myocytes, Cardiac / immunology*
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Myocytes, Cardiac / metabolism
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Myocytes, Cardiac / pathology
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Pregnancy
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RNA, Small Cytoplasmic / genetics
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RNA, Small Cytoplasmic / immunology
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RNA, Small Cytoplasmic / metabolism*
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / immunology
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RNA-Binding Proteins / metabolism*
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Ribonucleoproteins / genetics
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Ribonucleoproteins / immunology
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Ribonucleoproteins / metabolism*
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Signal Transduction / genetics
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Signal Transduction / immunology
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Toll-Like Receptor 7 / antagonists & inhibitors
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Toll-Like Receptor 7 / metabolism
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Toll-Like Receptor 8 / antagonists & inhibitors
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Toll-Like Receptor 8 / metabolism
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Toll-Like Receptors / antagonists & inhibitors
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Toll-Like Receptors / metabolism*
Substances
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Antibodies, Antinuclear
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Autoantigens
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Inflammation Mediators
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Ligands
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RNA, Small Cytoplasmic
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RNA-Binding Proteins
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RO60 protein, human
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Ribonucleoproteins
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SS-A antibodies
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TLR7 protein, human
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TLR8 protein, human
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Toll-Like Receptor 7
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Toll-Like Receptor 8
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Toll-Like Receptors