Ro60-Associated Single-Stranded RNA Links Inflammation with Fetal Cardiac Fibrosis via Ligation of TLRs: A Novel Pathway to Autoimmune-Associated Heart Block

Abstract

Activation of TLR by ssRNA after FcγR-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-α secretion, an effect not observed using control RNAs or normal IgG. Dependence on TLR was supported by the significant inhibition of TNF-α release by IRS661 and chloroquine. The requirement for FcγRIIIa-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.

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).…”

“…Human Y3 RNA was prepared from hY3 plasmids, kindly provided by Dr. Sandra Wolin (Yale University School of Medicine), as described (4,17). Single-stranded oligoribonucleotides that specifically activate TLR7 or TLR8 and a modified oligodeoxynucleotide that inhibits TLR7/8/9 were synthesized and purified at Idera Pharmaceuticals as described previously (18 -20).…”

“…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.…”

“…This pathologic process is facilitated by circulating autoantibodies against nucleic antigens such as nucleosomes, histones, DNA, and ribonucleoproteins, a hallmark of disease (1)(2)(3). Autoantibody-dependent, Fc␥R-mediated uptake permits nucleic acids to activate dendritic cells and macrophages through the ligation of endosomal Toll-like receptors (TLR) (1,4). For example, the SSA/Ro autoantigen is an RNA binding protein targeted by 40 -60% of patients with lupus (5), and, together as a complex with hY3 RNA, it has been shown to activate TLR7/8 in macrophages (4,6).…”

“…Autoantibody-dependent, Fc␥R-mediated uptake permits nucleic acids to activate dendritic cells and macrophages through the ligation of endosomal Toll-like receptors (TLR) (1,4). For example, the SSA/Ro autoantigen is an RNA binding protein targeted by 40 -60% of patients with lupus (5), and, together as a complex with hY3 RNA, it has been shown to activate TLR7/8 in macrophages (4,6). One of the strongest clinical associations with reactivity to this ribonucleoprotein is observed in mothers of children with neonatal lupus, a fetal/neonatal disease that may share in common with SLE the pathologic activation of effector cells by nucleic acids.…”