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 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.

FIGURE 1

Stimulation of macrophages by hY3 and mpre-5S RNAs is TLR-dependent. A, IFN-γ–primed macrophages express TLR7 and TLR8 (FACS, RT-PCR). B, TNF-α was measured in the supernatants generated from human primed macrophages transfected with hY3, mpre-5S RNA or SSRNA41 under varied conditions. Treatments include coincubations in the presence or absence of IRS661, chloroquine, RNase as well as alkaline phosphatase. Bars represent means ± SEM.

FIGURE 2

Stimulation of macrophages by ICs composed of Ro60-associated ssRNA is TLR- and FcγRIIIa-dependent. In A, TNF-α was measured in the supernatants generated from human macrophages incubated with native Ro60 in complex with hY3 or hY3 A/U, and CHB IgG or nIgG. Treatments included coincubations in the presence or absence of chloroquine or IRS661 or anti-CD16a or an isotype Ab (control). B, TNF-α was measured in supernatants generated from human macrophages incubated with native Ro60 in complex with hY3 and affinity purified anti-Ro60 (anti-Ro60 IgG) or monoclonal anti-Ro (anti-Ro60 ScFv). Treatments included coincubations with chloroquine, IRS661, RNase, or RNase plus RNase inhibitor. Bars represent means ± SEM.

FIGURE 3

Transdifferentiation of human fetal cardiac fibroblasts exposed to supernatants from macrophages transfected with hY3 ssRNA is TLR-dependent. Human fetal cardiac fibroblasts were prepared as monolayers. Cells were incubated with supernatants of macrophages diluted 1:1 with fibroblast medium. The supernatants were generated from human macrophages transfected with hY3, m-pre5S RNA or ssRNA41, or hY3 in the presence of IRS661 or chloroquine (shown in Fig. 1). Fibroblasts were then stained with Hoechst and anti–SMAc-Cy3, and analyzed by fluorescence microscopy (original magnifications ×10 and ×40). Results are representative of three experiments.

FIGURE 4

Transdifferentiation of human fetal cardiac fibroblasts exposed to supernatants from macrophages incubated with ICs composed of Ro60-associated ssRNA is TLR- and FcγRIIIa-dependent. Human fetal cardiac fibroblasts were prepared as monolayers. Cells were incubated with supernatants of macrophages diluted 1:1 with fibroblast media. The supernatants were generated from human macrophages incubated with a complex of native Ro60 plus hY3 or hY3 A/U and CHB IgG, and Ro hY3 CHB IgG with or without IRS661, chloroquine and anti-CD16a (as shown in Fig. 2). Fibroblasts were then stained with Hoechst and anti–SMAc-Cy3, and analyzed by fluorescence microscopy (original magnification ×10 and ×40). Results are representative of three experiments.

FIGURE 5

Collagen secretion by human fetal cardiac fibroblasts exposed to supernatants from macrophages transfected with hY3 ssRNA is TLR-dependent. Human fetal cardiac fibroblasts were prepared as monolayers and treated using conditions described in Fig. 3. Cells were plated into four-chamber slides (24 h) and serum starved (24 h). A, Conditions match those in Fig. 3. B, To examine the effect of neutralizing Abs on transdifferentiation of cardiac fibroblasts, cells were incubated with supernatants in the absence or presence of a TNF-α–neutralizing Ab (1 µg/ml) or a TGF-β–neutralizing Ab (1 µg/ml) for 24 h. After the 24-h incubation, supernatants of fibroblast cultures were retrieved and total collagen was detected by the Sircol assay. Results are representative of three experiments. Bars represent means ± SEM.

FIGURE 6

Collagen secretion by human fetal cardiac fibroblasts exposed to supernatants from macrophages incubated with ICs composed of Ro60-associated ssRNA is FcγRIIIa-dependent. A, Human fetal cardiac fibroblasts were prepared as monolayers and treated using conditions described in Fig. 4. Cells were plated into four-chamber slides (24 h) and serum starved (24 h). B, Cells were incubated with supernatants in the absence or presence of a TNF-α–neutralizing Ab (1 µg/ml) or a TGF-β–neutralizing Ab (1 µg/ml) for 24 h. After the 24-h incubation, supernatants of fibroblast cultures were retrieved and total collagen was detected by the Sircol assay. Results are representative of three experiments. Bars represent means ± SEM.

FIGURE 7

TLR7 infiltrating mononuclear cells in heart tissue obtained from a fetus dying with CHB. Sections from the septal region of a 22-wk fetus with CHB and an age-matched control electively terminated were stained with anti-TLR7, rabbit IgG (isotype control for anti-TLR7), anti-CD45, or mouse IgG (isotype control for anti-CD45) as primary Ab. Stains are visualized using anti-rabbit IgG alkaline phosphatase (brown) or anti-mouse IgG peroxidase (red; original magnification ×40).