Cardiac fibroblast transcriptome analyses support a role for interferogenic, profibrotic, and inflammatory genes in anti-SSA/Ro-associated congenital heart block

Abstract

The signature lesion of SSA/Ro autoantibody-associated congenital heart block (CHB) is fibrosis and a macrophage infiltrate, supporting an experimental focus on cues influencing the fibroblast component. The transcriptomes of human fetal cardiac fibroblasts were analyzed using two complementary approaches. Cardiac injury conditions were simulated in vitro by incubating human fetal cardiac fibroblasts with supernatants from macrophages transfected with the SSA/Ro-associated noncoding Y ssRNA. The top 10 upregulated transcripts in the stimulated fibroblasts reflected a type I interferon (IFN) response [e.g., IFN-induced protein 44-like (IFI44L), of MX dynamin-like GTPase (MX)1, MX2, and radical S-adenosyl methionine domain containing 2 (Rsad2)]. Within the fibrotic pathway, transcript levels of endothelin-1 (EDN1), phosphodiesterase (PDE)4D, chemokine (C-X-C motif) ligand (CXCL)2, and CXCL3 were upregulated, while others, including adenomedullin, RAP guanine nucleotide exchange factor 3 (RAPGEF3), tissue inhibitor of metalloproteinase (TIMP)1, TIMP3, and dual specificity phosphatase 1, were downregulated. Agnostic Database for Annotation, Visualization and Integrated Discovery analysis revealed a significant increase in inflammatory genes, including complement C3A receptor 1 (C3AR1), F2R-like thrombin/trypsin receptor 3, and neutrophil cytosolic factor 2. In addition, stimulated fibroblasts expressed high levels of phospho-MADS box transcription enhancer factor 2 [a substrate of MAPK5 (ERK5)], which was inhibited by BIX-02189, a specific inhibitor of ERK5. Translation to human disease leveraged an unprecedented opportunity to interrogate the transcriptome of fibroblasts freshly isolated and cell sorted without stimulation from a fetal heart with CHB and a matched healthy heart. Consistent with the in vitro data, five IFN response genes were among the top 10 most highly expressed transcripts in CHB fibroblasts. In addition, the expression of matrix-related genes reflected fibrosis. These data support the novel finding that cardiac injury in CHB may occur secondary to abnormal remodeling due in part to upregulation of type 1 IFN response genes.NEW & NOTEWORTHY Congenital heart block is a rare disease of the fetal heart associated with maternal anti-Ro autoantibodies which can result in death and for survivors, lifelong pacing. This study provides in vivo and in vitro transcriptome-support that injury may be mediated by an effect of Type I Interferon on fetal fibroblasts.

Keywords: RNASeq; autoimmune congenital heart block; cardiac fibroblast.

Fig. 1.

Differential expression of targeted and agnostically determined candidate genes of fibroblasts treated with supernatants from human SSA/Ro-associated noncoding Y ssRNA [hY3; a component of SSA/Ro 60-associated ssRNA]-transfected macrophages. Interferon (IFN) genes (A), inflammatory genes (B), fibrotic genes (C), and antifibrotic genes (D) are displayed as a heat map of fibroblasts treated with hY3 macrophage supernatant (MΦ; n = 1) or untreated (mean, n = 2) and a bar graph showing the log₂-transformed ratio (y-axis) versus targeted gene (x-axis). An inspection of transcripts ranked by fold change in expression yielded genes reflecting a type I IFN response. By agnostic Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis, inflammatory genes were also highly expressed. For targeted candidates, fibroblasts treated with hY3 macrophage supernatants expressed a pattern of fibrotic genes that is consistent with a path toward fibrosis.

Fig. 2.

Assessment of activity of the IFN-α pathway in hY3 macrophage supernatants (Mac Sups). To authenticate the presence of IFN-α in hY3 macrophage supernatants, WISH cells were incubated without treatment, with macrophage supernatants, and with hY3-treated macrophage supernatants overnight. RNA was isolated from WISH cells, and an evaluation of the transcripts IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) was performed. Values on the y-axis are the log₂-transformed values of $2^{-\mathrm{\Delta \Delta }{\text{C}}_{\text{T}}}$ (where C_T is threshold cycle); the x-axis shows the treatment of WISH cells. Values appearing above the dashed line represent IFIT1 levels greater than means + 2SD. The results showed that IFIT1 was highly abundant within the transcriptome reported by WISH cells given hY3 macrophage supernatants but not untreated macrophage supernatants (P = 0.02, Fisher’s exact test).

Fig. 3.

Evidence of ERK5 activation and its inhibition on the expression of IFN, inflammatory, fibrotic, and antifibrotic genes in stimulated fibroblasts. Human fetal fibroblasts at equal cell numbers were evaluated for the expression of phospho-MADS box transcription enhancer factor 2 (MEF2C), a proxy of ERK5 activation. It was noted that phospho-MEF2C was detected by fibroblasts treated with hY3 macrophage supernatants (A) (results are representative of 3 experimentsm magnification: ×40). Inflammatory, fibrotic, and antifibrotic genes (B) and IFN genes (C) are displayed as log₂-transformed ratios (y-axis) versus the targeted gene (x-axis) in stimulated fibroblasts in the presence and absence of BIX-02189, an ERK5 inhibitor. The robust expression of IFN genes in stimulated fibroblasts was not inhibited by BIX-02189. However, cotreatment of fibroblasts with BIX-02189 yielded a profile of attenuated fibrotic genes and increased antifibrotic genes.

Fig. 4.

Effect of ERK5 inhibition on the expression of MX dynamin-like GTPase 1 (MX1), endothelin-1 (EDN1), and RAP guanine nucleotide exchange factor 3 (RAPGEF3) in stimulated fibroblasts. The upregulation of the EDN1 transcript in response to hY3 macrophage supernatants for 18 h was attenuated, whereas the downregulation of RAPGEF3 was reversed by BIX-02189. In contrast, the expression of MX1 was unaffected by cotreatment with BIX-02189. Shown on the y-axis are $2^{-\mathrm{\Delta \Delta }{\text{C}}_{\text{T}}}$ (transcript normalized to GAPDH) values, and shown on the x-axis are conditions including untreated, BIX-02189, hY3 macrophage supernatants, and hY3 macrophage supernatants + BIX-02189. Results are representative of three experiments. Bars represent means ± SE.

Fig. 5.

Authentication of enriched leukocytes, endothelial cells, and fibroblasts based on flow sorting and transcriptome analysis. CHB heart (A and B) and control heart (C and D) were stained following the protocols outlined in materials and methods. After the selection of viable (DAPI negative) cells (not shown), anti-CD45 and anti-CD31 were used to separate leukocytes and endothelial cells, respectively (A and C). Note within the same panels the utility of a portion of the unstained cells (gate, black) to further separate isolated cells using anti-podoplanin (B and D). Flow panels show the gating, as indicated on the labels of y- and x-axes of A–D, to isolate CD45, CD31, and podoplanin fractions that are enriched populations of leukocytes, endothelial cells, and fibroblasts (E). As shown in E, RNA abundance of varied isolated cells after flow sorting (which was derived from RNASeq) was consistent with the lineage assignment.

Fig. 6.

Comparison of expression of targeted and agnostically determined candidate genes of flow-sorted (fl) fibroblasts derived from CHB and healthy (control) human fetal hearts. IFN genes (A), inflammatory genes (B), fibrotic genes (C), and antifibrotic genes (D) are displayed as heat maps and bar graphs showing log₂-transformed ratios (y-axis) versus the targeted gene (x-axis). Flow CHB fibroblasts expressed high levels of IFN, inflammatory, and fibrotic genes and low levels of antifibrotic genes compared with flow healthy fibroblasts.

Fig. 7.

Interferogenic, profibrotic and inflammatory genes and their impact on the phenotype of fibroblast toward fibrosis pathways of autoimmune CHB. Upstream pathological events result in the release of IFNs, fibrotic, and inflammatory mediators by human macrophages treated with immune complexes of Ro60-hY3 and anti-Ro60. The highly reactive fibroblast transcriptome reflects an hY3 macrophage supernatant-enriched environment. For IFNs and inflammatory mediators in addition to well-documented antiviral and pro-inflammatory actions, it has been shown that type I IFN and inflammatory C3A promotes the expression of key component-controlling metabolic gene expression, which, when upregulated, may contribute to the cellular metabolism that is needed to support fibrosis. While “canonical pathways” represent common properties related to the SMAD3 pathway, noncanonical pathways, such as MAPKs, inclusive of MEK5/ERK5, are those that deviated from the canonical paradigm. In our model, for the association of phenotype and fibroblasts treated with hY3 macrophage supernatants, transforming growth factor (TGF)-β signaling by noncanonical is supportive. Specific inhibition of ERK5 with the small molecule BIX-02189 may forestall fibrosis.