II-11 Siglec-1 macrophages and the contribution of IFN to the development of autoimmune congenital heart block

Abstract

Background Given that diseases associated with anti-Ro such as SLE and Sjogren’s syndrome associate with an upregulation of type I interferons, recent attention has focused on a potential role for IFN in the pathogenesis of congenital heart block (CHB). Based on the consistent demonstration of macrophages and multinucleated giant cells in areas of injury, it is relevant that Sialic Acid Binding Ig Like Lectin 1 (SIGLEC1), a receptor on monocytes/macrophages is upregulated by IFN. Functionally, Siglec-1 expressing macrophages might play an important role as effector cells in fibrosis. Accordingly, this study leveraged both autopsy tissue and freshly isolated macrophages from a fetal heart dying with CHB to address whether IFN-α contributes to the pathogenesis of CHB by regulating activated macrophages in affected cardiac tissue. Methods Three approaches were taken to evaluate Siglec-1 expression. Transcriptomic analysis was performed on macrophages freshly isolated from a fetal heart dying with CHB at 19 weeks and a heart from an otherwise healthy electively terminated fetus using (DAPI negative cells with isolation by flow using antibodies to CD45). Immunohistochemistry was performed on another fetal heart dying with CHB. In vitro experiments utilized cultured healthy human macrophages transfected with anti-SSA/Ro- associated ssRNA as a proxy for the in vivo conditions. Results Transcriptomes of the two hearts for each isolated leukocyte fraction were compared. By following 213 IFN inducible genes, there was enrichment of targeted transcripts in CHB vs control (p=0.0001) and SIGLEC1, which was 200-fold more abundant in CHB vs control and ranked among the top three differentially expressed candidates. In another fetal heart dying with CHB, Siglec1 staining as detected by antibody HPA053457 was prominent in areas of injury. By morphology, the two cell types expressing Siglec 1 were macrophages and dendritic cells. In vitro experiments were performed in accordance with previous laboratory work, in which a model of anti-SSA/Ro-associated injury exploits macrophages stimulated with the ssRNA component (hY3) of the SSA/Ro immune complex. IFN inducible genes (15 transcripts) were among the 30 most highly upregulated genes in hY3 stimulated conditions and SIGLEC1 was two-fold more abundant in CHB vs control. Given the enrichment of type I IFN-responsive genes in the macrophage transcriptome, a WISH cell line was selected to evaluate supernatants from macrophages transfected with hY3. IFIT1 MX1, and EIF2AK2 transcripts were significantly increased in the WISH cells treated with hY3 macrophage supernatants, but not macrophage supernatants alone (n=7, p=0.02). Conclusion These data now provide a link between IFN and the inflammatory and possibly fibrosing component of CHB and position Siglec-1 positive macrophages as integral to the process.