Background/Purpose: Since one of the strongest associations with antibodies (abs) to SSA/Ro (Ro60) is the development of congenital heart block (CHB), this model provides an exceptional opportunity to define novel insights that link maternal abs with an inflammatory cellular response which eventuates in fibrotic replacement of the AV node. We recently compiled risk genes based on an agnostic transcriptomic survey of macrophages isolated from hearts of fetuses dying with CHB and healthy aged matched fetuses electively terminated, noting that IFN related genes (IRGs), including IFN induced Protein with Tetratricopeptide Repeats 1(IFIT1) and Sialic Acid Binding Ig Like Lectin 1 (SIGLEC1), are highly upregulated in the CHB hearts.  Accordingly, this study addressed the hypothesis that IRGs contribute to CHB pathogenesis.

Methods: hY3 RNA, a noncoding ssRNA and TLR7/8 agonist, was used as a proxy of the Ro60 immune complex. Human derivatives included healthy peripheral blood macrophages and fibroblasts isolated from a healthy human fetal heart. Neutralizing IFNα and IFNβ abs  were used to assess the contribution of the respective cytokines to the model.  Macrophage readouts included the expression of IFIT1 and SIGLEC1 transcripts (qPCR, units, fold change based on 2-ΔΔCT, relative expression of transcript normalized to GAPDH) and myofibroblast phenotype (EdU imaging and SMAc by IF, respectively).

Results: As expected, exposure of macrophages to IFNα resulted in a significant  upregulation of IFIT1 and SIGLEC1 compared to untreated macrophages (70±25 vs 1, and 17±9, vs 1, respectively with both N=3, P< 0.05).  Similarly, exposure to IFNβ also resulted in the upregulation of these transcripts (254±237 vs 1, p=0.03, and 21±14 vs 1, respectively with both N=4, p< 0.03). The expression of these transcripts by IFNα- and IFNβ-treated macrophages was completely attenuated by co-treatment using respective Type I IFN-specific neutralizing antibodies. In parallel, transfection of human macrophages with hY3 also resulted in upregulation of IFIT1 (112±30 vs 1, p=0.02, N=3) and SIGLEC (13±7 vs 1, N=3). To confirm TLR7/8 dependency of IRGs, the addition  of TLR7/8 antagonist IRS661 to our in vitro model resulted in a significant decrease of IFIT1 expression to 14% (14±10, n=6) and SIGLEC1 to 54% (7±5, n=7, both P=0.03).  Co-treatment with neutralizing antibody against IFNα reduced the expression  of  IFIT1 to 9% (10±9, n=3) and SIGLEC1 to 35% (5±3, n=3). Co-treatment with neutralizing antibody against IFNβ also reduced the expression  of  IFIT1 to 24% (24±6, n=2) and SIGLEC1 to 59% (3±5, n=3). For a survey of direct effects of type I IFN, IFNα and IFNβ were shown  sharing the capacity to stimulate fibroblast proliferation (EdU, % positive) yielding a result of untreated (16%), IFNα (40%), and IFNβ (48%). In addition, exposure of human fibroblasts to IFNα as well as IFNβ induced expression of the myofibroblast marker, SMAc (IF) versus no expression by the untreated fibroblasts.

Conclusion: These results suggest that type I IFN contributes to the inflammatory and profibrosing milieu associated with the development of CHB. Feed forward expression of IFN related genes in response to TLR signaling may provide new targets towards the prevention of disease. 

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/linking-toll-like-receptor-signaling-and-type-i-interferons-to-inflammation-and-fibrosis-in-a-macrophage-fibroblast-model-of-congenital-heart-block/