Background/Purpose:

The role of Type I IFN in anti-SSA/Ro-associated Congenital Heart Block (CHB) may include an adverse consequence to the cardiac vasculature. One scenario consistent with injury is that the phenotype of cardiac endothelial cells contributes to Caspase-1/Interleukin-1 converting enzyme (inflammasome) to account for chronic levels of inflammatory cytokines and oxidative stress. This study sought to test the hypothesis that anti-SSA/Ro-mediated injury to the vasculature occurs via a Type 1 IFN-enriched environment, which acts on the endothelial cells to upregulate the inflammasome along with metabolical changes contributing to damage by decreasing oxygen to the tissue.

Methods:

Two sources of endothelial cells were studied. In an in vitro approach, cultured HUVECs were evaluated in the presence and absence of supernatants generated from macrophages treated with ssRNA (hY3) since previous studies have identified macrophages as a potential source of IFN in CHB. Both transcriptomic analyses and cellular metabolism as reflected in the extracellular acidification rate (ECAR) and mitochondrial oxygen consumption (OCR) were analyzed using the Seahorse platform. A second in vivo approach used freshly obtained endothelial cells (DAPI negative cells isolated by flow using antibodies to CD31) from a 19 week fetus dying with CHB and an otherwise healthy 22 week heart.

Results:

For the in vitro experiments, IFIT1 (IFN response gene) was significantly increased by 163-fold in HUVECs incubated with hY3 macrophage supernatants compared to HUVECS treated with macrophage supernatants alone (p=0.023, qPCR). The inflammasome components NLRP3 and CASP1 were also significantly increased by 2.8 fold and 2.1 fold in HUVECS treated with hY3 macrophage supernatants vs macrophage supernatants alone (p=0.025, p=0.05, respectively). Reflecting oxidant stress, significant increases in ECAR were observed in the HUVECS treated with hY3 macrophage supernatants compared to macrophage supernatants alone (15±4 mpH/min vs 5±3 mpH/min, p=0.05, N = 3). For bioenergetic health index (BHI), a composite of spare capacity, coupling efficiency, Proton leak and non mitochondrial respiration, there was a trend to be lower EC + hY3 macrophage supernatants vs EC + macrophage supernatants (2.0 vs 3.3, p=0.2, respectively) .

Transcriptomes of the two hearts for each isolated endothelial cell fraction were compared. Based on DAVID annotation, data were organized into clusters of closely related genes. The top GO category was the type I IFN signaling pathway with 5 IFN inducible genes in the top 10. Regarding targeted genes with greater than two-fold upregulation (CHB vs control), there were genes within the inflammasome pathway, including IFN inducible NLRC5, which serves to interact with NLRP3, along with well characterized multiprotein oligomer of the inflammasome such as NLRP3, IRF7, IRF9 & CASP1. Inflammasome precursors including IL18, IL1B, & IL1A were also upregulated.

Conclusion:

Anti-SSA/Ro-induced cardiac injury may include a previously unappreciated effect on the vasculature mediated by IFN. This vascular effect is reflected in the upregulation of metabolic and inflammasome genes.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cardiac-endothelial-cell-transcriptome-analyses-support-a-pathological-role-of-metabolic-and-inflammasome-genes-in-anti-ssaro-associated-congenital-heart-block/