Interleukin-6 is critical in the development of pulmonary vascular disease in Gcn2-deficient mice.

Abstract

Biallelic mutations in EIF2AK4, encoding Eukaryotic Translation Initiation Factor 2α kinase 4 or General Control Nonderepressible 2 (GCN2), cause pulmonary veno-occlusive disease (PVOD), a fatal form of pulmonary hypertension. The mechanisms linking GCN2 deficiency with pulmonary vascular pathology are poorly understood. To investigate this, we developed two mouse models: genetic ablation of Gcn2, to mirror GCN2-mutation positive PVOD, and a pharmacological model using mitomycin C, a drug which can cause PVOD as an idiosyncratic drug reaction. Both models were phenotyped, and lungs from wild-type and Gcn2-deficient mice were analysed using single-cell RNA sequencing. We show that homozygous loss of Gcn2 is sufficient to induce mild pulmonary hypertension in mice. Single-cell transcriptomic profiling identified adventitial fibroblasts as the cell population exhibiting the most Gcn2-dependent transcriptional changes. Pathway analysis revealed upregulation of inflammatory signalling in Gcn2−/− adventitial fibroblasts. Consistent with this, we demonstrate a pro-inflammatory phenotype in Gcn2−/− mouse fibroblasts and in Gcn2−/− mice. Using a novel mitomycin C-induced murine model, genetic deletion of interleukin-6 (Il6) rescued the pulmonary vascular phenotype. Furthermore, chronic lipopolysaccharide exposure exaggerated pulmonary hypertension in Gcn2−/− mice, and Il6 ablation rescued both baseline and lipopolysaccharide-exacerbated disease. Pharmacological inhibition or genetic ablation of the Integrated Stress Response, which can be driven by GCN2-activation, phenocopies Gcn2-deficiency. Therefore, we establish a regulatory effect of an intact GCN2-Integrated Stress Response on IL-6 signalling. Together, we show that interleukin-6 is a critical mediator of both Gcn2-deficiency-associated and mitomycin C-triggered pulmonary vascular disease in mice, and highlight IL-6-dependent pathways as potential therapeutic targets.