Gene expression profile at single cell level of lung from TRAF5 knockout mouse with Systemic Lupus Erythematosus-associated Pulmonary Arterial Hypertension (SLE-PAH)

Pulmonary arterial hypertension (PAH) is a severe complication that significantly impacts the prognosis of patients with systemic lupus erythematosus (SLE). SLE-PAH has high mortality and morbidity but poorly understood pathogenesis. Our goal is to identify novel susceptibility genes associated with SLE-PAH by performing genetic analyses on large cohorts and validating the functional significance. On the genetic level, Whole Exome Sequencing (WES) and Sanger sequencing were performed on the discovery and validation cohorts to identify susceptibility genes associated with SLE-PAH; At the transcriptional level, the expression of susceptibility gene was examined by RNA-seq and RT-qPCR. TRAF5 knockout mice were compared with control after induction of the previously established SLE-PAH model, and the protective effects of TRAF5 overexpression were evaluated. Single-cell RNA sequencing (scRNA-seq) analyses were conducted on lung tissues to investigate pulmonary cellular population. In vitro interventional studies involving TRAF5 knockdown in pulmonary arterial endothelial cells (PAECs) were performed to further investigate its functions. Supporting data from both the discovery and validation cohorts revealed a total of 7 deleterious variants on TRAF5 in 8 cases of SLE-PAH (n=226), with none found in the health control group (HC, n=934). There was decreased TRAF5 expression at the transcriptional level (n=120) compared to SLE-nonPAH (n=116) and HC (n=20). Mice with TRAF5 knockout exhibited more severe pulmonary hypertension, right ventricular hypertrophy, and early mortality. scRNA-seq analyses of lung tissue in knockout mice revealed significantly decreased TRAF5 expression and increased apoptosis in arterial ECs, with distinct pathways identified. In vitro interventional studies confirmed that TRAF5 deficiency caused dysfunction in PAECs, regulating the BMP/TGF-β pathway. Endothelial-targeted overexpression of TRAF5 attenuated PH in model mice. Single cell RNA sequencing (scRNA-seq) was conducted in Novogene Technology Co., Ltd, China. Lung tissue from WT_PtHx and KO_PtHx mice (n=1, respectively) were harvested. Cell preparation followed by 10x Genomics® Cell Preparation Guide. The cell suspension was loaded into Chromium microfluidic chips with 3’ chemistry and barcoded with a 10× Chromium Controller (10X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell 3’ reagent kit (10X Genomics) according to the manufacturer’s instructions. Sequencing was performed with Illumina (NovaSeq), according to the manufacturer’s instructions (Illumina). scRNA-seq library was constructed according to the instruction of the Single Cell 3’ Reagent Kit v3.1 (10x Genomics). Cell Ranger (v6.0.1) was used to process raw sequencing data and generate the count matrix. Next, data were normalized and scaled by NormalizeData and ScaleData function, then integrated into an un-banched matrix. Cell clustering were performed and visualized by Uniform Manifold Approximation and Projection (UMAP) algorithm, and identified the distinct cell types were according to the relative expression levels of canonical markers. The differentially expressed genes (DEGs) between KO_PtHx and WT_PtHx groups in each cell type were identified using wilcoxon rank-sum test with (1) |log2 fold change| > 0.25; (2) adjusted P value < 0.05. R package clusterProfiler (version 3.18.1) was used to perform gene ontology (GO) analysis and GSEA enrichment analysis with the default parameter. Additionally, the gene set scores were calculated by AddModuleScore function in R package Seurat.