Association Between Circulating CD4+ T Cell Methylation Signatures of Network-Oriented SOCS3 Gene and Hemodynamics in Patients Suffering Pulmonary Arterial Hypertension

Abstract

Pathogenic DNA methylation changes may be involved in pulmonary arterial hypertension (PAH) onset and its progression, but there is no data on potential associations with patient-derived hemodynamic parameters. The reduced representation bisulfite sequencing (RRBS) platform identified N = 631 differentially methylated CpG sites which annotated to N = 408 genes (DMGs) in circulating CD4⁺ T cells isolated from PAH patients vs. healthy controls (CTRLs). A promoter-restricted network analysis established the PAH subnetwork that included 5 hub DMGs (SOCS3, GNAS, ITGAL, NCOR2, NFIC) and 5 non-hub DMGs (NR4A2, GRM2, PGK1, STMN1, LIMS2). The functional analysis revealed that the SOCS3 gene was the most recurrent among the top ten significant pathways enriching the PAH subnetwork, including the growth hormone receptor and the interleukin-6 signaling. Correlation analysis showed that the promoter methylation levels of each network-oriented DMG were associated individually with hemodynamic parameters. In particular, SOCS3 hypomethylation was negatively associated with right atrial pressure (RAP) and positively associated with cardiac index (CI) (|r|≥ 0.6). A significant upregulation of the SOCS3, ITGAL, NFIC, NCOR2, and PGK1 mRNA levels (qRT-PCR) in peripheral blood mononuclear cells from PAH patients vs. CTRLs was found (P ≤ 0.05). By immunoblotting, a significant upregulation of the SOCS3 protein was confirmed in PAH patients vs. CTRLs (P < 0.01). This is the first network-oriented study which integrates circulating CD4⁺ T cell DNA methylation signatures, hemodynamic parameters, and validation experiments in PAH patients at first diagnosis or early follow-up. Our data suggests that SOCS3 gene might be involved in PAH pathogenesis and serve as potential prognostic biomarker.

Keywords: CD4+ T cells; DNA Methylation; Hemodynamic Parameters; Network Analysis; Pulmonary Arterial Hypertension.

Fig. 1

Study design. This flowchart shows the network-oriented strategy used to build the PAH subnetwork via integrating circulating CD4⁺ T cell DNA methylation signatures and hemodynamic parameters. Abbreviations: CTRLs, controls; DMGs, differentially methylated genes; gDNA, genomic DNA; PAH, pulmonary arterial hypertension; PBMCs, peripheral blood mononuclear cells; RRBS, reduced representation bisulfite sequencing

Fig. 2

The PAH subnetwork. The interactome shows 5 hub DMGs and 5 non-hub DMGs (green circles) linked through physical interactions (grey links) to specific known PAH-related genes as per DisGeNET (light blue diamonds). The size of both circles and diamonds is scaled according to the centrality degree and betweenness centrality measures. Original source: Cytoscape v.3.7.2 software

Fig. 3

Overrepresentation analysis (ORA). This figure shows the top ten significant pathways enriched by the genes of the PAH subnetwork through REACTOME database. Each pathway is indicated by a red or orange circle according to their p value (red circles correspond to the most significant p values). The circle size corresponds to the number of genes enriching that pathway. The small light blue circles represent the input gene list (54 nodes) and are located in the network periphery. ORA counts the number of genes shared by an input gene set and each annotated gene set, and applies a statistical test (the cumulative hyper-geometric test) to calculate the statistical significance of the overlap. A p value cutoff (p < 0.05) is then applied to select the annotated gene sets that have statistically significant overlaps with the input gene set. The IL-6 signaling axis, the growth hormone receptor signaling, and the interleukin signaling (highlighted in red) reached the highest statistical significance. The network-oriented SOCS3 gene was the most recurrent in the top significant pathways. Original source: NetworkAnalyst 3.0 software

Fig. 4

Association between network-oriented promoter DNA methylation levels and hemodynamic parameters. A Unsupervised hierarchical clustering shows the mean methylation of the dmCpGs associated with the hub and non-hub DMGs. DNA methylation values of each dmCpG (rows) are averaged within the pre-classified group (column), including CTRLs (N = 7) vs. IPAH (N = 4) and Associated-PAH (N = 3). The color key indicates DNA methylation status; red/orange represents hypomethylated DMGs and yellow/white represents hypermethylated DMGs. Correlograms show the Pearson’s correlation between promoter DNA methylation levels of each network-oriented DMG and hemodynamic parameters in IPAH (B) or Associated-PAH (C). Circle size is scaled by the correlation coefficient. Blue and red colors designate, respectively, positive and negative correlations. The asterisk (*) indicates significant correlations (p ≤ 0.05). Abbreviations: PAH, pulmonary arterial hypertension; CI, cardiac index; ITGAL, integrin subunit alpha L; GNAS guanine nucleotide binding protein (G protein), alpha stimulating activity; GRM2, glutamate metabotropic receptor 2; LIMS2, LIM zinc finger domain containing 2; mPAP, mean pulmonary arterial pressure; NCOR2, nuclear receptor corepressor 2; NFIC, nuclear factor I C; NR4A2, nuclear receptor subfamily 4 group A member 2; PGK1, phosphoglycerate kinase 1; PVR, pulmonary vascular pressure; RAP, right atrial pressure; SOCS3, suppressor of cytokine signaling 3; STMN1, stathmin

Fig. 5

Network-oriented transcriptional profiles. Dot plots show the relative expression (FC) of hypomethylated and hypermethylated network-oriented DMGs and IL-6/STAT3 signaling axis in PBMCs from N = 20 PAH patients vs. N = 10 CTRLs (Wilcoxon rank sum exact test, *p < 0.05 vs. controls; **p < 0.01 vs. controls). Dot plots were performed using GraphPad Prism version 9.0.0 software. Abbreviations: ITGAL integrin subunit alpha L; GNAS, guanine nucleotide binding protein (G protein), alpha stimulating activity; GRM2, glutamate metabotropic receptor 2; LIMS2, LIM zinc finger domain containing 2; NCOR2, nuclear receptor corepressor 2; NFIC, nuclear factor I C; NR4A2, nuclear receptor subfamily 4 group A member 2; PAH, pulmonary arterial hypertension; PGK1, phosphoglycerate kinase 1; SOCS3, suppressor of cytokine signaling 3

Fig. 6

Network-oriented SOCS3 expression profile. A Western blot analysis of SOCS3 protein expression in PBMCs isolated from CTRLs (N = 5) vs. IPAH (N = 5) and Associated-PAH (N = 7). The dashed black lines correspond to the cuts in the original film. B The dot plot shows the absolute values of the SOCS3/GAPDH ratio for each study participant. SOCS3 protein expression was significantly higher in PAH patients vs. CTRLs (two-tailed t-test; **p < 0.01). Dot plot was performed using GraphPad Prism version 9.0.0 software. Abbreviations: CTRLs, controls; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IPAH, idiopathic pulmonary arterial hypertension; SOCS3, suppressor of cytokine signaling 3