Proteogenomic Data Integration Reveals CXCL10 as a Potentially Downstream Causal Mediator for IL-6 Signaling on Atherosclerosis

Abstract

Background: Genetic and experimental studies support a causal involvement of IL-6 (interleukin-6) signaling in atheroprogression. Although trials targeting IL-6 signaling are underway, any benefits must be balanced against an impaired host immune response. Dissecting the mechanisms that mediate the effects of IL-6 signaling on atherosclerosis could offer insights about novel drug targets with more specific effects.

Methods: Leveraging data from 522 681 individuals, we constructed a genetic instrument of 26 variants in the gene encoding the IL-6R (IL-6 receptor) that proxied for pharmacological IL-6R inhibition. Using Mendelian randomization, we assessed its effects on 3281 plasma proteins quantified with an aptamer-based assay in the INTERVAL cohort (n=3301). Using mediation Mendelian randomization, we explored proteomic mediators of the effects of genetically proxied IL-6 signaling on coronary artery disease, large artery atherosclerotic stroke, and peripheral artery disease. For significant mediators, we tested associations of their circulating levels with incident cardiovascular events in a population-based study (n=1704) and explored the histological, transcriptomic, and cellular phenotypes correlated with their expression levels in samples from human atherosclerotic lesions.

Results: We found significant effects of genetically proxied IL-6 signaling on 70 circulating proteins involved in cytokine production/regulation and immune cell recruitment/differentiation, which correlated with the proteomic effects of pharmacological IL-6R inhibition in a clinical trial. Among the 70 significant proteins, genetically proxied circulating levels of CXCL10 (C-X-C motif chemokine ligand 10) were associated with risk of coronary artery disease, large artery atherosclerotic stroke, and peripheral artery disease, with up to 67% of the effects of genetically downregulated IL-6 signaling on these end points mediated by decreases in CXCL10. Higher midlife circulating CXCL10 levels were associated with a larger number of cardiovascular events over 20 years, whereas higher CXCL10 expression in human atherosclerotic lesions correlated with a larger lipid core and a transcriptomic profile reflecting immune cell infiltration, adaptive immune system activation, and cytokine signaling.

Conclusions: Integrating multiomics data, we found a proteomic signature of IL-6 signaling activation and mediators of its effects on cardiovascular disease. Our analyses suggest the interferon-γ-inducible chemokine CXCL10 to be a potentially causal mediator for atherosclerosis in 3 vascular compartments and, as such, could serve as a promising drug target for atheroprotection.

Keywords: Mendelian randomization analysis; atherosclerosis; interleukin-6.

Figure 1.. Study overview.

Overview of major analytical approaches and data sources used in the current study to identify proteins up- or downregulated by genetically proxied IL-6 signaling as well as mediators of its effect on atherosclerotic disease.

Figure 2.. Plasma proteomic changes in association with genetically proxied IL-6 signaling.

(a) Schematic representation of the study design and data sources. (b) Volcano plot of the associations of genetically downregulated IL-6 signaling with plasma proteins in the INTERVAL study (n=3,301). The results are derived from random-effects inverse-variance weighted Mendelian randomization analyses. Log p-value in the y axis refers to the log base 10 logarithm. The dotted line corresponds to a false discovery rate (FDR)-corrected p-value<0.05. (c) Significant (FDR-corrected p-value<0.05) Gene Ontology (GO) Pathway enrichment analysis for significant proteins. (d) Correlation between Mendelian randomization estimates for proteins associated with genetically downregulated signaling and estimates from linear regression for pharmacological IL-6R inhibition among 24 individuals treated with tocilizumab versus 24 individuals treated with placebo in the Norwegian tocilizumab NSTEMI study (the blue and red dots correspond to proteins that were found to be down- and upregulated, respectively in the genetic analysis of panel B).

Figure 3.. Genetically proxied levels of proteins associated with genetically downregulated IL-6 signaling and atherosclerotic cardiovascular disease.

(a) Associations of genetically proxied levels of proteins associated with genetically downregulated IL-6 signaling with peripheral artery disease (PAD), large artery atherosclerotic stroke (LAS), and coronary artery disease (CAD), as derived from inverse-variance weighted Mendelian randomization analyses. The stars indicate significant associations at an FDR-corrected p<0.05, whereas the box around significant associations highlights associations that were also significant in cis-MR analyses. (b) Regional association plots at the IL-6R locus for associations with soluble IL-6 receptor levels (upper part) and CXCL10 levels (lower part) demonstrating colocalization of the signal. (c) Mediation Mendelian randomization analysis for the total effects of genetically downregulated IL-6 signaling on PAD, LAS, and CAD, as well as the indirect effects mediated through changes in CXCL10 levels.

Figure 4.. Circulating CXCL10 levels in association with circulating IL-6 and major adverse cardiovascular events in the population-based MONICA/KORA cohort.

(a) Correlations between circulating IL-6 and CXCL10 levels among 1,704 participants of the MONICA/KORA cohort. (b) Kaplan-Meier curve of the associations between baseline circulating CXCL10 levels and risk of CAD or stroke over a follow-up period extending up to 30 years. (c) Hazard ratios of the associations of circulating IL-6 and CXCL10 levels with risk of CAD or stroke in models adjusted for age, sex, and baseline survey (Model 1), models adjusted for age, sex, baseline survey, and vascular risk factors (Model 2), and a model adjusted for all variables of Model 2 and both proteins simultaneously included (Model 3).

Figure 5.. Expression of CXCL10 within atherosclerotic lesions.

(a) Schematic representation of CXCL10 mRNA quantification among 623 carotid atherosclerotic plaques from patients with carotid stenosis in the Athero-EXPRESS Biobank, who underwent endarterectomy. (b) Associations of plaque CXCL10 expression with histological features of plaque vulnerability. (c) Co-expression of CXCL10 with 16,214 other genes in atherosclerotic aortic root tissue from 514 participants in the STARNET network. (d) Pathway enrichment analysis of genes co-expressed with CXCL10, as derived from Reactome. (e) Cell-specific expression of top 42 genes co-expressed with CXCL10 at an r>0.3 in aortic tissue, as derived from single-nuclei RNA sequencing analysis in human aorta samples.