IL-6R Protective Variant rs7529229 Reduces Interleukin-6 Signaling and Contributes To A Decreased Ischemic Stroke Risk

Background Interleukin-6 (IL-6) signaling is associated with an increased risk of coronary artery disease (CAD) and ischemic stroke (IS). Growing evidence shows that the minor alleles of IL-6 receptor gene (IL-6R) variants rs2228145, rs7529229, and rs4129267 signicantly increase soluble IL-6R levels and reduce CAD risk. However, the role of IL-6R variants in IS is largely unknown, prompting us to perform a comprehensive analysis. we a of three study from to evaluate the association of we to examine the effects of on IL-6R expression in neuropathologically individuals from the UK Brain Expression Consortium, and the we used a tissue-specic gene expression analysis to evaluate differences in IL-6R expression across human tissues using gene expression data from GTEx. we conducted a case–control gene expression analysis to explore the differential expression of IL-6R in the whole blood of IS patients and healthy controls. Results We found that: (1) the rs7529229 minor allele signicantly reduced the risk of developing IS (odds ratio=0.97, 95% condence interval 0.95–0.99, P=2.30E-03); (2) the rs7529229 minor allele signicantly reduced IL-6R expression in relevant tissues especially in blood vessels and whole blood; (3) IL-6R was mainly expressed in skeletal muscle and whole blood; and (4) IL-6R expression was signicantly reduced in the whole blood of healthy controls compared with IS patients. Importantly, the biological senses in stages 1–4 were all convergent. Machine-learning identies a of gene expression blood accurately

Mendelian randomization (MR) studies have been conducted to evaluate the association between IL-6 signaling and CVD [11][12]. For example, Rosa et al. applied a reduced IL-6 signaling, increased sIL-6R pattern [11], while Georgakis et al. selected a reduced IL-6 signaling, lower C-reactive protein (CRP) pattern [12]. Interestingly, both MR studies found that increased IL-6 signaling was causally associated with an increased risk of CAD and IS, although inconsistent ndings for IS subtypes were reported [11][12].
Hence, the inhibition of IL-6 signaling might be effective in treating CVD or lowering its risk.
Tocilizumab, a monoclonal antibody that blocks both membrane-bound and circulating IL-6R, has been licensed for the treatment of rheumatoid arthritis (RA) to reduce systemic and articular in ammation including CRP and brinogen concentrations [9,[13][14][15]. Tocilizumab binds to the IL-6 binding site of human IL-6R and competitively inhibits IL-6 signaling [16]. Hence, patients receiving tocilizumab treatment show signi cantly increased serum IL-6 and sIL-6R levels [16]. Evidence from epidemiological studies indicates that RA patients have a 50% increased risk of cardiovascular-related morbidity and mortality [17]. Therefore, it is important to clarify whether CVD patients may bene t from treatment with IL-6R blockade using tocilizumab.
The association between IL-6R genetic variant minor alleles and reduced CAD risk is well established and randomized controlled trials (RCTs) have been performed. In 2016, Kleveland et al. conducted a doubleblind, randomized, placebo-controlled phase 2 trial of 117 acute non-ST elevation myocardial infarction (NSTEMI) patients (placebo, n = 59; tocilizumab, n = 58) [34]. They demonstrated that IL-6 inhibition using tocilizumab attenuated systemic in ammation and troponin T-release in NSTEMI patients, with no signi cant difference in major safety concerns between the two groups [34]. In 2020, they further found that tocilizumab treatment of NSTEMI patients increased circulating levels of the neutrophil extracellular traps marker citrullinated histone H3, indicating that tocilizumab could enhance NETosis [35].
While these ndings show that human genetic variants can be used to repurpose existing targets for new therapeutic uses [9,18,[36][37], the role of IL-6R genetic variants in IS is largely unknown. In a recent genome-wide association study (GWAS), all three IL-6R variants, rs2228145 (P = 0.03351), rs7529229 (P = 0.01204), and rs4129267 (P = 0.0264), were identi ed as signi cantly associated with IS risk, with rs7529229 showing the highest association [38]. Hence, we investigated the rs7529229 variant in our current comprehensive analysis. In stage 1, we evaluated the association of rs7529229 with IS using three GWAS datasets [19,[38][39]. In stage 2, we examined the effects of rs7529229 on IL-6R expression in neuropathologically healthy individuals using three independent expression quantitative trait loci (eQTLs) dataset resources [40][41][42]. In stage 3, we evaluated potential IL-6R expression differences across different human tissues [43]. In stage 4, we explored the differential expression of IL-6R in IS cases and controls using a gene expression dataset [44].

Study design
This study is based on large-scale GWAS summary datasets, eQTLs datasets, expression datasets, and case-control expression datasets. All participants gave their informed consent in the corresponding original studies. All relevant data, analytic methods, and study materials are within the paper. This study does not use animal models.

GWAS datasets
We selected three different IS GWAS dataset resources. The rst is from the largest multi-ancestry meta- . The MEGASTROKE IS GWAS dataset is publicly available from http://www.megastroke.org/index.html. The second IS GWAS dataset resource is from UK Biobank and is publicly available from PheWeb (http://pheweb.sph.umich.edu/SAIGE-UKB/). It includes 1,501 IS cases with cerebral artery occlusion and 399,017 controls [39]. The third IS GWAS dataset resource is from the Million Veteran Program (MVP), and includes 1,198 IS cases with cerebral artery occlusion and 331,601 controls [19].
As a comparison we selected large-scale GWAS datasets for RA and CAD. The RA GWAS dataset is from a large-scale meta-analysis of individuals with European ancestry including 14,361 RA cases and 43,923 controls [45], which is publicly available from http://plaza.umin.ac.jp/~yokada/datasource/software.htm. The CAD GWAS dataset is from the CARDIoGRAMplusC4D consortium ((Coronary ARtery DIsease Genome wide Replication and Metaanalysis (CARDIoGRAM) and The Coronary Artery Disease (C4D) Genetics) including 60,801 CAD cases and 123,504 controls, most of European ancestry [46]. It is publicly available from http://www.cardiogramplusc4d.org/data-downloads/. C4D GWAS is a meta-analysis of GWAS studies of individuals of European and South Asian descent (PROCARDIS, HPS, PROMIS, and LOLIPOP) involving 15,420 CAD cases and 15,062 controls [47].

eQTLs datasets
We examined the association between rs7529229 and IL-6R expression using multiple eQTLs dataset resources. The rst eQTLs dataset resource is from the UK Brain Expression Consortium, which is publicly available from the Brain eQTL Almanac (Braineac) database [40]. Gene expression levels were measured using Affymetrix GeneChip Human exon 1.0 ST arrays [40]. Braineac includes 10 eQTLs datasets of 10 brain tissues from 134 neuropathologically healthy individuals of European descent [40].
The third eQTLs resource is from the eQTLGen Consortium [42]. This consortium conducted a large-scale meta-analysis in 31,684 human whole blood samples from 37 cohorts, with the majority of European ancestry [42]. Gene expression levels were pro led by Illumina, Affymetrix U291, Affymetrix HuEx v1.0 ST expression arrays, and RNA-seq [42].

IS case-control gene expression dataset
To evaluate the differential expression of IL-6R, we performed an IS case-control gene expression analysis in whole blood using a gene expression dataset from the Gene Expression Omnibus (GEO) database (GSE16561). In this dataset, gene expression pro ling was measured in the peripheral whole blood of 39 IS patients (17 men and 22 women) and 24 healthy controls (10 men and 14 women) using Illumina microarrays [44]. All 63 participants were of European ancestry [44].
Genetic association analysisof IL-6R rs7529229 We rst extracted corresponding summary statistics of the rs7529229 variant from three IS GWAS dataset resources including MEGASTROKE, the UK Biobank, and MVP. We then conducted a metaanalysis to evaluate the association between rs7529229 and IS using R Package (meta: General Package for Meta-Analysis). The overall odds ratio (OR) was calculated by the xed effects model (Mantel-Haenszel) or random effects model (DerSimonian-Laird), which was determined by the level of heterogeneity among these three resources [48]. We further investigated the association of rs7529229 with IS subtypes (LAS, CES, and SVS), RA, and CAD using corresponding GWAS summary statistics. The statistical signi cance for the association between rs7529229 and one speci c phenotype was a Bonferroni-corrected threshold 0.05/6=0.0083. Original P values between 0.0083 and 0.05 were considered to be suggestive of an association.

eQTLs analysis
In Braineac, we rst downloaded IL-6R expression data and genotype data of variants within 1 Mb upstream of the transcription start site and 1 Mb downstream of the transcription end site [40]. We then evaluated the potential association between rs7529229 and IL-6R expression using linear regression analysis under an additive model by adjusting for several critical covariates including the brain bank, gender, and batch effects in Partek's Genomics Suite v6.6 [40].
In GTEx, eQTLs analysis was performed using FastQTL with the following covariates: top ve genotyping principal components, a set of covariates identi ed using the Probabilistic Estimation of Expression Residuals (PEER) method (the number of PEER factors was determined as a function of sample size [N]: 15 factors for N<150, 30 factors for 150≤ N<250, 45 factors for 250≤ N<350, and 60 factors for N≥350), sequencing platform (Illumina HiSeq 2000 or HiSeq X), sequencing protocol (PCR-based or PCR-free), and sex [43]. Detailed information for laboratory and analytical methods was provided in the original paper and the GTEx website (https://www.gtexportal.org/home) [43].
In eQTLGen, a data-driven method was used to integrate gene expression data from platforms [42]. For a given single nucleotide polymorphism (SNP), genes within 1 Mb up/downstream were selected according to the central position of the gene [42]. eQTLs analysis was conducted by a Spearman correlation [42].

Gene expression analysis of IL-6R in GTEx
We conducted gene expression analysis to investigate IL-6R expression differences in different human tissues using gene expression data in GTEx (version 8). The gene expression level was quanti ed by transcripts per million (TPM) based on the GENCODE 26 annotation, then collapsed to a single transcript model for each gene using a custom isoform collapsing procedure [43]. Here, we selected the T test or analysis of variance method to evaluate the potential difference of IL-6R expression in different human tissues. Statistical signi cance was set at P < 0.05. IS case-control gene expression analysis We performed a differential expression analysis using the NCBI web application GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/) [49] to invoke the Bioconductor R packages to transform and analyze GEO datasets [49]. Evidence has shown the existence of sex differences in IS epidemiology, presentations, and outcomes [50]. Hence, we further conducted a subgroup analysis by sex. We de ned P < 0.05 as the signi cance level of differential expression of IL-6R in IS patients and healthy controls.

Results
Genetic association analysis of IL-6R rs7529229 We found no signi cant heterogeneity among the three IS GWAS dataset resources using Cochran's Q test (P = 0.12), so the overall OR was calculated using the xed effects model. We found that the rs7529229 variant C allele was signi cantly associated with a 3% reduced IS risk (OR = 0.97, 95% CI 0.95-0.99, P = 2.30E-03). The single point estimate of each study and overall effect size are provided in Fig. 1, a forest plot for the meta-analysis of rs7529229 in MEGASTROKE, UK Biobank, and MVP. This nding is consistent with RA and CAD GWAS datasets, which showed that the rs7529229 variant C allele reduced the RA risk by 7% (OR = 0.93, 95% CI 0.91-0.96, P = 1.90E-05) and the CAD risk by 4% (OR = 0.96, 95% CI 0.94-0.98, P = 2.50E-07) ( Table 1). Using GWAS summary datasets from MEGASTROKE, as provided in Table 1, the rs7529229 variant C allele showed a possible association with reducing the CES risk by 4% (P = 2.15E-02), and the SVS risk by 5% (P = 4.15E-02), but not with the LAS risk (P = 3.32E-01). The statistical signi cance for the association between rs7529229 and one speci c phenotype is a Bonferroni-corrected threshold 0.05/6 = 0.0083.

Gene expression analysis of IL-6R in GTEx
Gene expression analysis showed that IL-6R is mainly expressed in skeletal muscle (TPM median = 67.24) and whole blood (TPM median = 52.69), with no signi cant difference in expression between these two tissues. However, IL-6R expression in skeletal muscle and whole blood was signi cantly higher than in other tissues (P < 0.05) such as the liver (TPM median =

Case-control gene expression analysis
In human whole blood, we identi ed signi cant dysregulation of IL-6R expression in IS cases compared with controls (P<0.05, Table 4). Importantly, we only found signi cantly increased IL-6R expression in IS cases (Table 4). For the two IL-6R transcripts, ILMN_1754753 and ILMN_1696394, IS cases showed 37% (P=2.33E-05) and 30% (P=7.90E-04) increased IL-6R expression compared with controls, respectively. This was further supported by subgroup analysis by sex, as shown in Table 4. The signi cance level is de ned to be P < 0.05.

Discussion
IL-6 signaling has previously been associated with an increased risk of CAD and stroke [6-8]. Hence, inhibiting IL-6 signaling through blockade of the IL-6R may reduce in ammation, such as by treatment with tocilizumab which is licensed for RA therapy [9,[13][14][15]. Some individuals with the minor allele of the IL-6R rs7529229 variant not receiving tocilizumab treatment show similar biomarker pro les to those receiving tocilizumab treatment [9,18], suggesting that this allele could reduce the risk of CAD, and that CAD patients may bene t from treatment with an IL-6R blockade [9,[18][19]. However, the role of IL-6R genetic variants in stroke remains unclear. We performed a comprehensive analysis using large-scale GWAS datasets, eQTLs datasets, gene expression datasets, and IS case-control gene expression datasets and discuss our ndings by comprehensive comparisons with previous studies. controls. This nding is consistent with previous GWAS in RA and CAD [45][46], as well as with earlier ndings from candidate variant analysis and PheWAS analysis [9,18,19].
In stage 2, we demonstrated that the IL-6R rs7529229 variant minor allele C signi cantly reduced IL-6R expression in the aorta, tibial artery, and whole blood using large-scale eQTLs datasets from Braineac, GTEx (version 8), and eQTLGen. In 2012, the IL6R Genetics Consortium Emerging Risk Factors Collaboration found that the rs2228145 minor allele was not associated with IL-6R mRNA levels or IL-6 production in monocytes [18]. High expression of IL-6R in relevant tissues or organs is associated with increased IL-6R signaling and CAD risk [11,51]. Hence, ndings from genetic association analyses and eQTLs analysis are consistent in suggesting that the rs7529229 variant C allele may rst reduce IL-6R expression in relevant tissues or organs, further diminish IL-6 signaling, and eventually lower the risk of CAD and stroke. We consider this an explanation for why individuals with IL-6R variant minor alleles not receiving tocilizumab treatment have similar biomarker pro les to those with tocilizumab treatment [9, 18].
In stage 3, we detected the highest IL-6R expression in skeletal muscle and whole blood using gene expression data from GTEx (version 8). IL-6R expression in both tissues was signi cantly higher than in the liver, lung, esophagus muscularis, sigmoid colon, esophagus gastroesophageal junction, spleen, uterus, and small intestine. The lowest IL-6R expression was found in human brain tissues, which may explain why eQTLs analysis detected no signi cant association between rs7529229 and IL-6R expression in human brain tissues.
In stage 4, we identi ed signi cantly increased IL-6R expression in IS cases compared with healthy controls using whole blood IS case-control gene expression data. High IL-6R expression was previously reported to be associated with increased IL-6 signaling [11,51]. Therefore, our ndings are consistent with recent Mendelian randomization studies, which identi ed IL-6 signaling as causally associated with an increased risk of CAD and IS [9,[11][12]. In stages 1 and 2, we found that the rs7529229 variant C allele lowered the risk of IS, and reduced IL-6R expression in relevant tissues of healthy individuals, respectively. The observed signi cantly reduced expression of IL-6R in healthy controls indicates that the biological senses in stages 1-4 were convergent.
Our current study had several limitations. First, we limited our analysis to participants of European ancestry including GWAS summary datasets, eQTLs datasets, expression datasets, and case-control expression datasets. However, the association of the rs7529229 variant with IS risk and IL-6R expression may differ among different ancestries. Therefore, our ndings should be validated in other ancestries using independent datasets where available. Second, evidence from observational studies using mouse models supports the role of tocilizumab in treating IS [52][53]. However, this has not been con rmed using RCTs, although RCT evidence found no increased risk of adverse cardiovascular events among RA patients treated with tocilizumab compared with those receiving other biological disease-modifying antirheumatic drugs [54][55][56].

Conclusions
Our comprehensive analysis using large-scale GWAS datasets, eQTLs datasets, gene expression datasets, and IS case-control gene expression datasets demonstrated that: (1) the rs7529229 variant minor allele signi cantly alleviated the risk of IS; (2) the rs7529229 variant minor allele signi cantly reduced the expression of IL-6R in relevant tissues or organs especially blood vessels and whole blood; (3) IL-6R was mainly expressed in skeletal muscle and whole blood; and (4) IL-6R expression was signi cantly decreased in the whole blood of healthy controls compared with IS patients. Importantly, the biological senses in the above ndings are convergent. Hence, IL-6R may be a potential therapeutic target for IS, and blocking IL-6 signaling by means such as tocilizumab might be an effective treatment for IS or method of lowering its risk. This warrants further testing in suitably powered RCTs.
Abbreviations IS, ischemic stroke; IL-6, Interleukin-6; IL-6R, IL-6 receptor; sIL-6R, soluble IL-6R; gp130, glycoprotein 130; CVD, cardiovascular diseases; CAD, coronary artery disease; RA, rheumatoid arthritis; CRP, C-reactive  Box plots for the expression of IL-6R gene in different tissues in GTEx The gene expression values are shown in transcripts per million (TPM). The gene expression level was quanti ed byTPM based on the GENCODE 26 annotation, collapsed to a single transcript model for each gene using a custom isoform collapsing procedure [43].