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Cited by 317Cytokine Signaling Pathways and InteractionsGenetic Associations and EpidemiologyRheumatoid Arthritis Research and Therapies

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Functional IL6R 358Ala Allele Impairs Classical IL-6 Receptor Signaling and Influences Risk of Diverse Inflammatory Diseases

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Ferreira, Ricardo C., Freitag, Daniel F., Cutler, Antony J., Howson, Joanna M. M., Rainbow, Daniel B., Smyth, Deborah J., Kaptoge, Stephen, Clarke, Pamela, Boreham, Charlotte, Coulson, Richard M., Pekalski, Marcin L., Chen, Wei-Min, Onengut-Gumuscu, Suna, Rich, Stephen S., Butterworth, Adam S., Malarstig, Anders, Danesh, John, Todd, John A.·2013·PLoS Genetics
DOI 10.1371/journal.pgen.1003444PMC PMC3617094
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Abstract

Inflammation, which is directly regulated by interleukin-6 (IL-6) signaling, is implicated in the etiology of several chronic diseases. Although a common, non-synonymous variant in the IL-6 receptor gene (IL6R Asp358Ala; rs2228145 A>C) is associated with the risk of several common diseases, with the 358Ala allele conferring protection from coronary heart disease (CHD), rheumatoid arthritis (RA), atrial fibrillation (AF), abdominal aortic aneurysm (AAA), and increased susceptibility to asthma, the variant's effect on IL-6 signaling is not known. Here we provide evidence for the association of this non-synonymous variant with the risk of type 1 diabetes (T1D) in two independent populations and confirm that rs2228145 is the major determinant of the concentration of circulating soluble IL-6R (sIL-6R) levels (34.6% increase in sIL-6R per copy of the minor allele 358Ala; rs2228145 [C]). To further investigate the molecular mechanism of this variant, we analyzed expression of IL-6R in peripheral blood mononuclear cells (PBMCs) in 128 volunteers from the Cambridge BioResource. We demonstrate that, although 358Ala increases transcription of the soluble IL6R isoform (P = 8.3×10⁻²²) and not the membrane-bound isoform, 358Ala reduces surface expression of IL-6R on CD4+ T cells and monocytes (up to 28% reduction per allele; P≤5.6×10⁻²²). Importantly, reduced expression of membrane-bound IL-6R resulted in impaired IL-6 responsiveness, as measured by decreased phosphorylation of the transcription factors STAT3 and STAT1 following stimulation with IL-6 (P≤5.2×10⁻⁷). Our findings elucidate the regulation of IL-6 signaling by IL-6R, which is causally relevant to several complex diseases, identify mechanisms for new approaches to target the IL-6/IL-6R axis, and anticipate differences in treatment response to IL-6 therapies based on this common IL6R variant.

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Figure 1 rs2228145 is the major determinant of circulating sIL-6R levels at the IL6R locus. (A) X2 statistics for the association (additive allelic effects model of inheritance; see Methods) of 45 SNPs genotyped using the Illumina ImmunoChip with sIL-6R concentrations are plotted against the physical position of the SNPs on chromosome 1 (hg.19). Recombination rates and linkage disequilibrium (r2) values are based on CEU HapMap. Inset depicts exonic structure of the membrane-bound IL-6R (fl-IL6R) and the differentially spliced soluble isoform (ds-IL6R). (B) Results from a regression model containing all three independent IL6R SNPs. Mean differences in sIL-6R concentration (%) compared to the common homozygote reference groups are plotted for the 3 SNPs independently associated with sIL-6R levels. Box size is proportional to the number of individuals in each group and error bars represent the standard error of the mean. SE = standard error of the mean difference. R2 = variance explained by the individual SNP.
Figure 1 rs2228145 is the major determinant of circulating sIL-6R levels at the IL6R locus. (A) X2 statistics for the association (additive allelic effects model of inheritance; see Methods) of 45 SNPs genotyped using the Illumina ImmunoChip with sIL-6R concentrations are plotted against the physical position of the SNPs on chromosome 1 (hg.19). Recombination rates and linkage disequilibrium (r2) values are based on CEU HapMap. Inset depicts exonic structure of the membrane-bound IL-6R (fl-IL6R) and the differentially spliced soluble isoform (ds-IL6R). (B) Results from a regression model containing all three independent IL6R SNPs. Mean differences in sIL-6R concentration (%) compared to the common homozygote reference groups are plotted for the 3 SNPs independently associated with sIL-6R levels. Box size is proportional to the number of individuals in each group and error bars represent the standard error of the mean. SE = standard error of the mean difference. R2 = variance explained by the individual SNP.CC BY · PubMed Central
Figure 2 rs2228145 regulates the expression of the ds-IL6R but not the fl-IL6R isoform. (A) Expression of fl-IL6R and (B) ds-IL6R relative to the housekeeping gene B2M was measured by quantitative real-time PCR of RNA purified from PBMCs of 88 healthy volunteers from the Cambridge BioResource. Individual expression levels and their mean (±SEM) are plotted according to rs2228145 genotype. Differences in the mean expression levels relative to the common homozygotes group (Asp/Asp) are indicated above the black horizontal lines. P-values represent test for an association of rs2228145 with the expression levels of fl-IL6R or ds-IL6R, using an additive allelic effects model. AU, arbitrary units.
Figure 2 rs2228145 regulates the expression of the ds-IL6R but not the fl-IL6R isoform. (A) Expression of fl-IL6R and (B) ds-IL6R relative to the housekeeping gene B2M was measured by quantitative real-time PCR of RNA purified from PBMCs of 88 healthy volunteers from the Cambridge BioResource. Individual expression levels and their mean (±SEM) are plotted according to rs2228145 genotype. Differences in the mean expression levels relative to the common homozygotes group (Asp/Asp) are indicated above the black horizontal lines. P-values represent test for an association of rs2228145 with the expression levels of fl-IL6R or ds-IL6R, using an additive allelic effects model. AU, arbitrary units.CC BY · PubMed Central
Figure 3 The 358Ala allele is associated with decreased levels of membrane-bound IL-6R. Surface expression of IL-6R was quantified by flow cytometry in cryopreserved PBMCs from 128 volunteers from the Cambridge BioResource. Donors were sampled according to rs2228145 genotype. IL-6R surface expression was measured in four distinct immune cell subsets: CD4+ naïve and memory T cells, CD4+ regulatory T cells (Treg) and monocytes. Scatter plots depict the individual normalized IL-6R fluorescence intensity values measured as molecules of equivalent fluorochrome (MEF; see Methods for details). Error bars represent the standard error of the mean as shown by the middle horizontal line. The horizontal grey dotted reference line represents the average background fluorescence signal of the isotype control group. Differences in the mean expression levels, relative to the common homozygote group (Asp/Asp) are indicated above the horizontal black lines. P-values represent test for an association of rs2228145 with surface IL-6R levels, using an additive allelic effects model (see Methods for details).
Figure 3 The 358Ala allele is associated with decreased levels of membrane-bound IL-6R. Surface expression of IL-6R was quantified by flow cytometry in cryopreserved PBMCs from 128 volunteers from the Cambridge BioResource. Donors were sampled according to rs2228145 genotype. IL-6R surface expression was measured in four distinct immune cell subsets: CD4+ naïve and memory T cells, CD4+ regulatory T cells (Treg) and monocytes. Scatter plots depict the individual normalized IL-6R fluorescence intensity values measured as molecules of equivalent fluorochrome (MEF; see Methods for details). Error bars represent the standard error of the mean as shown by the middle horizontal line. The horizontal grey dotted reference line represents the average background fluorescence signal of the isotype control group. Differences in the mean expression levels, relative to the common homozygote group (Asp/Asp) are indicated above the horizontal black lines. P-values represent test for an association of rs2228145 with surface IL-6R levels, using an additive allelic effects model (see Methods for details).CC BY · PubMed Central
Figure 4 The 358Ala allele is associated with reduced IL-6 signaling potential. (A) Frequency of pSTAT3 and (B) pSTAT1 positive cells following stimulation of PBMCs with 0, 0.1, 1 or 10 ng/ml of IL-6. Intracellular levels of pSTAT3 and pSTAT1 were measured by flow cytometry in three distinct immune cell subsets: CD4+ naïve T cells, CD4+ memory T cells and monocytes in 14 Asp/Asp and 14 Ala/Ala volunteers from the Cambridge BioResource. Median and interquartile range of the distribution of the frequency of pSTAT3 and pSTAT1 positive events in the two genotype groups for each dose of IL-6 stimulation are plotted. P-values represent tests for differences between rs2228145 genotype groups in pSTAT activation compared to control across doses. (see Methods and Figure S6 for details).
Figure 4 The 358Ala allele is associated with reduced IL-6 signaling potential. (A) Frequency of pSTAT3 and (B) pSTAT1 positive cells following stimulation of PBMCs with 0, 0.1, 1 or 10 ng/ml of IL-6. Intracellular levels of pSTAT3 and pSTAT1 were measured by flow cytometry in three distinct immune cell subsets: CD4+ naïve T cells, CD4+ memory T cells and monocytes in 14 Asp/Asp and 14 Ala/Ala volunteers from the Cambridge BioResource. Median and interquartile range of the distribution of the frequency of pSTAT3 and pSTAT1 positive events in the two genotype groups for each dose of IL-6 stimulation are plotted. P-values represent tests for differences between rs2228145 genotype groups in pSTAT activation compared to control across doses. (see Methods and Figure S6 for details).CC BY · PubMed Central

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Metadata

Authors
Ferreira, R. C., Freitag, D. F., Cutler, A. J., Howson, J. M. M., Rainbow, D. B., Smyth, D. J., Kaptoge, S., Clarke, P., Boreham, C., Coulson, R. M., Pekalski, M. L., Chen, W.-M., Onengut-Gumuscu, S., Rich, S. S., Butterworth, A. S., Malarstig, A., Danesh, J., & Todd, J. A.
Affiliations
  • University of Cambridge (GB)
  • University of Virginia (US)
Corresponding author
Todd, John A.
ORCID
Venue
PLoS Genetics
Publisher
Public Library of Science (PLoS)
Published
2013-04-04
Volume
9
Issue
4
Pages
e1003444
ISSN
  • 1553-7390 (ISSN-L)
  • 1553-7404
DOI
10.1371/journal.pgen.1003444
PMC
PMC3617094
OpenAlex
W1991747007
Cited by
316
References
56
Citations by year
2026: 23 · 2025: 34 · 2024: 19 · 2023: 32 · 2022: 30 · 2021: 27 · 2020: 26 · 2019: 26 · 2018: 19 · 2017: 26 · 2016: 18 · 2015: 15 · 2014: 13 · 2013: 8
FWCI
9.65
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS Genetics (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
Funders
  • Juvenile Diabetes Research Foundation International (9-2011-253, U01 DK062418, 068545/Z/02)
  • Pfizer
  • GlaxoSmithKline
  • Wellcome Trust (068545/Z/02), FP7/2007–2013, 091157, 096388, G0000934, 061858, 100140, 068545, 9-2011-253, 076113, 076113/C/04/Z)
  • National Institute for Health and Care Research (NF-SI-0512-10165, FP7/2007–2013, G0000934, NF-SI-0508-10275)
  • British Heart Foundation (FS/09/050, RG/08/014, 068545/Z/02, G0000934, RG/08/014/24067)
  • Bupa Foundation
  • University of Cambridge
  • University of Leicester
  • European Commission (241447, 2007–2013, FP7 2007-2013, FP7/2007)
  • University of Bristol
  • Evelyn Trust
  • Merck Sharp and Dohme
  • Medical Research Council (FP7/20072013, MR/L003120/1, 068545/Z/02, G0000934)
  • Cambridge Institute for Medical Research, University of Cambridge (100140)
  • NIHR Cambridge Biomedical Research Centre
  • National Heart, Lung, and Blood Institute (068545/Z/02, G0000934)
  • National Human Genome Research Institute
  • National Institute of Allergy and Infectious Diseases
  • Fogarty International Center
  • National Institute of Diabetes and Digestive and Kidney Diseases (DK062418, U01DK062418)
  • National Institute of Child Health and Human Development
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Cytokine Signaling Pathways and Interactions
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Associations and Epidemiology
  • Health Sciences › Medicine › Rheumatology › Rheumatoid Arthritis Research and Therapies
Keywords
IL-6 receptor, rs2228145, soluble IL-6 receptor, IL-6 signaling, type 1 diabetes, STAT3 phosphorylation, STAT1 phosphorylation, CD4+ T cells, monocytes, peripheral blood mononuclear cells, coronary artery disease, rheumatoid arthritis, atrial fibrillation, abdominal aortic aneurysm, asthma
MeSH terms
Alleles, Diabetes Mellitus, Type 1, Humans, Inflammation, Leukocytes, Mononuclear, Mutation, Phosphorylation, Risk Factors, Signal Transduction, Amino Acid Substitution, Receptors, Interleukin-6, Protein Isoforms, Genetic Association Studies

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