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

<div><p>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 (<i>IL6R</i> 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 <i>IL6R</i> isoform (<i>P</i> = 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; <i>P</i>≤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 (<i>P</i>≤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 <i>IL6R</i> variant.</p> </div

The 358Ala allele is associated with decreased levels of membrane-bound IL-6R.

<p>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. <i>P</i>-values represent test for an association of rs2228145 with surface IL-6R levels, using an additive allelic effects model (see Methods for details).</p

The 358Ala allele is associated with reduced IL-6 signaling potential.

<p>(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. <i>P</i>-values represent tests for differences between rs2228145 genotype groups in pSTAT activation compared to control across doses. (see Methods and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003444#pgen.1003444.s006" target="_blank">Figure S6</a> for details).</p