<i>N. brasiliensis</i>-induced and IL-4Rα-mediated intestinal hypercontractility is impaired in iLck^creIL-4Rα^−/lox mice.

<p>Jejunum pieces (1 cm) of non-infected and <i>N. brasiliensis</i> infected (PI day 7 and 10) mice were stimulated with KCl and contractility was measured (A). Comparison of the different mouse strains in response to acetylcholine is also shown for naïve, day 7 or day 10 infected IL-4Rα^−/lox, IL-4Rα^−/− and iLck^creIL-4Rα^−/lox mice (B). Contractility is shown as a mean value ± SEM for individual dose points. Graphs show three independent experiments with n = 12 in total. One-Way-ANOVA, *,# <i>P<.05</i>; **,## <i>P<.01</i>; ***,### <i>P<.001</i>. *indicates statistical significant differences between IL-4Rα^−/lox and IL-4Rα^−/−, # shows differences between IL-4Rα^−/lox and iLck^creIL-4Rα^−/lox mice.</p

<i>N. brasiliensis</i> induced smooth muscle cell hypertrophy/hyperplasia is unaffected in iLck^creIL-4Rα^−/lox mice.

<p>Haematoxylin and eosin stained sections were used to determine the smooth muscle cell layer thickness from Day 3, 7 and 10 <i>N. brasiliensis-</i>infected iLck^creIL-4Rα^−/lox and control mice. Representative photomicrographs are shown from control mice at days 3, 7 and 10 at 40× magnification. Also shown is a photomicrograph at 200× showing the longitudinal and circular smooth muscle layers included in the measurement (A). Measurements are shown in a bar graph (B) with mean values+SEM and represent 2 independent experiments with n = 4 or 5 mice per group. Ns = not significant. One-Way-ANOVA, ***<i>P<.001</i>.</p

Reduced IL-4 response in <i>N. brasiliensis-</i>infected iLck^creIL-4Rα^−/lox and IL-4Rα^−/− mice.

<p>Mice were infected with 750 <i>N. brasiliensis</i> L3 larvae and at days 7 and 10 PI CD4⁺ cells from pooled mesenteric lymph nodes were isolated by negative selection (purity>90%) then restimulated with anti-CD3 for 48 hours and IL-4, IL-13, INF-γ, IL-17 cytokine concentration of the supernatant determined by ELISA (A). Further, IL-4 and IL-13 concentrations were determined in homogenates of the jejunum (B). The graphs show mean values+SEM and are representative of the results of three independent experiments with IL-17 only determined in one experiment for CD4⁺ T cells and IL-13 in two independent experiments for homogenates, with n = 4 or 5 per group. One-Way-ANOVA, *<i>P<.05</i>, **<i>P<.01</i>, ***<i>P<.001</i>.</p

IL-4 responsive T cells are not needed for expulsion of <i>N. brasiliensis.</i>

<p>iLck^creIL-4Rα^−/lox and control mice were infected with 750 <i>N. brasiliensis</i> L3 larvae. Faeces were collected from day 6 to 14 post infection (PI) and egg production was calculated using the modified McMaster technique (A). At days 7 and 10 PI the worm burden in the small intestine was assessed (pooled from 3 experiments) (B). Intestinal goblet cell hyperplasia was assessed by determining the total number of PAS-positive goblet cells per 5 villi in histological sections of the small intestine at day 7 and 10 PI (C). Mucus and PAS staining at days 7 and 10 PI. Representative photomicrographs are shown from individual mice and <i>N. brasiliensis</i> is indicated with a black arrow (D). Total IgE production in the serum was measured by ELISA at day 7 and 10 PI (E). The graphs show mean values ± SEM and represent the results of three independent experiments, except B and E where 2–3 independent experiments were combined with n = 4 or 5 mice per group. ND, not detected. One-Way-ANOVA, *<i>P<.05</i>, **<i>P<.01</i>, ***<i>P<.001</i> for all experiments.</p

rs2228145 regulates the expression of the <i>ds-IL6R</i> but not the <i>fl-IL6R</i> isoform.

<p>(A) Expression of <i>fl-IL6R</i> and (B) <i>ds-IL6R</i> relative to the housekeeping gene <i>B2M</i> 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. <i>P</i>-values represent test for an association of rs2228145 with the expression levels of <i>fl-IL6R</i> or <i>ds-IL6R</i>, using an additive allelic effects model. AU, arbitrary units.</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

rs2228145 is the major determinant of circulating sIL-6R levels at the <i>IL6R</i> locus.

<p>(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 (<i>fl-IL6R</i>) and the differentially spliced soluble isoform (<i>ds-IL6R</i>). (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.</p

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

Regulatory T Cell Responses in Participants with Type 1 Diabetes after a Single Dose of Interleukin-2: A Non-Randomised, Open Label, Adaptive Dose-Finding Trial

BackgroundInterleukin-2 (IL-2) has an essential role in the expansion and function of CD4+ regulatory T cells (Tregs). Tregs reduce tissue damage by limiting the immune response following infection and regulate autoreactive CD4+ effector T cells (Teffs) to prevent autoimmune diseases, such as type 1 diabetes (T1D). Genetic susceptibility to T1D causes alterations in the IL-2 pathway, a finding that supports Tregs as a cellular therapeutic target. Aldesleukin (Proleukin; recombinant human IL-2), which is administered at high doses to activate the immune system in cancer immunotherapy, is now being repositioned to treat inflammatory and autoimmune disorders at lower doses by targeting Tregs.Methods and FindingsTo define the aldesleukin dose response for Tregs and to find doses that increase Tregs physiologically for treatment of T1D, a statistical and systematic approach was taken by analysing the pharmacokinetics and pharmacodynamics of single doses of subcutaneous aldesleukin in the Adaptive Study of IL-2 Dose on Regulatory T Cells in Type 1 Diabetes (DILT1D), a single centre, non-randomised, open label, adaptive dose-finding trial with 40 adult participants with recently diagnosed T1D. The primary endpoint was the maximum percentage increase in Tregs (defined as CD3+CD4+CD25highCD127low) from the baseline frequency in each participant measured over the 7 d following treatment. There was an initial learning phase with five pairs of participants, each pair receiving one of five pre-assigned single doses from 0.04 × 106 to 1.5 × 106 IU/m2, in order to model the dose-response curve. Results from each participant were then incorporated into interim statistical modelling to target the two doses most likely to induce 10% and 20% increases in Treg frequencies. Primary analysis of the evaluable population (n = 39) found that the optimal doses of aldesleukin to induce 10% and 20% increases in Tregs were 0.101 × 106 IU/m2 (standard error [SE] = 0.078, 95% CI = −0.052, 0.254) and 0.497 × 106 IU/m2 (SE = 0.092, 95% CI = 0.316, 0.678), respectively. On analysis of secondary outcomes, using a highly sensitive IL-2 assay, the observed plasma concentrations of the drug at 90 min exceeded the hypothetical Treg-specific therapeutic window determined in vitro (0.015–0.24 IU/ml), even at the lowest doses (0.040 × 106 and 0.045 × 106 IU/m2) administered. A rapid decrease in Treg frequency in the circulation was observed at 90 min and at day 1, which was dose dependent (mean decrease 11.6%, SE = 2.3%, range 10.0%–48.2%, n = 37), rebounding at day 2 and increasing to frequencies above baseline over 7 d. Teffs, natural killer cells, and eosinophils also responded, with their frequencies rapidly and dose-dependently decreased in the blood, then returning to, or exceeding, pretreatment levels. Furthermore, there was a dose-dependent down modulation of one of the two signalling subunits of the IL-2 receptor, the β chain (CD122) (mean decrease = 58.0%, SE = 2.8%, range 9.8%–85.5%, n = 33), on Tregs and a reduction in their sensitivity to aldesleukin at 90 min and day 1 and 2 post-treatment. Due to blood volume requirements as well as ethical and practical considerations, the study was limited to adults and to analysis of peripheral blood only.ConclusionsThe DILT1D trial results, most notably the early altered trafficking and desensitisation of Tregs induced by a single ultra-low dose of aldesleukin that resolves within 2–3 d, inform the design of the next trial to determine a repeat dosing regimen aimed at establishing a steady-state Treg frequency increase of 20%–50%, with the eventual goal of preventing T1D.Trial RegistrationISRCTN Registry ISRCTN27852285; ClinicalTrials.gov NCT01827735</div