IL-4Rα Expression Is Impaired on Smooth Muscle Cells in SM-MHC^CreIL-4Rα^−/lox Mice

<div><p>(A) Genomic integrity of the SM-MHC^CreIL-4Rα^−/lox hemizygous mice was established by PCR.</p><p>(B) Smooth muscle cells were identified by intracellular α-actin (i) staining versus isotype control (ii). IL-4Rα surface expression was analyzed on gated α-actin–positive cells (iii).</p><p>(C) cDNA levels in trachea and small intestine of IL-4Rα^−/lox (black bars) and SM-MHC^CreIL-4Rα^−/lox (hatched bars). Data are derived from pooled tissue samples from three mice and are representative of two experiments.</p><p>(D) IL-4Rα expression on lymphocyte subpopulations of T cells and B cells is unaffected in SM-MHC^CreIL-4Rα^−/lox mice.</p></div

SM-MHC^CreIL-4Rα^−/lox Mice Have a Delayed Adult Worm Expulsion from the Intestine

<div><p>(A) N. brasiliensis egg production in infected mice was assessed daily from day 5 PI using the modified McMaster technique.</p><p>(B) Worm burden was established on days 4, 7, and 10 PIn by counting worms in intestines removed from infected mice.</p><p>(C) Serum IgE antibody responses in IL-4Rα^−/lox and SM-MHC^CreIL-4Rα^−/lox mice are equivalent. Serum from infected mice was taken on days 7 and 10 PI and analyzed for antibody production by ELISA as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030001#s4" target="_blank">Materials and Methods</a>. *Significant differences from IL-4Rα^−/lox mice (<i>p</i> < 0.05); data are representative of four separate experiments.</p></div

Intestinal Goblet Cell Hyperplasia Is Delayed in SM-MHC^CreIL-4Rα^−/lox Mice following Infection with N. brasiliensis

<p>Mucus-producing goblet cells were visualized using periodic−acid Schiff reagent staining at days 4, 7, and 10 PI. The number of hyperplasic goblet cells per five villi was calculated. Values indicate mean ± SD, with *, x, +, and ++ indicating significant differences between groups (<i>p</i> < 0.05). *Significant decrease in hyperplasic goblet cells in IL-4Rα^−/lox mice at day 10 PI compared to IL-4Rα^−/lox mice at day 7 PI. x, Significantly less hyperplasic goblet cells in IL-4Rα^−/− mice than in IL-4Rα^−/lox mice at day 7 PI. +, SM-MHC^CreIL-4Rα^−/lox mice had significantly more hyperplasic goblet cells at day 10 PI than did SM-MHC^CreIL-4Rα^−/lox mice at day 7 PI. ++, SM-MHC^CreIL-4Rα^−/lox mice had significantly more hyperplasic goblet cells than did IL-4Rα^−/lox mice at day 10 PI. Data are representative of four separate experiments.</p

Intestinal Expression of M3 Receptor Is Inhibited by Disrupted Smooth Muscle Cell IL-4Rα Expression

<p>mRNA was extracted from intestines of N. brasiliensis infected mice at days 4, 7, and 10 PI. Synthesized cDNA was probed with primers to M3. Increases are normalized against α-actin. Data are representative of two to five experiments per time point. <i>n</i> ≥ 4 mice per group. *<i>p</i> < 0.05.</p

Intestinal IL-13 Levels Are Disrupted in SM-MHC^CreIL-4Rα^−/lox Mice

<p>Intestinal supernatants were analyzed for IL-13 cytokine production by ELISA as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030001#s4" target="_blank">Materials and Methods</a>. *Significant difference (<i>p</i> < 0.05) from IL-4Rα^−/lox mice; +significant difference from day 10 PI IL-4Rα^−/− mice. Data are representative of three repeated experiments.</p

Role of Smooth Muscle IL-4Rα in N. brasiliensis Infection

<p>Solid arrows represent demonstrated effects of smooth muscle IL-4Rα on the host response to N. brasiliensis infection. Dotted arrows indicate other potential and/or likely interactions.</p

Comparison of results from stochastic GUESSFM and stepwise searches.

<p><i>p</i> values are shown for the stepwise search and compare the listed model to the model above (or the null model, for single SNPs). Bayesian Information Criterion (BIC) is shown for stepwise models and index models found through GUESSFM search.</p

Haplotype analysis of MS association using the index SNPs for the one and two SNP models selected.

<p>Minor alleles are indicated by bold font. Fq = haplotype frequency.</p

Comparison of of several multivariate methods for fine mapping using simulated data.

<p>We simulated quantitative phenotype data with between two and five causal variants using genotype data from the T1D dataset for the <i>IL2RA</i> region. The simulated data sets were analysed using forward stepwise regression, GUESSFM, the lasso, the group lasso and the elastic net. GUESSFM produces credible sets for each variant chosen using the snp.picker algorithm described in Materials and Methods. We defined pseudo “credible sets” for the other approaches as the set of SNPs with <i>r</i>² > 0.8 with a selected SNP. We calculated the discovery rate (the proportion of causal variants within at least one credible set, y axis) and false discovery rate (proportion of detected variants whose credible sets did not contain any causal variant, x axis) at different thresholds for the stepwise <i>p</i> value, the group marginal posterior probability of inclusion (gMPPI) for GUESSFM and the regularization parameter(s) across simulated datasets (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005272#sec004" target="_blank">Methods</a> for details). GUESSFM-3 and GUESSFM-5 refer to GUESSFM run with a prior expectation of three or five causal variants per region, respectively. Results are averaged over 1000 replicates.</p

The proportion of naive CD4⁺ T cells that express CD25 (log scale) increases with age.

<p>The MS protective allele for the M2 SNP rs41295055:C > T associates with fewer CD4⁺ T cells expressing CD25 across all ages (<i>p</i> = 3.45 × 10⁻⁸), and is statistically preferred to the previously reported M1 SNP, rs2104286:T > C (<i>p</i> = 2.56 × 10⁻⁶; Δ BIC = 8.43). S and P are used to represent the (common) MS-susceptible and (rare) MS-protective alleles respectively at each SNP. These SNPs are in limited LD (<i>r</i>² = 0.3).</p