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