E small intestine resulted in similar number per villi between control and iLckcreIL-4Ra2/lox mice (Figure 1C and D) with significantly lower intestinal mucus production in global IL-4Ra2/2 mice, (as previously shown) (20,24). Whereas total IgE antibody concentration was below detection limit in the sera of global IL-4Ra2/2 mice, IgE A 196 antibodies were present in naive iLckcreIL-4Ra2/lox mice and increased during infection, though to a lesser extent than infected control mice (Figure 1E). Together, this indicates that sufficient IL-4 is present for IL-4Ra-dependent type 2 B-cell responses. As N. brasiliensis is known to cause intestinal smooth muscle hyperplasia/hypertrophy we measured the thickness of this layer in the intestine of all mouse groups. Indeed we detected a significant thickening of this muscle layer when comparing day 3 (before the worms have reached the intestine) with day 7 and 10 post infection (Figure 2A and B). However, there was no significant difference between all mouse groups suggesting that the thickening is independent of IL-4Ra.IL-4 and IL-13 Production in the Jejunum is Abrogated in Infected T Cell-specific IL-4Ra Deficient MiceIn order to determine T helper cytokine responses, mesenteric lymph node CD4+ T cells were isolated at days 7 and 10 PI, then restimulated with anti-CD3. As expected, IL-4Ra-responsive CD4+ T cells from IL-4Ra2/lox control mice secreted high levelsIL-4Ra-Mediated Intestinal HypercontractilityFigure 1. IL-4 responsive T cells are not needed for expulsion of N. brasiliensis. iLckcreIL-4Ra2/lox and control mice were infected with 750 N. brasiliensis L3 larvae. Faeces were collected from day 6 to 14 16574785 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 POR-8 web cellIL-4Ra-Mediated Intestinal Hypercontractilityhyperplasia 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 N. brasiliensis 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 6 SEM and represent the results of three independent experiments, except B and E where 2? independent experiments were combined with n = 4 or 5 mice per group. ND, not detected. One-Way-ANOVA, *P,.05, **P,.01, ***P,.001 for all experiments. doi:10.1371/journal.pone.0052211.gFigure 2. N. brasiliensis induced smooth muscle 
cell hypertrophy/hyperplasia is unaffected in iLckcreIL-4Ra2/lox mice. Haematoxylin and eosin stained sections were used to determine the smooth muscle cell layer thickness from Day 3, 7 and 10 N. brasiliensis-infected iLckcreIL-4Ra2/ lox and control mice. Representative photomicrographs are shown from control mice at days 3, 7 and 10 at 406 magnification. Also shown is a photomicrograph at 2006showing 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-WayANOVA, ***P,.001. doi:10.1371/journal.pone.0052211.gIL-4Ra-Mediated Intestinal HypercontractilityFigure 3. Reduced IL-4 response in N. brasi.E small intestine resulted in similar number per villi between control and iLckcreIL-4Ra2/lox mice (Figure 1C and D) with significantly lower intestinal mucus production in global IL-4Ra2/2 mice, (as previously shown) (20,24). Whereas total IgE antibody concentration was below detection limit in the sera of global IL-4Ra2/2 mice, IgE antibodies were present in naive iLckcreIL-4Ra2/lox mice and increased during infection, though to a lesser extent than infected control mice (Figure 1E). Together, this indicates that sufficient IL-4 is present for IL-4Ra-dependent type 2 B-cell responses. As N. brasiliensis is known to cause intestinal smooth muscle hyperplasia/hypertrophy we measured the thickness of this layer in the intestine of all mouse groups. Indeed 
we detected a significant thickening of this muscle layer when comparing day 3 (before the worms have reached the intestine) with day 7 and 10 post infection (Figure 2A and B). However, there was no significant difference between all mouse groups suggesting that the thickening is independent of IL-4Ra.IL-4 and IL-13 Production in the Jejunum is Abrogated in Infected T Cell-specific IL-4Ra Deficient MiceIn order to determine T helper cytokine responses, mesenteric lymph node CD4+ T cells were isolated at days 7 and 10 PI, then restimulated with anti-CD3. As expected, IL-4Ra-responsive CD4+ T cells from IL-4Ra2/lox control mice secreted high levelsIL-4Ra-Mediated Intestinal HypercontractilityFigure 1. IL-4 responsive T cells are not needed for expulsion of N. brasiliensis. iLckcreIL-4Ra2/lox and control mice were infected with 750 N. brasiliensis L3 larvae. Faeces were collected from day 6 to 14 16574785 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 cellIL-4Ra-Mediated Intestinal Hypercontractilityhyperplasia 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 N. brasiliensis 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 6 SEM and represent the results of three independent experiments, except B and E where 2? independent experiments were combined with n = 4 or 5 mice per group. ND, not detected. One-Way-ANOVA, *P,.05, **P,.01, ***P,.001 for all experiments. doi:10.1371/journal.pone.0052211.gFigure 2. N. brasiliensis induced smooth muscle cell hypertrophy/hyperplasia is unaffected in iLckcreIL-4Ra2/lox mice. Haematoxylin and eosin stained sections were used to determine the smooth muscle cell layer thickness from Day 3, 7 and 10 N. brasiliensis-infected iLckcreIL-4Ra2/ lox and control mice. Representative photomicrographs are shown from control mice at days 3, 7 and 10 at 406 magnification. Also shown is a photomicrograph at 2006showing 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-WayANOVA, ***P,.001. doi:10.1371/journal.pone.0052211.gIL-4Ra-Mediated Intestinal HypercontractilityFigure 3. Reduced IL-4 response in N. brasi.
