Per group. One-Way-ANOVA, *P,.05, **P,.01, ***P,.001. doi:10.1371/journal.pone.0052211.Title Loaded From File gIL-4Ra-Mediated Intestinal HypercontractilityFigure 4. N. brasiliensis-induced and IL-4Ra-mediated intestinal hypercontractility is impaired in iLckcreIL-4Ra2/lox mice. Jejunum pieces (1 cm) of non-infected and N. 1326631 brasiliensis 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 naive, day 7 or day 10 infected IL-4Ra2/lox, IL-4Ra2/2 and iLckcreIL-4Ra2/lox mice (B). Contractility is shown as a mean value 6 SEM for individual dose points. Graphs show three independent experiments with n = 12 in total. One-Way-ANOVA, *,# P,.05; **,## P,.01; ***,### P,.001. *indicates statistical significant differences between IL-4Ra2/lox and IL4Ra2/2, # shows differences between IL-4Ra2/lox and iLckcreIL-4Ra2/lox mice. doi:10.1371/journal.pone.0052211.gneeded for optimal N. brasiliensis-induced smooth muscle cell hypercontractility.DiscussionMorphological and physiological Title Loaded From File changes in the gastrointestinal system during nematode infections may be important contributors to host defence and pathology. These responses have previously been demonstrated to be controlled by the TH2 immunity associated with infection. Non-haematopoietic contributions by IL-4Ra responsive smooth muscle 1379592 cells have been previously demonstrated [12]. It is however important to understand the molecular requirements of haematopoietic cell populations to contribute to this striking physiological response. Using a mouse model with impaired IL-4Ra expression on specific T cell populations, we demonstrated roles for IL-4 responsive T cells in intestinal hypercontractile responses to N. brasiliensis. In this study the impact of IL-4Ra-responsive T cells on smooth muscle cell hypercontraction and their contribution to clearance of N. brasiliensis infection was defined. We showed that IL-4Ra-responsive T cells are required for optimal N. brasiliensis-induced intestinal hypercontractility, but not for worm expulsion. Wild type mice resist infection with N. brasiliensis and develop polarized TH2 responses with high IL-4/IL-13 and low IFN-c production [38?0]. Well-established TH2 induced effector mechanisms following N. brasiliensis infection are eosinophilia [4,41] mucosal mastocytosis [6], pathogen specific antibodies including IgE and IgG1 [4,5] goblet cell hyperplasia and promotion of TH2 cytokine responses. IL-4 has been implicated in driving the polarized TH2 response against N. brasiliensis,demonstrated by diminished type 2 responses in IL-42/2, IL4Ra2/2 and STAT-62/2 mice [16,24,28,37,42,43]. Although it is known that both IL-4Ra [13] and CD4+ T cells [20] are involved in worm clearance, more recent studies from us and others have shown that IL-4 responsive CD4+ T cells [28] or signalling through the STAT-6 pathway in these cells is not needed for worm expulsion. Indeed, IL-4 receptor expression by non-bone marrowderived cells is required to expel N. brasiliensis [44]. However, type 2 immunity is controlled by IL-4/IL-13 expression in haematopoetic non-eosinophil cells of the innate immune system [45]. This is consistent with the findings in our present study, as infected mice deficient in IL-4Ra expression on all T cell subpopulations showed impaired TH2 responses but still presentedIL-13 production in the mesenteric lymph nodes, able to respond with goblet cell hyperplasia.Per group. One-Way-ANOVA, *P,.05, **P,.01, ***P,.001. doi:10.1371/journal.pone.0052211.gIL-4Ra-Mediated Intestinal HypercontractilityFigure 4. N. brasiliensis-induced and IL-4Ra-mediated intestinal hypercontractility is impaired in iLckcreIL-4Ra2/lox mice. Jejunum pieces (1 cm) of non-infected and N. 1326631 brasiliensis 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 naive, day 7 or day 10 infected IL-4Ra2/lox, IL-4Ra2/2 and iLckcreIL-4Ra2/lox mice (B). Contractility is shown as a mean value 6 SEM for individual dose points. Graphs show three independent experiments with n = 12 in total. One-Way-ANOVA, *,# P,.05; **,## P,.01; ***,### P,.001. *indicates statistical significant differences between IL-4Ra2/lox and IL4Ra2/2, # shows differences between IL-4Ra2/lox and iLckcreIL-4Ra2/lox mice. doi:10.1371/journal.pone.0052211.gneeded for optimal N. brasiliensis-induced smooth muscle cell hypercontractility.DiscussionMorphological and physiological changes in the gastrointestinal system during nematode infections may be important contributors to host defence and pathology. These responses have previously been demonstrated to be controlled by the TH2 immunity associated with infection. Non-haematopoietic contributions by IL-4Ra responsive smooth muscle 1379592 cells have been previously demonstrated [12]. It is however important to understand the molecular requirements of haematopoietic cell populations to contribute to this striking physiological response. Using a mouse model with impaired IL-4Ra expression on specific T cell populations, we demonstrated roles for IL-4 responsive T cells in intestinal hypercontractile responses to N. brasiliensis. In this study the impact of IL-4Ra-responsive T cells on smooth muscle cell hypercontraction and their contribution to clearance of N. brasiliensis infection was defined. We showed that IL-4Ra-responsive T cells are required for optimal N. brasiliensis-induced intestinal hypercontractility, but not for worm expulsion. Wild type mice resist infection with N. brasiliensis and develop polarized TH2 responses with high IL-4/IL-13 and low IFN-c production [38?0]. Well-established TH2 induced effector mechanisms following N. brasiliensis infection are eosinophilia [4,41] mucosal mastocytosis [6], pathogen specific antibodies including IgE and IgG1 [4,5] goblet cell hyperplasia and promotion of TH2 cytokine responses. IL-4 has been implicated in driving the polarized TH2 response against N. brasiliensis,demonstrated by diminished type 2 responses in IL-42/2, IL4Ra2/2 and STAT-62/2 mice [16,24,28,37,42,43]. Although it is known that both IL-4Ra [13] and CD4+ T cells [20] are involved in worm clearance, more recent studies from us and others have shown that IL-4 responsive CD4+ T cells [28] or signalling through the STAT-6 pathway in these cells is not needed for worm expulsion. Indeed, IL-4 receptor expression by non-bone marrowderived cells is required to expel N. brasiliensis [44]. However, type 2 immunity is controlled by IL-4/IL-13 expression in haematopoetic non-eosinophil cells of the innate immune system [45]. This is consistent with the findings in our present study, as infected mice deficient in IL-4Ra expression on all T cell subpopulations showed impaired TH2 responses but still presentedIL-13 production in the mesenteric lymph nodes, able to respond with goblet cell hyperplasia.
