Type 3 Muscarinic Receptors (M3R) Contribute to Expulsion of Nippostrongylus brasiliensis Through Induction of Th2 Cytokines

Abstract

Introduction: Lower rates of helminth infection in persons from developed countries are associated with a higher prevalence of autoimmune diseases including IBD (Weinstock & Elliott, 2009). In mice, infection with the enteric nematode Nippostrongylus brasiliensis (Nb) induces a Th2 cytokine response in small intestine (SI) and characteristic STAT6/IL-13 dependent changes in gut function including increased permeability, smooth muscle hypercontractility, decreased fluid absorption and secretion, and goblet cell expansion. We showed previously that M3R promotes clearance of the enteric bacterium Citrobacter rodentium (Cr). The contribution of M3R to expulsion of enteric nematodes is unclear. Aim: To investigate the hypothesis that M3R affects clearance of Nb. Methods: WT and M3R-/- mice were infected with 500 third-stage Nb larvae. Worm and fecal egg counts were performed to assess clearance. Expression of the Th2 cytokines IL-4 and IL-13, the Th1/Th17 cytokines IFN-γ, TNF-α, and IL-17, and the T-cell marker CD3 was assessed by real-time PCR. Epithelial permeability, secretion and absorption in response to acetylcholine and glucose, smooth muscle tension, and goblet cell expansion were determined. Results: Worm and fecal egg counts were significantly higher in Nb-infected M3R-/- mice. Expression of IFN-γ was modestly, but significantly, upregulated in Nb-infected M3R-/- SI. TNF-α and IL-17 expression were similar. Expression of IL-4 and IL-13 was upregulated significantly in Nb-infected WT SI, but abrogated in Nb-infected M3R-/- SI (Table). These changes were not due to altered T cell recruitment to M3R-/- SI as expression of CD3 was similar in Nb-infected WT and M3R-/- SI. Characteristic alterations in gut function occurred in Nb-infected WT mice, but not Nb-infected M3R-/- mice. Summary: Clearance of Nb is delayed from M3R-/- mice and is associated with absent upregulation of Th2 cytokines and a modest upregulation of IFN-γ. The impaired Th2 response in M3R-/- mice is associated with absent downstream STAT6/IL-13 dependent alterations in gut physiology that promote worm expulsion. Conclusion: We previously reported that M3R-deficiency is associated with delayed clearance of Cr, but an intact Th1/Th17 cytokine response. Our findings here support a role for M3R in the induction of Th2 cytokines in response to enteric nematode infection and suggest a differential role for M3R in the regulation of and response to pathogens that trigger Th1/Th17 (Cr) versus Th2 (Nb) immune responses.Table 1: Expression of IL-4 and IL-13 in uninfected and Nb-infected WT and M3R-/- small intestine