Type 3 Muscarinic Receptors Contribute to Homeostasis and Host Defense within the Gastrointestinal Tract

Abstract

Although activation of the alpha 7 nicotinic receptor exerts anti-inflammatory effects on immune cells, the contribution of muscarinic receptors to mucosal homeostasis, response to enteric pathogens, and modulation of immune cell function is undefined. WT and type 3 muscarinic receptor (M3R)-deficient (Chrm3<super>-/-</super>) mice were infected with <super>Citrobacter rodentium</super>, an attaching/effacing gram-negative bacterium that infects murine colon and evokes a highly polarized T_H1/T_H17 response, or Nippostrongylus brasiliensis, an enteric nematode that infects murine small intestine and induces a stereotypic T_H2 response. Permeability and expression of T_H1/T_H17 cytokines is increased in Chrm3<super>-/-</super> small intestine; however, no changes in permeability or cytokine expression occur in Chrm3<super>-/-</super> colon. Clearance of both C. rodentium and N. brasiliensis was delayed from Chrm3<super>-/-</super> mice; however, Chrm3<super>-/-</super> mice infected with C. rodentium retained their ability to mount a T_H1/T_H17 response whereas upregulation of T_H2 cytokines in response to N. brasiliensis was abrogated. Delayed clearance of C. rodentium from Chrm3<super>-/-</super> mice is associated with decreased goblet cell number and mucin 2 expression. Within Chrm3<super>-/-</super> small intestine, T_H2-dependent changes in gut function (smooth muscle hypercontractility, increased permeability, decreased secretion and absorption, and goblet cell expansion) did not occur despite infection with N. brasiliensis. Furthermore, treatment of bone-marrow derived macrophages (BMDM) with bethanechol, a muscarinic-specific agonist, induces a classically activated macrophage phenotype, an effect dependent on M3R. Chrm3<super>-/-</super> BMDM retain their ability to attain a classically activated phenotype when exposed to the T_H1 cytokine IFN-γ. Our data establish a role for M3R in host defense against enteric pathogens. Interestingly, in Chrm3<super>-/-</super> mice cytokine generation is intact in the setting of a T_H1/T_H17 inducing stimulus, but abrogated in the setting of a T_H2 inducing stimulus. In addition, M3R exerts pro-inflammatory effects on macrophage phenotype and function and contributes to barrier function and mucosal homeostasis in uninfected small intestine. These findings support an expanding role for cholinergic receptors not only in mucosal homeostasis and the response to inflammatory stimuli, but also in clearance of enteric pathogens.