Type 3 Muscarinic Receptors Modulate Macrophage Phenotype in the Presence of Th2 Cytokines

Abstract

Purpose: Macrophages (Mφ) play a critical role in IBD. Resident Mφs within the intestinal lamina propria are exposed to many antigens, but are tolerant to stimulation and prevent inflammation (Heinsbroek & Gordon, 2009). We showed previously that stimulation of type 3 (M3R) muscarinic receptors (MR) encoded by Chrm3 promoted the development of pro-inflammatory classically activated Mφs (CAM) (McLean, et al, 2012). Whether M3R plays a role in the development of alternatively activated Mφs (AAM), which promote wound healing and tissue repair, is unclear. Our aim was to test the hypothesis that M3R plays a role in AAM development. Methods: Bone marrow cells were isolated from WT and Chrm3-/- mice and differentiated in the presence of monocyte colony stimulating factor (M-CSF) for 7 days. Flow cytometry was used to analyze cell populations. After 24 hour treatment with a muscarinic receptor-selective agonist, bethanechol (BTH), +/- the Th2 cytokine IL-4, cells were harvested and mRNA expression of the AAM marker Arg-1 assess by real-time PCR. Results: We confirmed that ˜95% of our cell population was positive for both CD11b and F4/80, indicating that these cells are Mφs. These Mφs express mRNA for M1R, M2R, M3R and M4R. In WT Mφs, BTH treatment significantly attenuated Arg-1 expression, indicating a constitutive bias against AAM development. Treatment of naïve WT Mφs with IL-4 significantly up-regulated Arg-1 expression; addition of BTH to IL-4 did not enhance this effect. In contrast, in Chrm3-/- Mφs up-regulation of Arg-1 in response to IL-4 was significantly greater than that observed in WT Mφs. Moreover, addition of BTH to IL-4 potentiated this up-regulation of Arg-1 in Chrm3-/- Mφs. Conclusion: Treatment with BTH alone attenuated the development of AAMs from naïve WT Mφs. As expected, treatment of WT Mφs with IL-4 induced the development of AAMs. In WT Mφs, addition of BTH to IL-4 did not modulate this effect. In M3R-deficient Mφs, IL-4 further polarized Mφs to the AAM phenotype. Notably, exposing M3R-deficient Mφs to both IL-4 & BTH further enhances AAM polarization. We previously reported that M3R expression exerts pro-inflammatory effects on Mφs. Our findings suggest that in the presence of IL-4, M3R activation attenuates the development of AAMs and, in the presence of IL-4, further polarizes M3R-deficient Mφs to an AAM phenotype, possibly by acting at M1R, M2R, and/or M4R. This effect is not observed in WT Mφs, likely due to M3R activity. These data further support a pro-inflammatory role of M3R. Pharmacologic attempts to manipulate Mφ phenotypes should focus on blocking M3R expression or activation to promote AAMs, a strategy that may reduce disease activity or maintain remission in IBD patients.