PMC

(A) Gene expression of Nos-2 in WT (black bars) and Chrm3^−/− (white bars) uninfected and C. rodentium-infected colon. N = 3–5 mice per genotype per time point.
(B) Gene expression of CD3 in WT (black bars) and Chrm3^−/− (white bars) uninfected and C. rodentium-infected colon. N = 3–5 mice per genotype per time point.
A one-way ANOVA with post hoc analysis for multiple comparisons was used to determine differences between comparisons as indicated below.
WT: Wild type. Chrm3^−/−: M3R-deficient. V: vehicle. 13: 13 DPI. 21: 21 DPI. ** p < 0.01 vs. WT vehicle. * p < 0.05 vs. WT vehicle. ## p < 0.01 vs. WT 13 DPI. # p < 0.05 vs. WT 13 DPI. ξξ p < 0.01 vs. Chrm3^−/− vehicle. ξ p < 0.05 vs. Chrm3^−/− vehicle.

(A) Gene expression of IFN-γ in WT (black bars) and Chrm3^−/− (white bars) uninfected and C. rodentium-infected colon. N = 3–5 mice per genotype per time point.
(B) Gene expression of TNF-α in WT (black bars) and Chrm3^−/− (white bars) uninfected and C. rodentium-infected colon. N = 3–5 mice per genotype per time point.
(C) Gene expression of IL-17A in WT (black bars) and Chrm3^−/− (white bars) uninfected and C. rodentium-infected colon. N = 3–5 mice per genotype per time point.
(D) Gene expression of IL-22 in WT (black bars) and Chrm3^−/− (white bars) uninfected and C. rodentium-infected colon. N = 3–5 mice per genotype per time point.
A one-way ANOVA with post hoc analysis for multiple comparisons was used to determine differences between comparisons as indicated below.
WT: Wild type. Chrm3^−/−: M3R-deficient. V: vehicle. 13: 13 DPI. 21: 21 DPI. ** p < 0.01 vs. WT vehicle. * p < 0.05 vs. WT vehicle. ## p < 0.01 vs. WT 13 DPI. ξξ p < 0.01 vs. Chrm3^−/− V.

(A) Gene expression of Chrm3 in WT BMDM treated with vehicle (black bar) or bethanechol 1–100 nM (white bars). Data are representative of three experiments.
(B) Gene expression of Chrm3 in vehicle, IFN-γ (10 ng/mL), and IL-4 (20 ng/mL) treated WT BMDM. Data are representative of three experiments.
(C) Gene expression of Chrm1, Chrm2, Chrm4, and α7 in vehicle (black bars) and IFN-γ (10 ng/mL, white bars) treated WT BMDM. Data are representative of three experiments. A one-way ANOVA with post hoc analysis for multiple comparisons was used to determine differences between comparisons as indicated below (A&B). Student’s t-test was used to determine differences in cholinergic receptor expression between WT and Chrm3^−/− macrophages (C).
** p < 0.01 vs. WT vehicle.

(A) Gene expression of Nos-2 in WT and Chrm3^−/− BMDM treated with vehicle (black bars) or bethanechol 100 nM (white bars). Data are representative of three experiments.
(B) Gene expression of Arg-1 in WT BMDM treated with vehicle (black bar) or bethanechol (100 nM, white bar). Data are representative of three experiments.
(C) Gene expression of Nos-2 in WT and Chrm3^−/− BMDM treated with vehicle (black bars) or atropine 50 nM (white bars). Data are representative of three experiments.
(D) Gene expression of Nos-2 in WT and Chrm3^−/− BMDM treated with vehicle or IFN-γ (10 ng/mL, white bars). Data are representative of three experiments.
Student’s t-test was used to determine differences in Nos-2/Arg-1 expression between WT and Chrm3^−/− macrophages (A, B, C).
WT: Wild type. Chrm3^−/−: M3R-deficient. * p < 0.05 vs. WT vehicle. ** p < 0.01 vs. WT vehicle.
(E) Gene expression of Nos-2 in WT BMDM treated with IFN-γ 10 ng/mL (black bars), IFN-γ 10 ng/mL and bethanechol 100 nM (white bars), and IFN-γ 10 ng/mL, bethanechol 10 nM, and atropine 50 nM (hatched bars). Data are representative of three experiments.
(F) Gene expression of Nos-2 in Chrm3^−/− BMDM treated with IFN-γ 10 ng/mL (black bars), IFN-γ 10 ng/mL and bethanechol 100 nM (white bars), and IFN-γ 10 ng/mL, bethanechol 10 nM, and atropine 50 nM (hatched bars). Data are representative of three experiments.
A one-way ANOVA with post hoc analysis for multiple comparisons was used to determine differences between comparisons as indicated below (E, F).
WT: Wild type. Chrm3^−/−: M3R-deficient. IFN: interferon-γ. BTH: bethanechol. ATR: atropine.
* p < 0.05 vs. IFN-γ.

(A) BrdU-labeled cells in uninfected and C. rodentium-infected WT and Chrm3^−/− colon. Relative number of BrdU-positive cells is increased in Chrm3^−/− colon 13 DPI. Images are representative of two experiments.
WT: Wild type. Chrm3^−/−: M3R-deficient. BrdU: bromodeoxyuridine.
(B) Mean ± SEM of BrdU-positive cells per crypt in uninfected and C. rodentium-infected WT (black bars) and Chrm3^−/− (white bars) colon mucosa. Twenty crypts counted per genotype per time point.
A one-way ANOVA with post hoc analysis for multiple comparisons was used to determine differences between comparisons as indicated below.
WT: Wild type. Chrm3^−/−: M3R-deficient. SEM: Standard error. V: vehicle. 13: 13 DPI. 21: 21 DPI. ** p < 0.01 vs. WT vehicle. * p < 0.05 vs. WT vehicle. ## p < 0.01 vs. WT 13 DPI. ξξ p < 0.01 vs. Chrm3^−/− vehicle.

Proposed model of effects of M3R on macrophage phenotype.
(A) In the absence of an inflammatory stimulus, acetylcholine acts at both α7 and M3R with net effect driving the macrophage towards an AAM phenotype.
(B) In the presence of T_H1/T_H17 cytokines, M3R expression on macrophages is increased and acetylcholine acts at relatively greater number of M3R, causing the macrophage to attain a more classically activated phenotype (or a less alternatively activated phenotype).
α7: alpha 7 nicotinic receptor. M3: type 3 muscarinic receptor. C. rod: C. rodentium

(A) Goblet cells per crypt in uninfected and C. rodentium-infected WT (black bars) and Chrm3^−/− (white bars) mice. N = 3 mice per genotype per time point with 10 crypts counted per mouse.
(B) Gene expression of CLCA3 in WT (black bars) and Chrm3^−/− (white bars) uninfected and C. rodentium-infected colon. N = 3–5 mice per genotype per time point.
A one-way ANOVA with post hoc analysis for multiple comparisons was used to determine differences between comparisons as indicated below.
(C) Gene expression of Muc2 in WT (black bars) and Chrm3^−/− (white bars) uninfected and C. rodentium-infected colon. N = 3–5 mice/genotype.
(D) Gene expression of Muc5AC in WT (black bars) and Chrm3^−/− (white bars) uninfected and C. rodentium-infected colon. N = 3–5 mice/genotype.
A one-way ANOVA with post hoc analysis for multiple comparisons was used to determine differences between comparisons as indicated below.
(E) H&E stained sections of uninfected and C. rodentium-infected WT and Chrm3^−/− colon.
WT: Wild type. Chrm3^−/−: M3R-deficient. V: vehicle. 13: 13 DPI. 21: 21 DPI. ** p < 0.01 vs. WT vehicle. * p < 0.05 vs. WT vehicle. ## p < 0.01 vs. Chrm3^−/− vehicle. # p < 0.05 vs. Chrm3^−/− vehicle. H&E: hematoxylin and eosin.

(A) Spleen weight per body weight in C. rodentium-infected Chrm3^−/− mice 13 DPI is increased compared to spleen weight per body weight in WT mice 13 DPI and uninfected Chrm3^−/− mice. Each symbol represents one mouse. A one-way ANOVA with post hoc analysis for multiple comparisons was used to determine differences between comparisons as indicated below.
WT: Wild type. Chrm3^−/−: M3R-deficient. V: vehicle. 13: 13 DPI. 21: 21 DPI. ** p < 0.01 vs. WT 13. ξξ p < 0.01 vs. WT V. # p < 0.05 vs. Chrm3^−/− V.
(B) At 13 DPI C. rodentium was detected in 1 of 4 WT spleens and 3 of 4 Chrm3^−/− spleens. Each symbol represents one mouse. The dashed line indicates the limit of detection for the assay.
CFU: colony forming unit. WT: Wild type. Chrm3^−/−: M3R-deficient. V: vehicle.

(A) Fecal bacterial content of C. rodentium infected WT (black bars) and Chrm3^−/− (white bars) mice at 6, 9, 12, 14, 16, and 19 DPI. N = 8–10 mice per strain at 6, 9, 12 DPI. N = 3–5 mice per strain at 14, 16, 19 DPI. Student’s t-test was used to determine differences in bacterial content between WT and Chrm3^−/− mice at each time point. The dashed line indicates the limit of detection for the assay.
CFU: colony forming unit. WT: Wild type. Chrm3^−/−: M3R-deficient. * p < 0.05 vs. WT. CR: C. rodentium. DPI: days post-infection.
(B) Immunofluorescent staining demonstrating persistent adherence of C. rodentium in Chrm3^−/− colon mucosa 13 DPI. Uninfected WT and Chrm3^−/− mucosa serve as negative controls. Images are representative of two experiments.
WT: Wild type. Chrm3^−/−: M3R-deficient. DAPI: 4’,6-diamidino-2-phenylindole.