Table 1.
Antibodies used for confocal microscopy.
Fig 1.
Infection with C. rodentium recruits ChAT+ T-cells to the colon.
(A) Confocal microscopy was conducted on colonic tissues from LB or C. rodentium infected ChAT-GFP reporter mice using DAPI, anti-CD3, and anti-GFP. (B) These CD3+ ChAT-GFP+ cells (inset “I”) where then quantified in 5 random fields from LB control, or 6, 10, 21, or 30 days post-infection with C. rodentium. * P < 0.05 ANOVA, with n = 5–6 mice per timepoint.
Fig 2.
Flow cytometric characterization of ChAT-GFP+ T-cells.
(A) Lamina propria lymphocytes were isolated from ChAT-GFP mice 10 days after C. rodentium infection, or (LB gavaged) controls. Representative gating strategy depicts analysis of (lymphocytes, single, cells, live CD3+). CD4+ ChAT-GFP+ or CD4+ ChAT-GFP- cells expressing IFNγ, IL-17A, or IL-22 in each of these populations (light grey: non-stimulated control, green; ChAT-GFP+ cells stimulated with PMA/ionomycin, dark grey: ChAT-GFP- cells stimulated with PMA/ionomycin). The amount of each cytokine was quantified by MFI (B), and the frequency of these cells per live CD3+ cell (C). * P < 0.05 ANOVA, with n = 4–6 mice per group.
Fig 3.
Increased C. rodentium colonization in ChAT T-cell cKO mice without increased histopathology.
(A) The number of colonic tissue adherent bacteria were assessed in WT and ChAT T-cell cKO mice 10 days p.i. (B) Confocal imaging of uninfected and infected WT and ChAT T-cell cKO mice demonstrates increased adherent bacteria in the luminal surface of intestinal epithelial cells (IEC, CDH1+ DAPI+). (C) Infection of WT and ChAT T-cell cKO mice induces crypt hyperplasia as indicated by significantly increased crypt lengths. (D&E) and Ki67+ IEC compared to uninfected controls. Data are representative from 3 separate experiments with 3–5 mice per group. ND = not detected, *, # P < 0.05 ANOVA.
Fig 4.
Conditional ablation of ChAT in T-cells increases C. rodentium-induced innate inflammatory genes.
The immune response during infection was assessed through qRT-PCR conducted on colonic tissues from LB or C. rodentium infected WT or ChAT T-cell cKO mice. Expression of the cytokines Il1-β, Il-6, and Tnfα, Ifnγ, Il-17a and Il-22 was evaluated in naïve and infected WT and ChAT T-cell cKO mice. *, # P < 0.05 ANOVA, with n = 8–10 mice per group.
Fig 5.
Loss of T-cell derived ACh reduces Nos2 expression during C. rodentium infection.
Expression of genes characteristic of macrophage polarization were assessed by qRT-PCR on colonic tissue from LB or C. rodentium infected WT or ChAT T-cell cKO mice. *, # P < 0.05 ANOVA, with n = 8–10 mice per group.
Fig 6.
NOS2 is reduced in C. rodentium infected Intestinal epithelial cells from ChAT T-cell cKO mice.
(A) Localization and quantification of NOS2 was conducted on fixed colonic tissues from LB and C. rodentium infected WT and ChAT T-cell cKO mice, 10 days p.i. Confocal microscopy was conducted using DAPI and antibodies directed against epithelial e-cadherin (CDH1), and NOS2. (B) Expression of NOS2 per IECs (DAPI+ CDH1+ cells) was then quantified using Imaris. (C) In a separate cohort of mice, NOS2 expression was evaluated 10 days p.i in isolated IEC by flow cytometry. IEC were identified as live/single/CD45- EpCAM+ cells. Expression of NOS2 was determined by MFI and is summarized in (D). *P <0.05 ANOVA n = 4–8 mice/group.
Fig 7.
Nos2 expression induced by IFNγ in IEC is enhanced by cholinergic agonists.
CMT-93 cells were stimulated with vehicle (control), Carbachol (‘Cch’ 100 μM), IFNγ (1 ng/mL), or IFNγ (1ng/mL) + Cch (100 μM) for 3h. Expression of IFNγ-induced genes Ciita, Irf1 and Nos2 was quantified by qRT-PCR. *, # P<0.05, **P < 0.01, ANOVA, n = 4 independent experiments.