TRPV1 controls innate immunity during Citrobacter rodentium enteric infection

Abstract

Mucosal immunity is critical to host protection from enteric pathogens and must be carefully controlled to prevent immunopathology. Regulation of immune responses can occur through a diverse range of mechanisms including bi-directional communication with the neurons. Among which include specialized sensory neurons that detect noxious stimuli due to the expression of transient receptor potential vanilloid receptor 1 (TRPV1) ion channel and have a significant role in the coordination of host-protective responses to enteric bacterial pathogens. Here we have used the mouse-adapted attaching and effacing pathogen Citrobacter rodentium to assess the specific role of the TRPV1 channel in coordinating the host response. TRPV1 knockout (TRPV1^-/-) mice had a significantly higher C. rodentium burden in the distal colon and fecal pellets compared to wild-type (WT) mice. Increased bacterial burden was correlated with significantly increased colonic crypt hyperplasia and proliferating intestinal epithelial cells in TRPV1^-/- mice compared to WT. Despite the increased C. rodentium burden and histopathology, the recruitment of colonic T cells producing IFNγ, IL-17, or IL-22 was similar between TRPV1^-/- and WT mice. In evaluating the innate immune response, we identified that colonic neutrophil recruitment in C. rodentium infected TRPV1^-/- mice was significantly reduced compared to WT mice; however, this was independent of neutrophil development and maturation within the bone marrow compartment. TRPV1^-/- mice were found to have significantly decreased expression of the neutrophil-specific chemokine Cxcl6 and the adhesion molecules Icam1 in the distal colon compared to WT mice. Corroborating these findings, a significant reduction in ICAM-1 and VCAM-1, but not MAdCAM-1 protein on the surface of colonic blood endothelial cells from C. rodentium infected TRPV1^-/- mice compared to WT was observed. These findings demonstrate the critical role of TRPV1 in regulating the host protective responses to enteric bacterial pathogens, and mucosal immune responses.

Fig. 1. TRPV1^−/− mice have an increased bacterial burden and colonic crypt hyperplasia during C. rodentium infection.

Wild-type (WT; open circles) and TRPV1^−/− mice (black circles) were infected with C. rodentium or given vehicle control (LB) by oral gavage. The number of fecal and colonic tissue adherent bacteria were assessed in WT and TRPV1^−/− mice at (A) 10 days p.i. and (B) 29 days p.i. (C) Hematoxylin and eosin (H&E) stained paraffin-embedded cross-sections of colon tissue from non-infected control, or 10, 29 days p.i. (Scale bar: 100μm), with (D) crypt length measured using FIJI. (E) Confocal images of distal colon tissue from non-infected, 10, or 29 days p.i. mice stained with Ki67 (red), CDH1 (green), and DAPI (blue), with (F) quantification of Ki67⁺ CDH1⁺ DAPI⁺ cells. Data are from 7-12 mice per group in 4 separate experiments, and presented as mean ± standard error of the mean: *, P < 0 .05, **, P < 0.01, and ***, P < 0.001 vs uninfected mice; #, P < .05, # #, P< 0.01 compared with WT mice; Student’s t test (A & B) and two-way ANOVA (D & F) with post-hoc analysis using Tukey’s multiple comparisons test. LB, Luria-Bertai; p.i., post-infection.

Fig. 2. T cell recruitment and cytokine production are unaffected by TRPV1 deficiency during C. rodentium infection.

(A & B) Colonic tissue sections were assessed for CD3⁺ (red) DAPI⁺ (blue) T cell infiltration in vehicle control (LB) and C. rodentium infected wild-type (WT) and TRPV1^−/− mice after 10-or 29-days p.i. (C) Lamina propria lymphocytes were assessed by flow cytometry to enumerate live CD45⁺ CD3⁺ CD4⁺ T cells and determine the frequency of (D) IFNγ⁺, (E) IL-17A⁺, or F) IL-22⁺ T cells in control and infected WT and TRPV1^−/− mice 10 days p.i. Data are presented as mean ± standard error of the mean: *, P < 0 .05, **, P < 0.01 and ***, P < 0.001; one-way ANOVA with post-hoc analysis using Tukey’s multiple comparisons test, with 4–11 animals per group. LB, Luria-Bertai; p.i., post-infection.

Fig. 3. Select innate immune responses are reduced in C. rodentium-infected TRPV1^−/− mice.

The host immune response during infection was assessed through qPCR conducted on colonic tissue from control or C. rodentium infected wild-type (WT) or TRPV1^−/− mice. These include the expression of proinflammatory cytokines (A) Il6, (B) Tnfα, (C) Il1β, (D) the antimicrobial peptide RegIIIy, (E) inducible nitric oxide synthase (Nos2), and (F) the neutrophil chemokine receptor Cxcr2. Data are presented as mean ± standard error of the mean, n= 7-13 animals/group: *, P < 0.05, **, P < 0.01, and ***, P < 0.0001 versus uninfected mice; #, P < 0.05 and ###, P < 0.0001 compared with WT C. rodentium-infected mice; one-way ANOVA with post-hoc analysis using Tukey’s multiple comparisons test. p.i., post-infection.

Fig. 4. Neutrophil recruitment is impaired in TRPV1^−/− mice during C. rodentium infection.

(A) Common neutrophil chemokines Cxcl1, Cxcl2, Cxcl3, and Cxcl6 were assessed by qPCR performed on colonic tissue from control or C. rodentium infected wild-type (WT) or TRPV1^−/− mice. (B-D) Neutrophil recruitment was determined by flow cytometry for live CD45⁺ CD11b⁺ Ly6G⁺ cells in the whole colon after epithelial cells were removed from WT and TRPV1^−/− mice given vehicle control (LB) and 10 days p.i. with C. rodentium (B) Representative flow plots and cumulative data of (C) frequency of live neutrophils and (D) total cell number of live neutrophils in the colon. (E-G) Cumulative data of frequency of live (E) Ly6C⁺ monocytes, (F) CD64⁺ macrophages, and (G) CD11c^hi conventional dendritic cells (cDC) in the lamina propria of the colon of WT and TRPV1^−/− mice at baseline and 10 days p.i. Data are presented as mean ± standard error of the mean: *, P < 0.05, **, P < 0.01 and ***, P < 0.001 compared with uninfected mice; #, P < 0.05 compared with WT C. rodentium-infected mice (A) *, P < 0.05, **, P < 0.01, and ***, P < 0.0001 (C-G); one-way ANOVA with post-hoc analysis using Tukey’s multiple comparisons test. 4–11 animals per group. LB, Luria-Bertani; p.i., post-infection.

Fig. 5. TRPV1 dependent neutrophil recruitment is driven by upregulation of ICAM-1 and VCAM-1 on blood endothelial cells.

(A & B) Colonic blood endothelial cells from wild-type (WT) and TRPV1^−/− mice were assessed for their expression of ICAM-1, VCAM-1, and MAdCAM-1 by flow cytometry at baseline and 10 days p.i. (C) Icam1, Vcam1, and Madcam1 mRNA were assessed by qPCR of the colonic tissue at baseline and 10 days p.i. Data are presented as mean ± standard error of the mean: *, P < 0.05, **, P < 0.01 and ***, P < 0.001; one-way ANOVA with post-hoc analysis using Tukey’s multiple comparisons test. 4–10 animals per group. LB, Luria-Bertani; p.i., post-infection.

Fig. 6. Neuronal TRPV1 signaling coordinates host protective immune responses during C. rodentium infection.

TRPV1⁺ neurons promote the recruitment of neutrophils by upregulating cell adhesion molecules, ICAM-1 and VCAM-1, on blood endothelial cells and by promoting the release of the chemokine, CXCL6. These factors collectively promote extravasation of neutrophils from the blood vessel into the site of infection for clearance of C. rodentium. Mice lacking TRPV1 exhibit dramatic decreases in recruitment of neutrophils necessary for C. rodentium clearance from the colon.