A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ dendritic cells to the colon in response to Trichuris muris infection

Abstract

The ability of the colon to generate an immune response to pathogens, such as the model pathogen Trichuris muris, is a fundamental and critical defense mechanism. Resistance to T. muris infection is associated with the rapid recruitment of dendritic cells (DCs) to the colonic epithelium via epithelial chemokine production. However, the epithelial-pathogen interactions that drive chemokine production are not known. We addressed the role of the cytosolic pattern recognition receptor Nod2. In response to infection, there was a rapid influx of CD103(+)CD11c(+) DCs into the colonic epithelium in wild-type (WT) mice, whereas this was absent in Nod2(-/-) animals. In vitro chemotaxis assays and in vivo experiments using bone marrow chimeras of WT mice reconstituted with Nod2(-/-) bone marrow and infected with T. muris demonstrated that the migratory function of Nod2(-/-) DCs was normal. Investigation of colonic epithelial cell (CEC) innate responses revealed a significant reduction in epithelial production of the chemokines CCL2 and CCL5 but not CCL20 by Nod2-deficient CECs. Collectively, these data demonstrate the importance of Nod2 in CEC responses to infection and the requirement for functional Nod2 in initiating host epithelial chemokine-mediated responses and subsequent DC recruitment and T-cell responses following infection.

Figure 1. Impaired recruitment of CD103⁺ DCs to the colonic epithelium in Nod2^−/− mice in response to T. muris

WT and Nod2^−/− mice were infected orally with approximately 175 embryonated T. muris eggs. Colonic epithelial cells from WT mice were analysed before and after infection by qPCR for Nod2 (A) and Rip2 (B) mRNA n=3-12 Data for time points D0 and D1 are pooled from two individual experiments. Lamina propria and intraepithelial cells were isolated from the large intestine and stained for CD45, MHCII, CD11c, CD103 and F4/80 on D0, D1, D2, D5, D7 and D9 post infection. (C) Gating strategy for CD103⁺ and F4/80 positive cells isolated from the large intestine. (D) Percentage of MHCII⁺CD11c⁺CD103⁺F4/80⁻ DCs were calculated as a percentage of the CD45⁺ cell population, (E) Percentage of MHCII+CD11c+CD103⁻ F4/80⁺ macrophages were calculated as a percentage of the CD45⁺ cell population. Data is representative of at least 3-4 mice each from 3 experiments (total n=9-12) with the exception of D7 and D9 which are representative of 1 experiment (n=4). (F)Mice infected with T. muris were injected with BRDU 16 hours before sacrifice. Lamina propria and intraepithelial cells were isolated from the large intestine and stained for CD45, MHCII, CD11c, CD103 and BrdU and the number of BRDU⁺CD103⁺ of CD45⁺ population quantified.n= 5 *P= <0.05, **P<0.001.

Figure 2. Impaired recruitment of CD103⁺ DCs to the colonic epithelium in Nod2^−/− mice in response to T. muris

Frozen caecal and colon sections were taken at autopsy, sectioned and stained for nuclei (blue), cytokeratin (green) and CD11c (red). Representative images shown in A (Scale bar for naïves = 100μm. D2 p.i. = 50 μm). (B) Quantification of CD11c⁺ cells in Nod2^−/− and C57BL/6 mice. Frozen caecal and colon sections were taken at autopsy, sectioned and stained for nuclei (Dapi, blue) CD11c (red) and CD103 or F4/80 (green). Co-localised cells are shown in yellow. (C) Representative image of CD103 staining. (Scale bar = 50μm (insert= 30 μm)). (D) Quantification of dual stained CD103⁺ CD11c⁺ cells. (E)Representative image of F4/80 staining. (Scale bar = 50μm (insert= 40 μm)). (F) Quantification of dual stained F4/80 CD11c cells. n = 3 (C57BL/6) n=4 (Nod2^−/−)/timepoint. Data shown are mean +/-SEM.

Figure 3. Delayed expulsion kinetics of T. muris in Nod2^−/− mice

Mice were infected orally with approximately 175 embryonated T. muris eggs and worm burdens were assessed at D21 post infection (A). Data shown are for individual mice with mean values per group. Data is representative of two independent experiments with n=4 (Nod2^−/−) and n=5 (C57BL/6) in each experiment. (B) Mesenteric lymph nodes cells re-stimulated with E/S at day 21p.i. IL-13 (B) and IFN-γ (C) levels in supernatants were then assayed by cytokine bead array. MLN cells were harvested from naïve mice and at D7, 10 and 18 post-infection and the numbers of T cells enumerated (D and E) and the number of gut homing (CCR9+) CD4 T cells (F) and activated/memory CD62L^loCD44^hi cells (G) enumerated by flow cytometry. Analysis of Data is representative of two independent experiments each with n=2-4 (naïve animals) and n=5 (infected animals). *= P<0.05

Figure 4. Increased CD103⁺ cells in the large intestine is not due to in situ proliferation of DCs and Nod2 has no role in basophil recruitment to the large intestine

Mice were infected orally with approximately 175 embryonated T. muris eggs. Lamina propria and intraepithelial cells were isolated from the large intestine and stained for Lineage markers (CD4, CD8α, B220, CD19), c-kit and FcεR. (A)Gating strategy for basophils (Lin⁻ C-kit⁻ FcεR⁺) cells. (B) Quantification of Lin⁻ c-kit⁻ FcεR⁺ cells in the large intestine and MLN. n= 5 (C57BL/6) n= 4 (Nod2^−/−).

Figure 5. Nod2^−/− dendritic cells express chemokine receptors and can migrate effectively in vitro

Mice were infected orally with approximately 175 embryonated T. muris eggs. Lamina propria and intraepithelial cells were isolated from the large intestine Dendritic cells isolated from the large intestine were stained with chemokine receptors CCR5 (A) and CCR2 (B). (C) A chemotaxis assay was performed using colonic DCs and CCL2 (10ng/ml) and CCL5 (1ng/ml) n= 5 (C57BL/6) n= 3-4 (Nod2^−/−). (D) BMDC were cultured with medium (negative control), LPS 5μg/ml) or T. muris antigen (5μg/ml) for 24 hours before being stained for CD45, CD11c and MHC-II and analysed by flow cytometry. Data shown is the average of three replicates and is representative of two experiments.

Figure 6. Nod2^−/− dendritic cells can migrate to a Nod2^+/+ epithelium

(A) A schematic representation of generation of bone marrow chimeras. (B) Successful reconstitution was determined by harvesting bone marrow from C57BL/6^Nod−/− mice and stimulating with medium (negative control), MDP (1.0μg/ml) and LPS (100ng/ml, positive control). DC responses to the various ligands were analysed by assessing the level of maturation by CD86, CD40 and MHC-II up-regulation by flow cytometry. The graphs show the percentage of MHCII^hiCD40⁺ CD86⁺ CD11c cells in each culture condition. (C) Lamina propria and intraepithelial cells were isolated from the large intestine of C57BL/6^Nod−/− and C57BL/6^wt mice and stained for CD45, MHC-II, CD11c, CD103 and F4/80 on D5 post infection and measured as a percentage of the CD45⁺ population. (n=4-5) (D) A defect in CD45 cell recruitment was also observed in Nod2^wt mice compared with controls. (E) Immunohistochemistry staining revealed a defect in CD11c⁺CD103⁺ DC recruitment in Nod2^wt mice compared with C57BL/6^Nod−/− mice. * p=0.01.

Figure 7. Nod2^−/− colonic epithelial cells are unable to produce the chemokines CCL2 CCL5 and CCL20

Colonic epithelial cells were cultured from C57BL/6 and Nod2^−/− mice and the levels of CCL2, CCL5, CCL20 and IL33 were measured by ELISA (A-D) (n=8 and data is representative of two independent experiments). Colonic epithelial cells were harvested from the colon of naïve and infected C57BL/6 and Nod2^−/− mice on D1 post infection. mRNA levels of CCL5, CCL2 and IL-33, were measured by qPCR (E-G) and data shows relative expression (n=11-13 and is pooled from two individual experiments).