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. 2016 Apr 12:6:23820.
doi: 10.1038/srep23820.

Ablating the aryl hydrocarbon receptor (AhR) in CD11c+ cells perturbs intestinal epithelium development and intestinal immunity

Song Hui Chng  1   2   3 , Parag Kundu  2 , Carmen Dominguez-Brauer  4 , Wei Ling Teo  5 , Kaname Kawajiri  6 , Yoshiaki Fujii-Kuriyama  7 , Tak Wah Mak  4 , Sven Pettersson  2   3   8
Affiliations

Ablating the aryl hydrocarbon receptor (AhR) in CD11c+ cells perturbs intestinal epithelium development and intestinal immunity

Song Hui Chng et al. Sci Rep. .

Abstract

Diet and microbiome derived indole derivatives are known to activate the ligand induced transcription factor, the Aryl hydrocarbon Receptor (AhR). While the current understanding of AhR biology has confirmed its role in mucosal lymphocytes, its function in intestinal antigen presenting cells (APCs) is poorly understood. Here, we report that Cre-mediated deletion of AhR in CD11c-expressing cells in C57/BL6 mice is associated with altered intestinal epithelial morphogenesis in vivo. Moreover, when co-cultured with AhR-deficient DCs ex vivo, intestinal organoids showed reduced SRY (sex determining region Y)-box 9 and increased Mucin 2 expression, which correlates with reduced Paneth cells and increased goblet cell differentiation, similar to the data obtained in vivo. Further, characterization of intestinal APC subsets, devoid of AhR, revealed an expression pattern associated with aberrant intrinsic Wnt pathway regulation. At a functional level, the loss of AhR in APCs resulted in a dysfunctional epithelial barrier, associated with a more aggressive chemically induced colitis compared to wild type animals. Our results are consistent with a model whereby the AhR signalling pathway may participate in the regulation of innate immunity through intestinal epithelium development and mucosal immunity.

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Figures

Figure 1
Figure 1. 11cAhR−/− mice are more susceptible to DSS-induced colitis.
(a) The body weight of each mouse within various groups of mice were recorded daily and expressed as percentage of initial weight in grams over the course of the 2% DSS induced acute colitis. Data points are shown as mean ± SEM (n ≥ 8 per group). (b) Colon lengths of mice from various groups were measured in centimeters at end-point. Each symbol represents an individual mouse and horizontal lines show the mean ± SEM (n ≥ 8 per group), Student’s t test: **P < 0.01; ***P < 0.001. (c) Quantitative RT-PCR analysis of acute phase proteins genes expression in the liver post-DSS treatment. Bar graphs depict mean ± SEM (n ≥ 8 per group), Mann-Whitney test: *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Figure 2
Figure 2. Altered intestinal epithelium morphogenesis in adult 11cAhR−/− mice.
(a) Quantitative RT-PCR analysis on Wnt-target genes expression from ileum epithelial scrapings. Data were pooled from 3 independent experiments and presented as mean ± SEM. Each symbol represents a single mouse. (b) In situ hybridization (ISH) and Periodic acid–Schiff (PAS) staining performed on paraffin-embedded sections of the ileum. Arrows point at stained goblet cells in the villus. (c) Quantification of intestinal stem cell and Paneth cell numbers. Graphs depict mean ± SEM of counted cells per crypt. More than 30 crypts were counted per animal (n = 4). (d) Quantification of goblet cells and villus length. Goblet cell numbers were counted and presented as a function of its respective villus length. Graphs show mean ± SEM (n = 3). Student’s t-test: *P < 0.05; ****P < 0.0001.
Figure 3
Figure 3. Differentiation of progenitors into secretory cell types are reduced in organoids co-cultured with AhR-deficient DCs.
(a–d) CD11c+MHCIIhi DCs from the MLNs of 11cAhR +/+ or 11cAhR−/− mice were pooled (n = 3) and co-cultured with isolated small intestinal crypts from AhRfl/fl mice. Co-cultures were kept for five days. (a) Relative expression of epithelial lineage markers, transcription factors and proliferative markers were examined via quantitative RT-PCR. Each symbol represents a single biological replicate and data presented as mean ± SEM. Dataset shown was one out of two independent experiments performed with similar results. (b) Bright-field images of co-cultures are shown at different optical zooms. (c) Schematic showing how sizes of organoids were measured. (d) Size of organoids co-cultured presented as ±SEM from the two groups. Student’s t-test: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 4
Figure 4. Classical cell-surface markers expressions in intestinal APCs were down regulated in the absence of AhR.
(a) Live/MHCIIhi cells were gated and the three major subsets of APCs in the SI LP are presented as shown based on their expression of markers: CD11c, CD11b, CD103 and F4/80. (b–c) Quantification of the three major subsets of APCs as defined in (b), shown as percentages among total live/MHCIIhi cells. Results are mean ± SEM pooled from 5 independent experiments (n ≥ 10). (d) Quantification of MLN CD103+ DCs as mean percentages ± SEM is shown (n = 6). (e) Mean fluorescent intensity (MFI) representing expression levels of various cell surface markers of gated populations of all three groups are shown. Data are mean ± SEM pooled from 4–5 independent experiments (n ≥ 9). Mann-Whitney test: *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Figure 5
Figure 5. Intestinal LP APCs from 11cAhR−/− mice exhibit aberrant expression of Wnt signalling constituents.
Quantitative RT-PCR performed on cDNA generated from FACS sorted populations corresponding to R1, R2 and R3 APC subsets. Each symbol represents data from one single mouse. Data presented as ±SEM were pooled from 3–5 independent experiments. Mann-Whitney test: *P < 0.05; **P < 0.01; ns, not significant.
Figure 6
Figure 6. AhR-deficiency in intestinal APC subsets did not result in overt mucosal immunity.
(a,b) Isolated lymphocytes from the small intestinal LP or the MLN re-stimulated for 4 hours ex vivo. (a) Representative FACS plots showing intracellular staining for Foxp3, IFNγ or IL-17 A in CD4+ T cells. (b) Graphs illustrate mean percentages within the CD4+ population ±SEM. Each symbol represents one mouse. Data were pooled from 3–4 independent experiments. Mann-Whitney test: *P < 0.05; **P > 0.01; ns, not significant.
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