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. 2014 Apr 1;306(7):G582-93.
doi: 10.1152/ajpgi.00218.2013. Epub 2014 Feb 6.

γδ T-cell-deficient mice show alterations in mucin expression, glycosylation, and goblet cells but maintain an intact mucus layer

Olivia I Kober  1 , David Ahl, Carmen Pin, Lena Holm, Simon R Carding, Nathalie Juge
Affiliations

γδ T-cell-deficient mice show alterations in mucin expression, glycosylation, and goblet cells but maintain an intact mucus layer

Olivia I Kober et al. Am J Physiol Gastrointest Liver Physiol. .

Abstract

Intestinal homeostasis is maintained by a hierarchy of immune defenses acting in concert to minimize contact between luminal microorganisms and the intestinal epithelial cell surface. The intestinal mucus layer, covering the gastrointestinal tract epithelial cells, contributes to mucosal homeostasis by limiting bacterial invasion. In this study, we used γδ T-cell-deficient (TCRδ(-/-)) mice to examine whether and how γδ T-cells modulate the properties of the intestinal mucus layer. Increased susceptibility of TCRδ(-/-) mice to dextran sodium sulfate (DSS)-induced colitis is associated with a reduced number of goblet cells. Alterations in the number of goblet cells and crypt lengths were observed in the small intestine and colon of TCRδ(-/-) mice compared with C57BL/6 wild-type (WT) mice. Addition of keratinocyte growth factor to small intestinal organoid cultures from TCRδ(-/-) mice showed a marked increase in crypt growth and in both goblet cell number and redistribution along the crypts. There was no apparent difference in the thickness or organization of the mucus layer between TCRδ(-/-) and WT mice, as measured in vivo. However, γδ T-cell deficiency led to reduced sialylated mucins in association with increased gene expression of gel-secreting Muc2 and membrane-bound mucins, including Muc13 and Muc17. Collectively, these data provide evidence that γδ T cells play an important role in the maintenance of mucosal homeostasis by regulating mucin expression and promoting goblet cell function in the small intestine.

Keywords: T cell receptor-γδ; intestinal intraepithelial lymphocytes; mouse mucin.

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Figures

Fig. 1.
Fig. 1.
TCRδ−/− mice are more susceptible to dextran sodium sulfate (DSS)-induced colitis than wild-type (WT) mice. TCRδ−/− and WT mice (n = 13) were given 2.5% DSS in drinking water for 7 days. The disease activity index (DAI) score for all 4 groups of mice (WT untreated, WT DSS-treated, TCRδ−/− untreated, and TCRδ−/− DSS-treated) was calculated daily on the basis of stool consistency, fecal blood content, and weight loss (A). Tissue sections for all 4 groups of mice were stained with hematoxylin and eosin Y (H&E) for histological analysis (B). Colon length was measured with a millimeter ruler on the final day of the DSS study (C). The extent of epithelial injury was scored blindly from H&E-stained tissue sections of the distal colon of DSS-treated WT and TCRδ−/− mice (D). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 2.
Fig. 2.
Mucin-filled goblet cell counts and crypt depth measurements in the small intestine (SI) and colon (C) of TCRδ−/− and WT mice. SI and colon tissue of WT and TCRδ−/− mice (n = 7) was stained with periodic acid-Schiff and Alcian blue (PAS/AB) (A). Average mucin-filled goblet cell (arrow indicates mucin-filled goblet cell) number (B) and crypt depth (C) were calculated from 10 crypts per mouse tissue, for 7 mice. SI and colon tissue of WT and TCRδ−/− mice (n = 3–5) was stained with anti-Ki67 (D) and the percentage of proliferating cells was determined from this (E). Magnification, ×400; scale bars, 50 μm. *P < 0.05; **P < 0.01.
Fig. 3.
Fig. 3.
Mucin-filled goblet cell counts and Muc2 staining in the colon of WT and TCRδ−/− mice following DSS treatment. Average mucin-filled goblet cell numbers (n = 6) were calculated from tissue area measurements. *P < 0.05. Distal colon sections of DSS-treated WT and TCRδ−/− mice (n = 3) were stained with anti-MUC2 (green) and counterstained with PNA and WGA (red) and DAPI (blue). Rabbit IgG represents the isotype control. Magnification, ×200; scale bars, 100 μm; Inset magnification, ×400.
Fig. 4.
Fig. 4.
In vivo mucus measurements in the ileum and distal colon of TCRδ−/− and WT mice. Mucus thickness (n = 7) was measured in vivo using a micropipette connected to a micromanipulator. Total mucus thickness (Total) indicates the thickness of the firm and loose layer. After removal of the loose layer, firm layer thickness was immediately measured (Firm 1). The mucus layer was allowed to regenerate and measured (60 min). The firm mucus layer was measured again after a second removal (Firm 2) to confirm its steady state.
Fig. 5.
Fig. 5.
Sialic acid and O-glycan concentration analysis in the SI and colon of WT and TCRδ−/− mouse mucus. Sialic acid concentration was determined by ninhydrin colorimetric assay for SI and C tissue (n = 5) (A). O-glycan concentration was determined by alkaline borohydrate colorimetric assay for SI and C tissue (n = 6) (B).
Fig. 6.
Fig. 6.
Glycosyltransferase and mucin expression analysis in the SI and colon of WT and TCRδ−/− mice. Glycosyltransferase (GT) gene expression of the SI of WT and TCRδ−/− mice (n = 3) was assessed by quantitative RT-PCR (qRT-PCR) and is represented as relative gene expression (2−ΔCt) (A). Gene expression of Muc1, Muc2, Muc3, Muc4, Muc5AC, Muc6, Muc12, Muc13, Muc17, and Muc19 was analyzed in the SI and colon of WT and TCRδ−/− mice (n = 3) by qRT-PCR, and represented as the relative gene expression (2−ΔCt) (B). *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 7.
Fig. 7.
TCRδ−/− SI crypts display similar growth patterns and differentiation and integrity characteristics compared with WT organoids. Images of SI crypts (n = 30–40) that form a cyst (D1) and then further develop through budding of new crypts (arrows) from the cyst body (D2–D7). A: day 0–day 7 images of C57BL/6 WT crypts. (B) day 0–day 7 images of TCRδ−/− crypts. Magnification ×200; scale bars, 50 μm. SI organoids from both groups of mice (n = 30–40) were stained with anti-chromogranin A (ChromA, Ci), anti-MUC2 (Cii), anti-lysozyme (Ciii), PNA and WGA (Civ), and SNA-I and MAA (Cv) lectins. Organoids were counterstained with DAPI (blue). Magnification ×400.
Fig. 8.
Fig. 8.
Treatment of WT and TCRδ−/− SI organoids with keratinocyte growth factor (KGF). MUC2-stained organoids grown in normal culture medium (control) and culture medium supplemented with KGF for 24 h. Organoids were counterstained with DAPI (blue) (A). Magnification, ×400. Crypt depth (μm) (B), number of crypts per organoid (C), and counts of goblet cells per crypt (D), measured in in vitro organoids from WT and TCRδ−/− mice at the initial experimental time and after 24 h in culture medium with and without KGF. Different symbols represent measurements obtained in each experimental condition (n ≈ 30) of the 3 biological replicated experiments. Symbols in C and D are stacked up to represent the number of organoids or crypts with that count. Red cross and vertical bars represent the average and standard deviation, respectively. ***P < 0.001.
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