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. 2011 Dec;131(12):2409-18.
doi: 10.1038/jid.2011.241. Epub 2011 Aug 11.

Dysfunctional γδ T cells contribute to impaired keratinocyte homeostasis in mouse models of obesity

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Dysfunctional γδ T cells contribute to impaired keratinocyte homeostasis in mouse models of obesity

Kristen R Taylor et al. J Invest Dermatol. 2011 Dec.

Abstract

Skin complications and chronic non-healing wounds are common in obesity, metabolic disease, and type 2 diabetes. Epidermal γδ T cells normally produce keratinocyte growth factors, participate in wound repair, and are necessary for keratinocyte homeostasis. We have determined that in γδ T cell-deficient mice, there are reduced numbers of keratinocytes and the epidermis exhibits a flattened, thinner structure with fewer basal keratinocytes. This is important in obesity, where skin-resident γδ T cells are reduced and rendered dysfunctional. Similar to γδ T cell-deficient mice, keratinocytes are reduced and the epidermal structure is altered in two obese mouse models. Even in regions where γδ T cells are present, there are fewer keratinocytes in obese mice, indicating that dysfunctional γδ T cells are unable to regulate keratinocyte homeostasis. The impact of absent or impaired γδ T cells on epidermal structure is exacerbated in obesity as E-cadherin localization and expression are additionally altered. These studies reveal that γδ T cells are unable to regulate keratinocyte homeostasis in obesity and that the obese environment further impairs skin structure by altering cell-cell adhesion. Together, impaired keratinocyte homeostasis and epidermal barrier function through direct and indirect mechanisms result in susceptibility to skin complications, chronic wounds, and infection.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. γδ T cells are required for keratinocyte homeostasis
(a) DAPI staining and quantification (mean ± SD) of WT and TCRδ−/− epidermal sheets (×200 images, scale bar = 0.05μm). (b) Immunofluorescence microscopy of γδ and αβ T cells (green) and keratinocytes (blue) in epidermal sheets (×200 images, scale bar = 0.05μm). Quantification (mean ± SD) of keratinocytes in 1 in2 area, correlating to the number of skin-resident T cells per area (x-axis). (c) Immunofluorescent staining of frozen skin sections with keratin 5 (green), keratin 1 (red) and DAPI (blue) (×1000 images, scale bar = 10μm). A minimum of three independent experiments were performed for each set of mice, *p<0.0001. For all microscopy experiments, a minimum of 20 fields were examined per experiment.
Figure 2
Figure 2. Keratinocytes are reduced in obese db/db and HFD mice
(a) DAPI staining of epidermal sheets isolated from 6-, 10- and 14-week old db/+ and db/db mice. Quantification of keratinocyte numbers in the epidermis of 6- to 20-week old db/+ and db/db mice. (b) DAPI staining and quantification of keratinocytes in epidermal sheets isolated from NCD and HFD mice. Data (mean ± SD) are representative of three independent experiments for each set of mice, *p<0.0001. All microscopy images were acquired at ×200, a minimum of 20 fields were counted per experiment. Scale bar = 0.05μm.
Figure 3
Figure 3. Keratinocyte numbers decline when neighboring γδ T cells are either absent or dysfunctional
Immunofluorescence microscopy of γδ T cells (red) and keratinocytes (blue) in epidermal sheets of (a) 12-week old db/+ and db/db mice and (b) NCD and HFD mice. Quantification of the number of keratinocytes in 1 in2 area, correlating to the number of γδ T cells per area (x-axis), in (a) 12-week old db/+ and db/db mice and (b) NCD and HFD mice. A minimum of three independent experiments were performed, Data (mean ± SD) are representative of three independent experiments for each set of mice, *p<0.0001. All microscopy images were acquired at ×200 and a minimum of 20 fields were examined per experiment. Scale bar = 0.05μm.
Figure 4
Figure 4. Disorganized epidermal structure and altered keratinocyte morphology in obese mice
Immunofluorescence microscopy of frozen skin sections from (a) 6-week db/+ and db/db, (b) 12-week db/+ and db/db and (c) NCD and HFD mice stained with keratin 5 (green), keratin 1 (red) and DAPI (blue). Microscopy images were acquired at ×1000, scale bar = 10μm. The dashed line represents the epidermal-dermal boundary. Arrows point to regions of aberrant keratin staining and localization. A minimum of three independent experiments were performed and a minimum of 20 images per experiment were examined, shown is one representative image.
Figure 5
Figure 5. Altered keratin protein expression and decreased proliferation in obese mice
(a) Immunoblots and densitometry for keratin 5 and keratin 1 expression in 6-week db/+ and db/db, 12-week db/+ and db/db and NCD and HFD epidermis. Blots were normalized for total protein content. β-tubulin was used as a loading control. A minimum of three independent experiments were performed. (b) Multiparameter flow cytometry of BrdU incorporation by Thy1.2 keratinocytes isolated from 10-week old db/+ and db/db mice. Mice were treated with BrdU in their drinking water for 7 days (upper panel). The same number of events is presented; numbers indicate the percent of keratinocytes that have incorporated BrdU. A total of four independent experiments were performed with one mouse per genotype and treatment per experiment.
Figure 6
Figure 6. Keratinocyte E-cadherin localization and expression is altered in obesity
Immunofluorescence microscopy of epithelial sheets for E-cadherin (red) and nuclei (blue) in (a) 12-week old db/+ and db/db and (b) NCD and HFD mice. Arrows highlight regions of organized (single arrows) or disorganized (double arrows) E-cadherin staining. All microscopy images were acquired at ×1000, a minimum of 20 fields were examined per experiment. Scale bar = 10μm. Quantification of junction width measuring E-cadherin staining, *p<0.0001. (c) Western blot analysis and densitometry of E-cadherin expression in db/+ and db/db and NCD and HFD epidermis. Blots were normalized for total protein content. β-tubulin expression was used as a loading control. A minimum of three independent experiments were performed and a representative image or blot is shown.

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