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

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.

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. 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. 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. 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. 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. 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.