Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jul 2;5(7):e11422.
doi: 10.1371/journal.pone.0011422.

Gammadelta T cells are reduced and rendered unresponsive by hyperglycemia and chronic TNFalpha in mouse models of obesity and metabolic disease

Kristen R Taylor  1 , Robyn E Mills, Anne E Costanzo, Julie M Jameson
Affiliations

Gammadelta T cells are reduced and rendered unresponsive by hyperglycemia and chronic TNFalpha in mouse models of obesity and metabolic disease

Kristen R Taylor et al. PLoS One. .

Abstract

Epithelial cells provide an initial line of defense against damage and pathogens in barrier tissues such as the skin; however this balance is disrupted in obesity and metabolic disease. Skin gammadelta T cells recognize epithelial damage, and release cytokines and growth factors that facilitate wound repair. We report here that hyperglycemia results in impaired skin gammadelta T cell proliferation due to altered STAT5 signaling, ultimately resulting in half the number of gammadelta T cells populating the epidermis. Skin gammadelta T cells that overcome this hyperglycemic state are unresponsive to epithelial cell damage due to chronic inflammatory mediators, including TNFalpha. Cytokine and growth factor production at the site of tissue damage was partially restored by administering neutralizing TNFalpha antibodies in vivo. Thus, metabolic disease negatively impacts homeostasis and functionality of skin gammadelta T cells, rendering host defense mechanisms vulnerable to injury and infection.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Reduced numbers of skin γδ T cells during obesity and metabolic disease is associated with hyperglycemia.
(A) γδ TCR immunofluorescence staining of epidermal sheets from BKS db/+ and db/db mice at 6-, 10- and 14-weeks of age. (B) Graphical representation of the number of epidermal γδ T cells in db/+ (solid line) and db/db (dashed line) mice at each age. *p<0.005. (C) Epidermal sheets from 10-week old BKS db/+ and db/db mice immunostained for the LC marker, langerin. (D) Graphical representation of the number of Langerhans cells at 10-weeks of age. All microscopy images were acquired at ×200. The bar represents 0.05 µm. Data (mean ± SEM) are representative of three independent experiments for each age group and a minimum of 15 fields per mouse.
Figure 2
Figure 2. Regulation of skin γδ T cell proliferation by glucose is associated with decreased STAT5B phosphorylation.
(A) In vitro growth of γδ 7–17 T cell line treated with 33.3 mM glucose at 0, 24 and 48 hours. Data (mean ± SD) presented as the % of glucose treated cells to untreated control cells. (B, C) Proliferation of (B) γδ 7–17 T cells and (C) freshly isolated γδ T cells sorted from wild-type B6 mice in IL-2 containing growth media supplemented with elevated glucose concentrations. Each experiment was performed in triplicate, data presented as mean ± SD. (D) The kinetics of expression of phosphorylated STAT5A and STAT5B following 40U/ml IL-2 stimulation in untreated and glucose treated γδ 7–17 T cells. Total STAT5 expression demonstrates even loading and expression. (E) Multiparameter flow cytometry of BrdU incorporation by skin γδ T cells isolated from 6- and 10-week old BKS db/+ and db/db mice treated with BrdU for 7 days. The same number of events is presented for each dot plot, numbers indicate the percent of γδ T cells that have incorporated BrdU. Epidermal cells were gated on live Thy1.2+ events for γδ T cells. (F) Graphical representation of the ratio of BrdU incorporation by γδ T cells in BKS db/+ to db/db mice at 6-weeks and 10-weeks of age, n = 3 per strain and age. Shown are black dots to represent the ratio of each experiment, the black line represents the average of three experiments. (G) Multiparameter flow cytometry of BrdU incorporation by keratinocytes isolated from 6-week old BKS db/+ and db/db mice treated with BrdU for 7 days. The same number of events is presented for each dot plot, numbers on the right indicate the percent of keratinocytes that have incorporated BrdU. Epidermal cells were gated on live γδ TCR− events for keratinocytes. Data are representative of five (A, B) or three (C–G) separate experiments.
Figure 3
Figure 3. Obese mice display impaired skin γδ T cell wound healing functions after injury.
(A) Immunofluorescent staining for γδ TCR in 12-week old lean BKS db/+ and obese db/db mice four hours post-wounding. The white dashed line represents the wound edge and arrowheads depict γδ T cells that have rounded near the wound edge. A minimum of 10 images was acquired at the wound edge for each experiment and the number of dendrites was determined per cell, a minimum of 300 total cells were counted. (B) Shown is the percentage of γδ T cells with 0 dendrites per cell (mean ± SEM). All images were acquired at ×200 magnification. (C, D) Skin γδ T cell production of TNFα in non-wounded and wounded skin tissue of (C) 12-week old BKS db/+ and db/db mice and (D) 27-week old B6 NCD and HFD mice. The numbers represent the percentage of cells expressing TNFα. Skin γδ T cell production of TGFβ1 in non-wounded and wounded skin tissue of (E) 12-week old BKS db/+ and db/db mice and (F) 27-week old B6 NCD and HFD mice. The numbers represent the percentage of cells expressing TGFβ1. Epidermal cells were gated on live Thy1.2+ and γδ TCR+ to distinguish γδ T cells. Shown is one representative experiment, a minimum of three experiments were performed with similar results (A–F).
Figure 4
Figure 4. Impaired activation and nutrient sensing by skin γδ T cells in obese mice.
(A, B) Microarray analysis of skin γδ T cells isolated from 10-week old BKS db/+ and db/db mice. Shown is gene expression of molecules associated with (A) insulin sensitivity and (B) PI3K/Akt/mTOR signaling. Data is presented as the mean of two independent experiments ± SEM. (C) Multiparameter flow cytometry of CD69, CD25 and CD103 on the cell surface of γδ T cells isolated from BKS db/+ and db/db in mice at 10-weeks of age. Numbers in the top right corners indicate percent of γδ T cells. (D) γδ TCR expression on γδ T cells isolated from BKS db/+ (solid line) and db/db (shaded gray) at 10-weeks of age. Dotted lines represent unstained controls. Epidermal cells were gated on live Thy1.2+ to distinguish γδ T cells. A minimum of three experiments were performed per age, shown is one representative experiment for each, the same number of events is presented for each dot plot.
Figure 5
Figure 5. γδ TCR does not contribute to defective skin γδ T cells in obese mice.
(A) δ−/− db/db mice were generated by breeding C57BL/6J δ−/− mice with C57BL/6J db/+ mice. (B) Weight of δ−/− db/db and B6 db/db obese mice compared to their δ−/− db/+ and B6 db/+ lean littermates. Data is presented as the mean weight ± SD. Between two and eleven mice were weighed per age per strain. (C) Graphical representation of the number of epidermal T cells at 14-weeks of age. Skin γδ T cells were counted in epidermal ear sheets from three δ−/− db/+ mice and four δ−/− db/db mice. The mean was determined for each experiment (black dots) and the black line represents the average of all the experiments. A minimum of 15 fields were counted for each mouse per experiment, with a minimum of 500 cells per experiment, a minimum of three independent experiments were performed. (D) Multiparameter flow cytometry of CD69 expression on the cell surface of γδ T cells isolated from B6 db/+ and db/db and δ−/− db/+ and db/db mice at 14-weeks of age. Numbers on the top right corner indicate percent of αβ T cells. Epidermal cells were gated on live CD3+ and Thy1.2+ to distinguish epidermal T cells. A minimum of three experiments were performed, shown is one representative experiment, the same number of events is presented for each dot plot.
Figure 6
Figure 6. The obese environment inhibits skin γδ T cell function.
(A) Skin γδ T cell morphology changes in epidermal sheets isolated from 10- to 14-week old BKS db/+ and db/db following in vitro stimulation with 10 µg/ml anti-CD3ε antibody compared to unstimulated control. All microscopy images were acquired at ×200 and the bar represents 0.05 µm. (B) Shown is a graphical representation of the percentage of skin γδ T cells with 0, 1, 2 or ≥3 dendrites, which represent the degree of γδ T cell rounding (mean ± SEM), in epidermal ear sheets from 10- to 14-week old BKS db/+ and obese db/db animals stimulated with 10 µg/ml anti-CD3ε antibody. Three independent experiments were performed, a minimum of 10 fields were counted for each, and this data represents the average of all 35 fields and approximately 1000 total cells. (C) Multiparameter flow cytometry of TNFα production and CD25 expression by γδ T cells isolated from 10- to 14-week old BKS db/+ and db/db mice following overnight stimulation with 1 µg/ml anti-CD3ε. Numbers in the upper right corner indicate percent γδ T cells. Epidermal cells gated on live Thy1.2+ events. Data are representative of at least three independent experiments.
Figure 7
Figure 7. Neutralization of TNFα rescues skin γδ T cell function at the wound site.
(A, B) Microarray analysis of skin γδ T cells isolated from 10-week old BKS db/+ and obese db/db mice. Shown is gene expression of molecules associated with TNFα signaling. Data is presented as the mean of two independent experiments ± SEM. (C) Epidermal sheets isolated from 10- to 14-week old BKS db/+ and obese db/db animals either unstimulated or stimulated with 10 µg/ml anti-CD3ε antibody and 100 ng/ml TNFα. All microscopy images were acquired at ×200 and the bar represents 0.05 µm. (D) Quantification of the percentage of skin γδ T cells with 0, 1, 2 or ≥3 dendrites, which represent the degree of γδ T cell rounding (mean ± SEM), in epidermal ear sheets from 10- to 14-week old BKS db/+ and obese db/db animals stimulated with 10 µg/ml anti-CD3ε antibody and 100 ng/ml TNFα. Three independent experiments were performed, a minimum of 10 fields were counted for each, and this data represents the average of all 35 fields and approximately 1000 total cells. (E, F) Fold change in MFI of (E) TGFβ1 and (F) TNFα expression in skin γδ T cells isolated from the wound edge compared to non-wound edge cells. Skin γδ T cells from 10- to 14-week old BKS db/+ mice were used as a positive control, 10- to 14-week old db/db mice were either treated with 1 mg/kg IgG control antibody or anti-TNFα antibody for a minimum of four days. Shown in fold change in MFI for 3 separate experiments, significance was determined by t-test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jameson J, Ugarte K, Chen N, Yachi P, Fuchs E, et al. A role for skin γδ T cells in wound repair. Science. 2002;296:747–749. - PubMed
    1. Cheroutre H. IELs: enforcing law and order in the court of the intestinal epithelium. Immunol Rev. 2005;206:114–131. - PubMed
    1. Komori HK, Meehan TF, Havran WL. Epithelial and mucosal γδ T cells. Curr Opin Immunol. 2006;18:534–538. - PubMed
    1. Xiong N, Raulet DH. Development and selection of γδ T cells. Immunol Rev. 2007;215:15–31. - PubMed
    1. Born WK, Jin N, Aydintug MK, Wands JM, French JD, et al. γδ T lymphocytes-selectable cells within the innate system? J Clin Immunol. 2007;27:133–144. - PubMed

Publication types

MeSH terms