γδ T cells promote inflammation and insulin resistance during high fat diet-induced obesity in mice - PubMed (original) (raw)

γδ T cells promote inflammation and insulin resistance during high fat diet-induced obesity in mice

Pooja Mehta et al. J Leukoc Biol. 2015 Jan.

Abstract

γδ T cells are resident in AT and increase during diet-induced obesity. Their possible contribution to the inflammatory response that accompanies diet-induced obesity was investigated in mice after a 5 to 10 week milk HFD. The HFD resulted in significant increases in CD44(hi), CD62L(lo), and TNF-α(+) γδ T cells in eAT of WT mice. Mice deficient in all γδ T cells (TCRδ(-/-)) or only Vγ4 and Vγ6 subsets (Vγ4/6(-/-)) were compared with WT mice with regard to proinflammatory cytokine production and macrophage accumulation in eAT. Obesity among these mouse strains did not differ, but obese TCRδ(-/-) and Vγ4/6(-/-) mice had significantly reduced eAT expression of F4/80, a macrophage marker, and inflammatory mediators CCL2 and IL-6 compared with WT mice. Obese TCRδ(-/-) mice had significantly reduced CD11c(+) and TNF-α(+) macrophage accumulation in eAT after 5 and 10 weeks on the HFD, and obese Vγ4/6(-/-) mice had significantly increased CD206(+) macrophages in eAT after 5 weeks on the diet and significantly reduced macrophages after 10 weeks. Obese TCRδ(-/-) mice had significant reductions in systemic insulin resistance and inflammation in liver and skeletal muscle after longer-term HFD feeding (10 and 24 weeks). In vitro studies revealed that isolated γδ T cells directly stimulated RAW264.7 macrophage TNF-α expression but did not stimulate inflammatory mediator expression in 3T3-L1 adipocytes. These findings are consistent with a role for γδ T cells in the proinflammatory response that accompanies diet-induced obesity.

Keywords: CCL2; IL-6; adipose tissue; liver; macrophage; skeletal muscle.

© Society for Leukocyte Biology.

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Figures

Figure 1.

Figure 1.. Characterization of γδ T cells in eAT of mice after 5 weeks of HFD.

(A) Analysis of CD3+GL3+ γδ T cells in SVF isolated by collagenase digestion of eAT from male C57BL/6J mice on 5 weeks of ND (white bars) or HFD (black bars). Cells were gated on the CD3+ population and analyzed for coexpression of GL3 by flow cytometry. Data are represented as total number of γδ T cells in eAT/mouse (# of cells/fat pads) and number of cells relative to grams of AT mass (# of cells/fat pads/g fat) and as percentage of total leukocytes in eAT, and numbers inside boxes refer to percentage of γδ T cells of total T cells; n = 8–10 mice in each group. *P < 0.05, ns, Not significant. (B) Flow cytometric analysis of CD69, CD62L and CD44 expression on γδ T cells in eAT of mice after 5 weeks of ND or HFD. (C) Flow cytometric analysis of CD27 and CCR6 expression on γδ T cells in eAT of mice after 5 weeks of ND or HFD. (D) Flow cytometric analysis of intracellular cytokine IFN-γ, IL-17, IL-6, and TNF-α expression by γδ T cells in eAT after 5 weeks of ND or HFD. SVF cells were isolated from eAT and cultured overnight in DMEM containing Brefeldin A. (B–D) Data are represented as number of cells/fats pads and number of cells/fat pads/gram of fat and as a percentage of γδ T cells in eAT (numbers inside box). *P < 0.05, and **P < 0.01 comparing ND- and HFD-fed groups of mice; n = 4–5 mice in each group. (E) PCR analysis to determine the γ and δ TCR chains expressed in eAT of C57BL/6J mice after 5 weeks of HFD. Representative gel picture of at least 3 repeat experiments. (F and G) Flow cytometric analysis of γδ T cells in (F) spleen and (G) whole blood after 5 weeks of ND or HFD, represented as number of cells/tissue and as a percent of total T cells; n = 3–5 mice in each group. Values are mean ±

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. *P < 0.05 by Student’s _t_-test.

Figure 2.

Figure 2.. CCL2, IL-6, and TNF-α expression in eAT of HFD-fed TCR_δ_−/− mice, V_γ_4/6−/− mice, and mice treated with anti-TCR_γδ_ antibody.

(A) mRNA expression of CCL2, IL-6, and TNF-α in eAT of TCR_δ_−/− and TCR_δ_+/+ mice after 5 weeks of ND or HFD, as determined by qPCR analysis, expressed relative to housekeeping gene GAPDH; n = 6–9 mice in each group. (B) mRNA expression of CCL2 and IL-6 in eAT of HFD-fed C57BL/6J mice treated with IgG isotype control antibody (white bar) or anti-TCR_γδ_ antibody (black bar), as determined by qPCR analysis; n = 5 mice in each group. *P < 0.05 by Student’s t_-test. (C) mRNA expression of CCL2, IL-6, and TNF-α in eAT of TCR_δ_−/− and TCR_δ+/+ mice after 10 weeks of ND or HFD, as determined by qPCR analysis; n = 5–6 mice in each group. (D) mRNA expression of CCL2 and IL-6 in eAT of V_γ_4/6−/− and WT mice after 5 weeks of ND or HFD, as determined by qPCR; n = 4–6 mice in each group. (E) mRNA expression of TNF-α in eAT of V_γ_4/6−/− and WT mice after 10 weeks of ND or HFD, as determined by qPCR; n = 4–6 mice in each group. Values are mean ±

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, (A, C–E) *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test. One representative cohort of 2 independent cohorts of mice.

Figure 3.

Figure 3.. Effect of γδ T cells on cytokine expression by 3T3-L1 adipocytes and RAW264.7 macrophages in vitro.

(A) qPCR gene expression of CCL2 and IL-6 by differentiated 3T3-L1 adipocytes treated for 24 h with eAT explant media from TCR_δ_+/+ or TCR_δ_−/− mice after 5 weeks of ND or HFD; n = 3–5 replicates/group. (B) qPCR gene expression of CCL2 and IL-6 by differentiated 3T3-L1 adipocytes treated with CM from γδ T cells or cocultured with γδ T cells for 24 h. As control, γδ T cells were plated onto 3T3-L1 cells and washed away immediately before harvesting the 3T3-L1 cells for RNA isolation (0 h). γδ T cells were isolated from spleen of C57BL/6J mice fed HFD for 5 weeks; n = 3 replicates/group. (C) Preadipocyte (Pre-AD)-to-adipocyte differentiation of 3T3-L1 cells treated with eAT explant media from WT or TCR_δ_−/− mice fed ND or HFD for 5 weeks. Undifferentiated preadipocytes and adipocytes treated with DMEM or eAT explant control media or mouse recombinant TNF-α used as controls for 3T3-L1 adipocyte differentiation. Oil Red O staining performed on Day 8 of adipocyte differentiation; n = 6 replicates/group. (D) TNF-α gene expression by RAW264.7 murine macrophage cell line cocultured with γδ T cells from spleens of male WT mice on ND or HFD, as analyzed by qPCR. RAW264.7 cells were cocultured with γδ T cells at a ratio of 20:1 (macrophages:γδ T cells) for 24 h. As control, γδ T cells were plated onto RAW264.7 cells and washed away immediately before harvesting the RAW264.7 cells for RNA isolation; n = 3 replicates/group. Data from a representative experiment of 3 independent experiments. Values are mean ±

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*P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test.

Figure 4.

Figure 4.. HFD-fed TCR_δ_−/− mice have fewer proinflammatory macrophages in eAT after 5 weeks of HFD.

(A) Flow cytometric analysis of F4/80+CD11b+ macrophages and F4/80+CD11c+ M1 macrophages in eAT of 5-week-old WT and TCR_δ_−/− mice at baseline before starting on HFD. Data are represented as number of cells/fat pads and number of cells/fat pads/gram fat; n = pool of eAT from 5 mice, 2–3 pools/group. (B) F4/80 mRNA expression in eAT of TCR_δ_−/− and TCR_δ_+/+ mice after 5 weeks of ND or HFD (n = 6–9 mice in each group) and in eAT of HFD-fed C57BL/6J mice treated with IgG isotype control antibody or anti-TCR_γδ_ antibody (n = 5 mice in each group). *P < 0.05 by Student’s t_-test, as determined by qPCR analysis. (C–E) Flow cytometric analysis of SVF cells in eAT of TCR_δ_−/− and TCR_δ+/+ mice after 5 weeks of ND or HFD. (C) Total F4/80+ macrophages, represented as percentage of cells within macrophage gate based on scatter pattern, (D) TNF-α+ macrophages, and (E) F4/80+CD11c+CD206– M1, F4/80+CD11c–CD206+ M2, and F4/80+CD11c–CD206– DN macrophages; n = 6–9 mice in each group. Data from a representative experiment of 2 independent experiments. Data are represented as number of cells/fat pads and number of cells/fat pads/gram of fat and as a percentage of macrophages. Values are mean ±

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*P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test. Numbers inside boxes refer to percent of cells, and *P < 0.05 comparing HFD-fed groups of mice.

Figure 5.

Figure 5.. Quantification of macrophages in eAT of TCR_δ_−/− mice after 10 weeks of HFD.

(A) mRNA expression of F4/80 and CD11c in eAT of TCR_δ_−/− and TCR_δ_+/+ mice after 10 weeks of ND or HFD, as determined by qPCR analysis; n = 5–6 mice in each group. (B–D) Flow cytometric analysis of SVF cells in eAT of TCR_δ_−/− and WT mice after 10 weeks of ND or HFD. (B) Total F4/80+ macrophages, represented as percentage of cells within macrophage gate based on scatter pattern, (C) TNF-α+ macrophages, and (D) F4/80+CD11c+CD206– M1, F4/80+CD11c–CD206+ M2, and F4/80+CD11c–CD206– DN macrophages; n = 4–5 mice in each group. Data from a representative experiment of 2 independent experiments. Data are represented as number of cells/fat pads and number of cells/fat pads/gram of fat. Values are mean ±

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*P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test.

Figure 6.

Figure 6.. HFD-fed V_γ_4/6−/− mice have increased CD206+ M2 macrophages in eAT.

(A) Quantification of γδ T cells in eAT of V_γ_4/6−/− and WT mice after 5 weeks of ND or HFD by flow cytometry analysis (n = 4–5 mice in each group; data from a representative experiment of 3 independent experiments) and PCR analysis to determine the γ and δ TCR chains expressed in eAT of V_γ_4/6−/− mice after 5 weeks of HFD (representative gel picture of at least 3 repeat experiments). (B) mRNA expression of F4/80 in eAT of V_γ_4/6−/− and WT mice after 5 weeks of ND or HFD, as determined by qPCR; n = 4–6 mice in each group. (C–E) Flow cytometric analysis of SVF cells in eAT of V_γ_4/6−/− and WT mice after 5 weeks of ND or HFD. (C) Total F4/80+ macrophages, (D) F4/80+CD11c+CD206– M1, F4/80+CD11c–CD206+ M2, and F4/80+CD11c–CD206– DN macrophages, and (E) TNF-α+ macrophages; n = 4–5 mice in each group. Data from a representative experiment of 3 independent experiments. (F) mRNA expression of F4/80 and CD11c in eAT of V_γ_4/6−/− and WT mice after 10 weeks of ND or HFD, as determined by qPCR; n = 4–6 mice in each group. (G–I) Flow cytometric analysis of SVF cells in eAT of V_γ_4/6−/− and WT mice after 10 weeks of ND or HFD. (G) Total F4/80+ macrophages, (H) TNF-α+ macrophages, and (I) F4/80+CD11c+CD206– M1, F4/80+CD11c–CD206+ M2, and F4/80+CD11c–CD206– DN macrophages; n = 4–5 mice in each group. Data from a representative experiment of 2 independent experiments. Data are represented as number of cells/fat pads and number of cells/fat pads/gram of fat and as a percentage of macrophages. Values are mean ±

sem.

*P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test. Numbers inside boxes refer to percent of cells, arrow points to value of double negative quadrant, and *P < 0.05, and **P < 0.01 comparing WT and V_γ_4/6−/− mice on the same diet.

Figure 7.

Figure 7.. Obese TCR_δ_−/− mice but not V_γ_4/6−/− mice are more insulin sensitive than WT mice.

(A) Fasting insulin levels in plasma of nonlittermates TCR_δ_−/− and WT mice on 5 weeks of ND or HFD after a 6 h fast; n = 5–6 mice in each group. (B) Fasting blood glucose levels and (C) blood glucose levels during ITT (0.75 U insulin/kg body weight) of TCR_δ_−/− and TCR_δ_+/+ mice on 10 weeks of ND or HFD after a 6 h fast; n = 8–11 mice in each group. (D) Fasting plasma insulin levels of V_γ_4/6−/− and WT mice on 5 weeks of ND or HFD after a 6 h fast; n = 4 mice in each group. (E) Fasting blood glucose levels and (F) blood glucose levels during ITT (0.75 U insulin/kg body weight) of V_γ_4/6−/− and WT mice on 10 weeks of ND or HFD after a 6 h fast; n = 4 mice in each group. Values are mean ±

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, (A and D) *P < 0.05, and **P < 0.01 by one-way ANOVA with Tukey's post hoc test. (C and F) *P < 0.05 by two-way ANOVA with Bonferroni’s post hoc test.

Figure 8.

Figure 8.. Obese TCR_δ_−/− mice have reduced inflammation in skeletal muscle and liver after long-term HFD.

(A) mRNA expression of TNF-α, CCL2, and F4/80 in soleus muscle of TCR_δ_−/− and TCR_δ_+/+ mice after 10 weeks of ND or HFD, as determined by qPCR analysis; n = 5–6 mice in each group. *P < 0.05 by one-way ANOVA with Tukey’s post hoc test. (B) mRNA expression of CCL2, F4/80, and IL-6 in liver of TCR_δ_−/− and WT mice after 24 weeks on HFD; n = 5–6 mice in each group. *P < 0.05, and **P < 0.01 by Student’s _t_-test. (C) Flow cytometry analysis of CD3+GL3+ γδ T cells in liver of male C57BL/6J mice after 5 weeks of ND (white bars) or HFD (black bars). Data are represented as total number of γδ T cells in liver/mouse; n = 7–8 mice in each group. *P < 0.05 by Student’s _t_-test. Values are mean ±

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.

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