Type II NKT cells stimulate diet-induced obesity by mediating adipose tissue inflammation, steatohepatitis and insulin resistance

Abstract

The progression of obesity is accompanied by a chronic inflammatory process that involves both innate and acquired immunity. Natural killer T (NKT) cells recognize lipid antigens and are also distributed in adipose tissue. To examine the involvement of NKT cells in the development of obesity, C57BL/6 mice (wild type; WT), and two NKT-cell-deficient strains, Jα18(-/-) mice that lack the type I subset and CD1d(-/-) mice that lack both the type I and II subsets, were fed a high fat diet (HFD). CD1d(-/-) mice gained the least body weight with the least weight in perigonadal and brown adipose tissue as well as in the liver, compared to WT or Jα18(-/-) mice fed an HFD. Histologically, CD1d(-/-) mice had significantly smaller adipocytes and developed significantly milder hepatosteatosis than WT or Jα18(-/-) mice. The number of NK1.1(+)TCRβ(+) cells in adipose tissue increased when WT mice were fed an HFD and were mostly invariant Vα14Jα18-negative. CD11b(+) macrophages (Mφ) were another major subset of cells in adipose tissue infiltrates, and they were divided into F4/80(high) and F4/80(low) cells. The F4/80(low)-Mφ subset in adipose tissue was increased in CD1d(-/-) mice, and this population likely played an anti-inflammatory role. Glucose intolerance and insulin resistance in CD1d(-/-) mice were not aggravated as in WT or Jα18(-/-) mice fed an HFD, likely due to a lower grade of inflammation and adiposity. Collectively, our findings provide evidence that type II NKT cells initiate inflammation in the liver and adipose tissue and exacerbate the course of obesity that leads to insulin resistance.

Figure 1. Body weight and weight gain of three strains of mice fed an SFD or an HFD.

(A, B) Body weight (BW) of female (A) and male mice (B) fed a high-fat diet (HFD) or a standard-fat diet (SFD) at 8 wk and weighed weekly. (n = 3–7 in each group) (C, D) Representative data of three similar experiments are shown. Weight gain (ΔBW = BW_{n wk}−BW_{8 wk}; n≥9) of female (C) and male (D) mice. Each point represents mean ± standard deviation (s.d.). Statistical analysis was performed according to the Tukey-Kramer test. **p<0.01 (WT and Jα18^−/− vs CD1d^−/−).

Figure 2. Physiological parameters of three strains of female mice fed an SFD or an HFD.

(A) Food intake of mice in each group (g/mouse/day). (B–E) The body, liver, perigonadal adipose tissue (WAT), and brown adipose tissue (BAT) were weighed after 18 wk of feeding at 26 wk of age (n = 3–7 in each group). Representative data of three similar experiments are shown. The results are expressed as mean ± s.d. Statistical analysis was performed according to the Tukey-Kramer test. *p<0.05, **p<0.01.

Figure 3. Histology and cytokine production in the liver.

(A) A liver section was stained with ORO (frozen section) in SFD- and HFD-fed mice. (B) Red-stained lipid droplets in liver sections of HFD-fed mice were quantified with image analysis software. (C) Serum levels of ALT were quantified with the Drychem system. (D, E) The production of cytokines from HMNC of HFD-fed mice stimulated with LPS for 20 h (n = 3–4 female mice in each group). The results are expressed as mean ± s.d. Statistical analysis was performed according to the Tukey-Kramer test. *p<0.05, **p<0.01.

Figure 4. Histology of perigonadal adipose tissue and serum leptin level.

(A, B) A paraffin section of perigonadal adipose tissue was stained with HE, and the circumference of adipocytes was morphometrically analyzed (10×10, 300–400 adipocytes measured) in HFD mice. (C) The serum leptin level was quantified by ELISA (n = 3–4 female mice in each group). Representative data of two similar experiments are shown. The results are expressed as mean ± s.d. Statistical analysis was performed according to the Tukey-Kramer test. *p<0.05, **p<0.01.

Figure 5. Flow cytometric analyses of HMNC in mice fed an SFD or an HFD.

(A) A representative flow cytometric dot-plot defining the population of α-GalCer/CD1d dimer⁺TCRβ⁺ and NK1.1⁺TCRβ⁺ cells in the liver of SFD-fed mice (a, c) and HFD-fed mice (b, d). (B) The proportion of NK1.1⁺TCRβ⁺ cells (a), CD4⁺ (b), CD8⁺ (c), and CD4⁻CD8⁻ (d) NKT cells (n = 3–6 female mice in each group). Representative data of three similar experiments are shown. The results are expressed as mean ± s.d. Statistical analysis was performed according to the Tukey-Kramer test. *p<0.05, **p<0.01.

Figure 6. Flow cytometric analyses of NKT cells in adipose tissue in mice fed an SFD or an HFD.

(A) A representative flow cytometric dot-plot defining the population of α-GalCer/CD1d dimer⁺TCRβ⁺ and NK1.1⁺TCRβ⁺ cells in adipose tissue of SFD-fed mice (a, c) and HFD-fed mice (b, d). (B) The proportion of each subset in SVF. NK1.1⁺TCRβ⁺ cells (a), CD4⁺ (b), CD8⁺ (c), and CD4⁻CD8⁻ (d) NKT cells (n = 3–6 female mice in each group). Representative data of three similar experiments are shown. The results are expressed as mean ± s.d. Statistical analysis was performed according to the Tukey-Kramer test. *p<0.05, **p<0.01. (C) The correlation between BW and the percentage of NKT cells in SVF by Pearson's correlation coefficient (R²) test. WT (a), P = 0.000006; Jα18^−/− (b), P = 0.004; and CD1d^−/− (c), P = 0.2. Values with P<0 .05 were considered statistically significant.

Figure 7. Flow cytometric analyses of infiltrated Mφ in perigonadal adipose tissue.

(A) A representative flow cytometric dot-plot defining the F4/80⁺CD11b⁺ population in perigonadal adipose tissue in HFD-fed mice. F4/80^hi (Population 1; red gate) and F4/80^low∼− (Population 2; blue gate) are set to a staining with isotype control mAb. (B) The percentage of cells of CD11b⁺ (total) (a), Population 1 (b) and Population 2 (c). (C) CD1d expression by the Population 1 and Population 2 (a), MFI in WT (b) and Jα18^−/− (c) mice fed an HFD (n = 3–4 male mice in each group). The results are expressed as mean ± s.d. Statistical analysis was performed according to the Tukey-Kramer test. *p<0.05, **p<0.01.

Figure 8. Cytokine production by Mϕ in perigonadal adipose tissue upon stimulation with LPS.

(A–C) IL-10, GM-CSF and TNF-α concentrations after stimulation of Mϕ in perigonadal adipose tissue with LPS (1 µg/ml) for 20 h (n = 3–4 male mice in each group). Representative data of three similar experiments are shown. The results are expressed as mean ± s.d. Statistical analysis was performed according to the Tukey-Kramer test. *p<0.05, **p<0.01.

Figure 9. Glucose and insulin tolerance.

(A) (IPGTT). Glucose (1 g/kg BW) was administered i.p. to female mice fed an SFD or an HFD. (B) The serum level of insulin 0 min and 120 min after glucose administration (i.p.) to HFD-fed mice was quantified by ELISA. (C) ITT. Insulin (0.75 U/kg BW) was administered i.p. to HFD-fed mice (n = 3–4 female mice in each group). Representative data of three similar experiments are shown. The results are expressed as mean ± s.d. Statistical analysis was performed according to the Tukey-Kramer test. *p<0.05 (WT versus CD1d^−/−); ^#p<0.05 (Jα18^−/− versus CD1d^−/−).