Type II NKT Cells Stimulate Diet-Induced Obesity by Mediating Adipose Tissue Inflammation, Steatohepatitis and Insulin Resistance

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 F4/80high and F4/80low cells. The F4/80low-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

Additional file 1: Figure S1. of Cooperation of Sox4 with β-catenin/p300 complex in transcriptional regulation of the Slug gene during divergent sarcomatous differentiation in uterine carcinosarcoma

(A) Ishikawa (Ish) cells were transfected with Sox4, Sox7, and Sox9 reporter constructs, together with either Sox4, Sox7, or Sox9. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiment was performed in duplicate. (B) Western blot analysis for the indicated proteins from Ishikawa, Hec251, and Hec6 cells cultured in STK2. (C) Western blot analysis for the indicated proteins from H6SL#8 and mock cells. (TIF 2816 kb

Additional file 3: Figure S3. of Cooperation of Sox4 with β-catenin/p300 complex in transcriptional regulation of the Slug gene during divergent sarcomatous differentiation in uterine carcinosarcoma

(A) Hec251 (left) and Hec6 cells (right) were transfected with Slug reporter constructs, together with β-cateninΔS45 (β-cat), Sox4, and p300. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiment was performed in duplicate. (B) After transfection of HA-β-cateninΔS45 and GFP-Sox4, cells were stained with anti-HA antibody. Immunopositive cell is indicated by arrow. Nuclei were stained by DAPI. (C,D) Hec251 (C) and Hec6 (D) cells were transfected with pM-β-catenin (left) or pM-Sox4 (right), along with pGL5 luc, Sox4, Sox7, Sox9, and p300. The experiment was performed in duplicate. (TIF 2017 kb

Glucose and insulin tolerance.

<p>(A) (IPGTT). Glucose (1 g/kg BW) was administered <i>i.p.</i> to female mice fed an SFD or an HFD. (B) The serum level of insulin 0 min and 120 min after glucose administration (<i>i.p.</i>) to HFD-fed mice was quantified by ELISA. (C) ITT. Insulin (0.75 U/kg BW) was administered <i>i.p.</i> 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. *<i>p</i><0.05 (WT versus CD1d^−/−); ^#p<0.05 (Jα18^−/− versus CD1d^−/−).</p

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

<p>(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. *<i>p</i><0.05, **<i>p</i><0.01.</p

Flow cytometric analyses of infiltrated Mφ in perigonadal adipose tissue.

<p>(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. *<i>p</i><0.05, **<i>p</i><0.01.</p

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

<p>(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. *<i>p</i><0.05, **<i>p</i><0.01.</p