Masashi Satoh

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.

Adipocyte-specific CD1d-deficiency mitigates diet-induced obesity and insulin resistance in mice

It has been shown that CD1d expression and glycolipid-reactive, CD1d-restricted NKT cells exacerbate the development of obesity and insulin resistance in mice. However, the relevant CD1d-expressing cells that influence the effects of NKT cells on the progression of obesity remain incompletely defined. In this study, we have demonstrated that 3T3-L1 adipocytes can present endogenous ligands to NKT cells, leading to IFN-γ production, which in turn, stimulated 3T3-L1 adipocytes to enhance expression of CD1d and CCL2, and decrease expression of adiponectin. Furthermore, adipocyte-specific CD1d deletion decreased the size of the visceral adipose tissue mass and enhanced insulin sensitivity in mice fed a high-fat diet (HFD). Accordingly, NKT cells were less activated, IFN-γ production was significantly reduced, and levels of adiponectin were increased in these animals as compared with control mice on HFD. Importantly, macrophage recruitment into the adipose tissue of adipocyte-specific CD1d-deficient mice was significantly blunted. These findings indicate that interactions between NKT cells and CD1d-expressing adipocytes producing endogenous NKT cell ligands play a critical role in the induction of inflammation and functional modulation of adipose tissue that leads to obesity.

CD11c+ macrophages and levels of TNF‐α and MMP‐3 are increased in synovial and adipose tissues of osteoarthritic mice with hyperlipidaemia

To understand more clearly the link between osteoarthritis and hyperlipidaemia, we investigated the inflammatory macrophage subsets and macrophage‐regulated matrix metalloprotease‐3 (MMP‐3) and A disintegrin and metalloprotease with thrombospondin motifs‐4 (ADAMTS4) in synovial (ST) and adipose tissues (AT) of osteoarthritic mice with hyperlipidaemia (STR/Ort). CD11c+F4/80+CD11b+ macrophage populations in the ST and AT of 9‐month‐old STR/Ort and C57BL/6J mice were characterized and compared by flow cytometry and real‐time polymerase chain reaction (PCR) analyses. Expression of tumour necrosis factor (TNF)‐α, MMP‐3 and ADAMTS4, and the response of these factors to anionic liposomal clodronate induced‐macrophage depletion were also evaluated by real‐time PCR. Expression of TNF‐α in CD11c+ cells, which were isolated by magnetic beads, was compared to CD11c– cells. In addition, the effect of TNF‐α on cultured synovial fibroblasts and adipocytes was investigated. CD11c+F4/80+CD11b+ macrophages were increased in ST and AT of STR/Ort mice. The CD11c+ cell fraction highly expressed TNF‐α. Expression of TNF‐α and MMP3 was increased in ST and AT, and was decreased upon macrophage depletion. TNF‐α treatment of cultured synovial fibroblasts and adipocytes markedly up‐regulated MMP‐3. CD11c+F4/80+CD11b+ macrophages were identified as a common inflammatory subset in the AT and ST of STR/Ort mice with hyperlipidaemia. The induction of inflammation in AT and ST may be part of a common mechanism that regulates MMP3 expression through TNF‐α. Our findings suggest that increased numbers of CD11c+ macrophages and elevated levels of TNF‐α and MMP‐3 in AT and ST may explain the relationship between hyperlipidaemia and OA.

Natural killer T cells are required for lipopolysaccharide-mediated enhancement of atherosclerosis in apolipoprotein E-deficient mice

Lipopolysaccharide (LPS) has been shown to accelerate atherosclerosis and to increase the prevalence of IL-4-producing natural killer T (NKT) cells in various tissues. However, the role of NKT cells in the development of LPS-induced atherosclerotic lesions has not been fully tested in NKT cell-deficient mice. Here, we examined the lesion development in apolipoprotein E knockout (apoE-KO) mice and apoE-KO mice on an NKT cell-deficient, CD1d knockout (CD1d-KO) background (apoE-CD1d double knockout; DKO). LPS (0.5 μg/g body weight/wk) or phosphate-buffered saline (PBS) was intraperitoneally administered to apoE-KO and DKO mice (8-wk old) for 5 wk and atherosclerotic lesion areas were quantified thereafter. Consistent with prior reports, NKT cell-deficient DKO mice showed milder atherosclerotic lesions than apoE-KO mice. Notably, LPS administration significantly increased the lesion size in apoE-KO, but not in DKO mice, compared to PBS controls. Our findings suggest that LPS, and possibly LPS-producing bacteria, aggravate the development of atherosclerosis primarily through NKT cell activation and subsequent collaboration with NK cells.

Communication between natural killer T cells and adipocytes in obesity

ABSTRACT Adipose tissue contains various types of immunocompetent cells, and these cells of innate and adaptive immunity control adipose tissue inflammation that blunts insulin sensitivity. Recent studies have shown that adipocytes express CD1d and present lipid antigen(s) to activate natural killer T (NKT) cells. The function of adipocytes is in turn modulated by cytokines that NKT cells produce to alter the expression of anti-inflammatory adipokine(s) and the production of inflammatory and chemoattractant cytokines. These in vitro studies imply that the interaction between adipocytes and NKT cells might affect the development of not only obesity but also obesity-related diseases. To test the importance of the interaction between NKT cells and adipocytes, we examined whether an adipocyte-specific CD1d deletion affected the development of obesity, which had been demonstrated with B6.CD1d−/− (CD1d KO). We found that the interaction is indeed important to induce adipose tissue inflammation and insulin resistance in response to lipid excess. In this commentary, the advances and controversies on NKT cells and obesity are discussed based on our recent report that NKT cells play a pivotal role in the regulation of adipose tissue by communicating with adipocytes via CD1d.

TNF-α production in NKT cell hybridoma is regulated by sphingosine-1-phosphate: implications for inflammation in atherosclerosis.

ObjectivesNatural killer T (NKT) cells are unique T lymphocytes that recognize glycolipid antigen and produce various cytokines. NKT cells accelerate atherosclerosis in mice. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid and regulates T-lymphocyte trafficking. We aimed to determine the effects of S1P on the production of proinflammatory cytokine, tumor necrosis factor (TNF)-&agr;, in NKT cell hybridomas and mouse NKT cells. Materials and methodsNKT cell hybridomas and sorted mouse NKT cells were stimulated with S1P and &agr;-galactosylceramide (&agr;-GalCer), the major ligand to produce cytokines in NKT cells. TNF-&agr; mRNA expression and protein production were determined by real-time PCR and ELISA, respectively. Cell migration was assayed using chemotaxicell. Plasma S1P was measured using HPLC. ResultsHybridomas expressed S1P receptors, S1P1, S1P2, and S1P4. S1P and &agr;-GalCer increased TNF-&agr; mRNA expression and protein production. S1P enhanced TNF-&agr; induction by &agr;-GalCer. S1P receptor antagonists decreased the TNF-&agr; mRNA expression induced by S1P. FTY720, an immunosuppressive S1P receptor modulator, also decreased the TNF-&agr; mRNA expression. The migration of NKT cell hybridomas was increased by S1P. FTY720 reduced the migration induced by S1P. S1P also increased the TNF-&agr; mRNA expression in mouse NKT cells. Plasma TNF-&agr; levels in patients with high plasma S1P (≥500 nmol/l) were higher than those in patients with low S1P (<500 nmol/l). ConclusionS1P binds to S1P receptors in NKT cells and enhances TNF-&agr; production. TNF-&agr; overproduction may induce atherogenic inflammatory responses. S1P may serve as a novel therapeutic target for amelioration of vascular inflammatory diseases.

Increase of circulating CD11b(+)Gr1(+) cells and recruitment into the synovium in osteoarthritic mice with hyperlipidemia.

Although recent studies suggest that hyperlipidemia is a risk factor for osteoarthritis (OA), the link between OA and hyperlipidemia is not fully understood. As the number of activated, circulating myeloid cells is increased during hyperlipidemia, we speculate that myeloid cells contribute to the pathology of OA. Here, we characterized myeloid cells in STR/Ort mice, a murine osteoarthritis model, under hyperlipidemic conditions. Ratios of myeloid cells in bone marrow, the spleen, and peripheral blood were determined by flow cytometry. To examine the influence of the hematopoietic environment, including abnormal stem cells, on the hematopoietic profile of STR/Ort mice, bone marrow transplantations were performed. The relationship between hyperlipidemia and abnormal hematopoiesis was examined by evaluating biochemical parameters and spleen weight of F2 animals (STR/Ort x C57BL/6J). In STR/Ort mice, the ratio of CD11b+Gr1+ cells in spleens and peripheral blood was increased, and CD11b+Gr1+ cells were also present in synovial tissue. Splenomegaly was observed and correlated with the ratio of CD11b+Gr1+ cells. When bone marrow from GFP-expressing mice was transplanted into STR/Ort mice, no difference in the percentage of CD11b+Gr1+ cells was observed between transplanted and age-matched STR/Ort mice. Analysis of biochemical parameters in F2 mice showed that spleen weight correlated with serum total cholesterol. These results suggest that the increase in circulating and splenic CD11b+Gr1+ cells in STR/Ort mice originates from hypercholesterolemia. Further investigation of the function of CD11b+Gr1+ cells in synovial tissue may reveal the pathology of OA in STR/Ort mice.

Nerve growth factor regulation and production by macrophages in osteoarthritic synovium

Nerve growth factor (NGF) functions to modulate osteoarthritis (OA)‐associated pain. Although recent studies suggest that tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β mediate NGF activity in human synovial fibroblasts, the regulation of NGF expression in human synovial macrophages remains unclear. Here, we examined the role of macrophages in the production and regulation of synovial (SYN) NGF in osteoarthritic knee joints by examining the mRNA expression of TNF‐α and IL‐1β in freshly isolated CD14‐positive (macrophage‐rich fraction) and CD14‐negative cells (fibroblast‐rich fraction) in synovial tissue from OA patients by quantitative polymerase chain reaction. We also examined the effects of IL‐1β and TNF‐α on NGF mRNA expression in cultured CD14‐positive (macrophage‐rich fraction) and CD14‐negative cells (fibroblast‐rich fraction). In addition, to examine the contribution of macrophages to NGF, TNF‐α and IL‐1β expression, we injected clodronate liposomes systemically into STR/Ort mice, an osteoarthritis animal model, to deplete macrophages. TNF‐α and IL‐1β mRNA levels in CD14‐positive cells from the SYN of OA patients was significantly higher than that in CD14‐negative cells, while NGF expression did not differ markedly between the two cell fractions. In addition, treatment of human cultured CD14‐positive and ‐negative cells with IL‐1β and TNF‐α enhanced NGF mRNA and protein levels. Expression of NGF, IL‐1β and TNF‐α was also reduced significantly in STR/Ort mice upon macrophage depletion. These findings suggest that IL‐1β and TNF‐α regulate NGF expression and production in synovial macrophages and fibroblasts in osteoarthritic joints.

Dendritic cell-derived TNF-α is responsible for development of IL-10-producing CD4+ T cells

Immature dendritic cells (DCs) appear to be involved in peripheral immune tolerance via induction of IL-10-producing CD4(+) T cells. We examined the role of TNF-alpha in generation of the IL-10-producing CD4(+) T cells by immature DCs. Immature bone marrow-derived DCs from wild type (WT) or TNF-alpha(-/-) mice were cocultured with CD4(+) T cells from OVA specific TCR transgenic mice (OT-II) in the presence of OVA(323-339) peptide. The WT DCs efficiently induced the antigen-specific IL-10-producing CD4(+) T cells, while the ability of the TNF-alpha(-/-) DCs to induce these CD4(+) T cells was considerably depressed. Addition of exogenous TNF-alpha recovered the impaired ability of the TNF-alpha(-/-) DCs to induce IL-10-producing T cells. However, no difference in this ability was observed between TNF-alpha(-/-) and WT DCs after their maturation by LPS. Thus, TNF-alpha appears to be critical for the generation of IL-10-producing CD4(+) T cells during the antigen presentation by immature DCs.

A Novel Mouse Model of iNKT Cell-deficiency Generated by CRISPR/Cas9 Reveals a Pathogenic Role of iNKT Cells in Metabolic Disease

AbstractiNKT cells play important roles in immune regulation by bridging the innate and acquired immune systems. The functions of iNKT cells have been investigated in mice lacking the Traj18 gene segment that were generated by traditional embryonic stem cell technology, but these animals contain a biased T cell receptor (TCR) repertoire that might affect immune responses. To circumvent this confounding factor, we have generated a new strain of iNKT cell-deficient mice by deleting the Traj18 locus using CRISPR/Cas9 technology, and these animals contain an unbiased TCR repertoire. We employed these mice to investigate the contribution of iNKT cells to metabolic disease and found a pathogenic role of these cells in obesity-associated insulin-resistance. The new Traj18-deficient mouse strain will assist in studies of iNKT cell biology.