Deficiency of Stat1 in CD11c+ Cells Alters Adipose Tissue Inflammation and Improves Metabolic Dysfunctions in Mice Fed a High-Fat Diet.

CD11c+ macrophages/dendritic cells (MDCs) are increased and display the classically activated M1-like phenotype in obese adipose tissue (AT) and may contribute to AT inflammation and insulin resistance. Stat1 is a key transcription factor for MDC polarization into the M1-like phenotype. Here, we examined the role of Stat1 in obesity-induced AT MDC polarization and inflammation and insulin resistance using mice with specific knockout of Stat1 in MDCs (cKO). Stat1 was upregulated and phosphorylated, indicating activation, early and persistently in AT and AT MDCs of wild-type mice fed a high-fat diet (HFD). Compared with littermate controls, cKO mice fed an HFD (16 weeks) had reductions in MDC (mainly CD11c+ macrophage) M1-like polarization and interferon-γ-expressing T-helper type 1 (Th1) cells but increases in interleukin 5-expressing Th2 cells and eosinophils in perigonadal and inguinal AT, and enhanced inguinal AT browning, with increased energy expenditure. cKO mice compared with controls also had significant reductions in triglyceride content in the liver and skeletal muscle and exhibited improved insulin sensitivity and glucose tolerance. Taken together, our results demonstrate that Stat1 in MDCs plays an important role in obesity-induced MDC M1-like polarization and AT inflammation and contributes to insulin resistance and metabolic dysfunctions in obese mice.

Foamy monocytes form early and contribute to nascent atherosclerosis in mice with hypercholesterolemia.

OBJECTIVE: To examine infiltration of blood foamy monocytes, containing intracellular lipid droplets, into early atherosclerotic lesions and its contribution to development of nascent atherosclerosis. APPROACH AND RESULTS: In apoE(-/-) mice fed Western high-fat diet (WD), >10% of circulating monocytes became foamy monocytes at 3 days on WD and >20% of monocytes at 1 week. Foamy monocytes also formed early in blood of Ldlr(-/-)Apobec1(-/-) (LDb) mice on WD. Based on CD11c and CD36, mouse monocytes were categorized as CD11c(-)CD36(-), CD11c(-)CD36(+), and CD11c(+)CD36(+). The majority of foamy monocytes were CD11c(+)CD36(+), whereas most nonfoamy monocytes were CD11c(-)CD36(-) or CD11c(-)CD36(+) in apoE(-/-) mice on WD. In wild-type mice, CD11c(+)CD36(+) and CD11c(-)CD36(+), but few CD11c(-)CD36(-), monocytes took up cholesteryl ester-rich very low-density lipoproteins (CE-VLDLs) isolated from apoE(-/-) mice on WD, and CE-VLDL uptake accelerated CD11c(-)CD36(+) to CD11c(+)CD36(+) monocyte differentiation. Ablation of CD36 decreased monocyte uptake of CE-VLDLs. Intravenous injection of DiI-CE-VLDLs in apoE(-/-) mice on WD specifically labeled CD11c(+)CD36(+) foamy monocytes, which infiltrated into nascent atherosclerotic lesions and became CD11c(+) cells that were selectively localized in atherosclerotic lesions. CD11c deficiency reduced foamy monocyte infiltration into atherosclerotic lesions. Specific and consistent depletion of foamy monocytes (for 3 weeks) by daily intravenous injections of low-dose clodrosome reduced development of nascent atherosclerosis. CONCLUSIONS: Foamy monocytes, which form early in blood of mice with hypercholesterolemia, infiltrate into early atherosclerotic lesions in a CD11c-dependent manner and play crucial roles in nascent atherosclerosis development.

Attenuated adipose tissue and skeletal muscle inflammation in obese mice with combined CD4+ and CD8+ T cell deficiency.

OBJECTIVES: High-fat diet (HFD) feeding in mice is characterized by accumulation of αß T cells in adipose tissue. However, the contribution of αß T cells to obesity-induced inflammation of skeletal muscle, a major organ of glucose uptake, is unknown. This study was undertaken to evaluate the effect of αß T cells on insulin sensitivity and inflammatory state of skeletal muscle and adipose tissue in obesity. Furthermore, we investigated whether CD4+IFNγ+ (TH1) cells are involved in skeletal muscle and adipose tissue metabolic dysfunction that accompanies obesity. METHODS: Mice lacking αß T cells (T cell receptor beta chain-deficient [TCRb-/-] mice) were fed HFD for 12 weeks. Obesity-induced skeletal muscle and adipose tissue inflammation was assessed by flow cytometry and quantitative RT-PCR. To investigate the effect of TH1 cells on skeletal muscle and adipose tissue inflammation and metabolic functions, we injected 5×10(5) TH1 cells or PBS weekly over 12 weeks into HFD-fed TCRb-/- mice. We also cultured C2C12 myofibers and 3T3-L1 adipocytes with TH1-conditioned medium. RESULTS: We showed that similar to adipose tissue, skeletal muscle of obese mice have higher αß T cell content, including TH1 cells. TCRb-/- mice were protected against obesity-induced hyperglycemia and insulin resistance. We also demonstrated suppressed macrophage infiltration and reduced inflammatory cytokine expression in skeletal muscle and adipose tissue of TCRb-/- mice on HFD compared to wild-type obese controls. Adoptive transfer of TH1 cells into HFD-fed TCRb-/- mice resulted in increased skeletal muscle and adipose tissue inflammation and impaired glucose metabolism. TH1 cells directly impaired functions of C2C12 myotubes and 3T3-L1 adipocytes in vitro. CONCLUSIONS: We conclude that reduced adipose tissue and skeletal muscle inflammation in obese TCRb-/- mice is partially attributable to the absence of TH1 cells. Our results suggest an important role of TH1 cells in regulating inflammation and insulin resistance in obesity.

Essential role of CD11a in CD8+ T-cell accumulation and activation in adipose tissue.

OBJECTIVE: T cells, particularly CD8(+) T cells, are major participants in obesity-linked adipose tissue (AT) inflammation. We examined the mechanisms of CD8(+) T-cell accumulation and activation in AT and the role of CD11a, a ß2 integrin. APPROACH AND RESULTS: CD8(+) T cells in AT of obese mice showed activated phenotypes with increased proliferation and interferon-γ expression. In vitro, CD8(+) T cells from mouse AT displayed increased interferon-γ expression and proliferation to stimulation with interleukin-12 and interleukin-18, which were increased in obese AT. CD11a was upregulated in CD8(+) T cells in obese mice. Ablation of CD11a in obese mice dramatically reduced T-cell accumulation, activation, and proliferation in AT. Adoptive transfer showed that CD8(+) T cells from wild-type mice, but not from CD11a-deficient mice, infiltrated into AT of recipient obese wild-type mice. CD11a deficiency also reduced tumor necrosis factor-α-producing and interleukin-12-producing macrophages in AT and improved insulin resistance. CONCLUSIONS: Combined action of cytokines in obese AT induces proliferative response of CD8(+) T cells locally, which, along with increased infiltration, contributes to CD8(+) T-cell accumulation and activation in AT. CD11a plays a crucial role in AT inflammation by participating in T-cell infiltration and activation.

ApoE and the role of very low density lipoproteins in adipose tissue inflammation.

OBJECTIVE: To identify the role of triglyceride-rich lipoproteins (TGRLs) and apoE, a major apolipoprotein in TGRLs, in adipose tissue inflammation with high-fat diet (HFD)-induced obesity. METHODS: Male apoE(-/-) and C57BL/6J wild-type (WT) mice fed HFD for 12 weeks were assessed for metabolic and inflammatory parameters. ApoE(-/-) and WT mice were orally gavaged with [(3)H]palmitic acid to examine the role of apoE in fat delivery to adipose tissue. VLDL from obese apoE(-/-) mice were intravenously injected into lean WT or apoE(-/-) mice to test potential contribution of TGRLs-derived fat delivery to inflammation in adipose tissue and the role of apoE. RESULTS: ApoE(-/-) mice gained less body weight, and had less fat mass and lower triglyceride levels in skeletal muscle than WT. ApoE(-/-) mice on HFD had better insulin sensitivity than WT even when comparing body weight-matched mice. Compared to WT mice, apoE(-/-) mice on HFD had lower levels of inflammatory cytokines/chemokines and CD11c in adipose tissue, and lower levels of inflammatory markers in skeletal muscle. At 6 h after oral gavage with [(3)H]palmitic acid, incorporation of [(3)H]palmitic acid into adipose tissue and skeletal muscle was lower in apoE(-/-) mice. After repeated daily injection for 3 days, VLDL from obese apoE(-/-) mice induced inflammation in adipose tissue of recipient WT but not apoE(-/-) mice. CONCLUSION: In HFD-induced obesity, apoE plays an important role in inflammation in adipose tissue and skeletal muscle, likely by mediating TGRL-derived fat delivery to these tissues.

Differential effect of weight loss with low-fat diet or high-fat diet restriction on inflammation in the liver and adipose tissue of mice with diet-induced obesity.

OBJECTIVE: We studied the effects of weight loss induced by either a low-fat normal diet (ND) or restriction of high-fat diet (HFD) on hepatic steatosis, inflammation in the liver and adipose tissue (AT), and blood monocytes of obese mice. METHODS: In mice with HFD-induced obesity, weight loss was achieved by switching from HFD to ND and maintaining on ND ad libitum or by restricting HFD intake to match body weight of mice with ND-induced weight loss. After diet interventions for 4 weeks, hepatic steatosis, hepatic and AT inflammation, and blood CD11c(+) monocytes were examined. RESULTS: At 4 weeks after switching diets, body weight was reduced by 23% from baseline. To achieve the same reduced body weight required restricting calorie intake from HFD. Weight loss with either ND or HFD restriction decreased body fat mass and ameliorated liver steatosis; both effects were greater with ND-induced weight loss than HFD restriction-induced weight loss. Weight loss with ND but not HFD restriction normalized blood CD11c(+) monocytes and attenuated hepatic inflammation assessed by chemokine and CD11c expression. In contrast, weight loss with HFD restriction significantly reduced chemokine levels and CD11c(+) cells in AT compared to obese controls, and tended to reduce AT chemokines and CD11c(+) cells more than ND-induced weight loss. CONCLUSION: In mice with diet-induced obesity, weight loss with ND was superior in alleviating hepatic inflammation and steatosis, whereas weight loss with HFD calorie restriction provided greater amelioration of AT inflammation.

Effect of the cannabinoid receptor-1 antagonist rimonabant on inflammation in mice with diet-induced obesity.

We studied whether cannabinoid receptor (CB1) blockade with rimonabant has an anti-inflammatory effect in obese mice, and whether this effect depends on weight loss and/or diet consumption. High-fat diet (HFD)-induced obese mice were treated orally with rimonabant (HFD-R) or vehicle (HFD-V) for 4 weeks. Paired-feeding was conducted in two additional groups of obese mice to achieve either the same body weight (HFD-BW) or the same HFD intake (HFD DI) as HFD-R. All these groups of mice were maintained on HFD throughout, with mice on normal diet (ND) throughout as lean controls. Rimonabant treatment of obese mice induced marked diet-intake reduction and weight loss during the first week, which was followed by maintenance of low body weight but not diet-intake reduction. Lower HFD intake was required to reach the same degree of weight loss in HFD-BW. HFD-DI had similar weight loss initially, but then started to gain weight, reaching a higher body weight than HFD-R. Despite the same degree of weight loss, HFD-R had less fat mass and lower adipogenic gene expression than HFD-BW. Compared to HFD-V or HFD-DI, HFD-R had reduced inflammation in adipose tissue (AT) and/or liver indicated primarily by lower monocyte chemoattractant protein-1 (MCP-1) levels. However, MCP-1 levels were not significantly different between HFD-R and HFD-BW. In vitro incubation of rimonabant with AT explants did not change MCP-1 levels. Thus, rimonabant induced weight loss in obese mice by diet-intake-dependent and -independent fashions. Rimonabant decreased inflammation in obese mice, possibly through a primary effect on weight reduction.

CD11c expression in adipose tissue and blood and its role in diet-induced obesity.

OBJECTIVE: To examine CD11c, a beta(2)-integrin, on adipose tissue (AT) leukocytes and blood monocytes and its role in diet-induced obesity. METHODS AND RESULTS: High-fat diet-induced obese C57BL/6 mice, CD11c-deficient mice, and obese humans were studied. CD11c, leukocytes, and chemokines/cytokines were examined in AT and/or blood by flow cytometry, RNase protection assay, quantitative polymerase chain reaction, or enzyme-linked immunosorbent assay. Obese C57BL/6 mice had increased CD11c in AT and blood compared with lean controls. CD11c messenger RNA positively correlated with monocyte chemoattractant protein 1 in human visceral AT. Obese humans with metabolic syndrome had a higher CD11c level on blood monocytes compared with lean humans. Low-fat diet-induced weight loss reduced blood monocyte CD11c in obese mice and humans. Mouse and human monocyte CD11c levels and mouse AT CD11c messenger RNA correlated with insulin resistance. CD11c deficiency in mice did not alter weight gain but decreased inflammation, evidenced by a lower T-cell number and reduced levels of major histocompatibility complex class II, C-C chemokine ligand 2 (CCL5), CCL4, and interferon gamma in AT, and ameliorated insulin resistance and glucose intolerance associated with diet-induced obesity. CONCLUSIONS: Diet-induced obesity increased CD11c in both AT and blood in mice and humans. CD11c plays an important role in T-cell accumulation and activation in AT, and contributes to insulin resistance associated with obesity.

T-cell accumulation and regulated on activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity.

BACKGROUND: Obesity is associated with chronic inflammation, which includes increased macrophage accumulation in adipose tissue (AT) and upregulation of chemokines and cytokines. T cells also play important roles in chronic inflammatory diseases such as atherosclerosis but have not been well studied in obesity. METHODS AND RESULTS: Flow cytometric analysis showed higher numbers of T cells and macrophages in AT of diet-induced obese insulin-resistant male mice than in lean mice and obese females (P<0.05). RNase protection assay, ELISA, and flow cytometry indicated gender-dependent upregulation of mRNA and protein levels of regulated on activation, normal T cell expressed and secreted (RANTES) and its receptor CCR5 in AT of obese mice. Adipocytes, stromal/vascular cells from mouse AT, and human and murine adipocytes expressed RANTES. RANTES mRNA levels were negatively correlated with adiponectin in mouse AT. Adiponectin-deficient mice fed high-fat diet showed higher RANTES mRNA levels in AT than wild-type mice. Activated T cells coincubated with preadipocytes in vitro significantly suppressed preadipocyte-to-adipocyte differentiation. Obese humans with metabolic syndrome had higher mRNA levels of RANTES and CCR5 in subcutaneous AT than lean humans. RANTES and CCR5 mRNA levels were significantly higher in visceral than subcutaneous AT of morbidly obese humans. RANTES mRNA levels were positively correlated with CD3 and CD11b in human visceral AT. CONCLUSIONS: Obesity is associated with increased accumulation of T cells and macrophages in AT, which may play important roles in obesity-related disease by influencing preadipocyte/adipocyte functions. RANTES is an adipokine that is upregulated in AT by obesity in both mice and humans.

Deficiency of CD11b or CD11d results in reduced staphylococcal enterotoxin-induced T cell response and T cell phenotypic changes.

The beta(2) integrin CD11a is involved in T cell-APC interactions, but the roles of CD11b, CD11c, and CD11d in such interactions have not been examined. To evaluate the roles of each CD11/CD18 integrin in T cell-APC interactions, we tested the ability of splenocytes of CD11-knockout (KO) mice to respond to staphylococcal enterotoxins (SEs), a commonly used superantigen. The defect in T cell proliferation with SEA was more severe in splenocytes from mice deficient in CD18, CD11b, or CD11d than in CD11a-deficient splenocytes, with a normal response in CD11c-deficient splenocytes. Mixing experiments showed that the defect of both CD11b-KO and CD11d-KO splenocytes was, unexpectedly, in T cells rather than in APC. Cytometric analysis failed to detect CD11b or CD11d on resting or activated T cells or on thymocytes of wild-type adult mice, nor did Abs directed to these integrins block responses in culture, suggesting that T cells educated in CD11b-KO or CD11d-KO mice were phenotypically altered. Consistent with this hypothesis, T cells from CD11b-KO and CD11d-KO splenocytes exhibited reduced intensity of CD3 and CD28 expression and decreased ratios of CD4/CD8 cells, and CD4(+) T cells were reduced among CD11b-KO and CD11d-KO thymocytes. CD11b and CD11d were coexpressed on a subset of early wild-type fetal thymocytes. We postulate that transient thymocyte expression of both CD11b and CD11d is nonredundantly required for normal thymocyte and T cell development, leading to phenotypic changes in T cells that result in the reduced response to SE stimulation.

Biodistribution and safety studies of hDel-1 plasmid-based gene therapy in mouse and rabbit models.

A plasmid encoding the human developmentally regulated endothelial locus-1 (hDel-1) protein formulated with poloxamer 188 is a potential gene therapy for peripheral arterial disease in man. As a prelude to clinical trials, the biodistribution and safety of this therapy were evaluated after intramuscular and intravenous administration in mice and rabbits. In mice, plasmid DNA persisted at the intramuscular injection site for at least 28 days, but was barely detectable in distal tissues by 24 h and essentially cleared by 28 days. By 24 h after intravenous administration, plasmid DNA was readily detected in blood, muscle, and lungs but sporadically and at low levels in other tissues. At 28 days, plasmid DNA was readily detectable only at the intravenous injection site (tail) after low- and high-dose administration, and sporadically in blood and muscle after high-dose administration. In rabbits, the highest intramuscular (4.2 mg kg(-1)) or intravenous (3.7 mg kg(-1)) dose caused no deaths; no treatment-related clinical signs; no changes in body weight, clinical pathology parameters, ophthalmology, ECG, or histopathology; and no detectable increase in antinuclear antibodies by 28 days. The results supported testing of hDel-1 plasmid-based gene therapy in phase I clinical trials.