Supplementary MaterialsData_Sheet_1. and insulin tolerance, decreased cholesterol, triglyceride, serum glutamic-oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT) levels that were comparable to NCD controls. However, despite weight loss, increased frequencies, but not total figures, of IL-17+ and IL-22+ CD4+ T cells, IFN-+ and TNF+ CD8+ T cells and IL-17+ and IL-22+ CD8+ T cells were observed in the adipose cells of mice switched from HFD to NCD compared to NCD and even HFD LY294002 fed mice. Further, in the liver, IFN-+ and TNF+ CD8+ T cell, IL-17+ and IL-22+ CD8+ T cell, macrophage frequencies and their manifestation of antigen showing molecules were improved. To determine if macrophages are the major determinants of the sustained inflammation observed during weight loss, we depleted macrophages, which significantly reduced IFN-+, TNF+, IL-17+, and IL-22+ CD8+ T cell frequencies in the liver and the adipose cells. In conclusion, we display that although excess weight loss enhances the metabolic profile, there is an active and ongoing CD8+ T cell swelling in liver and adipose tissue mediated by macrophages. 0.05, ** 0.01, *** 0.001 compared to NCD. $ 0.05, $$ 0.01, $$$ 0.001 compared to HFD to NCD. (G) Serum lipid profile and (H) and liver function enzymes between NCD, HFD and HFD to NCD groups. Pooled data from = 2C3 experiments, 3C5 mice each. Statistical significance was tested by Kruskal-Wallis followed by Dunn’s test (C,D,G,H) or 2-Way ANOVA followed by Tukey’s multiple comparisons test (B,E,F). In a separate experiment, after switching to NCD, macrophages were depleted by intravenous (i.v.) injection of 150 l of clodronate liposomes (Clodronate Liposomes Foundation; Netherlands; http://clodronate.liposomes.com) and the control mice received equal volumes of PBS liposomes. Glucose Tolerance and Insulin Tolerance Test, Lipid Profile and Liver Enzymes Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were carried out as described elsewhere (11). In brief, 6 h after fasting, mice were intraperitoneally (i.p.) injected with 1 g/kg body weight of glucose solution. At 0, 30, 60, and 120 min blood glucose levels were measured by a glucometer (AccuCheck Advantage; Roche Diagnostics GmbH, Mannheim, Germany). Four hours after fasting, ITT was Mouse monoclonal to FLT4 performed. Briefly, human insulin (Sanofi-Aventis, Frankfurt, Germany) 1 U of insulin/kg body weight was i.p. injected and at 0, 30, 60, and 120 min blood glucose levels were measured. The LY294002 area under the curve (AUC) was derived by calculating the area between the x-axis and a given curve using GraphPad Prism software (version 8.3; GraphPad Software, San Diego, Calif., USA). Lipid profiles and liver enzymesserum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT)were measured using Reflotron (Roche Diagnostics GmbH) according to the manufacturer’s protocol. Isolation of Stromal Vascular Fraction From Adipose Tissue and Leucocytes From Liver Mice were deeply anesthetized by i.p. injection of 10 mg/kg xylazin (Rompun? Bayer, Germany) + 100 mg/ml ketamine (Ratiopharm GmbH Germany). Mice were intracardially perfused with 1x PBS for 5 min to remove circulating and non-adhered blood leukocytes from the organs (12). After perfusion, adipose tissue stromal vascular fraction (SVF) and liver lymphocytes were isolated. In brief, the excised epididymal adipose tissue from the mice was digested with 0.2 mg/ml of collagenase (Sigma-Aldrich; Taufkirchen, Germany) LY294002 in DMEM medium at 37C for 40 min. After the digestion, the adipocytes were removed and SVF pellet was filtered by passing through a 40 m filter after red blood cell lysis (Invitrogen, Thermo Fisher Scientific; Carlsbad, CA, USA). To isolate cells from the liver, the liver was minced into small pieces followed by digestion with 0.5 mg/ml collagenase A (Roche, Basel, Switzerland) at 37C for 30 min. After the digestion single cell suspension was generated by passing the digested tissue through a 70 m filter. Lymphocytes were enriched from the LY294002 homogenate using a percoll gradient. Cell Culture After cell enumeration from SVF and liver single cell suspension, isolated cells were cultured in 12-well tissue culture at concentrations of 1 1 106 cells/ml in the presence of phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (1 g/ml) for 6 h in RPMI-1640 moderate (Gibco, Thermo Fischer medical) at 37C. After.