Metabolic inhibitors

Freshly isolated splenic CD4⁺ T cells (5 × 10⁵ per well) were polarized for 3 d with plate-bound anti-CD3e (2 ug/ml) and soluble anti-CD28 (1 ug/ml) in RPMI. Th1 polarization was performed by adding IL-12 (10 ng/ml) and anti-IL-4 (10 ug/ml), and Th17 polarization was performed by adding TGFβ (3 ng/ml), IL-6 (50 ng/ml), 6-Formylindolo (3,2-b) carbazole (FICZ, 300 nM; Enzo Life Sciences), anti-IL-4 and anti-IFNγ (10 ug/ml each). Metabolic inhibitors (all from Sigma), Met (1 mM), 2DG (1 mM), DCA (10 mM), UK5099 (10 uM) or troglitazone (10 uM), were added to the cell cultures at the beginning of polarization. Gene expression for metabolic enzymes was measured by quantitative RT-PCR using Sybr Green incorporation as previously indicated (19 ).

TC mice have been described previously (18 (link)). B6 and B6.MRL-Fas^lpr/J (B6.lpr) mice were originally purchased from the Jackson Laboratory. Only female mice were used in this study at the age indicated for each experiment. Treatment was performed with metabolic inhibitors (all from Sigma) dissolved in drinking water: Met (3 mg/mL), 2DG (5 mg/mL), dichloroacetate (DCA, 2mg/ml) or a combination of two of these drugs for the duration indicated for each study. For each treatment study, contemporaneous age-matched control mice received plain drinking water. Preventive treatments were performed in 2 month old mice, and reversal treatments were performed in mice at least 7 month of age and all anti-dsDNA IgG positive for TC mice, and 4 month of age for B6.lpr mice. Peripheral blood was collected to analyze autoantibody production; body weight and blood sugar levels were monitored weekly and biweekly respectively. At the end of the treatment, spleens were collected for flow cytometry and metabolic analysis of CD4⁺ T cells, and kidneys were evaluated for renal pathology. All experiments were conducted according to protocols approved by the University of Florida IACUC.