exogenous leptin  (R&D Systems)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 4. Inhibition of mTOR with rapamycin inhibits the expression of leptin and LepR in Teffs and at the systemic level. (A) Proliferation of hu- man Teffs, pretreated or not with rapamycin or anti- leptin neutralizing mAb or both, before anti-CD3/ CD28 stimulation. Data are mean 6 SD (n = 20). *p , 0.005, **p , 0.0001. (B) Proliferation of Teffs, pretreated or not with rapamycin before treatment with leptin (100 ng/ml), before anti-CD3/ CD28 stimulation. Data are mean 6 SD (n = 10). *p , 0.005, **p , 0.0001. (C) Confocal micros- copy of human Teffs, pretreated or not with rapamycin in the presence of anti-CD3/CD28 stimulation, and stained for leptin (green) and LepR (red). Representative of three independent experi- ments. Immunofluorescence images were acquired in the green, red, and blue channels at a resolution of 1024 3 1024 pixels. (D) Real-time PCR for leptin in human Teffs, pretreated or not with rapa- mycin, in the presence of anti-CD3/CD28 stimu- lation. Data are mean 6 SD (n = 5). *p , 0.001. (E) Serum leptin levels in patients with acquired cystic kidney disease, chronically treated with rapamycin, analyzed after 6 and 12 mo of mTOR inhibition treatment (black line). BMI in all of the treated patients at the different time points is represented by the gray bars. Data are mean 6 SD (n = 15 patients/group). *p , 0.05. (F) Serum leptin levels in vehicle or rapamycin-treated mice analyzed at different time points. Data are mean 6 SD (n = 5 mice/group). *p , 0.05.

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: Inhibition, Expressing, Staining, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 5. Leptin activates the mTOR pathway and controls Teff pro- liferation. (A) Immunoblot for p-STAT3, p-AKT, and p-S6 on Baf/3 cells stably transfected with the long form of human leptin receptor, treated or not with leptin for 30 or 60 min, in the presence or absence of LY294002 or rapamycin. One representative of three independent experiments. (B) Im- munoblot for p-mTOR, p-p70S6K, p-S6, and p-STAT3 on unstimulated Teffs treated or not with recombinant leptin for 1 h. One representative of five independent experiments. (C) Immunoblot for p-mTOR, p-p70S6K, p-S6, and p-STAT3 on Teffs in the presence or absence of anti-CD3/28 stimula- tion and treated or not with recombinant leptin or leptin-neutralizing mAb for 1 h. One representative of five independent experiments. (D) Ex vivo p-S6 expression in Teffs from db/+ mice or db/db mice (n = 3 mice/ group, representative of three independent experiments). (E) Ex vivo p-S6 expression in Teffs from ob/ob mice treated or not with recombinant leptin after 2 h from i.p. injection (n = 3 mice/group, representative of three independent experiments). (F) Flow cytometry for BrdU incorpo- ration in Teffs from the lymph nodes of ad libitum fed, 48-h fasted, and 48-h fasted + leptin eGFP-Foxp3 mice. Representative of two independent experiments (n = 3). The gray shaded graph represents the isotype-matched negative control. *p , 0.05 versus 48-h fasted mice, **p , 0.001 versus ad

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: Western Blot, Stable Transfection, Transfection, Recombinant, Ex Vivo, Expressing, Injection, Flow Cytometry, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 6. In vivo leptin enhances Teff proliferation through mTOR activation. (A) Schematic model of the experimental design. Briefly, mice were treated daily with BrdU in basal conditions and upon Ag immunization with CFA; they were injected with a single dose of rapamycin or vehicle or leptin or rapamycin plus leptin 12 h before CFA priming, and the proliferation of Teffs was followed over time. Blood samples were obtained at day 5, and draining lymph nodes were harvested at days 8 and 12. Percentage (B) and absolute number (C) of Teffs gated on CD4+ cells in the lymph nodes from all of the groups of mice immunized with CFA. Data are mean 6 SD (n = 6). *p , 0.05, **p , 0.001. (D) Flow cytometry for BrdU incorporation in Teffs from the lymph nodes of mice pretreated in vivo with vehicle, rapamycin (RAPA), leptin, or RAPA and leptin 12 d after immunization with CFA. Representative of three independent experiments (n = 3 mice/group). *p , 0.005 versus RAPA pretreatment, **p , 0.001 versus vehicle. No significant difference between vehicle versus leptin or vehicle versus RAPA + leptin was detected. The gray shaded graph represents the isotype-matched negative control. (E) p-S6 expression in Teffs from lymph nodes of all groups of mice. Representative of three independent experiments (n = 3 mice/group). *p , 0.005 versus RAPA pretreatment, **p , 0.001 versus vehicle.

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: In Vivo, Activation Assay, Injection, Flow Cytometry, BrdU Incorporation Assay, Negative Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 8. Schematic model of leptin-induced mTOR activation in CD4+CD252FOXP32 Teffs. Under normal conditions (left panel), leptin, by binding its receptor (LepR), activates the mTOR pathway in CD4+CD252FOXP32 Teffs, resulting in an increase in T cell proliferation, Th1/Th17 cytokine se- cretion, and TCR signaling activation. The mTOR pathway itself is responsible for the autocrine leptin secretion by Teffs, which, in turn, sustains their own proliferation. Rapamycin pretreatment (middle) inhibits activation of the leptin-mediated mTOR pathway, resulting in the inhibition of Teff proliferation, a decrease in Th1/Th17 cytokine production, and TCR activation. Moreover, mTOR inhibition deceases leptin production and secretion by Teffs. Similar results can be detected in LepR-deficient Teffs, in which mTOR-pathway activity is impaired because of a lack of proper leptin signaling. These data sustain the hypothesis that rapamycin pretreatment and LepR deficiency share a common cellular, biochemical, and gene-expression profile, suggesting the presence of a convergence between leptin and mTOR at the signaling-pathway level to drive and control Teff responses.

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: Activation Assay, Binding Assay, Inhibition, Activity Assay, Gene Expression, Control