1. Coinhibitory Suppression of T Cell Activation by CD40 Protects Against Obesity and Adipose Tissue Inflammation in MiceCLINICAL PERSPECTIVE
by Dennis Wolf, Felix Jehle, Nathaly Anto Michel, Eva Nora Bukosza, Jennifer Rivera, Yung Chih Chen, Natalie Hoppe, Bianca Dufner, Alexandra Ortiz Rodriguez, Christian Colberg, Leandro Nieto, Benjamin Rupprecht, Ansgar Wiedemann, Lisa Schulte, Alexander Peikert, Nicole Bassler, Andrey Lozhkin, Sonja Patricia Hergeth, Peter Stachon, Ingo Hilgendorf, Florian Willecke, Constantin von zur Mühlen, Dominik von Elverfeldt, Christoph J. Binder, Peter Aichele, Nerea Varo, Mark A. Febbraio, Peter Libby, Christoph Bode, Karlheinz Peter, and Andreas Zirlik
Circulation
Volume 129(23):
June 10, 2014
Copyright © American Heart Association, Inc. All rights reserved.

2. CD40 is preferably expressed by adipocytes in obese adipose tissue.
CD40 is preferably expressed by adipocytes in obese adipose tissue. Wild-type (WT) animals were fed a high-fat diet (HFD) to induce obesity or a standard diet (low-fat diet [LFD]) for 20 weeks. Plasma levels of soluble CD40 (sCD40) were determined by ELISA (A). CD40 mRNA in lysates of visceral adipose tissue (VAT; B). Expression of CD40 in protein lysates of the stromal vascular fraction (SVF) or the adipocyte fraction was quantified by ELISA (C) and Western blot (D). Mean expression of CD40 (mean fluorescent intensity) was quantified on VAT leukocytes, and relative change of CD40 expression induced by HFD was plotted as percent of LFD (E and F) or circulating leukocytes (G). Data are presented as mean±SD. Statistical significance was calculated by unpaired 2-tailed Student t test.
Dennis Wolf et al. Circulation. 2014;129:
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3. CD40 deficiency aggravates the metabolic syndrome in mice.
CD40 deficiency aggravates the metabolic syndrome in mice. Wild-type (WT) and CD40−/− mice consumed a high-fat diet (HFD) for 20 weeks. Relative increases in body weight (BW; percent increase from starting body weight) and total body fat, as assessed by magnetic resonance imaging–based body composition analysis, are shown for the indicated time points (A and B). Weight of peripheral fat depositions is displayed as percent body weight (C). Leptin levels were determined by ELISA (D). Glucose tolerance testing (E) and insulin tolerance testing (F) were performed after intraperitoneal injection of glucose or insulin. Plasma glucose, cholesterol, free fatty acids (FAAs), and triglycerides were determined in animals fasting overnight (G and H). Liver cholesterol and triglycerides were quantified in whole-tissue lysates (H). Liver sections were stained with lipid-specific Oil Red O (I). mRNA of peroxisome proliferator-activated receptor-α (PPARα), carbohydrate-responsive element-binding protein (ChREBP), sterol regulatory element binding protein-1 (SREBP1c), free fatty acid synthase (FAS), acetyl-CoA-carboxylase 1 (ACC1), and apolipoprotein-B100 (ApoB100) was determined by real-time polymerase chain reaction in whole-liver tissue (J). Data are depicted as mean±SD. Statistical significance was calculated by repeated 2-way ANOVA (E and F) or unpaired 2-tailed Student t test between the indicated groups. *P<0.05.
Dennis Wolf et al. Circulation. 2014;129:
Copyright © American Heart Association, Inc. All rights reserved.

4. Lack of CD40 potentiates immune cell invasion and inflammatory gene expression in visceral adipose tissue (VAT).
Lack of CD40 potentiates immune cell invasion and inflammatory gene expression in visceral adipose tissue (VAT). Wild-type (WT) and CD40−/− mice consumed a high-fat diet for 20 weeks. Mean adipocyte diameter was quantified by image processing software in VAT sections (A). VAT sections were stained for the T-cell antigen CD8 (B). Infiltration of leukocytes in VAT was identified by flow cytometry and expressed as percentage of all VAT cells or as a ratio of CD8+/CD4+ cells (C). mRNA preparations of VAT from 3 animals per group were analyzed by mRNA chip array. Results are displayed as heat map based on the row Z score (D). Data are depicted as mean±SD. Statistical significance was calculated by unpaired 2-tailed Student t test.
Dennis Wolf et al. Circulation. 2014;129:
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5. Lymphocyte-expressed CD40 accounts for its proinflammatory phenotype during diet-induced obesity.
Lymphocyte-expressed CD40 accounts for its proinflammatory phenotype during diet-induced obesity. Eight-week-old wild-type (WT) mice were lethally irradiated and reconstituted with either bone-marrow–derived cells (BM) from WT mice or a mixture of BM cells from CD40−/− and Rag1−/− mice (Rag1/CD40; A). Mice were subsequently pair-fed with HFD for 12 weeks. Relative increase in body weight (BW) is shown in B. Weight of peripheral fat pads was quantified at the end of the study (C). Plasma glucose was determined in animals fasting overnight (D). Insulin tolerance testing (E) and glucose tolerance testing (F) were performed after intraperitoneal injection of glucose or insulin. Liver lipids were quantified by Oil Red O (ORO) staining of liver sections (G) and quantification of ORO-positive area (H). Triglycerides were determined in fasting animals (I). Leukocyte infiltration into adipose tissue is shown as a percentage of all visceral adipose tissue (VAT) cells (J). Data are depicted as mean±SD. Statistical significance was calculated by repeated 2-way ANOVA (E and F) or unpaired 2-tailed Student t test between the indicated groups. *P<0.05.
Dennis Wolf et al. Circulation. 2014;129:
Copyright © American Heart Association, Inc. All rights reserved.

6. Activation of CD40 receptor signaling protects against the metabolic syndrome.
Activation of CD40 receptor signaling protects against the metabolic syndrome. Wild-type (WT) mice were fed a high-fat diet (HFD) for 6 weeks. Mice were subsequently treated with the stimulating anti-CD40 antibody FGK45 or a corresponding isotype control (rat IgG2a) for 6 weeks. Increase in weight is shown as percent of starting body (A). Fat depositions were imaged by magnetic resonance imaging (MRI; B). Intraperitoneal glucose tolerance testing (C) and insulin tolerance testing (D) were performed. Glucose levels (E) were measured after overnight starvation. Visceral adipose tissue (VAT) leukocytes were quantified by flow cytometry (F). Representative histograms of CD11c-expressing F4/80+ M1 macrophages are shown in G. SAT indicates subcutaneous adipose tissue. Data are depicted as mean±SD. Statistical significance was calculated by repeated 2-way ANOVA (C and D) or unpaired 2-tailed Student t test. *P<0.05.
Dennis Wolf et al. Circulation. 2014;129:
Copyright © American Heart Association, Inc. All rights reserved.

7. CD40 signaling dampens proinflammatory cytokine release in T cells.
CD40 signaling dampens proinflammatory cytokine release in T cells. Splenocytes were isolated from wild-type (WT) mice, stained with carboxyfluorescein succinimidyl ester (CFSE), and stimulated in the presence of anti-CD3/28 antibodies and FGK45 or an IgG control for 72 hours. CFSE loading as a marker of proliferation (A) and intracellular cytokines such as interferon-γ (IFNγ; B) or tumor necrosis factor-α (TNFα; C) were quantified by flow cytometry. CD4+ and CD8+ T cells (D), CD19+ B cells (E), and CD11c+ macrophages (F) were isolated from visceral adipose tissue (VAT) of obese WT mice by bead separation and incubated with IgG or FGK45 for 24 hours. Activation markers and intracellular cytokine expression are expressed as percent of IgG (D–F). Relative change in cytokines in the supernatant of CD8+ T cells induced by FGK45 (percent change in IgG-treated sample) is shown in G. Data are depicted as mean±SD. Statistical significance was calculated by unpaired 2-tailed Student t test.
Dennis Wolf et al. Circulation. 2014;129:
Copyright © American Heart Association, Inc. All rights reserved.

8. Adoptive transfer of CD40−/− T cells transfers dysmetabolism and inflammation to Rag1−/− mice.
Adoptive transfer of CD40−/− T cells transfers dysmetabolism and inflammation to Rag1−/− mice. Rag1−/− mice were injected intraperitoneally with 5×106 purified T cells from wild-type (WT) or CD40−/− mice or without cells (none; A). After 12 weeks of high-fat-diet weight gain (B), peripheral fat depositions were quantified (C). Glucose tolerance testing (D) and insulin tolerance testing (E) were performed after intraperitoneal injection of glucose or insulin. Inflammatory cell recruitment (F) and activation of T cells (G) into visceral adipose tissue (VAT) were characterized by flow cytometry (F). T-cell subsets were defined as CD62L−CD44+ effector-memory T cells (TEM), CD62L+CD44+ central memory T cells (TCM), and CD62L+CD44− naïve T cells (Tnaïve, H). BW indicates body weight; and FP, fat pads. Data are depicted as mean±SD. Statistical significance was calculated by repeated 2-way ANOVA (D and E) or unpaired 2-tailed Student t test.
Dennis Wolf et al. Circulation. 2014;129:
Copyright © American Heart Association, Inc. All rights reserved.

9. CD40 is associated with obesity in humans.
CD40 is associated with obesity in humans. RNA from human subcutaneous fat tissue was extracted and tested for CD40 mRNA expression. Values were normalized for GAPDH and plotted for the body mass index (BMI) of the tested individuals (A). In a clinical cohort, soluble CD40 (sCD40) levels were quantified in 183 patients and tested for association with BMI (B), waist circumference (WC; C), and leptin (D). High WC was defined as >88 cm for women and >102 cm for men. Median sCD40 plasma level was defined as the cutoff (D). Data are depicted as mean±SD. Statistical significance was calculated as indicated in the Methods section. P values are indicated in each graph.
Dennis Wolf et al. Circulation. 2014;129:
Copyright © American Heart Association, Inc. All rights reserved.