IgG is elevated in obese white adipose tissue but does not induce glucose intolerance via Fcγ-receptor or complement.

BACKGROUND/OBJECTIVES: In obesity, B cells accumulate in white adipose tissue (WAT) and produce IgG, which may contribute to the development of glucose intolerance. IgG signals by binding to Fcγ receptors (FcγR) and by activating the complement system. The aim of our study was to investigate whether activation of FcγR and/or complement C3 mediates the development of high-fat diet-induced glucose intolerance. METHODS: We studied mice lacking all four FcγRs (FcγRI/II/III/IV-/-), only the inhibitory FcγRIIb (FcγRIIb-/-), only the central component of the complement system C3 (C3-/-), and mice lacking both FcγRs and C3 (FcγRI/II/III/IV/C3-/-). All mouse models and wild-type controls were fed a high-fat diet (HFD) for 15 weeks to induce obesity. Glucose metabolism was assessed and adipose tissue was characterized for inflammation and adipocyte functionality. RESULTS: In obese WAT of wild-type mice, B cells (+142%, P<0.01) and IgG (+128% P<0.01) were increased compared to lean WAT. Macrophages of FcγRI/II/III/IV-/-mice released lower levels of cytokines compared to wild-type mice upon IgG stimulation. Only C3-/- mice showed reduced HFD-induced weight gain as compared to controls (-18%, P<0.01). Surprisingly, FcγRI/II/III/IV-/- mice had deteriorated glucose tolerance (AUC +125%, P<0.001) despite reduced leukocyte number (-30%, P<0.05) in gonadal WAT (gWAT), whereas glucose tolerance and leukocytes within gWAT in the other models were unaffected compared to controls. Although IgG in gWAT was increased (+44 to +174%, P<0.05) in all mouse models lacking FcγRIIb, only FcγRI/II/III/IV/C3-/- mice exhibited appreciable alterations in immune cells in gWAT, for example, increased macrophages (+36%, P<0.001). CONCLUSIONS: Lack of FcγRs reduces the activity of macrophages upon IgG stimulation, but neither FcγR nor C3 deficiency protects against HFD-induced glucose intolerance or reduces adipose tissue inflammation. This indicates that if obesity-induced IgG contributes to the development of glucose intolerance, this is not mediated by FcγR or complement activation.

Blocking CD40-TRAF6 interactions by small-molecule inhibitor 6860766 ameliorates the complications of diet-induced obesity in mice.

BACKGROUND: Immune processes contribute to the development of obesity and its complications, such as insulin resistance, type 2 diabetes mellitus and cardiovascular disease. Approaches that target the inflammatory response are promising therapeutic strategies for obesity. In this context, we recently demonstrated that the interaction between the costimulatory protein CD40 and its downstream adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes adipose tissue inflammation, insulin resistance and hepatic steatosis in mice in the course of diet-induced obesity (DIO). METHODS: Here we evaluated the effects of a small-molecule inhibitor (SMI) of the CD40-TRAF6 interaction, SMI 6860766, on the development of obesity and its complications in mice that were subjected to DIO. RESULTS: Treatment with SMI 6860766 did not result in differences in weight gain, but improved glucose tolerance. Moreover, SMI 6860766 treatment reduced the amount of CD45(+) leucocytes in the epididymal adipose tissue by 69%. Especially, the number of adipose tissue CD4(+) and CD8(+) T cells, as well as macrophages, was significantly decreased. CONCLUSIONS: Our results indicate that small-molecule-mediated inhibition of the CD40-TRAF6 interaction is a promising therapeutic strategy for the treatment of metabolic complications of obesity by improving glucose tolerance, by reducing the accumulation of immune cells to the adipose tissue and by skewing of the immune response towards a more anti-inflammatory profile.

Differentiation factors and cytokines in the atherosclerotic plaque micro-environment as a trigger for macrophage polarisation.

The phenotype of macrophages in atherosclerotic lesions can vary dramatically, from a large lipid laden foam cell to a small inflammatory cell. Classically, the concept of macrophage heterogeneity discriminates between two extremes called either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. Polarisation of plaque macrophages is predominantly determined by the local micro-environment present in the atherosclerotic lesion and is rather more complex than typically described by the M1/M2 paradigm. In this review we will discuss the role of various polarising factors in regulating the phenotypical state of plaque macrophages. We will focus on two main levels of phenotype regulation, one determined by differentiation factors produced in the lesion and the other determined by T-cell-derived polarising cytokines. With foam cell formation being a key characteristic of macrophages during atherosclerosis initiation and progression, these polarisation factors will also be linked to lipid handling of macrophages.

Disruption of hedgehog signalling in ApoE - /- mice reduces plasma lipid levels, but increases atherosclerosis due to enhanced lipid uptake by macrophages.

Embryonic pathways are often re-expressed in adult pathology. Here we investigated the role of the morphogen hedgehog (hh), which we found to be re-expressed in atherosclerotic plaques. Male ApoE - /- mice were treated for 12 weeks with an anti-hh antibody (5E1) or a control IgG (1E6) starting at the age of 6 or 18 weeks. Inhibition of hh signalling induced a significant increase in total plaque area in the aortic arch, a result of an increase (54% and 36%, respectively) in the area of advanced plaques (atheromata). In mice treated with anti-hh, plaques contained large (18-35% > ctrl), lipid-filled, sometimes multinucleated macrophage foam cells. Plasma cholesterol levels decreased after anti-hh treatment. In bone marrow-derived macrophages, foam cell formation was enhanced after inhibition of hh signalling. Anti-hh treatment caused a 54-75% increase in early oxLDL uptake (10-240 min), which was scavenger receptor-mediated. After 3-24 h of oxLDL incubation, intense Oil red O staining as well as increased amounts of cholesterol esters were present in these macrophages after anti-hh treatment. Activation of the HH-signalling cascade by recombinant Shh induced a decrease in oxLDL uptake. Here we show that the hh-signalling pathway is one of the morphogenic pathways that regulate plasma lipid levels and atherosclerosis development and progression.

Gene profiling of cathepsin K deficiency in atherogenesis: profibrotic but lipogenic.

Recently, we showed that cathepsin K deficiency reduces atherosclerotic plaque progression, induces plaque fibrosis, but aggravates macrophage foam cell formation in the ApoE -/- mouse. To obtain more insight into the molecular mechanisms by which cathepsin K disruption evokes the observed phenotypic changes, we used microarray analysis for gene expression profiling of aortic arches of CatK -/-/ApoE -/- and ApoE -/- mice on a mouse oligo microarray. Out of 20 280 reporters, 444 were significantly differentially expressed (p-value of < 0.05, fold change of > or = 1.4 or < or = - 1.4, and intensity value of > 2.5 times background in at least one channel). Ingenuity Pathway Analysis and GenMAPP revealed upregulation of genes involved in lipid uptake, trafficking, and intracellular storage, including caveolin - 1, - 2, - 3 and CD36, and profibrotic genes involved in transforming growth factor beta (TGFbeta) signalling, including TGFbeta2, latent TGFbeta binding protein-1 (LTBP1), and secreted protein, acidic and rich in cysteine (SPARC), in CatK -/-/ApoE -/- mice. Differential gene expression was confirmed at the mRNA and protein levels. In vitro modified low density lipoprotein (LDL) uptake assays, using bone marrow derived macrophages preincubated with caveolae and scavenger receptor inhibitors, confirmed the importance of caveolins and CD36 in increasing modified LDL uptake in the absence of cathepsin K. In conclusion, we suggest that cathepsin K deficiency alters plaque phenotype not only by decreasing proteolytic activity, but also by stimulating TGFbeta signalling. Besides this profibrotic effect, cathepsin K deficiency has a lipogenic effect owing to increased lipid uptake mediated by CD36 and caveolins.

Disruption of the cathepsin K gene reduces atherosclerosis progression and induces plaque fibrosis but accelerates macrophage foam cell formation.

BACKGROUND: Cathepsin K (catK), a lysosomal cysteine protease, was identified in a gene-profiling experiment that compared human early plaques, advanced stable plaques, and advanced atherosclerotic plaques containing a thrombus, where it was highly upregulated in advanced stable plaques. METHODS AND RESULTS: To assess the function of catK in atherosclerosis, catK(-/-)/apolipoprotein (apo) E(-/-) mice were generated. At 26 weeks of age, plaque area in the catK(-/-)/apoE(-/-) mice was reduced (41.8%) owing to a decrease in the number of advanced lesions as well as a decrease in individual advanced plaque area. This suggests an important role for catK in atherosclerosis progression. Advanced plaques of catK(-/-)/apoE(-/-) mice showed an increase in collagen content. Medial elastin fibers were less prone to rupture than those of apoE(-/-) mice. Although the relative macrophage content did not differ, individual macrophage size increased. In vitro studies of bone marrow derived-macrophages confirmed this observation. Scavenger receptor-mediated uptake (particularly by CD36) of modified LDL increased in the absence of catK, resulting in an increased macrophage size because of increased cellular storage of cholesterol esters, thereby enlarging the lysosomes. CONCLUSIONS: A deficiency of catK reduces plaque progression and induces plaque fibrosis but aggravates macrophage foam cell formation in atherosclerosis.

Leukocyte CD40L deficiency affects the CD25(+) CD4 T cell population but does not affect atherosclerosis.

Inhibition of CD40-CD40L interactions results in a reduction of innate regulatory T cells (Tregs) in CD40(-/-) mice and induces a stable plaque phenotype in atherosclerosis-prone mouse strains. Here we investigated the effects of leukocyte CD40L on the Treg population and on atherosclerosis. LDLR(-/-) mice were reconstituted with wild-type or CD40L(-/-) bone marrow (BM). These BM chimeras were analysed by flow cytometry for the presence of innate Tregs (CD45RB(low) CD25(+) CD4) in lymphoid organs and peripheral blood. As in CD40(-/-) mice, the CD45RB(high):CD45RB(low) CD4 T cell ratio significantly increased and the CD25(+) CD4(+) subpopulation significantly decreased in LDLR(-/-) mice receiving CD40L(-/-) BM compared to LDLR(-/-) mice receiving wild-type BM. However, atherosclerotic plaque progression and plaque phenotype did not change in LDLR(-/-) mice reconstituted with CD40L(-/-) BM. In conclusion, the present study shows that CD40-CD40L interactions on leukocytes are essential for the size of the CD45RB(low) CD25(+) CD4 Treg subpopulation. Nevertheless, CD40L deficiency on hemopoietic cells did not affect atherosclerosis, implying that CD40L expressing leukocytes alone are not responsible for the stable plaque phenotype observed after total CD40L blockade.