Diet-induced obesity in mice diminishes hematopoietic stem and progenitor cells in the bone marrow.

Obesity is associated with chronic low-grade inflammation, characterized by leukocytosis and inflammation in the adipose tissue. Continuous activation of the immune system is a stressor for hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). Here we studied how diet-induced obesity (DIO) affects HSPC population dynamics in the BM. Eight groups of age-matched C57Bl/6 mice received a high-fat diet (45% kilocalories from fat) ranging from 1 d up to 18 wk. The obesogenic diet caused decreased proliferation of lineage(-)Sca-1(+)c-Kit(+) (LSK) cells in the BM and a general suppression of progenitor cell populations including common lymphoid progenitors and common myeloid progenitors. Within the LSK population, DIO induced a shift in stem cells that are capable of self-renewal toward maturing multipotent progenitor cells. The higher differentiation potential resulted in increased lymphoid and myeloid ex vivo colony-forming capacity. In a competitive BM transplantation, BM from obese animals showed impaired multilineage reconstitution when transplanted into chow-fed mice. Our data demonstrate that obesity stimulates the differentiation and reduces proliferation of HSPCs in the BM, leading to a decreased HSPC population. This implies that the effects of obesity on HSPCs hampers proper functioning of the immune system.-Van den Berg, S. M., Seijkens, T. T. P., Kusters, P. J. H., Beckers, L., den Toom, M., Smeets, E., Levels, J., de Winther, M. P. J., Lutgens, E. Diet-induced obesity in mice diminishes hematopoietic stem and progenitor cells in the bone marrow.

Blocking CD40-TRAF6 signaling is a therapeutic target in obesity-associated insulin resistance.

The immune system plays an instrumental role in obesity and insulin resistance. Here, we unravel the role of the costimulatory molecule CD40 and its signaling intermediates, TNF receptor-associated factors (TRAFs), in diet-induced obesity (DIO). Although not exhibiting increased weight gain, male CD40(-/-) mice in DIO displayed worsened insulin resistance, compared with wild-type mice. This worsening was associated with excessive inflammation of adipose tissue (AT), characterized by increased accumulation of CD8(+) T cells and M1 macrophages, and enhanced hepatosteatosis. Mice with deficient CD40-TRAF2/3/5 signaling in MHCII(+) cells exhibited a similar phenotype in DIO as CD40(-/-) mice. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII(+) cells displayed no insulin resistance and showed a reduction in both AT inflammation and hepatosteatosis in DIO. To prove the therapeutic potential of inhibition of CD40-TRAF6 in obesity, DIO mice were treated with a small-molecule inhibitor that we designed to specifically block CD40-TRAF6 interactions; this compound improved insulin sensitivity, reduced AT inflammation, and decreased hepatosteatosis. Our study reveals that the CD40-TRAF2/3/5 signaling pathway in MHCII(+) cells protects against AT inflammation and metabolic complications associated with obesity whereas CD40-TRAF6 interactions in MHCII(+) cells aggravate these complications. Inhibition of CD40-TRAF6 signaling by our compound may provide a therapeutic option in obesity-associated insulin resistance.

Scattered Deletion of PKD1 in Kidneys Causes a Cystic Snowball Effect and Recapitulates Polycystic Kidney Disease.

In total, 1 in 1000 individuals carries a germline mutation in the PKD1 or PKD2 gene, which leads to autosomal dominant polycystic kidney disease (ADPKD). Cysts can form early in life and progressively increase in number and size during adulthood. Extensive research has led to the presumption that somatic inactivation of the remaining allele initiates the formation of cysts, and the progression is further accelerated by renal injury. However, this hypothesis is primarily on the basis of animal studies, in which the gene is inactivated simultaneously in large percentages of kidney cells. To mimic human ADPKD in mice more precisely, we reduced the percentage of Pkd1-deficient kidney cells to 8%. Notably, no pathologic changes occurred for 6 months after Pkd1 deletion, and additional renal injury increased the likelihood of cyst formation but never triggered rapid PKD. In mildly affected mice, cysts were not randomly distributed throughout the kidney but formed in clusters, which could be explained by increased PKD-related signaling in not only cystic epithelial cells but also, healthy-appearing tubules near cysts. In the majority of mice, these changes preceded a rapid and massive onset of severe PKD that was remarkably similar to human ADPKD. Our data suggest that initial cysts are the principal trigger for a snowball effect driving the formation of new cysts, leading to the progression of severe PKD. In addition, this approach is a suitable model for mimicking human ADPKD and can be used for preclinical testing.

Screening assay for blood vessel maturation inhibitors.

In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.

The acceptability of computer-aided cognitive behavioural therapy: a pragmatic study.

The clinical and cost-effectiveness of a computer-aided cognitive behavioural therapy (CCBT) programme, Beating the Blues, is indicated by a number of studies, but relatively little is known about its acceptability for patients with depression, anxiety, or both. This study investigated the acceptability of Beating the Blues offered on eight scheduled clinic visits with brief face-to-face support. Pre and posttreatment measures explored the relationship among programme acceptability, treatment continuation, and outcomes for people accessing the programme in routine care. Two hundred and nineteen patients with depression, anxiety, or both were offered Beating the Blues in 11 primary and secondary care practices. One hundred and ninety-one (87%) completed the pretreatment measures and 84 (38%) completed a treatment feedback questionnaire. Analysis of treatment acceptability for CCBT indicated a positive patient experience with the programme. Pretreatment expectancies predicted CCBT treatment completion but not outcomes. No differences were found between men and women on pretreatment measures. Posttreatment, women reported more favourable responses to the therapy, finding the programme more helpful and more satisfactory, than did men. No relationship between treatment acceptability and age was found. Study limitations, including research methods and attrition rates, and implications for future research are discussed. It is concluded that the Beating the Blues CCBT programme is an acceptable treatment for common mental health problems in routine care.

Deletion of hematopoietic Dectin-2 or CARD9 does not protect against atherosclerotic plaque formation in hyperlipidemic mice.

Inflammatory reactions activated by pattern recognition receptors (PRRs) on the membrane of innate immune cells play an important role in atherosclerosis. Whether the PRRs of the C-type lectin receptor (CLR) family including Dectin-2 may be involved in the pathogenesis of atherosclerosis remains largely unknown. Recently, the CLR-adaptor molecule caspase recruitment domain family member 9 (CARD9) has been suggested to play a role in cardiovascular pathologies as it provides the link between CLR activation and transcription of inflammatory cytokines as well as immune cell recruitment. We therefore evaluated whether hematopoietic deletion of Dectin-2 or CARD9 reduces inflammation and atherosclerosis development. Low-density lipoprotein receptor (Ldlr)-knockout mice were transplanted with bone marrow from wild-type, Dectin-2- or Card9-knockout mice and fed a Western-type diet containing 0.1% (w/w) cholesterol. After 10 weeks, lipid and inflammatory parameters were measured and atherosclerosis development was determined. Deletion of hematopoietic Dectin-2 or CARD9 did not influence plasma triglyceride and cholesterol levels. Deletion of hematopoietic Dectin-2 did not affect atherosclerotic lesion area, immune cell composition, ex vivo cytokine secretion by peritoneal cells or bone marrow derived macrophages. Unexpectedly, deletion of hematopoietic CARD9 increased atherosclerotic lesion formation and lesion severity. Deletion of hematopoietic CARD9 did also not influence circulating immune cell composition and peripheral cytokine secretion. Besides a tendency to a reduced macrophage content within these lesions, plasma MCP-1 levels decreased upon WTD feeding. Deletion of hematopoietic Dectin-2 did not influence atherosclerosis development in hyperlipidemic mice. The absence of CARD9 unexpectedly increased atherosclerotic lesion size and severity, suggesting that the presence of CARD9 may protect against initiation of atherosclerosis development.

High Fat Diet Increases Circulating Endocannabinoids Accompanied by Increased Synthesis Enzymes in Adipose Tissue.

The endocannabinoid system (ECS) controls energy balance by regulating both energy intake and energy expenditure. Endocannabinoid levels are elevated in obesity suggesting a potential causal relationship. This study aimed to elucidate the rate of dysregulation of the ECS, and the metabolic organs involved, in diet-induced obesity. Eight groups of age-matched male C57Bl/6J mice were randomized to receive a chow diet (control) or receive a high fat diet (HFD, 45% of calories derived from fat) ranging from 1 day up to 18 weeks before euthanasia. Plasma levels of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA), and related N-acylethanolamines, were quantified by UPLC-MS/MS and gene expression of components of the ECS was determined in liver, muscle, white adipose tissue (WAT) and brown adipose tissue (BAT) during the course of diet-induced obesity development. HFD feeding gradually increased 2-AG (+132% within 4 weeks, P < 0.05), accompanied by upregulated expression of its synthesizing enzymes Daglα and ß in WAT and BAT. HFD also rapidly increased AEA (+81% within 1 week, P < 0.01), accompanied by increased expression of its synthesizing enzyme Nape-pld, specifically in BAT. Interestingly, Nape-pld expression in BAT correlated with plasma AEA levels (R 2 = 0.171, ß = 0.276, P < 0.001). We conclude that a HFD rapidly activates adipose tissue depots to increase the synthesis pathways of endocannabinoids that may aggravate the development of HFD-induced obesity.

Immune Modulation of Brown(ing) Adipose Tissue in Obesity.

Obesity is associated with a variety of medical conditions such as type 2 diabetes and cardiovascular diseases and is therefore responsible for high morbidity and mortality rates. Increasing energy expenditure by brown adipose tissue (BAT) is a current novel strategy to reduce the excessive energy stores in obesity. Brown adipocytes burn energy to generate heat and are mainly activated upon cold exposure. As prolonged cold exposure is not a realistic therapy, researchers worldwide are searching for novel ways to activate BAT and/or induce beiging of white adipose tissue. Recently, the contribution of immune cells in the regulation of brown adipocyte activity and beiging of white adipose tissue has gained increased attention, with a prominent role for eosinophils and alternatively activated macrophages. This review discusses the rediscovery of BAT, presents an overview of modes of activation and differentiation of beige and brown adipocytes, and describes the recently discovered immunological pathways that are key in mediating brown/beige adipocyte development and function. Interventions in immunological pathways harbor the potential to provide novel strategies to increase beige and brown adipose tissue activity as a therapeutic target for obesity.

Atorvastatin accelerates clearance of lipoprotein remnants generated by activated brown fat to further reduce hypercholesterolemia and atherosclerosis.

BACKGROUND AND AIMS: Activation of brown adipose tissue (BAT) reduces both hyperlipidemia and atherosclerosis by increasing the uptake of triglyceride-derived fatty acids by BAT, accompanied by formation and clearance of lipoprotein remnants. We tested the hypothesis that the hepatic uptake of lipoprotein remnants generated by BAT activation would be accelerated by concomitant statin treatment, thereby further reducing hypercholesterolemia and atherosclerosis. METHODS: APOE*3-Leiden.CETP mice were fed a Western-type diet and treated without or with the selective ß3-adrenergic receptor (AR) agonist CL316,243 that activates BAT, atorvastatin (statin) or both. RESULTS: ß3-AR agonism increased energy expenditure as a result of an increased fat oxidation by activated BAT, which was not further enhanced by statin addition. Accordingly, statin treatment neither influenced the increased uptake of triglyceride-derived fatty acids from triglyceride-rich lipoprotein-like particles by BAT nor further lowered plasma triglyceride levels induced by ß3-AR agonism. Statin treatment increased the hepatic uptake of the formed cholesterol-enriched remnants generated by ß3-AR agonism. Consequently, statin treatment further lowered plasma cholesterol levels. Importantly, statin, in addition to ß3-AR agonism, also further reduced the atherosclerotic lesion size as compared to ß3-AR agonism alone, without altering lesion severity and composition. CONCLUSIONS: Statin treatment accelerates the hepatic uptake of remnants generated by BAT activation, thereby increasing the lipid-lowering and anti-atherogenic effects of BAT activation in an additive fashion. We postulate that, in clinical practice, combining statin treatment with BAT activation is a promising new avenue to combat hyperlipidemia and cardiovascular disease.

Helminth antigens counteract a rapid high-fat diet-induced decrease in adipose tissue eosinophils.

Brown adipose tissue (BAT) activation and white adipose tissue (WAT) beiging can increase energy expenditure and have the potential to reduce obesity and associated diseases. The immune system is a potential target in mediating brown and beige adipocyte activation. Type 2 and anti-inflammatory immune cells contribute to metabolic homeostasis within lean WAT, with a prominent role for eosinophils and interleukin (IL)-4-induced anti-inflammatory macrophages. We determined eosinophil numbers in epididymal WAT (EpAT), subcutaneous WAT (ScAT) and BAT after 1 day, 3 days or 1 week of high-fat diet (HFD) feeding in C57Bl/6 mice. One day of HFD resulted in a rapid drop in eosinophil numbers in EpAT and BAT, and after 3 days, in ScAT. In an attempt to restore this HFD-induced drop in adipose tissue eosinophils, we treated 1-week HFD-fed mice with helminth antigens from Schistosoma mansoni or Trichuris suis and evaluated whether the well-known protective metabolic effects of helminth antigens involves BAT activation or beiging. Indeed, antigens of both helminth species induced high numbers of eosinophils in EpAT, but failed to induce beiging. In ScAT, Schistosoma mansoni antigens induced mild eosinophilia, which was accompanied by slightly more beiging. No effects were observed in BAT. To study type 2 responses on brown adipocytes directly, T37i cells were stimulated with IL-4. This increased Ucp1 expression and strongly induced the production of eosinophil chemoattractant CCL11 (+26-fold), revealing that brown adipocytes themselves can attract eosinophils. Our findings indicate that helminth antigen-induced eosinophilia fails to induce profound beiging of white adipocytes.

BCG lowers plasma cholesterol levels and delays atherosclerotic lesion progression in mice.

BACKGROUND AND AIMS: Bacille-Calmette-Guérin (BCG), prepared from attenuated live Mycobacterium bovis, modulates atherosclerosis development as currently explained by immunomodulatory mechanisms. However, whether BCG is pro- or anti-atherogenic remains inconclusive as the effect of BCG on cholesterol metabolism, the main driver of atherosclerosis development, has remained underexposed in previous studies. Therefore, we aimed to elucidate the effect of BCG on cholesterol metabolism in addition to inflammation and atherosclerosis development in APOE*3-Leiden.CETP mice, a well-established model of human-like lipoprotein metabolism. METHODS: Hyperlipidemic APOE*3-Leiden.CETP mice were fed a Western-type diet containing 0.1% cholesterol and were terminated 6 weeks after a single intravenous injection with BCG (0.75 mg; 5 × 10(6) CFU). RESULTS: BCG-treated mice exhibited hepatic mycobacterial infection and hepatomegaly. The enlarged liver (+53%, p = 0.001) coincided with severe immune cell infiltration and a higher cholesterol content (+31%, p = 0.03). Moreover, BCG reduced plasma total cholesterol levels (-34%, p = 0.003), which was confined to reduced nonHDL-cholesterol levels (-36%, p = 0.002). This was due to accelerated plasma clearance of cholesterol from intravenously injected [(14)C]cholesteryl oleate-labelled VLDL-like particles (t½ -41%, p = 0.002) as a result of elevated hepatic uptake (+25%, p = 0.05) as well as reduced intestinal cholestanol and plant sterol absorption (up to -37%, p = 0.003). Ultimately, BCG decreased foam cell formation of peritoneal macrophages (-18%, p = 0.02) and delayed atherosclerotic lesion progression in the aortic root of the heart. BCG tended to decrease atherosclerotic lesion area (-59%, p = 0.08) and reduced lesion severity. CONCLUSIONS: BCG reduces plasma nonHDL-cholesterol levels and delays atherosclerotic lesion formation in hyperlipidemic mice.

Mitochondrial Dysfunction Prevents Repolarization of Inflammatory Macrophages.

Macrophages are innate immune cells that adopt diverse activation states in response to their microenvironment. Editing macrophage activation to dampen inflammatory diseases by promoting the repolarization of inflammatory (M1) macrophages to anti-inflammatory (M2) macrophages is of high interest. Here, we find that mouse and human M1 macrophages fail to convert into M2 cells upon IL-4 exposure in vitro and in vivo. In sharp contrast, M2 macrophages are more plastic and readily repolarized into an inflammatory M1 state. We identify M1-associated inhibition of mitochondrial oxidative phosphorylation as the factor responsible for preventing M1→M2 repolarization. Inhibiting nitric oxide production, a key effector molecule in M1 cells, dampens the decline in mitochondrial function to improve metabolic and phenotypic reprogramming to M2 macrophages. Thus, inflammatory macrophage activation blunts oxidative phosphorylation, thereby preventing repolarization. Therapeutically restoring mitochondrial function might be useful to improve the reprogramming of inflammatory macrophages into anti-inflammatory cells to control disease.

Salsalate activates brown adipose tissue in mice.

Salsalate improves glucose intolerance and dyslipidemia in type 2 diabetes patients, but the mechanism is still unknown. The aim of the current study was to unravel the molecular mechanisms involved in these beneficial metabolic effects of salsalate by treating mice with salsalate during and after development of high-fat diet-induced obesity. We found that salsalate attenuated and reversed high-fat diet-induced weight gain, in particular fat mass accumulation, improved glucose tolerance, and lowered plasma triglyceride levels. Mechanistically, salsalate selectively promoted the uptake of fatty acids from glycerol tri[(3)H]oleate-labeled lipoprotein-like emulsion particles by brown adipose tissue (BAT), decreased the intracellular lipid content in BAT, and increased rectal temperature, all pointing to more active BAT. The treatment of differentiated T37i brown adipocytes with salsalate increased uncoupled respiration. Moreover, salsalate upregulated Ucp1 expression and enhanced glycerol release, a dual effect that was abolished by the inhibition of cAMP-dependent protein kinase (PKA). In conclusion, salsalate activates BAT, presumably by directly activating brown adipocytes via the PKA pathway, suggesting a novel mechanism that may explain its beneficial metabolic effects in type 2 diabetes patients.

Smaller grey matter volumes in the anterior cingulate cortex and greater cerebellar volumes in patients with long-term remission of Cushing's disease: a case-control study.

OBJECTIVE: Patients with long-term remission of Cushing's disease (CD) have persistent psychological and cognitive impairments. It is unknown whether, and to what extent, these impairments are accompanied by structural abnormalities in the brain. We aim to investigate structural changes in the brain in patients with predominantly long-term remission of CD and to examine whether these changes are associated with psychological and cognitive dysfunction and clinical severity. DESIGN: A cross-sectional, case-control study. METHODS: In 25 patients with predominantly long-term remission of CD and 25 matched healthy controls, grey matter volumes in the regions of interest (hippocampus, amygdala, and anterior cingulate cortex (ACC)) and in the whole brain were examined, using 3T magnetic resonance imaging and a voxel-based morphometry approach. Psychological and cognitive functioning were assessed using validated questionnaires and clinical severity was assessed using the Cushing's syndrome severity index. RESULTS: Compared with controls, patients had smaller grey matter volumes of areas in the ACC (on average 14%, P<0.05) and greater volume of the left posterior lobe of the cerebellum (on average 34%, P<0.05). As expected, patients with remitted CD reported more depressive symptoms (P=0.005), more anxiety (P=0.003), more social phobia (P=0.034), more apathy (P=0.002), and more cognitive failure (P=0.023) compared with controls, but the differences in grey matter volumes were not associated with psychological or cognitive measures, nor with clinical severity. CONCLUSION: Patients with predominantly long-term remission of CD showed specific structural brain abnormalities, in the presence of psychological dysfunction. Our data form a basis for future work aimed at elucidating the relation of the structural brain abnormalities and the sustained psychological deficits after long-term exposure to high cortisol levels.