Deficiency of T cell CD40L has minor beneficial effects on obesity-induced metabolic dysfunction

Objective Obesity-associated metabolic dysfunction increases the risk of multiple diseases such as type 2 diabetes and cardiovascular disease. The importance of the co-stimulatory CD40-CD40L dyad in diet-induced obesity (DIO), with opposing phenotypes arising when either the receptor (aggravating) or the ligand (protective) is deleted, has been described previously. The functions of CD40 and CD40L are cell type dependent. As co-stimulation via T cell-mediated CD40L is essential for driving inflammation, we here investigate the role of T cell CD40L in DIO. Research design and methods CD4CreCD40L fl/fl mice on a C57BL/6 background were generated and subjected to DIO by administration of 15 weeks of high fat diet (HFD). Results HFD-fed CD4CreCD40L fl/fl mice had similar weight gain, adipocyte sizes, plasma cholesterol and triglyceride levels as their wild-type (WT) counterparts. Insulin and glucose tolerance were comparable, although CD4CreCD40L fl/fl mice did have a decreased plasma insulin concentration, suggesting a minor improvement of insulin resistance. Furthermore, although the degree of hepatosteatosis was similar in both genotypes, the gene expression of fatty acid synthase 1 and ATP-citrate lyase had decreased, whereas expression of peroxisome proliferator-activated receptor-α had increased in livers of CD4CreCD40L fl/fl mice, suggesting decreased hepatic lipid uptake in absence of T cell CD40L. Moreover, CD4CreCD40L fl/fl mice displayed significantly lower numbers of effector memory CD4 + T cells and regulatory T cells in blood and lymphoid organs compared with WT. However, immune cell composition and inflammatory status of the adipose tissue was similar in CD4CreCD40L fl/fl and WT mice. Conclusions T cell CD40L deficiency results in a minor improvement of insulin sensitivity and hepatic steatosis in DIO, despite the strong decrease in effector T cells and regulatory T cells in blood and lymphoid organs. Our data indicate that other CD40L-expressing cell types are more relevant in the pathogenesis of obesity-associated metabolic dysfunction

Macrophage CD40 plays a minor role in obesity-induced metabolic dysfunction.

Obesity is a low-grade inflammatory disease that increases the risk for metabolic disorders. CD40-CD40L signaling plays a central role in obesity-induced inflammation. Genetic deficiency of CD40L in diet-induced obesity (DIO) ameliorates adipose tissue inflammation, hepatic steatosis and increases insulin sensitivity. Unexpectedly, absence of CD40 worsened insulin resistance and caused excessive adipose tissue inflammation and hepatosteatosis. To investigate whether deficiency of macrophage CD40 is responsible for the phenotype observed in the CD40-/- mice, we generated CD40flflLysMcre and fed them a standard (SFD) and 54% high fat obesogenic diet (HFD) for 13 weeks. No differences in body weight, adipose tissue weight, adipocyte size, plasma cholesterol or triglyceride levels could be observed between CD40flflLysMcre and wild type (WT) mice. CD40flflLysMcre displayed no changes in glucose tolerance or insulin resistance, but had higher plasma adiponectin levels when fed a SFD. Liver weights, liver cholesterol and triglyceride levels, as well as the degree of hepatosteatosis were not affected by absence of macrophage CD40. CD40flflLysMcre mice displayed a minor increase in adipose tissue leukocyte infiltration on SFD and HFD, which did not result in differences in adipose tissue cytokine levels. We here show that loss of macrophage CD40 signaling does not affect obesity induced metabolic dysregulation and indicates that CD40-deficiency on other cell-types than the macrophage is responsible for the metabolic dysregulation, adipose tissue inflammation and hepatosteatosis that are observed in CD40-/- mice

Microgravity Effect on Pancreatic Islets

We previously demonstrated that boundary cap neural crest stem cells (BCs) induce the proliferation of beta-cells in vitro, increase survival of pancreatic islets (PIs) in vivo after transplantation, and themselves strongly increase their proliferation capacity after exposure to space conditions. Therefore, we asked if space conditions can induce the proliferation of beta-cells when PIs are alone or together with BCs in free-floating or 3D-printed form. During the MASER 15 sounding rocket experiment, half of the cells were exposed to 6 min of microgravity (mu g), whereas another group of cells were kept in 1 g conditions in a centrifuge onboard. The proliferation marker EdU was added to the cells just before the rocket reached mu g conditions. The morphological assessment revealed that PIs successfully survived and strongly proliferated, particularly in the free-floating condition, though the fusion of PIs hampered statistical analysis. Proliferation of beta-cells was displayed in 3D-printed islets two weeks after mu g exposure, suggesting that the effects of mu g may be delayed. Thus, PIs in 3D-printed scaffolds did not fuse, and this preparation is more suitable than free-floating specimens for morphological analysis in mu g studies. PIs maintained their increased proliferation capacity for weeks after mu g exposure, an effect that may not appear directly, but can emerge after a delay.De två första författarna delar förstaförfattarskapet</p

Peritoneal macrophages have an impaired immune response in obesity which can be reversed by subsequent weight loss

Introduction Obesity is recognized as a risk factor for various microbial infections. The immune system, which is affected by obesity, plays an important role in the pathophysiology of these infections and other obesity-related comorbidities. Weight loss is considered the most obvious treatment for obesity. However, multiple studies suggest that the comorbidities of obesity may persist after weight loss. Deregulation of immune cells including adipose tissue macrophages of obese individuals has been extensively studied, but how obesity and subsequent weight loss affect immune cell function outside adipose tissue is not well defined. Research design and methods Here we investigated the phenotype of non-adipose tissue macrophages by transcriptional characterization of thioglycollate-elicited peritoneal macrophages (PM) from mice with diet-induced obesity and type 2 diabetes (T2D). Subsequently, we defined the characteristics of PMs after weight loss and mimicked a bacterial infection by exposing PMs to lipopolysaccharide. Results and conclusions In contrast to the proinflammatory phenotype of adipose tissue macrophages in obesity and T2D, we found a deactivated state of PMs in obesity and T2D. Weight loss could reverse this deactivated macrophage phenotype. Anti-inflammatory characteristics of these non-adipose macrophages may explain why patients with obesity and T2D have an impaired immune response against pathogens. Our data also suggest that losing weight restores macrophage function and thus contributes to the reduction of immune-related comorbidities in patients

Macrophage CD40-signaling drives experimental autoimmune encephalomyelitis

The co-stimulatory CD40L-CD40 dyad plays a major role in multiple sclerosis (MS). CD40 is highly expressed on MHCII+ B cells, dendritic cells and macrophages in human MS lesions. Here we investigated the role of the CD40 downstream signaling intermediates TRAF2 and TRAF6 in MHCII+ cells in experimental autoimmune encephalomyelitis (EAE). Both MHCII-CD40-Traf2-/- and MHCII-CD40-Traf6-/- mice showed a reduction in clinical signs of EAE and prevented demyelination. However, only MHCII-CD40-Traf6-/- mice displayed a decrease in myeloid and lymphoid cell infiltration into the central nervous system (CNS) that was accompanied by reduced levels of TNF-α, IL-6, and IFN-γ. As CD40-TRAF6 interactions predominantly occur in macrophages, we subjected CD40flfl LysMcre mice to EAE. This myeloid specific deletion of CD40 resulted in a significant reduction in EAE severity, reduced CNS inflammation and demyelination. In conclusion, the CD40-TRAF6 signaling pathway in MHCII+ cells plays a key role in neuro-inflammation and demyelination during EAE. Concomitant with the fact that CD40-TRAF6 interactions are predominant in macrophages, depletion of myeloid CD40 also reduces neuro-inflammation. CD40-TRAF6 interactions thus represent a promising therapeutic target for multiple sclerosis

Adipocytes control haematopoiesis and inflammation through CD40 signaling

The co-stimulatory CD40-CD40L dyad plays an important role in chronic inflammatory diseases associated with ageing. Although CD40 is mainly expressed by immune cells, CD40 is also present on adipocytes. We aimed to delineate the role of adipocyte-CD40 in the aging haematopoietic system and evaluated the effects of adipocyte CD40 deficiency on cardiometabolic diseases. Adult adipocyte CD40-deficient mice (AdiCD40KO) mice had a decrease in bone marrow (BM) haematopoietic stem cells (Lin-Sca+cKit+, LSK) and common lymphoid progenitors, which was associated with increased BM adiposity and T-cell activation, along with elevated plasma corticosterone levels, a phenotype that became more pronounced with age. Atherosclerotic AdiCD40koApoE-/- (CD40AKO) mice also displayed changes in the LSK population, showing increased myeloid- and lymphoid multipotent progenitors, and augmented corticosterone levels. Increased T-cell activation could be observed in BM, spleen, and adipose tissue (AT), while B-cell numbers were decreased. Although atherosclerosis was reduced in CD40AKO mice, plaques contained more activated T-cells and larger necrotic cores. Analysis of peripheral AT in a diet-induced obesity model revealed that obese AdiCD40KO mice showed increased T-cell activation in AT and lymphoid organs, but exhibited decreased weight gain and improved insulin sensitivity, along with increased fat oxidation. In conclusion, adipocyte CD40 plays an important role in maintaining immune cell homeostasis in BM during ageing and chronic inflammatory diseases, particularly of the lymphoid populations. Although adipocyte CD40-deficiency reduces atherosclerosis burden and ameliorates diet-induced obesity, the accompanying T-cell activation may eventually aggravate cardiometabolic diseases

Apolipoprotein A1 deficiency in mice primes bone marrow stem cells for T cell lymphopoiesis

The bone marrow has emerged as a potentially important target in cardiovascular disease as it generates all leukocytes involved in atherogenesis. In the current study, we evaluated whether a change in bone marrow functionality underlies the increased atherosclerosis susceptibility associated with high-density lipoprotein (HDL) deficiency. We found that HDL deficiency in mice due to the genetic lack of hepatocyte-derived apolipoprotein A1 (APOA1) was associated with an increase in the Lin-Sca-1+Kit+ (LSK) bone marrow stem cell population and lymphoid-primed multipotent progenitor numbers, which translated into a higher production and systemic flux of T cell subsets. In accordance with APOA1 deficiency-associated priming of stem cells to increase T lymphocyte production, atherogenic diet-fed low-density lipoprotein receptor knockout mice transplanted with bone marrow from APOA1-knockout mice displayed marked lymphocytosis as compared to wild-type bone marrow recipients. However, atherosclerotic lesion sizes and collagen contents were similar in the two groups of bone marrow recipients. In conclusion, systemic lack of APOA1 primes bone marrow stem cells for T cell lymphopoiesis. Our data provide novel evidence for a regulatory role of HDL in bone marrow functioning in normolipidemic mice

Antibody-Mediated Inhibition of CTLA4 Aggravates Atherosclerotic Plaque Inflammation and Progression in Hyperlipidemic Mice

T cell-driven inflammation plays a critical role in the initiation and progression of atherosclerosis. The co-inhibitory protein Cytotoxic T-Lymphocyte Associated protein (CTLA) 4 is an important negative regulator of T cell activation. Here, we studied the effects of the antibody-mediated inhibition of CTLA4 on experimental atherosclerosis by treating 6-8-week-old Ldlr-/- mice, fed a 0.15% cholesterol diet for six weeks, biweekly with 200 μg of CTLA4 antibodies or isotype control for six weeks. 18F-fluorodeoxyglucose Positron Emission Tomography-Computed Tomography showed no effect of the CTLA4 inhibition of activity in the aorta, spleen, and bone marrow, indicating that monocyte/macrophage-driven inflammation was unaffected. Correspondingly, flow cytometry demonstrated that the antibody-mediated inhibition of CTLA4 did not affect the monocyte populations in the spleen. αCTLA4 treatment induced an activated T cell profile, characterized by a decrease in naïve CD44-CD62L+CD4+ T cells and an increase in CD44+CD62L- CD4+ and CD8+ T cells in the blood and lymphoid organs. Furthermore, αCTLA4 treatment induced endothelial activation, characterized by increased ICAM1 expression in the aortic endothelium. In the aortic arch, which mainly contained early atherosclerotic lesions at this time point, αCTLA4 treatment induced a 2.0-fold increase in the plaque area. These plaques had a more advanced morphological phenotype and an increased T cell/macrophage ratio, whereas the smooth muscle cell and collagen content decreased. In the aortic root, a site that contained more advanced plaques, αCTLA4 treatment increased the plaque T cell content. The short-term antibody-mediated inhibition of CTLA4 thus accelerated the progression of atherosclerosis by inducing a predominantly T cell-driven inflammation, and resulted in the formation of plaques with larger necrotic cores and less collagen. This indicates that existing therapies that are based on αCTLA4 antibodies may promote CVD development in patients

Targeting the ACOD1-itaconate axis stabilizes atherosclerotic plaques

Inflammatory macrophages are key drivers of atherosclerosis that can induce rupture-prone vulnerable plaques. Skewing the plaque macrophage population towards a more protective phenotype and reducing the occurrence of clinical events is thought to be a promising method of treating atherosclerotic patients. In the current study, we investigate the immunomodulatory properties of itaconate, an immunometabolite derived from the TCA cycle intermediate cis-aconitate and synthesised by the enzyme Aconitate Decarboxylase 1 (ACOD1, also known as IRG1), in the context of atherosclerosis. Ldlr−/− atherogenic mice transplanted with Acod1−/− bone marrow displayed a more stable plaque phenotype with smaller necrotic cores and showed increased recruitment of monocytes to the vessel intima. Macrophages from Acod1−/− mice contained more lipids whilst also displaying reduced induction of apoptosis. Using multi-omics approaches, we identify a metabolic shift towards purine metabolism, in addition to an altered glycolytic flux towards production of glycerol for triglyceride synthesis. Overall, our data highlight the potential of therapeutically blocking ACOD1 with the aim of stabilizing atherosclerotic plaques

Immune Checkpoint Inhibitor Therapy Aggravates T Cell–Driven Plaque Inflammation in Atherosclerosis

Background: Immunotherapy has revolutionized cancer treatment. However, immune checkpoint inhibitors (ICIs) that target PD-1 (programmed cell death protein-1) and/or CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) are commonly associated with acute immune-related adverse events. Accumulating evidence also suggests that ICIs aggravate existing inflammatory diseases. Objectives: As inflammation drives atherosclerotic cardiovascular disease, we studied the propensity of short-term ICI therapy to aggravate atherosclerosis. Methods: We used 18F-FDG (2-deoxy-2-[fluorine-18]fluoro-D-glucose) positron emission tomography–computed tomography to detect macrophage-driven vascular and systemic inflammation in pembrolizumab and nivolumab/ipilimumab–treated melanoma patients. In parallel, atherosclerotic Ldlr–/– mice were treated with CTLA-4 and PD-1 inhibition to study the proinflammatory consequences of immune checkpoint inhibition. Results: ICI treatment did not affect 18F-FDG uptake in the large arteries, spleen, and bone marrow of melanoma patients, nor myeloid cell activation in blood and lymphoid organs in hyperlipidemic mice. In contrast, we found marked changes in the adaptive immune response (i.e., increased CD4+ effector T cell and CD8+ cytotoxic T cell numbers in lymphoid organs and the arterial wall of our hyperlipidemic mice). Although plaque size was unaffected, plaques had progressed toward a lymphoid-based inflammatory phenotype, characterized by a 2.7-fold increase of CD8+ T cells and a 3.9-fold increase in necrotic core size. Increased endothelial activation was observed with a 2.2-fold and 1.6-fold increase in vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, respectively. Conclusions: This study demonstrates that combination therapy with anti-CTLA-4 and anti-PD-1 antibodies does not affect myeloid-driven vascular and systemic inflammation in melanoma patients and hyperlipidemic mice. However, short-term ICI therapy in mice induces T cell–mediated plaque inflammation and drives plaque progression