Adipocytes control hematopoiesis and inflammation through CD40 signaling.

The co-stimulatory CD40-CD40L dyad plays an important role in chronic inflammatory diseases associated with aging. 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 hematopoietic 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 hematopoietic stem cells (Lin-Sca+cKit+, LSK) and common lymphoid progenitors, which was associated with increased bone marrow 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 bone marrow, spleen, and adipose tissue, while the numbers of B cells were decreased. Although atherosclerosis was reduced in CD40AKO mice, plaques contained more activated T cells and larger necrotic cores. Analysis of peripheral adipose tissue in a diet-induced model of obesity revealed that obese AdiCD40KO mice had increased T-cell activation in adipose tissue and lymphoid organs, but 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 bone marrow during aging 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.

Deficiency of the T cell regulator Casitas B-cell lymphoma-B aggravates atherosclerosis by inducing CD8+ T cell-mediated macrophage death.

Aims: The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis. Methods and results: The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb-/-Apoe-/- mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb-/-Apoe-/- macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb-/-Apoe-/- CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb-/-Apoe-/- bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells. Conclusion: Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.

Malaria Parasite-Mediated Alteration of Macrophage Function and Increased Iron Availability Predispose to Disseminated Nontyphoidal Salmonella Infection.

Disseminated infections with nontyphoidal Salmonella (NTS) are a significant cause of child mortality in sub-Saharan Africa. NTS infection in children is clinically associated with malaria, suggesting that malaria compromises the control of disseminated NTS infection. To study the mechanistic basis for increased NTS susceptibility, we utilized a model of concurrent infection with Salmonella enterica serotype Typhimurium and Plasmodium yoelii nigeriensis (P. yoelii). Underlying malaria blunted monocyte expression of Ly6C, a marker for inflammatory activation, and impaired recruitment of inflammatory cells to the liver. Hepatic mononuclear phagocytes expressed lower levels of inducible nitric oxide synthase, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor and showed increased levels of production of interleukin-10 and heme oxygenase-1, indicating that the underlying malaria modifies the activation state and inflammatory response of mononuclear phagocytes to NTS. P. yoelii infection also increased intracellular iron levels in liver mononuclear cells, as evidenced by elevated levels of ferritin and by the rescue of an S Typhimurium tonB feoB mutant defective for iron uptake. In addition, concurrent P. yoelii infection partially rescued the systemic colonization defect of an S Typhimurium spiB mutant defective for type III secretion system 2 (T3SS-2), indicating that the ability of phagocytic cells to limit the spread of S Typhimurium is impaired during concurrent P. yoelii infection. These results show that concurrent malaria increases susceptibility to disseminated NTS infection by blunting macrophage bactericidal mechanisms and providing an essential nutrient that enhances bacterial growth.

Immuno-PET Imaging of Atherosclerotic Plaques with [89Zr]Zr-Anti-CD40 mAb-Proof of Concept.

Non-invasive imaging of atherosclerosis can help in the identification of vulnerable plaque lesions. CD40 is a co-stimulatory molecule present on various immune and non-immune cells in the plaques and is linked to inflammation and plaque instability. We hypothesize that a 89Zr-labeled anti-CD40 monoclonal antibody (mAb) tracer has the potential to bind to cells present in atherosclerotic lesions and that CD40 Positron Emission Tomography (PET) can contribute to the detection of vulnerable atherosclerotic plaque lesions. To study this, wild-type (WT) and ApoE-/- mice were fed a high cholesterol diet for 14 weeks to develop atherosclerosis. Mice were injected with [89Zr]Zr-anti-CD40 mAb and the aortic uptake was evaluated and quantified using PET/Computed Tomography (CT) imaging. Ex vivo biodistribution was performed post-PET imaging and the uptake in the aorta was assessed with autoradiography and compared with Oil red O staining to determine the tracer potential to detect atherosclerotic plaques. On day 3 and 7 post injection, analysis of [89Zr]Zr-anti-CD40 mAb PET/CT scans showed a more pronounced aortic signal in ApoE-/- compared to WT mice with an increased aorta-to-blood uptake ratio. Autoradiography revealed [89Zr]Zr-anti-CD40 mAb uptake in atherosclerotic plaque areas in ApoE-/- mice, while no signal was found in WT mice. Clear overlap was observed between plaque areas as identified by Oil red O staining and autoradiography signal of [89Zr]Zr-anti-CD40 mAb in ApoE-/- mice. In this proof of concept study, we showed that PET/CT with [89Zr]Zr-anti-CD40 mAb can detect atherosclerotic plaques. As CD40 is associated with plaque vulnerability, [89Zr]Zr-anti-CD40 mAb has the potential to become a tracer to detect vulnerable atherosclerotic plaques.

Immune checkpoint inhibitor treatment and atherosclerotic cardiovascular disease: an emerging clinical problem.

Antibody-mediated blockade of co-inhibitory molecules such as cytotoxic T lymphocyte-associated protein 4, PD1 and PDL1 elicits potent antitumor responses and improves the prognosis of many patients with cancer. As these immune checkpoint inhibitors (ICIs) are increasingly prescribed to a diverse patient population, a broad range of adverse effects is emerging. Atherosclerosis, a lipid-driven chronic inflammatory disease of the large arteries, may be aggravated by ICI treatment. In this review, we discuss recent clinical studies that analyze the correlation between ICI use and atherosclerotic cardiovascular disease (CVD). Indeed, several studies report an increased incidence of atherosclerotic CVD after ICI administration, with the occurrence of pathologies such as myocardial infarction, ischemic stroke and coronary artery disease significantly higher after ICI use. Increased awareness and better monitoring of ICI-treated patients can elucidate risk factors that contribute to ICI-induced aggravation of atherosclerosis and identify promising treatment strategies. For now, optimal cardiovascular risk assessment is required to protect ICI-receiving patients and long-term survivors of cancer from the detrimental effects of ICI therapy on atherosclerotic CVD.

E3 Ubiquitin Ligases as Immunotherapeutic Target in Atherosclerotic Cardiovascular Disease.

Chronic low-grade inflammation drives atherosclerosis and despite optimal pharmacological treatment of classical cardiovascular risk factors, one third of the patients with atherosclerotic cardiovascular disease has elevated inflammatory biomarkers. Additional anti-inflammatory strategies to target this residual inflammatory cardiovascular risk are therefore required. T-cells are a dominant cell type in human atherosclerotic lesions. Modulation of T-cell activation is therefore a potential strategy to target inflammation in atherosclerosis. Ubiquitination is an important regulatory mechanism of T-cell activation and several E3 ubiquitin ligases, including casitas B-lineage lymphoma proto-oncogene B (Cbl-B), itchy homolog (Itch), and gene related to anergy in lymphocytes (GRAIL), function as a natural brake on T-cell activation. In this review we discuss recent insights on the role of Cbl-B, Itch, and GRAIL in atherosclerosis and explore the therapeutic potential of these E3 ubiquitin ligases in cardiovascular medicine.

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.

Short-term regulation of hematopoiesis by lipoprotein(a) results in the production of pro-inflammatory monocytes.

BACKGROUND: Lipoproteins are important regulators of hematopoietic stem and progenitor cell (HSPC) biology, predominantly affecting myelopoiesis. Since myeloid cells, including monocytes and macrophages, promote the inflammatory response that propagates atherosclerosis, it is of interest whether the atherogenic low-density lipoprotein (LDL)-like particle lipoprotein(a) [Lp(a)] contributes to atherogenesis via stimulating myelopoiesis. METHODS & RESULTS: To assess the effects of Lp(a)-priming on long-term HSPC behavior we transplanted BM of Lp(a) transgenic mice, that had been exposed to elevated levels of Lp(a), into lethally-irradiated C57Bl6 mice and hematopoietic reconstitution was analyzed. No differences in HSPC populations or circulating myeloid cells were detected ten weeks after transplantation. Likewise, in vitro stimulation of C57Bl6 BM cells for 24 h with Lp(a) did not affect colony formation, total cell numbers or myeloid populations 7 days later. To assess the effects of elevated levels of Lp(a) on myelopoiesis, C57Bl6 bone marrow (BM) cells were stimulated with lp(a) for 24 h, and a marked increase in granulocyte-monocyte progenitors, pro-inflammatory Ly6high monocytes and macrophages was observed. Seven days of continuous exposure to Lp(a) increased colony formation and enhanced the formation of pro-inflammatory monocytes and macrophages. Antibody-mediated neutralization of oxidized phospholipids abolished the Lp(a)-induced effects on myelopoiesis. CONCLUSION: Lp(a) enhances the production of inflammatory monocytes at the bone marrow level but does not induce cell-intrinsic long-term priming of HSPCs. Given the short-term and direct nature of this effect, we postulate that Lp(a)-lowering treatment has the capacity to rapidly revert this multi-level inflammatory response.

Inhibition of PFKFB3 Hampers the Progression of Atherosclerosis and Promotes Plaque Stability.

AIMS: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3-mediated glycolysis is pivotal in driving macrophage- and endothelial cell activation and thereby inflammation. Once activated, these cells play a crucial role in the progression of atherosclerosis. Here, we analyzed the expression of PFKFB3 in human atherosclerotic lesions and investigated the therapeutic potential of pharmacological inhibition of PFKFB3 in experimental atherosclerosis by using the glycolytic inhibitor PFK158. METHODS AND RESULTS: PFKFB3 expression was higher in vulnerable human atheromatous carotid plaques when compared to stable fibrous plaques and predominantly expressed in plaque macrophages and endothelial cells. Analysis of advanced plaques of human coronary arteries revealed a positive correlation of PFKFB3 expression with necrotic core area. To further investigate the role of PFKFB3 in atherosclerotic disease progression, we treated 6-8 weeks old male Ldlr -/- mice. These mice were fed a high cholesterol diet for 13 weeks, of which they were treated for 5 weeks with the glycolytic inhibitor PFK158 to block PFKFB3 activity. The incidence of fibrous cap atheroma (advanced plaques) was reduced in PFK158-treated mice. Plaque phenotype altered markedly as both necrotic core area and intraplaque apoptosis decreased. This coincided with thickening of the fibrous cap and increased plaque stability after PFK158 treatment. Concomitantly, we observed a decrease in glycolysis in peripheral blood mononuclear cells compared to the untreated group, which alludes that changes in the intracellular metabolism of monocyte and macrophages is advantageous for plaque stabilization. CONCLUSION: High PFKFB3 expression is associated with vulnerable atheromatous human carotid and coronary plaques. In mice, high PFKFB3 expression is also associated with a vulnerable plaque phenotype, whereas inhibition of PFKFB3 activity leads to plaque stabilization. This data implies that inhibition of inducible glycolysis may reduce inflammation, which has the ability to subsequently attenuate atherogenesis.

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.