Acid ceramidase regulates innate immune memory.

Innate immune memory, also called "trained immunity," is a functional state of myeloid cells enabling enhanced immune responses. This phenomenon is important for host defense, but also plays a role in various immune-mediated conditions. We show that exogenously administered sphingolipids and inhibition of sphingolipid metabolizing enzymes modulate trained immunity. In particular, we reveal that acid ceramidase, an enzyme that converts ceramide to sphingosine, is a potent regulator of trained immunity. We show that acid ceramidase regulates the transcription of histone-modifying enzymes, resulting in profound changes in histone 3 lysine 27 acetylation and histone 3 lysine 4 trimethylation. We confirm our findings by identifying single-nucleotide polymorphisms in the region of ASAH1, the gene encoding acid ceramidase, that are associated with the trained immunity cytokine response. Our findings reveal an immunomodulatory effect of sphingolipids and identify acid ceramidase as a relevant therapeutic target to modulate trained immunity responses in innate immune-driven disorders.

Trained immunity: Target for prophylaxis and therapy.

Trained immunity is a de facto memory for innate immune responses, leading to long-term functional reprogramming of innate immune cells. In physiological conditions, trained immunity leads to adaptive states that enhance resistance against pathogens and contributes to immunosurveillance. Dysregulated trained immunity can however lead either to defective innate immune responses in severe infections or cancer or to inflammatory and autoimmune diseases if trained immunity is inappropriately activated. Here, we review the immunological and molecular mechanisms that mediate trained immunity induction and propose that trained immunity represents an important target for prophylactic and therapeutic approaches in human diseases. On the one hand, we argue that novel approaches that induce trained immunity may enhance vaccine efficacy. On the other hand, induction of trained immunity in cancer, and inhibition of exaggerated induction of trained immunity in inflammatory disorders, are viable targets amenable for new therapeutic approaches.

Cardiovascular Molecular Imaging With Fluorine-19 MRI: The Road to the Clinic.

Fluorine-19 (19F) magnetic resonance imaging is a unique quantitative molecular imaging modality that makes use of an injectable fluorine-containing tracer that generates the only visible 19F signal in the body. This hot spot imaging technique has recently been used to characterize a wide array of cardiovascular diseases and seen a broad range of technical improvements. Concurrently, its potential to be translated to the clinical setting is being explored. This review provides an overview of this emerging field and demonstrates its diagnostic potential, which shows promise for clinical translation. We will describe 19F magnetic resonance imaging hardware, pulse sequences, and tracers, followed by an overview of cardiovascular applications. Finally, the challenges on the road to clinical translation are discussed.

Multiparametric Immunoimaging Maps Inflammatory Signatures in Murine Myocardial Infarction Models.

In the past 2 decades, research on atherosclerotic cardiovascular disease has uncovered inflammation to be a key driver of the pathophysiological process. A pressing need therefore exists to quantitatively and longitudinally probe inflammation, in preclinical models and in cardiovascular disease patients, ideally using non-invasive methods and at multiple levels. Here, we developed and employed in vivo multiparametric imaging approaches to investigate the immune response following myocardial infarction. The myocardial infarction models encompassed either transient or permanent left anterior descending coronary artery occlusion in C57BL/6 and Apoe-/-mice. We performed nanotracer-based fluorine magnetic resonance imaging and positron emission tomography (PET) imaging using a CD11b-specific nanobody and a C-C motif chemokine receptor 2-binding probe. We found that immune cell influx in the infarct was more pronounced in the permanent occlusion model. Further, using 18F-fluorothymidine and 18F-fluorodeoxyglucose PET, we detected increased hematopoietic activity after myocardial infarction, with no difference between the models. Finally, we observed persistent systemic inflammation and exacerbated atherosclerosis in Apoe-/- mice, regardless of which infarction model was used. Taken together, we showed the strengths and capabilities of multiparametric imaging in detecting inflammatory activity in cardiovascular disease, which augments the development of clinical readouts.

Trained immunity in atherosclerotic cardiovascular disease.

Trained immunity, also known as innate immune memory, is a persistent hyper-responsive functional state of innate immune cells. Accumulating evidence implicates trained immunity as an underlying mechanism of chronic inflammation in atherosclerotic cardiovascular disease. In this context, trained immunity is induced by endogenous atherosclerosis-promoting factors, such as modified lipoproteins or hyperglycaemia, causing broad metabolic and epigenetic reprogramming of the myeloid cell compartment. In addition to traditional cardiovascular risk factors, lifestyle factors, including unhealthy diets, sedentary lifestyle, sleep deprivation and psychosocial stress, as well as inflammatory comorbidities, have been shown to activate trained immunity-like mechanisms in bone marrow haematopoietic stem cells. In this Review, we discuss the molecular and cellular mechanisms of trained immunity, its systemic regulation through haematopoietic progenitor cells in the bone marrow, and the activation of these mechanisms by cardiovascular disease risk factors. We also highlight other trained immunity features that are relevant for atherosclerotic cardiovascular disease, including the diverse cell types that show memory characteristics and transgenerational inheritance of trained immunity traits. Finally, we propose potential strategies for the therapeutic modulation of trained immunity to manage atherosclerotic cardiovascular disease.

Resolving sepsis-induced immunoparalysis via trained immunity by targeting interleukin-4 to myeloid cells.

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.

DNA storage in thermoresponsive microcapsules for repeated random multiplexed data access.

DNA has emerged as an attractive medium for archival data storage due to its durability and high information density. Scalable parallel random access to information is a desirable property of any storage system. For DNA-based storage systems, however, this still needs to be robustly established. Here we report on a thermoconfined polymerase chain reaction, which enables multiplexed, repeated random access to compartmentalized DNA files. The strategy is based on localizing biotin-functionalized oligonucleotides inside thermoresponsive, semipermeable microcapsules. At low temperatures, microcapsules are permeable to enzymes, primers and amplified products, whereas at high temperatures, membrane collapse prevents molecular crosstalk during amplification. Our data show that the platform outperforms non-compartmentalized DNA storage compared with repeated random access and reduces amplification bias tenfold during multiplex polymerase chain reaction. Using fluorescent sorting, we also demonstrate sample pooling and data retrieval by microcapsule barcoding. Therefore, the thermoresponsive microcapsule technology offers a scalable, sequence-agnostic approach for repeated random access to archival DNA files.

Engineering cytokine therapeutics.

Cytokines have pivotal roles in immunity, making them attractive as therapeutics for a variety of immune-related disorders. However, the widespread clinical use of cytokines has been limited by their short blood half-lives and severe side effects caused by low specificity and unfavourable biodistribution. Innovations in bioengineering have aided in advancing our knowledge of cytokine biology and yielded new technologies for cytokine engineering. In this Review, we discuss how the development of bioanalytical methods, such as sequencing and high-resolution imaging combined with genetic techniques, have facilitated a better understanding of cytokine biology. We then present an overview of therapeutics arising from cytokine re-engineering, targeting and delivery, mRNA therapeutics and cell therapy. We also highlight the application of these strategies to adjust the immunological imbalance in different immune-mediated disorders, including cancer, infection and autoimmune diseases. Finally, we look ahead to the hurdles that must be overcome before cytokine therapeutics can live up to their full potential.

PET/MR imaging of inflammation in atherosclerosis.

Myocardial infarction, stroke, mental disorders, neurodegenerative processes, autoimmune diseases, cancer and the human immunodeficiency virus impact the haematopoietic system, which through immunity and inflammation may aggravate pre-existing atherosclerosis. The interplay between the haematopoietic system and its modulation of atherosclerosis has been studied by imaging the cardiovascular system and the activation of haematopoietic organs via scanners integrating positron emission tomography and resonance imaging (PET/MRI). In this Perspective, we review the applicability of integrated whole-body PET/MRI for the study of immune-mediated phenomena associated with haematopoietic activity and cardiovascular disease, and discuss the translational opportunities and challenges of the technology.

Trained immunity - basic concepts and contributions to immunopathology.

Trained immunity is a functional state of the innate immune response and is characterized by long-term epigenetic reprogramming of innate immune cells. This concept originated in the field of infectious diseases - training of innate immune cells, such as monocytes, macrophages and/or natural killer cells, by infection or vaccination enhances immune responses against microbial pathogens after restimulation. Although initially reported in circulating monocytes and tissue macrophages (termed peripheral trained immunity), subsequent findings indicate that immune progenitor cells in the bone marrow can also be trained (that is, central trained immunity), which explains the long-term innate immunity-mediated protective effects of vaccination against heterologous infections. Although trained immunity is beneficial against infections, its inappropriate induction by endogenous stimuli can also lead to aberrant inflammation. For example, in systemic lupus erythematosus and systemic sclerosis, trained immunity might contribute to inflammatory activity, which promotes disease progression. In organ transplantation, trained immunity has been associated with acute rejection and suppression of trained immunity prolonged allograft survival. This novel concept provides a better understanding of the involvement of the innate immune response in different pathological conditions, and provides a new framework for the development of therapies and treatment strategies that target epigenetic and metabolic pathways of the innate immune system.

Self-gated, dynamic contrast-enhanced magnetic resonance imaging with compressed-sensing reconstruction for evaluating endothelial permeability in the aortic root of atherosclerotic mice.

High-risk atherosclerotic plaques are characterized by active inflammation and abundant leaky microvessels. We present a self-gated, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) acquisition with compressed sensing reconstruction and apply it to assess longitudinal changes in endothelial permeability in the aortic root of Apoe-/- atherosclerotic mice during natural disease progression. Twenty-four, 8-week-old, female Apoe-/- mice were divided into four groups (n = 6 each) and imaged with self-gated DCE-MRI at 4, 8, 12, and 16 weeks after high-fat diet initiation, and then euthanized for CD68 immunohistochemistry for macrophages. Eight additional mice were kept on a high-fat diet and imaged longitudinally at the same time points. Aortic-root pseudo-concentration curves were analyzed using a validated piecewise linear model. Contrast agent wash-in and washout slopes (b1 and b2 ) were measured as surrogates of aortic root endothelial permeability and compared with macrophage density by immunohistochemistry. b2 , indicating contrast agent washout, was significantly higher in mice kept on an high-fat diet for longer periods of time (p = 0.03). Group comparison revealed significant differences between mice on a high-fat diet for 4 versus 16 weeks (p = 0.03). Macrophage density also significantly increased with diet duration (p = 0.009). Spearman correlation between b2 from DCE-MRI and macrophage density indicated a weak relationship between the two parameters (r = 0.28, p = 0.20). Validated piecewise linear modeling of the DCE-MRI data showed that the aortic root contrast agent washout rate is significantly different during disease progression. Further development of this technique from a single-slice to a 3D acquisition may enable better investigation of the relationship between in vivo imaging of endothelial permeability and atherosclerotic plaques' genetic, molecular, and cellular makeup in this important model of disease.

Trained immunity: adaptation within innate immune mechanisms.

The mechanisms underlying innate immune memory have been extensively explored in the last decades but are in fact largely unknown. Although the specificity of adaptive immune memory in vertebrates is ensured through the recombination of immunoglobulin family genes and clonal expansion, the basic mechanisms of innate immune cells' nonspecific increased responsiveness rely on epigenetic, transcriptional, and metabolic programs after transient stimulation. Changes in these programs result in enhanced responsiveness to secondary challenges with a wide variety of stimuli. This phenomenon is termed "trained immunity" or "innate immune memory." On one hand, trained immunity improves the response to infections and vaccination, facilitating stronger innate immune responses and enhanced protection against a variety of microbial stimuli. Conversely, trained immunity may contribute to the pathophysiology of cardiovascular, autoinflammatory, and neurodegenerative diseases. In this review, we gather the current body of knowledge in this field and summarize the foundations and mechanisms of trained immunity, the different cell types involved, its consequences for health and disease, and the potential of its modulation as a therapeutic tool.

Reprogramming of myeloid cells and their progenitors in patients with non-medullary thyroid carcinoma.

Myeloid cells, crucial players in antitumoral defense, are affected by tumor-derived factors and treatment. The role of myeloid cells and their progenitors prior to tumor infiltration is poorly understood. Here we show single-cell transcriptomics and functional analyses of the myeloid cell lineage in patients with non-medullary thyroid carcinoma (TC) and multinodular goiter, before and after treatment with radioactive iodine compared to healthy controls. Integrative data analysis indicates that monocytes of TC patients have transcriptional upregulation of antigen presentation, reduced cytokine production capacity, and overproduction of reactive oxygen species. Interestingly, these cancer-related pathological changes are partially removed upon treatment. In bone marrow, TC patients tend to shift from myelopoiesis towards lymphopoiesis, reflected in transcriptional differences. Taken together, distinct transcriptional and functional changes in myeloid cells arise before their infiltration of the tumor and are already initiated in bone marrow, which suggests an active role in forming the tumor immune microenvironment.

Systematically evaluating DOTATATE and FDG as PET immuno-imaging tracers of cardiovascular inflammation.

In recent years, cardiovascular immuno-imaging by positron emission tomography (PET) has undergone tremendous progress in preclinical settings. Clinically, two approved PET tracers hold great potential for inflammation imaging in cardiovascular patients, namely FDG and DOTATATE. While the former is a widely applied metabolic tracer, DOTATATE is a relatively new PET tracer targeting the somatostatin receptor 2 (SST2). In the current study, we performed a detailed, head-to-head comparison of DOTATATE-based radiotracers and [18F]F-FDG in mouse and rabbit models of cardiovascular inflammation. For mouse experiments, we labeled DOTATATE with the long-lived isotope [64Cu]Cu to enable studying the tracer's mode of action by complementing in vivo PET/CT experiments with thorough ex vivo immunological analyses. For translational PET/MRI rabbit studies, we employed the more widely clinically used [68Ga]Ga-labeled DOTATATE, which was approved by the FDA in 2016. DOTATATE's pharmacokinetics and timed biodistribution were determined in control and atherosclerotic mice and rabbits by ex vivo gamma counting of blood and organs. Additionally, we performed in vivo PET/CT experiments in mice with atherosclerosis, mice subjected to myocardial infarction and control animals, using both [64Cu]Cu-DOTATATE and [18F]F-FDG. To evaluate differences in the tracers' cellular specificity, we performed ensuing ex vivo flow cytometry and gamma counting. In mice subjected to myocardial infarction, in vivo [64Cu]Cu-DOTATATE PET showed higher differential uptake between infarcted (SUVmax 1.3, IQR, 1.2-1.4, N = 4) and remote myocardium (SUVmax 0.7, IQR, 0.5-0.8, N = 4, p = 0.0286), and with respect to controls (SUVmax 0.6, IQR, 0.5-0.7, N = 4, p = 0.0286), than [18F]F-FDG PET. In atherosclerotic mice, [64Cu]Cu-DOTATATE PET aortic signal, but not [18F]F-FDG PET, was higher compared to controls (SUVmax 1.1, IQR, 0.9-1.3 and 0.5, IQR, 0.5-0.6, respectively, N = 4, p = 0.0286). In both models, [64Cu]Cu-DOTATATE demonstrated preferential accumulation in macrophages with respect to other myeloid cells, while [18F]F-FDG was taken up by macrophages and other leukocytes. In a translational PET/MRI study in atherosclerotic rabbits, we then compared [68Ga]Ga-DOTATATE and [18F]F-FDG for the assessment of aortic inflammation, combined with ex vivo radiometric assays and near-infrared imaging of macrophage burden. Rabbit experiments showed significantly higher aortic accumulation of both [68Ga]Ga-DOTATATE and [18F]F-FDG in atherosclerotic (SUVmax 0.415, IQR, 0.338-0.499, N = 32 and 0.446, IQR, 0.387-0.536, N = 27, respectively) compared to control animals (SUVmax 0.253, IQR, 0.197-0.285, p = 0.0002, N = 10 and 0.349, IQR, 0.299-0.423, p = 0.0159, N = 11, respectively). In conclusion, we present a detailed, head-to-head comparison of the novel SST2-specific tracer DOTATATE and the validated metabolic tracer [18F]F-FDG for the evaluation of inflammation in small animal models of cardiovascular disease. Our results support further investigations on the use of DOTATATE to assess cardiovascular inflammation as a complementary readout to the widely used [18F]F-FDG.

[Immune cell activation in cardiovascular disease: new pathophysiological mechanisms explained]. / Immuuncelactivatie bij hart- en vaatziekten.

Optimal treatment of classical risk factors, such as dyslipidemia, cannot completely prevent atherosclerotic cardiovascular disease, which is called residual cardiovascular risk. The anti-inflammatory drugs colchicine and canakinumab can lower this residual risk, illustrating the importance of inflammation in the pathophysiology of cardiovascular disease. Circulating granulocytes and activated monocytes are associated with atherosclerotic cardiovascular disease in observational studies. In the past few years, two mechanisms have been discovered that contribute to monocyte and neutrophil activation in the context of atherosclerosis, namely trained immunity and clonal hematopoiesis. In this Perspective, we discuss these mechanisms, since we foresee that in the near future knowledge of these mechanisms could improve cardiovascular risk prediction and provide targets for novel drugs to prevent or treat cardiovascular diseases.

Diverse ultrastructural landscape of atherosclerotic endothelium.

BACKGROUND AND AIMS: The endothelium plays a major role in atherosclerosis, yet the endothelial plaque surface is a largely uncharted territory. Here we hypothesize that atherosclerosis-driven remodeling of the endothelium is a dynamic process, involving both damaging and regenerative mechanisms. METHODS: Using scanning electron microscopy (SEM) and immuno-SEM, we studied endothelial junction ultrastructure, endothelial openings and immune cell-endothelium interactions in eight apoe-/- mice and two human carotid plaques. RESULTS: The surface of early mouse plaques (n = 11) displayed a broad range of morphological alterations, including junctional disruptions and large transcellular endothelial pores with the average diameter between 0.6 and 3 µm. The shoulder region of advanced atherosclerotic lesions (n = 7) had a more aggravated morphology with 8 µm-size paracellular openings at two-fold higher density. In contrast, the central apical surface of advanced plaques, i.e., the plaque body (n = 7), displayed endothelial normalization, as shown by a significantly higher frequency of intact endothelial junctions and a lower incidence of paracellular pores. This normalized endothelial phenotype correlated with low immune cell density (only 5 cells/mm2). The human carotid plaque surface (n = 2) displayed both well-organized and disrupted endothelium with similar features as described above. In addition, they were accompanied by extensive thrombotic areas. CONCLUSIONS: Our study unveils the spectrum of endothelial abnormalities associated with the development of atherosclerosis. These were highly abundant in early lesions and in the shoulder region of advanced plaques, while normalized at the advanced plaque's body. Similar endothelial features were observed in human atherosclerotic plaques, underlining the versatility of endothelial transformations in atherosclerosis.

Kinase Inhibitors' Effects on Innate Immunity in Solid Cancers.

Innate immune cells constitute a plastic and heterogeneous cell population of the tumor microenvironment. Because of their high tumor infiltration and close interaction with resident tumor cells, they are compelling targets for anti-cancer therapy through either ablation or functionally reprogramming. Kinase inhibitors (KIs) that target aberrant signaling pathways in tumor proliferation and angiogenesis have been shown to have additional immunological effects on myeloid cells that may contribute to a protective antitumor immune response. However, in patients with malignancies, these effects are poorly described, warranting meticulous research to identify KIs' optimal immunomodulatory effect to support developing targeted and more effective immunotherapy. As many of these KIs are currently in clinical trials awaiting approval for the treatment of several types of solid cancer, we evaluate here the information on this drug class's immunological effects and how such mechanisms can be harnessed to improve combined treatment regimens in cancer.