Universitäts-Herzzentrum Freiburg • Bad Krozingen

CORE PROJECT 1:

Defining the adaptive immune response to develop a vaccine against atherosclerosis

Most people worldwide die of cardiovascular disease, such as of heart attack and stroke. Often, the underlying cause is atherosclerosis, a disease that fuels the build-up of vessel-narrowing plaques in arteries that are vital because they supply the heart and the brain. During early disease, a subgroup of white blood cells called T cells infiltrate the plaque and drive its growth. Usually, these immune cells protect from infection, but in atherosclerosis some T cells attack the body itself. This is why atherosclerosis is now understood as auto-immune disease. They get attracted by the protein ApoB-100, which is a part of LDL-cholesterol in the plaque. However, little is known about the function of these self-reactive T cells and how to dampen their harmful action. Using innovative tools, such as 2-photon microscopy of explanted atherosclerotic plaques (see above, left), combined with classical atherosclerosis tools, such as quantification of atherosclerotic lesions in mice (middle), and MHC-multimers to track single auto-reactive T cells we are asking some intriguing questions: What is the frequency and function of autoreactive T cells? Which proteins and genes do they express? When transferred into another animal, how do these autoreactive T cells change atherosclerosis in the recipient? We are also trying to track auto-reactive T cells in humans with coronary artery disease. Deciphering how the immune system and the T cell response work in atherosclerosis will not only boost our understanding of auto-immunity in atherosclerosis but it will also build the basis to define new therapies. Our long-term goal is to define the properties of a vaccine against ApoB-100 to modify the immune system in a way that protective autoreactive T cells are boosted. Such therapy would represent the first causal preventive therapy against the atherosclerosis-related immune response in future.

Suggested reading:  

1. Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B100-Reactive CD4+ T-Regulatory Cells
   link: https://pubmed.ncbi.nlm.nih.gov/32703007/
2. Immunity and Inflammation in Atherosclerosis.
   link: https://pubmed.ncbi.nlm.nih.gov/30653442/
3. Regulatory CD4+ T Cells Recognize MHC-II-Restricted Peptide Epitopes of Apolipoprotein B
   link: https://www.ncbi.nlm.nih.gov/pubmed/29588316

CORE PROJECT 2:

Therapeutic targeting of integrins in inflammation

The recruitment of leukocytes to inflamed tissue is a necessary step to resolve inflammation and infection in tissues. However, this initially protective attempt is often dysregulated and initiates a vicious circle that attracts more leukocytes, drives inflammatory cytokine secretion, and eventually causes tissue damage. One solution to this problem is to block leukocyte recruitment by inhibiting the molecules on leukocytes that are required for leukocytes to attach to the endothelium and to transmigrate into the tissue. Yes, this may be problematic, because adhesion molecules, e.g. leukocyte integrins, are simultaneously needed for many beneficial functions, such as phagocytosis, bacterial clearance and cell-cell interactions. We found that the interaction of one leukocyte integrin, Mac-1, with the immune molecule CD40L specifically regulates harmful cell recruitment by binding to endothelial CD40L and serving as adhesion receptor (see schematic above) without interfering with Mac-1’s protective functions in host defense, thrombosis, and wound healing. We designed an antibody and a peptide to specifically neutralize this receptor-ligand interaction, while leaving other potentially beneficial functions of the leukocyte integrin untouched. Our work documents a novel therapeutic strategy that protects from atherosclerosis and sepsis without relevant side effects observed in conventional treatment strategies. Two international patents arose from this work, which now serve as basis to ultimately develop a novel leukocyte recruitment blocker for human disease. We are using with a variety of protein assays to monitor the interaction of proteins and inhibitors and testing these directly in vivo in intravital microscopy and several inflammatory models, such as in experimental sepsis or peritonitis. Currently, we are also testing whether the specific inhibitor of the CD40L/Mac-1 interaction prevents adverse cardiac remodeling after myocardial infarction.

Suggested reading:  

1. A ligand-specific blockade of the integrin Mac-1 selectively targets pathologic inflammation while maintaining protective host-defense
   ink: https://www.ncbi.nlm.nih.gov/pubmed/29410422
2. CD40L and Its Receptors in Atherothrombosis-An Update
   link: https://www.ncbi.nlm.nih.gov/pubmed/28676852
3. Binding of CD40L to Mac-1's I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis - but does not affect immunity and thrombosis in mice
   link: https://www.ncbi.nlm.nih.gov/pubmed/21998326

CORE PROJECT 3:

Finding inflammatory signaling pathways in cardio-metabolic disease

Atherosclerosis, its acute complication myocardial infarction, as well as other cardiovascular disease is initiated and maintained by cardiovascular risk factors, such as obesity, insulin resistance, hypertension, hyperlipidemia, often referred to as the Metabolic Syndrome. It is now well established that some components of the Metabolic Syndrome, such as obesity and insulin resistance, drive an inflammatory response in visceral adipose tissue. It is, however, not known to which extend adipose tissue inflammation contributes to atherosclerosis and whether mutual inflammatory pathways exist that are druggable to define therapies that target both: risk factors and vascular disease simultaneously. We have found that CD40L, a costimulatory molecule initially described on activated T cells, is expressed on adipocytes, particular in obese, inflamed adipose tissue. Functionally, mice with a genetic deficiency of CD40L are protected from inflammation, while a deficiency of its classical interaction partner CD40 induces a hyper-inflammatory phenotype in adipose tissue and aggravated dysmetabolism in mice. A deficiency of the signaling adaptor TRAF-1 that is functioning downstream of CD40- and TNF-signaling resembles this hyper inflammatory phenotype, but unexpectedly protects from dysmetabolism. These findings document a novel role of CD40 as co-inhibitory molecule on T cells and suggest that activating CD40 antibodies – as currently tested in cancer trials – can protect from cardio- metabolic disease. Targeting inflammation of visceral adipose tissue alone may, however, not suffice to improve metabolic risk factors. We have developed and used a broad repertoire of methods and techniques to study metabolism in mice, including monitoring fat depositions by MRI (see Figure above, left), histo-morphology of adipocytes (middle), metabolic chambers, and genetic tools to screen for the expression of inflammatory pathways in human fat tissue by gene enrichment analysis (right).

Suggested reading:  

1. Inflammatory Pathways Regulated by Tumor Necrosis Receptor-Associated Factor 1 Protect From Metabolic Consequences in Diet-Induced Obesity
   link: https://www.ncbi.nlm.nih.gov/pubmed/29358227
2. Coinhibitory suppression of T cell activation by CD40 protects against obesity and adipose tissue inflammation in mice
   link: https://www.ncbi.nlm.nih.gov/pubmed/24664276
3. CD40L deficiency attenuates diet-induced adipose tissue inflammation by impairing immune cell accumulation and production of pathogenic IgG-antibodies
   link: https://www.ncbi.nlm.nih.gov/pubmed/22412980

CORE PROJECT 4:

Depicting leukocyte heterogeneity in atherosclerosis

The development and progression of atherosclerosis is predominately driven by leukocyte infiltration and accumulation in atherosclerotic plaques. Experimentally, atherosclerosis can be prevented and treated by modulating the influx and the function of pro-inflammatory leukocytes. The exact contribution of different leukocyte lineages, their phenotypes, and the existence of atherosclerosis-specific leukocyte subsets, however, is largely unknown. Historical protocols to digest and phenotype leukocytes in atherosclerotic mouse aortas are based on classical flow cytometry and have been applied since 2006 to study leukocyte populations in atherosclerosis and other vascular disease models by many labs around the world. Since then, it became increasingly clear that not all leukocytes found in the atherosclerotic aorta are equal; in fact, many principal aortic leukocyte lineages, such as B- or T-cells, encompass both protective and pathogenic sub-populations. This diversity has great implications on understanding the definitive biology and to establish novel cell-based therapeutic strategies. Our inspiration came from recent observations that high-parameter phenotyping in other disease models has the potential to detect novel sub-populations with unexpected combinations of surface markers. These cells may therefore reflect disease-specific leukocytes and identify subsets beyond what flow cytometry and immunohistochemistry can do. We are using a combination of cell sorting of aortic leukocytes in atherosclerotic mouse aortas (see above, middle and left) and single cell RNA-sequencing. These new approaches are less biased, i.e., make fewer assumptions based on pre-conceived notion. Thus, they are effective, efficient and powerful screening tools. Currently, we are aiming to define leukocyte heterogeneity in human atherosclerotic plaques and to test whether particular leukocyte sub-populations in peripheral blood of humans correlate with cardiovascular disease.

Suggested reading:

1. Heterogeneity of T Cells in Atherosclerosis Defined by Single-Cell RNA-Sequencing and Cytometry by Time of Flight.
   link: https://pubmed.ncbi.nlm.nih.gov/33267666/
2. Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas.
   link: https://pubmed.ncbi.nlm.nih.gov/32673538/
3. Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry
   link: https://www.ncbi.nlm.nih.gov/pubmed/29545366

CORE PROJECT 5:

The role of air pollution in cardiovascular disease

Air pollution stands as the single largest environmental health risk factor, responsible for approximately 7 million premature deaths worldwide each year. Among the different components that are present in polluted air, airborne fine particulate matter below 2.5 μm in diameter (Feinstaub, PM_2.5) has been identified as the primary hazardous constituent. PM_2.5 mainly originates from fossil fuel combustion during power generation, industrial processes, and transportation. While the lungs are in first contact with PM_2.5 after inhalation, the main complications of breathing polluted air manifest as cardiovascular diseases, including myocardial infarction and stroke. PM_2.5 uptake by lung tissue resident alveolar macrophages triggers a local and systemic inflammatory response, which alters homeostasis at secondary locations, such as the blood vessels, the heart, the brain, and fat depots. Consequently, exposure to PM_2.5 aggravates myocardial infarction, impairs cardiac lesion remodelling, and favours the progression to heart failure. In the last decade, it has been increasingly recognized that PM_2.5 exposure accelerates the development of risk factors for cardiovascular disease (refer to the figure below), such as hypertension and diabetes. However, mechanistic links between PM_2.5-driven inflammation and metabolic derangements remain unclear. We are combining a variety of in vivo, ex vivo, and in vitro experimental models of exposure to PM_2.5 to unravel the contribution of air pollution to cardiovascular disease onset and progression, and to evaluate inhibitory approaches to deal with this high-impact public health issue.

Suggested reading:

1. Molecular mechanisms underlying NLRP3 inflammasome activation and IL-1β production in air pollution fine particulate matter (PM_2.5)-primed macrophages.
   link: https://pubmed.ncbi.nlm.nih.gov/38000727/
2. Redox and inflammatory mechanisms linking air pollution particulate matter with cardiometabolic derangements.
   link: https://pubmed.ncbi.nlm.nih.gov/37852544/
3. Pathogenic role of air pollution particulate matter in cardiometabolic disease: evidence from mice and humans.
   link: https://pubmed.ncbi.nlm.nih.gov/32403947/
4. Acute exposure to air pollution particulate matter aggravates experimental myocardial infarction in mice by potentiating cytokine secretion from lung macrophages.
   link: https://pubmed.ncbi.nlm.nih.gov/27240856/