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Universitäts-Herzzentrum Freiburg - Bad KrozingenUniversitäts-Herzzentrum Freiburg - Bad Krozingen
Our research


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.     Regulatory CD4+ T Cells Recognize MHC-II-Restricted Peptide Epitopes of 
        Apolipoprotein B
        link: https://www.ncbi.nlm.nih.gov/pubmed/29588316

2.     Atheroprotective vaccination with MHC-II-restricted ApoB peptides induces
        peritoneal IL-10 producing CD4 T cells.
        link: https://www.ncbi.nlm.nih.gov/pubmed/28087520

3.     Beyond vascular inflammation - recent advances in understanding
        link: https://www.ncbi.nlm.nih.gov/pubmed/26100516



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



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
        link: https://www.ncbi.nlm.nih.gov/pubmed/22412980



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.     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

2.     Single-Cell RNA-Seq Reveals the Transcriptional Landscape and Heterogeneity
        of Aortic Macrophages in Murine Atherosclerosis
        link: https://www.ncbi.nlm.nih.gov/pubmed/29545365

3.     Atherosclerosis in the single-cell era
        link: https://www.ncbi.nlm.nih.gov/pubmed/30020199


Five most important publications from the lab:  

1.     A ligand-specific blockade of the integrin Mac-1 selectively targets pathologic inflammation while maintaining protective host-defense. Wolf D, Anto-Michel N, Blankenbach H, Wiedemann A, Buscher K, Hohmann JD, Lim B, Bäuml M, Marki A, Mauler M, Duerschmied D, Fan Z, Winkels H, Sidler D, Diehl P, Zajonc DM, Hilgendorf I, Stachon P, Marchini T, Willecke F, Schell M, Sommer B, von Zur Muhlen C, Reinöhl J, Gerhardt T, Plow EF, Yakubenko V, Libby P, Bode C, Ley K, Peter K, Zirlik A.
Nature communications, 2018; 9(1):52

2.     Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA- Sequencing and Mass Cytometry. Winkels H, Ehinger E, Vassallo M, Buscher K, Dinh HQ, Kobiyama K, Hamers AAJ, Cochain C, Vafadarnejad E, Saliba AE, Zernecke A, Pramod AB, Ghosh AK, Anto Michel N, Hoppe N, Hilgendorf I, Zirlik A, Hedrick CC, Ley K, Wolf D.
Circulation Research. 2018; 122(12):1675-1688

3.     Inflammatory Pathways Regulated by Tumor Necrosis Receptor- Associated Factor 1 Protect From Metabolic Consequences in Diet-Induced Obesity.
Anto Michel N, Colberg C, Buscher K, Sommer B, Pramod AB, Ehinger E, Dufner B, Hoppe N, Pfeiffer K, Marchini T, Willecke F, Stachon P, Hilgendorf I, Heidt T, von Zur Muhlen C, von Elverfeldt D, Pfeifer D, Schüle R, Kintscher U, Brachs S, Ley K, Bode C, Zirlik A, Wolf D.
Circulation Research. 2018; 122(5):693-700

4.     Coinhibitory suppression of T cell activation by CD40 protects against obesity and adipose tissue inflammation in mice.
Wolf D, Jehle F, Michel NA, Bukosza EN, Rivera J, Chen YC, Hoppe N, Dufner B, Rodriguez AO, Colberg C, Nieto L, Rupprecht B, Wiedemann A, Schulte L, Peikert A, Bassler N, Lozhkin A, Hergeth SP, Stachon P, Hilgendorf I, Willecke F, von Zur Mühlen C, von Elverfeldt D, Binder CJ, Aichele P, Varo N, Febbraio MA, Libby P, Bode C, Peter K, Zirlik A.
Circulation. 2014; 129(23):2414-25

5.     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.
Wolf D, Hohmann JD, Wiedemann A, Bledzka K, Blankenbach H, Marchini T, Gutte K, Zeschky K, Bassler N, Hoppe N, Rodriguez AO, Herr N, Hilgendorf I, Stachon P, Willecke F, Duerschmied D, von zur Muhlen C, Soloviev DA, Zhang L, Bode C, Plow EF, Libby P, Peter K, Zirlik A.
Circulation Research. 2011; 109(11):1269-79

Full list of publications:

PD Dr. Dennis Wolf
Dr. Dennis Wolf

Lab:   +49761-270-70370
Klinik: +49761-270-34010