DEPARTMENT OF

SYSTEMS BIOLOGY

The homeostasis of an organism is regulated by soluble mediators such as hormones and cytokines. These messengers activate various intracellular signalling pathways that interact in complex networks. The temporal and spatial orchestration of these signalling pathways is strictly controlled. An unbalanced activation of the signalling pathways leads to severe immunological, inflammatory or proliferative diseases. Multi-site docking proteins such as Gab1 play a particularly important role in the interconnection of these signalling pathways. Misregulation of Gab1 has been described in breast and colon cancer and leukaemia, among others. Multi-site docking proteins do not have an enzymatic function but mediate the networking of various signalling pathways via protein-protein interactions between various other signalling molecules. "Multi-site docking proteins thus integrate different signalling pathways - almost like a molecular computer. This makes them promising targets in the development of new therapeutic approaches. The great importance of Gab1 in physiologically and patho-physiologically important cellular processes encourages us to focus on Gab1 as a therapeutic target.

The currently prevailing therapeutic strategies are based on the blockade of specific extracellular signaling components (e.g. receptors, mediators) with biologicals, such as therapeutic antibodies, or on the inhibition of intracellular signalling proteins (e.g. kinases) with cell-permeable pharmacological inhibitors. However, the use of such inhibitors is limited by non-specific "off-targets" and the primary effect against signalling proteins with enzymatic function. However, intracellular Gab1, which is essential for signal integration, cannot be inhibited by inhibitors because it has no enzymatic function. We therefore postulate the use of therapeutic peptides for the targeted control of Gab1 function. Peptides are short amino acid chains of small size. The high binding specificity of peptides and the possibility of interfering with non-enzymatic processes such as protein-protein interactions open up the possibility of specifically controlling the function of Gab1. Until now, the use of therapeutic peptides has been difficult due to their low cell permeability and short half-life and is primarily limited to extracellular applications.

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1. State-of the Art and Key Research Question(s): Coronary Artery Disease (CAD) pathophysiology is initiated when coronary arteries supplying blood to the heart are being blocked with the accumulating plaques, forming varying degree of fatty streaks, built from inflammatory cells, including monocytes derived macrophages and lymphocytes [3-5]. These blood patrolling immune cells attain an inflammatory state in response to the signals delivered by a dysfunctional endothelium, which is initially caused by deposition and subsequent physicochemical modification of circulating low-density lipoproteins (LDL) in subendothelial spaces. Further, the oxidized lipid ladened pathological macrophages residing at subendothelial spaces produce excessive reactive oxygen species (ROS) and proinflammatory cytokines, including IL-6, which decreases nitric oxide bioavailability and substantially increases oxidative stress in the plaque microenvironment. Under these circumstances, the damaged endothelium releases VWF, which is not only best known for its role in hemostasis and thrombosis, supporting platelet adhesion and aggregation, but also plays a central role in vascular inflammation, favoring leukocyte recruitment and extravasation. Such a constant stimulus, including oxidative stress, initiates endothelial senescence, the process characterized by cell cycle arrest and changes in pro-inflammatory gene expression, in the vasculature. With regard to vascular inflammation in CAD patients, we consider a shift from pro-inflammatory to the pro - senescence state of a vascular endothelium as a key decision point that must be focused and targeted to mitigate the conversion of senescence. This is highly essential because senescence associated with vascular endothelium secretes senescence associated secretory phenotypes (SASP), in addition to many dramatic changes occurring at the intracellular level. Further, endothelial-SASP aggravates and sustain chronic inflammation throughout the lifetime of a CAD patient, which lowers the quality of autologous vessel when used for coronary artery bypass graft (CABG) surgery, as CABG still is considered as a gold standard method for multivessel coronary artery disease. These autologous bypass grafts (vessels) are highly prone to getting occluded with thrombus and therefore exhibit a poor long-term potency, which evidently raises the question on the quality of autologous vessel. Of note, vascular endothelial senescence was evident in arterial diseases. Apart, it has been reported that the ligand for inducible costimulator (ICOS-L) were increasingly expressed on an activated endothelium, under the influence of proinflammatory cytokines. The ICOS-L is one of the immune checkpoint molecule that binds to ICOS, expressed on activated T cells, where the ICOS-L/ICOS axis exhibits multifaceted role in immune function, including polarization towards (i) TH₁ immunity; (ii) TH₂ immunity; (iii) TH₁₇ immunity; (iv) Tregs immunity; (v) germinal center formation and B cell immunity in antibody production. However, the potential role of ICOS-L in inducing or preventing endothelial senescence is not yet explored and is therefore largely unknown.
1.2 Unresolved key questions: Since cardiovascular inflammation diseases remains to be the first leading cause of death globally, we intend to stamp on critical window phase where the transformation of vascular endothelium occurs from pro-inflammatory to pro-senescence state, with the aim of preserving the quality of vessels, thereby avoiding further worsening from chronic inflammation due to senescence and thereby to subsequently increase the patency rate when used for CABG surgery. For this purpose, we explore an in-depth role of ICOS-L/ICOS axis in this above-mentioned decision phase in the presence of atherosclerotic progressive factors.
A. The atherosclerosis related soluble factors, including vWF, blood clotting factors and immune cell associated cytokines, IL-6, IL-1ß, IL-8 increase the endothelial transmembrane expression level of ICOS-L will be explored
B. Despite increased ICOS-L expression, the knowledge of its functional significance on vascular endothelium is largely unknown and will therefore be addressed during the state of (i) initial inflammation, (ii) progressive inflammation and (iii) transformation of inflammation to senescence. This will be achieved by determining the recruitment of intracellular anti-senescence molecules, including SIRTUIN-1 and FOXO1, where these pathways will be thoroughly investigated. Here, we intend to employ the ApoE^-/-atherosclerotic mouse model, ICOS-L transgenic and knockout mouse to investigate the senomorphic role of ICOS-L in vivo as well as in vitro, with endothelial cell culture systems, with ICOS-L overexpression and ICOS-L knockout using CRIPSR-Cas9 tools.

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SARS-CoV2 is highly infectious and causes the disease COVID-19. 10-20 % of patients infected with SARS-CoV2 develop severe symptoms. In these patients, SARS-CoV2 can trigger a cytokine storm that leads to the life-threatening Cytokine Release Syndrome (CRS). Among the cytokines released, Interleukin-6 (IL-6), a paradigm pro-inflammatory cytokine with deleterious functions, correlates strongly with and predicts the severity of COVID-19. Noteworthy, systemic vascular complications in critically ill COVID-19 patients represent a main risk. The expression of SARS-CoV2 entry factors on vascular cells in virtually all organs suggests that vascular damage could be a consequence of lytic viral infection of vascular cells. However, it is also discussed that impaired vessel function is mediated by loss of function of non-infected vascular cells exposed to systemically elevated levels of IL-6. In addition, SARS-CoV2 may locally affect IL-6 signalling pathways by controlling the expression and release of IL-6 receptor subunits and IL-6 itself. The suspected role of IL-6 in the development of COVID-19 is the basis for several ongoing clinical trials with approved drugs that either inhibit IL-6 function extracellularly or intervene in intracellular IL-6 signal processing. However, the molecular mechanisms and pathophysiological consequences of IL-6 and the causes of vascular damage in COVID-19 are still unknown.
Preliminary results from clinics show that immunosuppressive glucocorticoids (GC) reduce deaths in certain patient groups by for so far unknown reasons. Remarkedly, both extracellular and intracellular IL-6 signalling is influenced by GC and vice versa IL-6 influences GC signalling. To address the increasing concerns about the efficacy of GC treatment for COVID-19 and possible (adverse) effects of GCs on the vascular system, the molecular mechanisms of GC action in SARS-CoV2-infected cells and the crosstalk of GC and IL-6 must be elucidated.
The aim of this project is to gain profound translational knowledge about molecular mechanisms and pathophysiological consequences of IL-6 and GC action in SARS-CoV2-infected cells and non-infected vascular cells. For this purpose, we will use highly defined 2D and 3D in vitro vascular models and single cell techniques to define the consequences of SARS-CoV2 infection in the two integral vessel cell types, endothelial cells and smooth muscle cells. The results obtained will be a prerequisite for understanding SARS-CoV2 infection and targeted development of treatments to cope with COVID-19.

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Glucocorticoids (e.g. cortisol) are the body's own messenger substances that are released in stressful situations. Synthetic glucocorticoids (e.g. dexamethasone) have been used to treat inflammatory diseases since the 1950s. However, the exact mechanisms of their effects and side effects are still not understood. It is known, , that glucocorticoids increase the IL-6-induced expression of acute-phase proteins. Together with the research group of Prof. Dr. Bode (Düsseldorf), we were able to show that this is triggered, among other things, by increased JAK/STAT signal transduction. We were able to explain the hyperactivation of JAK/STAT signal transduction by a reduced expression of the IL-6-induced feed-back inhibitor SOCS3. We are investigating the molecular causes of this process to show at which level of protein expression SOCS3 synthesis is suppressed. In addition, we also study the molecular basis of the interplay between glandular hormones (prostaglandin, glucagon), as well as pro-inflammatory cytokines (e.g. interleukin 1) and IL-6.
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The interdisciplinary project aims to develop a systemic view of the complex biology of the cytokine interleukin-6 (IL-6), which is regarded as one of the most important inflammatory mediators. IL-6 is currently the target molecule of several therapeutic strategies for the treatment of autoimmune diseases. Two different mechanisms of IL-6 signal transduction initiation are known: classical signaling via membrane-bound IL-6 receptors (IL-6R) and trans-signaling via a soluble form of IL-6R (sIL-6R). Existing therapeutic approaches block both IL-6 pathways. Our cooperation partners (Prof. Rose-John CAU Kiel and Prof. Scheller HHU Düsseldorf) have discovered that IL-6 trans-signaling is responsible for the pro-inflammatory activities of IL-6, while the classical signaling is required for the defense against infections and for regenerative processes. A designer protein (sgp130Fc) was therefore developed that specifically inhibits IL-6-
Trans-signaling without affecting classical Signaling. Clinical testing of an optimized sgp130Fc variant began in June 2013. The InTraSig project will provide the basis for the design of personalized, anti-inflammatory intervention strategies using sgp130Fc proteins. To this end, factors and reactions will be identified that are critical for the specific dynamics of IL-6-induced classical signaling and trans-signaling under physiological and pathophysiological conditions. Deciphering the underlying molecular mechanisms requires new experimental approaches and modeling tools, as well as the combination of biological experiments, mathematical modeling and model-based analysis by the Chair of Systems Theory and Control Engineering at OvGU Magdeburg (Prof. Findeisen). Critical factors and reactions are experimentally verified as potential biomarkers and ultimately serve as the basis for the design of individualized therapeutic approaches by the industrial project partner CONARIS Research Institute AG.
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This project focuses on a new biological concept that assigns an important role to the cellular stress response in the regulation of the expression of inflammation-related cytokines. Within this framework, we want to investigate how stress regulates the expression of the inflammatory cytokine TNF-a and the versatile signal transduction inhibitor SOCS3. Together we will further investigate how this regulation is influenced by interleukin-6, the main mediator of the acute phase response, and by immunosuppressive glucocorticoids. This work is based on our discovery that the genes of inflammatory cytokines often contain highly potent intragenic RNA activators of protein kinase R (PKR). Activated PKR is one of the kinases that phosphorylate the eukaryotic initiation factor eIF2a and thus inhibit translation. This process is essential for the establishment of a complete cellular stress response. For example, IFN-g mRNA inhibits its own translation by causing local activation of PKR through a 5-proximal RNA structure. Furthermore, we were able to show that efficient splicing of TNF-a mRNA requires a short element in the 3-UTR of TNF-a mRNA, which also activates PKR. The activation of PKR leads to the phosphorylation of eIF2a, which is essential for the splicing of TNF-a mRNA. This mechanism represents a previously undescribed positive regulation of mRNA splicing by eIF2a. The expression of SOCS3 is also regulated by PKR and eIF2a phosphorylation as part of the cellular stress response. Under conditions that induce eIF2a phosphorylation, activation of PKR induces the expression of an N-terminally truncated SOCS3 isoform, delta N-SOCS3, which is longer-lived than SOCS3 and thus acts as a more potent inhibitor. Recently, we demonstrated that glucocorticoids enhance IL-6-dependent gene induction by inhibiting SOCS3 expression, but without affecting SOCS3 protein stability or SOCS3 mRNA quantity or stability. These observations suggest a repression of SOCS3 translation. We therefore wonder whether the PKR activation required for the synthesis of the more stable delta N-SOCS3 is achieved by intragenic SOCS3 RNA activators and whether glucocorticoids influence SOCS3 expression via regulation of PKR activity and eIF-2a phosphorylation. The activation of PKR and the phosphorylation of eIF2a thus control the expression of SOCS3 and TNF-a. Both the expression of SOCS3 and the expression of TNF-a are regulated by IL-6 and glucocorticoids. These observations form the basis of this research project. The results of these joint studies on the biological basis of the cellular stress response will be important for the understanding of inflammatory processes.
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The protein tyrosine phosphatase SHP2 has ambivalent functions in IL-6-induced signal transduction. On the one hand, it inhibits the JAK/STAT signaling pathway, on the other hand, it promotes the activation of the MAPK cascade. Both functions and their interaction are still not fully understood. In an interdisciplinary project with the working group of Prof. Dr. Mönnigmann (Bochum), we were able to show that SHP2 does not act as a negative regulator in early IL-6-induced signal transduction. In contrast, we postulate a previously unknown function of SHP2 as a repressor of a cytokine-independent activity of the JAK/STAT signaling pathway, which is detected, for example, in many proliferative diseases. Mutations in SHP2 are found in patients with various diseases (Noonan syndrome, LEOPARD syndrome and leukemias). We were able to show that these mutations in SHP2 alter the interaction of SHP2 with other molecules. We would like to understand the influence of these mutations and the resulting altered binding properties on IL-6-induced signal transduction. In further subprojects, we are investigating how SHP2 contributes to IL-6-induced activation of the MAPK cascade and how SHP2 and the feed-back inhibitor SOCS3 control the balance between the JAK/STAT signaling pathway and the MAPK cascade.
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Glucocorticoids (GC) are widely prescribed anti-inflammatory drugs. However, their exact mechanism of action is not yet sufficiently understood. We showed recently that one reason for the GC-function is an increased IL-6-induced JAK/STAT-signalling in the liver. The increase in STAT3 activation is caused by a reduced expression of the IL-6-induced feedback-inhibitor SOCS3. SOCS3 is known to be a major mediator of inflammation. Though, the exact function of SOCS3 is still under investigation. In many cell types a prolonged activation of STAT3, caused e.g. by a reduced SOCS3 expression, leads to severe diseases and overexpression of SOCS3 reduces progression of arthritis in mice.GC also affect antigen-presenting cells such as dendritic cells, where they promote the differentiation of a tolerogenic phenotype. These tolerogenic dendritic cells have a low costimulatory potential, secrete high levels of anti-inflammatory cytokines and exhibit an impaired potential to induce the differentiation of inflammatory T cells. However, the molecular mechanism of GC- modified dendritic cell maturation is still under investigation. Interestingly, the group of Prof. Yoshimura showed that SOCS3-deficient dendritic cells also develop into tolerogenic DC. Furthermore, other groups showed that the timely orchestrated expression of SOCS3 is important for the development of mature immunogenic dendritic cells and that SOCS3 is one of the master regulators defining the inflammatory phenotype of dendritic cells. Based on these studies we postulate that GC influence the development of tolerogenic DC by reducing the expression of SOCS3 in DC. This is an up to know unknown molecular mechanism that may contribute to the anti-inflammatory properties of GC.

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