Research interests

Upon cell damage and cell death molecular structures, so-called damage-associated molecular pattern (DAMPs) are released into the circulation and induce a systemic inflammatory response by ligation of pattern recognition receptors (PRR). DAMPs released from nucleated cells include- among others- extra-cellular (cell-free) DNA, DNA-binding proteins (e.g. Histones, HMGB-1) and heme. DAMPs released from erythrocytes comprise hemoglobin, iron and heme, respectively. Extracellular DNA, e.g nucleosomes, and HMGB1 have been demonstrated to induce an inflammatory response via Toll-like receptor (TLR) 9 and histones via TLR2 and TLR4, respectively. Heme is cytotoxic through the formation of highly active reactive oxygen species (ROS), an effect that is - in part - dependent on TLR4 and induction of the NFkappaB pathway. Complement activation upon damage of nucleated and non-nucleated cells propagate the inflammatory response induced by DAMPs and may lead by itself to cell destruction.

DAMPs may have detrimental effects by inducing a systemic inflammation resulting in fatality. In addition, the systemic inflammation may also facilitate and perpetuate (uncontrolled) activation of the adaptive immune response. Therefore the organism has multiple “self-defense” strategies to neutralize the potential fatal effects of DAMPs. These strategies include a tight regulation of DAMP release and the presence of systemic inhibitors (e.g. plasma proteins) to neutralize the toxic effects of DAMPs. The last years it became evident that DAMPs may play significant role in the inflammatory processes occurring in hematological malignancies, e.g. graft versus host disease (GvHD) and neutropenic fever, as well as in benign hematological diseases, such as sickle cell disease, paroxysomal nocturnal hemoglobinuria (PNH) and autoimmune hemolytic anemia (AIHA). The research of my group focuses on the mechanism on how DAMPs are released, on the regulation of this process with a special focus on the role of plasma proteins herein, the identification of the cellular source of the DAMPs, and the systemic biological effects of the released DAMPs.

 

Focus 1: mechanism, regulation and biological effects of DAMP-release from nucleated cells

Given the highly conserved structure of DAMPs and the cross-reactivity of antibodies reacting with DAMPs reliable systemic measurement of DAMPs is troublesome. We set up a couple of assays to measure DAMPs in plasma, e.g in the form of nucleosomes or using RT-PCR detecting cell-specific DNA. Combined measurement of neutrophilic proteases and nucleosomes turned out to be reliable measures in plasma for neutrophil activation in the form of neutrophil extracellular traps (NETs). Others and we showed NET formation to be involved in the pathogenesis in various diseases (e.g. sepsis, typhoid fever, melioidosis, sickle cell disease). We also showed NET formation to contribute to venous thrombosis. DAMP release from nucleated dead cells is highly regulated. We identified Factor VII-activating protease (FSAP), a plasmatic serine protease which is activated upon contact with dead cells, to “detoxify” apoptotic cells by removing the nuclear contents in form of nucleosomes and – in concert- with DNAse1 also from necrotic cells. We identified histone 1 (H1) to be the target in necrotic cells, which is cleaved by FSAP resulting in nucleosome release. We showed that plasma protein inhibitors, such as C1-inhibitor, α2-antiplasmin, plasminogen activator inhibitor (PAI)-1 and tissue factor pathway inhibitor (TFPI) inhibit FSAP activation and activity and hence the release of nucleosomes. We unraveled the mechanism on how TFPI precisely binds and inhibits FSAP activity. We recently demonstrated FSAP to be activated by core histones (H2A, H2B, H3 and H4) as well and subsequently to cleave core histones resulting in neutralization of histone cytotoxicity. Together these results suggest that FSAP protects the host form cytotoxic effects of histones released during inflammation.

 

Focus 2: DAMPs in hemolytic diseases Hemolytic diseases are characterized by a shortened survival of erythrocytes in the circulation due to intra- and/or –extravascular hemolysis. Upon hemolysis of erythrocytes, DAMPs (e.g. free hemoglobin and heme) are released into the circulation resulting in systemic inflammation. We study the mechanism on how erythrocytes are removed from and/or lysed in circulation with a special focus on the role of the complement system herein. We show that complement mediated hemolysis of erythrocytes occurs in a significant percentage of patients with autoimmune hemolytic anemia (AIHA), which is mainly IgM driven. To date we are characterizing these antibodies with regard to their glycosylation pattern, rearrangement and isotype structure (penta- vs hexameric forms). We demonstrated that complement inhibiting strategies, e.g. plasma-derived C1-inhibitor or inhibitors of C3 can efficiently block complement mediated erythrocyte destruction in AHIA patients. At the moment we are performing an investigator-initiated clinical phase 1 trial to test the efficacy of C1-inhibitor in complement-mediated destruction of erythrocytes in patients suffering from AIHA. We also study the systemic consequences of erythrocyte lysis. The release of heme induces a strong systemic inflammatory response among others by the activation of neutrophils through the formation of NETs. We demonstrated that neutrophil activation in form of NET is significantly involved in the pathogenesis of organ damage in sickle cell disease and is surprisingly driven by iron rather than heme. Interestingly, preliminary results from our group suggest that NET formation is also involved in the pathogenesis of AIHA. We actually study the efficacy of plasma proteins hemopexin, haptoglobin and apotransferrin to neutralize the proinflammatory effects of heme and iron, respectively. In addition we study the role of inducible heme-oxygenase-1 (HO-1) in systemic inflammation induced by infection and intravascular hemolysis.

Focus 3: Therapeutic plasma proteins to neutralize systemic effects of DAMPs

We have a solid track record in protein purification and structure-function analysis. During the last 15 years we purified many plasma proteins devoid of significant impurities, such as FSAP, C1-inhibitor, TFPI, hemopexin, haptoglobin and α1-chymotrypsin. We optimized protocols in order to facilitate up- scaling of purification processes for these proteins from plasma as well as from waste fractions of Cohn fractionation in order to produce therapeutic proteins on large scale. We generated monoclonal- and polyclonal antibodies to these proteins in order to setup assays to measure protein concentration, activity and protein variants. We also explored the production of recombinant proteins with potential therapeutic indications using techniques to select proteins with optimal glycosylation resulting in comparable biological half-life to the plasma-derived product as well as screening techniques selecting specific glycol-variants of the respective proteins.

Diseases of interest: AIHA, PNH, sickle cell disease, acute graft-vs host disease, transfusion complications, sepsis

Technologies
ELISA, protein purification, protein structure-function analysis, production of mono- and polyclonal antibodies, gel electrophoresis, western blot, molecular biology techniques, mouse models for inflammation, GvHD and transfusion; rat models for transfusion

ID: 107319