ERC FUNDED PROJECTS

Host cytokines, chemokines and type I IFNs are critical effectors of the innate immune response to viral and bacterial pathogens. Several classes of germ-line encoded pattern recognition receptors have been identified, which sense non-self nucleic acids and trigger these responses. Recently NLRP-3, a member of the NOD-like receptor (NLR) family, has been shown to sense endogenous danger signals, environmental insults and the DNA viruses adenovirus and HSV. Activation of NLRP-3 induces the formation of a large multiprotein complex in cells termed inflammasome , which controls the activity of pro-caspase-1 and the maturation of pro-IL-1² and pro-IL18 into their active forms. NLRP-3, however, does not regulate these responses to double stranded cytosolic DNA. We identified the cytosolic protein AIM2 as the missing receptor for cytosolic DNA. AIM2 contains a HIN200 domain, which binds to DNA and a pyrin domain, which associates with the adapter molecule ASC to activate both NF-ºB and caspase-1. Knock down of AIM2 down-regulates caspase-1-mediated IL-1² responses following DNA stimulation or vaccinia virus infection. Collectively, these observations demonstrate that AIM2 forms an inflammasome with the DNA ligand and ASC to activate caspase-1. Our underlying hypothesis for this proposal is that AIM2 plays a central role in host-defence to cytosolic microbial pathogens and also in DNA-triggered autoimmunity. The goals of this research proposal are to further characterize the DNA ligand for AIM2, to explore the molecular mechanisms of AIM2 activation, to define the contribution of AIM2 to host-defence against viral and bacterial pathogens and to assess its function in nucleic acid triggered autoimmune disease. The characterization of AIM2 and its role in innate immunity could open new avenues in the advancement of immunotherapy and treatment of autoimmune disease.

1 727 920 €

Start date: 2009-12-01, End date: 2014-11-30

"Archaea constitute the third domain of life and are believed to be close to the origin of life. They comprise a diverse group of micro-organisms that combine bacterial and eukaryotic features, but also employ many novel mechanisms. They possess a unique cell envelope with a cytoplasmic membrane of ether lipids surrounded by a proteinaceous S-layer and various cell appendages such as flagella, pili and more unusual structures. Studies have shown that the archaeal flagellum is an unique structure as it functionally resembles the bacterial flagellum, but structurally it is a simple type IV pilus. Moreover, we have shown that this type IV pilus can rotate. Therefore I propose to name the archaeal flagellum, the archaellum, as it is fundamentally different from the bacterial flagellum. In this proposal I aim to understand the assembly and mechanism of rotation of the archaellum of the thermocacidophilic crenarchaen Sulfolobus acidocaldarius by using biochemical, genetic and biophysical methods. The main milestons are: - Biochemical and structural characterization of all archaellum subunits - To understand the assembly pathway of the archaellum and the interactions of its different subunits - To understand how rotation of the filament is achieved and which subunits are important for this movement This work will identify a new, relatively simple motor complex that has evolved from primordial type IV pili assembly machineries and therefore uncover general principles of macromolecular assemblies at cellular surfaces and a novel mechanism to generate mechanical force that can be translated into movement."

1 464 317 €

Start date: 2013-02-01, End date: 2018-01-31

Immune systems are highly variable, but the sources of this variation are poorly understood. Genetic variation only explains a minor fraction of this, and we are unable to accurately predict the risk of immune mediated disease or severe infection in any given individual. I recently found that immune cells and proteins in healthy twins vary because of non-heritable influences (infections, vaccines, microbiota etc.), with only minor influences from heritable factors (Brodin, et al, Cell 2015). When and how such environmental influences shape our immune system is now important to understand. Birth represents the most transformational change in environment during the life of any individual. I propose, that environmental influences at birth, and during the first months of life could be particularly influential by imprinting on the regulatory mechanisms forming in the developing immune system. Adaptive changes in immune cell frequencies and functional states induced by early-life exposures could determine both the immune competence of the newborn, but potentially also its long-term trajectory towards immunological health or disease. Here, I propose a study of 1000 newborn children, followed longitudinally during their first 1000 days of life. By monitoring immune profiles and recording many environmental influences, we hope to understand how early life exposures can influence human immune system development. We have established a new assay based on Mass Cytometry and necessary data analysis tools (Brodin, et al, PNAS 2014), to simultaneously monitor the frequencies, phenotypes and functional states of more than 200 blood immune cell populations from only 100 microliters of blood. By monitoring environmental influences at regular follow-up visits, by questionnaires, serum measurements of infection, and gut microbiome sequencing, we aim to provide the most comprehensive analysis to date of immune system development in newborn children.

1 422 339 €

Start date: 2016-07-01, End date: 2021-06-30

Leukocytes are the key components of the immune system that fight infections and provide tissue repair, yet their migration patterns throughout the body over the course of a day are completely unknown. Circadian, ~24 hour rhythms are emerging as important novel regulators of immune cell migration and function, which impacts inflammatory diseases such as myocardial infarction and sepsis. Altering leukocyte tissue infiltration and activation at the proper times provides an option for therapy that would maximize the clinical impact of drugs and vaccinations and minimize side effects. We aim to create a four-dimensional map of leukocyte migration to organs in time and space and investigate with epigenetics techniques the molecular mechanisms that regulate cell-type specific rhythms. We will functionally define the daily oscillating molecular signature(s) of leukocytes and endothelial cells with novel proteomics approaches and thus identify a circadian traffic code that dictates the rhythmic migration of leukocyte subsets to specific organs under steady-state and inflammatory conditions with pharmacological and genetic tools. We will assess the impact of lineage-specific arrhythmicities on immune homeostasis and leukocyte trafficking using an innovative combination of novel genetic tools. Based on these data we will create a model predicting circadian leukocyte migration to tissues. The project combines the disciplines of immunology and chronobiology by obtaining unprecedented information in time and space of circadian leukocyte trafficking and investigating how immune-cell specific oscillations are generated at the molecular level, which is of broad impact for both fields. Our extensive experience in the rhythmic control of the immune system makes us well poised to characterize the molecular components that orchestrate circadian leukocyte distribution across the body.

1 497 688 €

Start date: 2015-09-01, End date: 2020-08-31

Macrophage differentiation programs are critical for the outcome of immunity against infection, chronic inflammatory diseases and cancer. How diverse inflammatory signals are translated to macrophage programs in the large range of human pathologies is largely unexplored. In the last years we focused on macrophage differentiation in granulomatous diseases. These affect millions worldwide, including young adults and children and tend to run a chronic course, with a high socioeconomic burden. Their common hallmark is the formation of granulomas, macrophage-driven structures of organized inflammation that replace healthy tissue. We revealed that macrophage precursors in granulomas experience a replication block and trigger the DNA Damage Response (DDR), a fundamental cellular process activated in response to genotoxic stress. This leads to the formation of multinucleated macrophages with tissue-remodelling signatures (Herrtwich, Cell 2016). Our work unravelled an intriguing link between genotoxic stress and granuloma-specific macrophage programs. The molecular pathways regulating DDR-driven macrophage differentiation and their role in chronic inflammatory pathologies remain however a black box. We hypothesize that the DDR promotes macrophage reprogramming to inflammation-maintaining modules. Such programs operate in granulomatous diseases and in chronic arthritis. Using state-of-the art genetic models, human tissues and an array of techniques crossing the fields of immunology, cell biology and cancer biology, our goal is to unravel the macrophage-specific response to genotoxic stress as an essential regulator of chronic inflammation-induced pathologies. The anticipated results will provide the scientific community with new knowledge on the role of genotoxic stress in immune dysregulation and will carry tremendous implications for the therapeutic targeting of macrophages in the context of chronic inflammatory diseases and cancer.

1 500 000 €

Start date: 2019-04-01, End date: 2024-03-31

"The incidence of chronic immune-mediated inflammatory diseases is continually increasing. Chronic inflammation has been linked to intestinal carcinogenesis, which is the second leading cause of cancer-related deaths. The cause of this increase could be the unprecedented dietary abundance typical of “Western” countries. Different types of diets shape the genetic composition and metabolic activity of human intestinal microorganisms; microbiota. There is a continuous cross talk between the microbiota and the immune system. For these reasons, the hypothesis that a “bad” diet promotes a chronic state of intestinal inflammation by shaping the microbiota and in turn carcinogenesis could be supported. However, this hypothesis and whether this is a reversible process remain to be tested. It has recently been shown that the composition and metabolism of the microbiota is plastic and it can be rapidly “reprogrammed” by switching to a healthier diet. This plastic behaviour has also been attributed to T helper cells. We have shown that Th17 cells, originally thought to be a stable T helper linage, can convert into a more pathogenic phenotype contributing to chronic inflammation or can acquire regulatory functions promoting the resolution of the inflammation. This project aims to reveal whether mouse and human Th17 cells can quickly adapt to the microbiota as the microbiota does to the diet and in turn mediate the diet effects. By using a unique set of sophisticated transgenic mice we will also test whether the immune system can be corrected by a “simple” change in diet – a widely held belief not yet substantiated. Studying the potential ""synchronized ballet"" of the diet and the immune system will reveal both the enormous dynamism and the revolutionary therapeutic opportunities intrinsic to T cell biology. This project will furthermore identify molecular targets for pharmacological treatments to reverse inflammatory diseases when a simple diet change no longer suffices."

1 499 695 €

Start date: 2016-12-01, End date: 2021-11-30

Environmental and internal stimuli are constantly sensed by the body’s two large sensory units, the nervous system and the immune system. Integration of these sensory signals and translation into effector responses are essential for maintaining body homeostasis. While some of the intrinsic pathways of the immune or nervous system have been investigated, how the two sensory interfaces coordinate their responses remains elusive. We have recently investigated neuro-immune interaction at the mucosa of the intestine, which is densely innervated by the enteric nervous system (ENS). Our research has exposed a previously unrecognized pathway used by enteric neurons to shape type 2 immunity at mucosal barriers. Cholinergic enteric neurons produce the neuropeptide Neuromedin U (NMU) to elicit potent activation of type 2 innate lymphoid cells (ILC2s) via Neuromedin U receptor 1, selectively expressed by ILC2s. Interestingly, NMU stimulated protective immunity against the parasite Nippostrongylus brasiliensis but also triggered allergic lung inflammation. Therefore, the NMU-NMUR1 axis provides an excellent opportunity to study how neurons and immune cells interact to regulate immune responses and maintain body homeostasis. We propose to generate and use elegant genetic tools, which will allow us to systematically investigate the consequences of neuro-immune crosstalk at mucosal surfaces in various disease models. These tools will enable us to selectively measure and interfere with neuronal and ILC2 gene expression and function, thereby leading to an unprecedented understanding of how the components of neuro-immune crosstalk contribute to parasite immunity or allergic disease development. Furthermore, we will progress into translational aspects of NMU-regulated immune activation for human immunology. Therefore, our research has the potential to develop basic concepts of mucosal immune regulation and such discoveries could also be harnessed for therapeutic intervention.

1 499 638 €

Start date: 2019-07-01, End date: 2024-06-30

FateMapB aims to understand how the unique differentiation potential of fetal hematopoietic stem and progenitor cells (HSPCs) contribute to functionally distinct cell types of the adult immune system. While most immune cells are replenished by HSPCs through life, others emerge during a limited window in fetal life and sustain through self-renewal in situ. The lineage identity of fetal HSPCs, and the extent of their contribution to the adult immune repertoire remain surprisingly unclear. I previously identified the fetal specific RNA binding protein Lin28b as a post-transcriptional molecular switch capable of inducing fetal-like hematopoiesis in adult bone marrow HSPCs (Yuan et al. Science, 2012). This discovery has afforded me with unique perspectives on the formation of the mammalian immune system. The concept that the mature immune system is a mosaic of fetal and adult derived cell types is addressed herein with an emphasis on the B cell lineage. We will use two complementary lineage-tracing technologies to stratify the immune system as a function of developmental time, generating fundamental insight into the division of labor between fetal and adult HSPCs that ultimately provides effective host protection. Aim 1. Determine the qualitative and quantitative contribution of fetal HSPCs to the mature immune repertoire in situ through Cre recombination mediated lineage-tracing. Aim 2. Resolve the disputed lineage relationship between fetal derived B1a cells and adult derived B2 cells by single cell lineage-tracing using cellular barcoding in vivo. Aim 3. Characterize the mechanism and effector functions of Lin28b induced B1a cell development for assessing the clinical utility of inducible fetal-like lymphopoiesis. The implications of FateMapB extend beyond normal development to immune regeneration and age-related features of leukemogenesis. Finally, our combinatorial lineage-tracing approach enables dissection of cell fates with previously unattainable resolution.

1 499 905 €

Start date: 2017-10-01, End date: 2022-09-30

Bacterial pathogens secrete effector proteins to manipulate host cells during infection. In Gram-negative bacteria the conserved type 3 secretion system (T3SS) delivers effector proteins to the host cell in a spatiotemporal manner. Although major T3SS constituting components were identified the function of this macromolecular complex remains elusive which is why the transport mechanism is not understood yet. Here, I present my research proposal of the functional and structural analysis of the T3SS with respect to the effector transport dynamics. The proposal is divided in three sections addressing the T3SS architecture, the cytosolic mechanism preceding effector molecule release and the molecular mechanisms of secretion regulation. Research focus of the first section is on the 3-dimensional structural analysis of isolated T3SS. The proposed studies should help detecting structural features related to the effector transport. A combination of electron microscopy (EM) and mass spectrometry is proposed to analyze the structure and surface properties of the transport channel together with bound effector molecule. I am also planning localization of bound lipids as well as to detect lipid induced structural changes in the T3SS by EM. Characterization of cytosolic processes, preceding the translocation is the focus of the second section. Effector molecule targeting, insertion into the T3SS channel and the chaperone function will be studied using a combination of biochemical and biophysical techniques.Finally, I propose experiments to analyze the T3SS regulation. Here, the research focus is on host signal reception and downstream posttranslational modifications inside bacteria as well as on conformational dynamics of the T3SS needle tip complex. The overall goal of the proposed work is to understand the molecular mechanisms of the T3SS. I believe these studies will impact both the understanding of bacterial pathogenesis as well as the transmembrane transport of proteins.

1 488 240 €

Start date: 2013-01-01, End date: 2017-12-31

Antibodies are destined to neutralize pathogens and can prevent and fight infectious diseases. Over the last years, advances in single B cell cloning resulted in the isolation of highly potent and broad HIV-1 neutralizing antibodies (bNAbs) that have been shown to prevent SHIV infection in non-human primates (NHPs). Recently, we have demonstrated that a combination of bNAbs can suppress HIV-1 replication in humanized mice, reducing viremia to undetectable levels. Moreover, bNAb therapy of SHIV-infected NHPs induced a rapid decline in viremia, followed by a prolonged control due to the long half-life of the antibodies. While these results strongly encourage the clinical evaluation of bNAbs in HIV-1 therapy, it is of critical importance to understand how the therapeutic potential of antibodies can be harnessed in the most effective way. Therefore, we aim to: I.) Identify exceptionally potent HIV-1 neutralizing antibodies that will be a crucial component of immunotherapy. By establishing novel methods for single-cell sorting and high-throughput sequencing we want to identify bNAbs targeting novel epitopes. II.) Prevent HIV-1 escape applying rationally designed treatment strategies targeting conserved functional sites for HIV-1 entry. III.) Evaluate immune markers and function in relation to bNAb administration in humans. Being at the forefront of one of the first clinical trials studying an HIV-1-directed bNAb, we will have the unique opportunity to investigate the interplay of antibody therapy and the host immune system. This proposal aims to strongly advance the field of HIV-1 antibody therapy and therefore enable the introduction of a new therapeutic modality for HIV-1, and will gain insights for antibody-mediated therapy in other infectious diseases.

1 500 000 €

Start date: 2016-01-01, End date: 2020-12-31

Chronic mucosal inflammation and tissue damage predisposes patients to the development of colorectal cancer. One hypothesis is that the same factors important for wound healing, if left unchecked, also promote tumorigenesis. Tight control by a sensor of tissue damage should induce these factors to promote tissue repair, while limiting their activity to prevent development of cancer. IL-22, a prototypical tissue repair factor, plays an important role in a wide variety of intestinal disease including infection, wound healing, colitis, and cancer. Indeed, IL-22 has protective and detrimental effects dependent on the milieu and disease suggesting that proper regulation is required. IL-22 expression is directly regulated, additionally a soluble IL-22 receptor (IL-22 binding protein; IL-22bp), can bind and neutralize IL-22. We reported recently that sensing of intestinal tissue damage and components of the microbiota via the NLRP3 or NLRP6 inflammasomes led to a down regulation of IL-22bp, thereby increasing bioavailability of IL-22. IL-22, which is induced during intestinal tissue damage, exerted protective properties during the peak of damage, but promoted tumor development if not controlled by IL-22bp during the recovery phase. Accordingly a spatial and temporal regulation of IL-22 is crucial. Hence, global administration or blockade of IL-22 is unlikely to be therapeutically beneficial. We are using several newly generated conditional knock-out (cCasp1-/-, cIL-18R-/-, cIL-18-/-, cIL-22R1-/-), knock-in (IL-22 BFP), and gnotobiotic mice, aiming to analyze the cellular and microbial network regulating the IL-22 – IL-22bp axis at a resolution previously unfeasible. Our results will provide novel insights into the network between microflora, epithelium, and immune system regulating tissue regeneration and tumor development, and can lead to therapies for potentially a wide variety of intestinal diseases, such as infection, colon cancer, IBD, or wound healing.

1 498 392 €

Start date: 2014-01-01, End date: 2018-12-31

Neutrophils are essential effector cells of the innate immune response. Intravital microscopy studies have recently changed our perspective on neutrophil tissue dynamics. They revealed swarm-like migration patterns in several models of inflammation and infection: Neutrophil populations show strikingly coordinated behavior with phases of highly directed chemotaxis and clustering at local sites of tissue damage. My previous work established that neutrophils self-amplify this swarming response by auto-signaling, which provided the first molecular basis for the collective nature of neutrophil swarms (Lämmermann et al., Nature 2013). However, we are still at the beginning of unraveling the molecular pathways behind this newly discovered phenomenon. Most importantly, we completely lack insight into the signals and mechanisms that stop neutrophil swarms in the resolution phase of an immune response. Since excess neutrophil accumulations cause deleterious tissue destruction in many inflammatory diseases, novel insights into the mechanisms, which prevent extensive swarm aggregation, might be of considerable therapeutic value. In accord with this, our proposal follows three aims: (i) dissecting the cellular and molecular mechanisms that control the resolution phase of neutrophil swarming, (ii) establishing a conceptual framework of how swarming immune cells adapt their dynamics to changing inflammatory milieus, and (iii) developing an integrated view on how neutrophil swarms are controlled by secondary waves of myeloid cell swarms. To achieve our goals, we will combine targeted mouse genetics with live cell imaging of immune cell dynamics in living tissues and the use of innovative mimics of physiological environments. Our future findings on innate immune cell swarms promise to (i) advance our knowledge on leukocyte navigation in complex inflammatory tissues and (ii) provide new avenues for the therapeutic modulation of tissue regeneration after inflammation and infection.

1 500 000 €

Start date: 2017-02-01, End date: 2022-01-31

Herpesviruses cause serious diseases in humans and animals. After initial lytic infection, herpesviruses establish a quiescent (latent) infection, which allows their persistence in the host for life. We and others recently identified a novel mechanism that allows maintenance of the genome of certain herpesviruses during latency, by integrating their complete genetic material into host telomeres. One of these viruses is human herpesvirus 6 (HHV-6) which is associated with seizures, encephalitis, and graft rejection in transplant patients. Sporadic reactivation of the integrated virus ensures continued evolution of the virus as it spreads to a new cadre of susceptible individuals. There are critical gaps in our knowledge regarding the fate of herpesvirus genomes during integration and reactivation as well as of viral and cellular factors involved in these processes. INTEGHER will make use of novel technologies to close these gaps and to devise new therapeutic approaches. Specifically, we will 1) determine the fate of the HHV-6 genome during latency by developing a novel reporter system that allows live-cell imaging of the virus genome in living cells and elucidate epigenetic changes of the HHV-6 genome during integration and reactivation; 2) identify viral and cellular factors that drive virus genome integration and reactivation, using recombinant viruses, drugs and CRISPR/Cas9 genome engineering 3) employ genome-editing tools to eliminate the virus genome integrated in host chromosomes in vitro and in an in vivo model. The proposal utilizes state-of-the-art technologies and pioneers new approaches, particularly with regard to visualization and excision of virus genomes in latently infected cells that are also present in (bone marrow) transplants. Altogether, these studies will define the mechanism of herpesvirus integration and reactivation and will provide new tools for therapeutic excision of virus genomes from living cells.

1 810 747 €

Start date: 2016-04-01, End date: 2021-03-31

The aim of this proposal is to examine the role of inflammatory signalling pathways in murine models of liver and biliary disease by application of conditional gene targeting using cre/loxP technology. Previous studies have provided evidence that the NF-kB pathway and its activating kinase complex – consisting of three subunits: IKK1, IKK2 and NEMO – are crucial regulators of liver physiology and pathology, but their differential, cell specific functions in the liver are currently only poorly understood. The first part of this proposal will focus on the evaluation of molecular mechanisms underlying the development of hepatocellular carcinoma in a setting of chronic hepatitis. By using a novel mouse model of spontaneous liver cancer based on conditional deletion of NEMO in hepatocytes, the functions of cytokines, specific intracellular signalling pathways, the innate and adaptive immune system and the role of hepatic stem cells in hepatitis and carcinogenesis will be examined. In the second part of this proposal, we will extend these studies by evaluating the function of NEMO/NF-kB in other hepatic cell compartments, specifically the function of NEMO in hepatic stellate cells and liver fibrosis, the endothelial function of NEMO/NF-kB in an in vivo model of hepatic ischemia-reperfusion injury and the role of the NF-kB pathway in biliary epithelial cells and inflammatory biliary diseases. Finally, in the third part of this proposal we will analyse the unknown intrahepatic role of non-canonical, IKK1-dependent signalling pathways and the function of TAK1 – a molecule at the interface between inflammatory and developmental pathways – in liver injury, fatty-liver-disease and insulin-resistance. Knowledge gained by these studies and the further understanding of the cell specific hepatic function of NF-kB and related pathways might build the basis for the development of novel pharmacological approaches for the future treatment of liver diseases and cancer in humans.

1 600 356 €

Start date: 2008-09-01, End date: 2014-08-31

Plasmodium sporozoites are the motile forms of the malaria parasite injected into the host by a mosquito. Sporozoite motility is essential for tissue penetration as well as host cell invasion and thus pathogenesis suggesting that blocking motility could potentially add a new way in controlling malaria. It is dependent on a parasite specific myosin, a highly divergent actin and plasma membrane proteins, adhesins that link the substrate to the actomyosin motor. We want to understand the molecular and biophysical basis that underlies the motility of sporozoites to eventually be able to block it. Consequently, we developed methods that allow a systematic probing of key variables important in motility in order to reveal the basic mechanisms of sporozoite locomotion and to screen for small molecules that inhibit motility. Using these assays, we made a number of groundbreaking observations on the cellular and molecular level that gave new insights into the mechanisms of sporozoite adhesion and motility. For example, the dynamic, actin-dependent turnover of adhesion sites was found to be a key factor in sporozoite motility. It is our ultimate goal to understand sporozoite motility to a degree that we can provide a comprehensive dynamic model of sporozoite movement. With the current proposal we aim at unravelling the initial molecular events leading to sporozoite motility focussing on three different adhesins that are known or suspected to be involved in motility. We hypothesize that outside-in signalling leading to actin rearrangements originates from the formation of homo- or heterodimers between these adhesins. Additionally we suggest that inside-out signalling contributes to modulation of adhesion strengths mediated by these adhesins. To test these hypotheses we will generate recombinant parasites that lack two adhesins or express fluorescently tagged adhesin fusions, chimeric or mutant adhesins and investigate these with our recently developed toolbox of novel assays.

1 453 800 €

Start date: 2012-04-01, End date: 2017-03-31

The Interleukin (IL)-1 family of pro-inflammatory cytokines are among the most potent pyrogens, and their excessive production can cause several auto-inflammatory syndromes. Additionally, overabundance of IL-1 cytokines can trigger, or contribute to a range of inflammatory and metabolic disorders. The expression of the key members of the IL-1 family, such as IL-1β and IL-18, is regulated at both the transcriptional and post-transcriptional levels. IL-1β and IL-18, are produced as inactive precursors, which require activation of caspase-1 by the inflammasomes for their maturation and release by from cells, occasionally at the cost of caspase-1 mediated-cell death. We have recently discovered that inflammasomes are released into the extracellular space where they remain active after the demise of activated cells, and that extracellular inflammasomes can amplify inflammation by sustaining extracellular production of IL-1β. However, the sources of extracellular pro-IL-1β are not known. Recent advances in platelet proteomics have revealed that these non-nucleated cells are able to produce their own cytokines, including soluble IL-1β and membrane-bound IL-1α, and are able to significantly magnify IL-1 production by immune cells. As platelets outnumber leukocytes by several folds, they could potentially be the major source of extracellular inflammasomes in the body, or be a major producer of IL-1 precursors that are cleaved by extracellular inflammasomes released from dying immune cells. In this proposal, we will investigate the mechanism(s) by which platelets produce IL-1, and the specific contribution of platelet-derived IL-1 to sterile inflammation, or host resistance to bacterial and viral infection. We believe that a deeper understanding of platelet-IL-1 and their interaction with immune cells during sterile inflammation, or infection might help to uncover new targets for immune-therapies.

1 488 854 €

Start date: 2017-03-01, End date: 2022-02-28

Quorum sensing (QS) is a bacterial cell–cell communication process involving the production, release, and detection of extracellular signal molecules called autoinducers. QS is key to all microbiology as it enables otherwise solitary bacteria to coordinate complex cooperative tasks such as biofilm formation and pathogenesis. Consequently, targeting QS is a promising new concept for antimicrobial therapy. However, for this concept to become reality, we must first identify QS systems in pathogenic bacteria, discover the relevant autoinducers and study the underlying regulatory principles. I recently identified a new QS pathway in Vibrio cholerae, the causative agent of cholera disease. The autoinducer of the system is DPO (3,5-dimethylpyrazin-2-ol), a new molecule to biology and the first pyrazine involved in QS. DPO production is widespread among microbes including pathogenic and commensal bacteria. V. cholerae synthesizes DPO from host mucins and our preliminary data show that DPO controls collective phenotypes, such as biofilm formation and toxin production in this major human pathogen. I therefore hypothesize that DPO connects virulence, QS and communication with the host microbiota in V. cholerae and related bacteria. The overarching goal of this project is to understand the roles of DPO in host-microbe interaction and collective behaviours. To this end, we will pursue three key research goals. First, we will study the molecular parameters underlying DPO-signalling and probe the global effects of DPO on gene expression. Second, we will focus on the role of DPO in virulence of V. cholerae and other pathogens. Third, we will probe the effect of DPO on microbial behaviours, such as swarming and biofilm formation. This combined work will provide a comprehensive model for DPO-signalling in bacteria, which will not only advance the fundamental understanding of QS-based communication strategies, but might also provide the framework for QS-inspired anti-infectives.

1 499 250 €

Start date: 2018-01-01, End date: 2022-12-31

All kingdoms possess a large fraction of RNA-based regulation. We identified several small non-coding regulatory RNAs (ncRNAs) in the human bacterial pathogen Listeria monocytogenes that controlled virulence by a direct RNA:RNA interaction. My group have also identified several 5´-untranslated RNAs (5´-UTRs) known to control expression of their downstream mRNA by a switch mechanism triggered by certain metabolites, specific compartments of the host or by different temperatures. In the suggested project, we will analyze the mechanism by how various RNA-species function on a molecular level by biochemical and genetic approaches. By constructing mutations (deletion and base-substitutions), the role of the regulatory RNAs and their targets during pathogenesis will be pin-pointed using different virulence model organisms. For 5´-UTRs binding specific metabolites, we will add non-metabolic analogs to examine if such molecules can block the function of the 5´-UTRs and hence infection. The core structure of one identified ncRNA will be used as a scaffold to develop an RNA interference system in bacteria. At least one RNA-helicase has been shown to be essential for bacterial motility and growth at 4°C. It is being purified to test its in vitro properties at mRNA targets and at different temperatures. Its in vivo role will be analyzed by genetic techniques. Bacterial resistance against different antibiotics is an increasing problem worldwide. We have identified one pyridine molecule specifically targeting listerial virulence gene expression and its mechanism of action will be revealed by genetic and biochemical techniques. A diffusible, although yet unknown molecule, with bacteriostatic activity was observed and its nature and mechanism will be revealed mainly by biochemical experiments. Our work will give important knowledge of how the bacterium uses RNA to sense its surroundings, but will also identifiy new types of antibacterial agents.

999 996 €

Start date: 2010-10-01, End date: 2015-09-30

During inflammation and infection, we are simultaneously confronted with both self and non-self in form of dying cells and microbes, respectively. Mechanisms that facilitate the non-immunogenic clearance of self-antigens derived from apoptotic and necrotic cells and that, in parallel, allow the initiation of an immune response against invading pathogens are incompletely understood. Recent data from our laboratory show that the immune system actively sorts apoptotic cells (ACs) and bacteria into distinct subspecies of macrophages and dendritic cells thereby enabling a segregated processing of self and non-self as well as a differential immune response against these two entities. Incorrect sorting and aberrant uptake of AC-derived self-antigens by pro-inflammatory and antigen-presenting dendritic cells, however, results in the break of self-tolerance and autoimmunity. Due to technical limitations, the identification and fate-mapping of specific phagocyte subsets that mediate the simultaneous clearance of dying cells and pathogens in vivo has remained largely elusive. We thus plan to develop novel tools that are based on cutting-edge technologies to comprehensively elucidate the sorting of dying cells and pathogens under inflammatory conditions in vivo. We plan to generate TAT-Cre transgenic mice and bacteria that will be used in conjunction with R26-eYFP reporter animals to permanently track phagocytes after ingestion of endogenously accumulated dying cells and pathogens, respectively. These approaches will enable us to characterize the involved phagocytes, study molecular mechanisms underlying the differential processing of self and non-self and follow the phagocyte’s migratory behaviour and its subsequent differentiation. The obtained data will not only provide insights into the pathogenesis of autoimmune and infectious diseases, but will also foster the development of novel therapeutic strategies for the treatment of such disorders.

1 479 781 €

Start date: 2015-05-01, End date: 2020-04-30