ERC FUNDED PROJECTS

Currently, more than 30% of all Europeans suffer from one or more allergic disorder but treatment is still mostly symptomatic due to a lack of understanding the underlying causality. Allergies are caused by type 2 immune responses triggered by recognition of harmless antigens. Both genetic and environmental factors have been proposed to favour allergic predisposition and both factors have a huge impact on the symbiotic microbiota and the intestinal immune system. Recently we and others showed that the transcription factor ROR(γt) seems to play a key role in mucosal tolerance in the gut and also regulates intestinal type 2 immune responses. Based on these results I postulate two major events in the gut for the development of an allergy in the lifetime of an individual: First, a failure to establish mucosal tolerance or anergy constitutes a necessity for the outbreak of allergic symptoms and allergic disease. Second, a certain ‘core’ microbiome or pathway of the intestinal microbiota predispose certain individuals for the later development of allergic disorders. Therefore, I will address the following aims: 1) Influence of ROR(γt) on mucosal tolerance induction and allergic disorders 2) Elucidate the T cell receptor repertoire of intestinal Th2 and ROR(γt)+ Tregs and assess the role of alternative NFκB pathway for induction of mucosal tolerance 3) Identification of ‘core’ microbiome signatures or metabolic pathways that favour allergic predisposition ALLERGUT will provide ground-breaking knowledge on molecular mechanisms of the failure of mucosal tolerance in the gut and will prove if the resident ROR(γt)+ T(reg) cells can function as a mechanistic starting point for molecular intervention strategies on the background of the hygiene hypothesis. The vision of ALLERGUT is to diagnose mucosal disbalance, prevent and treat allergic disorders even before outbreak and thereby promote Public Health initiative for better living.

1 498 175 €

Start date: 2017-07-01, End date: 2022-06-30

The immune system is a complex ensemble of diverse lineages. Studies on in-vivo-hematopoiesis have until now largely rested on transplantation. More physiological experiments have been limited by the inability to analyze hematopoietic stem (HSC) and progenitor cells in situ without cell isolation and other disruptive manipulations. We have developed mouse mutants in which a fluorescent marker can be switched on in HSC in situ (inducible fate mapping), and traced HSC lineage output under unperturbed conditions in vivo. These experiments uncovered marked differences comparing in situ and post-transplantation hematopoiesis. These new developments raise several important questions, notably on the developmental fates HSC realize in vivo (as opposed to their experimental potential), and on the structure (routes and nodes) of hematopoiesis from HSC to peripheral blood and immune lineages. Answers to these questions (and in fact the deconvolution of any tissue) require the development of non-invasive, high resolution barcoding systems. We have now designed, built and tested a DNA-based barcoding system, termed Polylox, that is based on an artificial recombination locus in which Cre recombinase can generate several hundred thousand genetic tags in mice. We chose the Cre-loxP system to link high resolution barcoding (i.e. the ability to barcode single cells and to fate map their progeny) to the zoo of tissue- or stage-specific, inducible Cre-driver mice. Here, I will present the principles of this endogenous barcoding system, demonstrate its experimental and analytical feasibilities and its power to resolve complex lineages. The work program addresses in a comprehensive manner major open questions on the structure of the hematopoietic system that builds and maintains the immune system. This project ultimately aims at an in depth dissection of unique or common lineage pathways emerging from HSC, and at resolving relationships within cell lineages of the immune system.

2 500 000 €

Start date: 2017-07-01, End date: 2022-06-30

"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

Herpes simplex virus 1 (HSV-1) is an important human pathogen, which intensively interacts with the cellular transcriptional machinery at multiple levels during lytic infection. Employing next-generation sequencing to study RNA synthesis, processing and translation in short intervals throughout lytic HSV-1 infection, my laboratory made the surprising observation that HSV-1 triggers widespread disruption of transcription termination of cellular but not viral genes. Transcription commonly extends for tens-of-thousands of nucleotides beyond poly(A)-sites and into downstream genes. In contrast to textbook knowledge, HSV-1 infection does not inhibit splicing but induces a broad range of aberrant splicing events associated with disruption of transcription termination. Exploring these fascinating phenomena will provide fundamental insights into RNA biology of human cells. The proposed work combines both hypothesis-driven and innovative unbiased screening approaches. I will utilise cutting-edge methodology ranging from high-throughput studies to advanced single molecule imaging. Thereby, I will detail the molecular mechanisms responsible for disruption of transcription termination and aberrant splicing. I will identify novel cellular proteins governing transcription termination using a genome-wide Cas9-knockout screen. I will develop RNA aptamer technology to visualise and track single RNA molecules suffering from poly(A) read-through. I will elucidate why transcription termination of some cellular and all viral genes remains unaltered throughout infection. I hypothesize that the alterations in RNA processing are depicted by specific changes in RNA Polymerase II CTD phosphorylation and in the associated proteins. I will characterise these dynamic changes using mNET-seq and quantitative proteomics. Finally, data-driven quantitative bioinformatic modelling will detail how the coupling of RNA synthesis, processing, export, stability and translation is orchestrated by HSV-1.

1 994 375 €

Start date: 2017-05-01, End date: 2022-04-30