FUNDING PHASE 1


Microbes and immune homeostasis

Projects aim at understanding the bi-directional cross-talk between the intestinal microbiota and the cellular components of mucosal immune system under physiologic conditions and to define influencing factors such as diet and host genotypes.

Modulation of the intestinal barrier by the intestinal microbiota – Role of dietetic factors and the mucosal immune system

The intestinal barrier is a complex functional entity consisting of mechanical components, but also immunological, neuronal and paracrine signals. On the one hand, it protects us against harmful components in the intestinal lumen, on the other, it allows absorption of fluids and nutrients required for survival. Moreover, it controls the intestinal microbiota, and in turn, the microbiota is one of the major regulators of the barrier function. Recently, data are cumulating that suggest a central role of the intestinal barrier for health and development of disease, the latter including both intestinal and extraintestinal diseases. Therefore, the interest in understanding this barrier in detail is now growing. In particular, we want to know how this barrier works, how we can assess barrier functions, and how we can support it. This is what is our project about.
The major objective of our project is to study the complex interaction between diet, commensal bacteria and the host’s mucosa in the intestine in order to understand how the intestinal barrier is regulated under normal and inflammatory conditions. In particular, we aim to define molecular mechanisms how the intestinal microbiota and the intestinal mucosal immune system, either alone or together, affect the intestinal barrier and – vice versa – how the barrier may shape the intestinal microbiota and the intestinal mucosal immune system. In this context, the role of environmental factors such as dietary components known to modulate the barrier either directly or via alterations of the composition and function of the intestinal microbiota will be studied. In the first three years, all experiments will be performed in appropriate mouse models; afterwards the project will be extended to humans and human diseases.
We expect new insights in dietetic, microbial and immunological factors modulating the intestinal barrier and therefore being of potential relevance for health and disease. From our approach, we also expect to define new strategies for future therapeutic interventions at the level of the intestinal barrier (e.g. dietary concepts, new probiotics, biologicals) with the potential to support barrier functions and thereby to prevent or treat diseases like IBD or intestinal infections. By doing this, we aim to increase our knowledge on how the intestinal barrier, the intestinal microbiota, and the intestinal immune system affect health and diseases.

Principal investigator:

Prof. Dr. Stephan Bischoff, Stuttgart
Universität Hohenheim

Impact of microbiota on the intestinal macrophage compartment

The pathogenesis of inflammatory bowel diseases (IBD) is incompletely understood. Remarkably the genome wide association studies explain only about one fourth of the disease cases. In parallel the incidence IBD has been increasing steadily since the Second World War, suggesting that environmental factors and ensuing changes in the intestinal microbiota contribute to this development. It may be concluded that intestinal microbiota play a crucial role in the homeostasis of the intestinal mucosa. Macrophage subpopulations strongly determine the balance of the mucosal immune system. While in health regulatory M2 subtypes are predominant, the inflammatory M1 subtype has primarily been found in intestinal inflammation such as inflammatory bowel diseases. With the present project we aim to define the role of microbiota in the development of macrophage subtypes in the intestinal compartment. Whether such effects are restricted to the neonatal phase or can also occur in the adult phase will be elucidated by comparing mice that stay germfree or are associated with microbiota directly after birth or at five weeks of age. To be able to define the functional relevance of microbiota-mediated changes in the intestinal macrophage compartment for IBD, various models of chronic intestinal inflammation will be included. Ultimately, single bacteria or bacterial compounds will be identified that have the potential to modulate the macrophage compartment to the regulatory subtype and thus maintain homeostasis. In parallel, a human in-vitro system with in vitro-polarized macrophages or intestinal macrophages from patients and controls will be used to transfer the findings from the mouse system to the human situation. This translational system will in addition make it possible to assess the functional impact of disease-associated mutations on the microbiota-dependent effects on macrophage subtypes.
These studies will characterize the role of defined microbiota in the development of the macrophage development in the intestinal mucosa. The obtained results are expected to help in the development of future therapeutic strategies for the treatment of IBD.

Principal investigators:

Prof. Dr. Michael Blaut, Nuthetal
Deutsches Institut für Ernährungsforschung
Potsdam-Rehbrücke (DIFE)

Prof. Dr. Britta Siegmund, Berlin
Charité - Universitätsmedizin Berlin

Reciprocal interactions between the intestinal microbiota and RORgamma-t-expressing innate lymphoid cells

It is becoming clear that the commensal microbiota is important in controlling differentiation and function of immune system components. In addition, it is believed that immune cells can control the composition of microbial communities residing on epithelial surfaces. Much of this complex crosstalk is not well understood. We and others have recently identified novel subsets of innate lymphocytes that are now widely referred to as innate lymphoid cells (ILC). Retinoic acid-related orphan receptor gamma-t-expressing (RORgt+) ILC, a subset of ILC, are potent producers of cytokines such as IL-22 and IL-17 and have been shown to fortify the intestinal epithelial barrier by directly controlling the expression of tissue-protective genes in Paneth cells and stem cells. Our preliminary data demonstrate that differentiation and function of RORgt+ ILC is controlled by the microbiota at various central checkpoints and that deficiency of RORgt+ ILC led to changes in the composition of microbial communities and increased susceptibility to inflammatory bowel diseases. Here, we will experimentally test our overall hypothesis that the intestinal microbiota controls differentiation and function of RORgt+ ILC and that RORgt+ ILC shape the composition of microbial communities. Using gnotobiotic approaches, we will identify specific classes of microbiota that control function and differentiation of RORgt+ ILC. It will also be determined if bacterial communities that increase or repress function of RORgt+ ILC have an impact on the development of intestinal inflammation. It is unknown which cell type(s) sense the presence of microbes as RORgt+ ILC do not express pattern recognition receptors. Our preliminary data show that the myeloid-derived cytokines IL-1 beta and IL-23 are central mediators for differentiation and function of RORgt+ ILC. However, the cellular sources of these cytokines are unknown. Using mice in which various subsets of myeloid cells can be ablated, we will identify the relevant populations controling differentiation and function of RORgt+ ILC. Finally, we will employ metagenomic sequencing analyses of bacterial 16S rRNA sequences to determine if'RORgt+ ILC control the composition of microbial communities residing in the intestine. Collectively, these studies will constitute an unprecedented interrogation of the crosstalk between microbial communities and components of the innate immune system possibly revealing evolutionary ancient circuits that protect the epithelial barrier. Such circuitry may be harnessed for the development of novel therapeutic options of chronic inflammatory disorders.

Principal investigator:

Prof. Dr. Andreas Diefenbach, Freiburg
Universitätsklinikum Freiburg

Role of bactericidal permeability increasing protein (BPI) in bidirectional hostmicrobiota communication in the gut

Bactericidal permeability increasing protein (BPI) has been characterized as LPS-binding protein with the highest affinity and displays strong antimicrobial activities against Gram negative bacteria. BPI is expressed by myeloid hematopoietic cells such as neutrophils and by epithelial cells including those of the gastrointestinal tract. Besides its features as very potent endogenous antibiotic, recent studies have shown that BPIneutralizing autoantibodies as well as BPI gene polymorphisms (Lys216Glu) are associated with inflammatory bowel diseases (IBD, Crohn´s disease and ulcerative colitis). We aim to investigate the role of BPI for stabilizing the interrelationship of gut microbiota and the gastrointestinal mucosal surface in homoeostatic and inflammatory conditions. Applying newly generated BPI gene-deficient as well as BPI-humanized BAC-transgenic mice I) the influence of microbiota on the mucosal expression of BPI as well as II) the role of BPI for shaping the microbiota composition, III) their spatial segregation from the host mucosa and IV) the control of bacteria-driven inflammatory processes in the gut will be addressed. A better understanding of the role of BPI in the bidirectional microbiota-host interaction will provide the basis for future therapeutic strategies in inflammatory bowel diseases.

Principal investigator:

Prof. Dr. André Gessner, Regensburg
Universität Regensburg

Host control of the enteric microbiota during the postnatal period

In the adult host, the enteric microbiota represents a complex, dense and competitive microbial ecosystem with the capacity to reconstitute after transient disturbance and to out-compete colonization by orally acquired bacteria. In contrast, the enteric microbiota of the neonate is largely determined by environmental exposure and exogenous factors. Although high bacterial numbers are reached shortly after birth, it displays a large individual variation and allows colonization by exogenous bacteria. The postnatal phase represents a critical period in the development of the mucosal immune system. Alterations of the newborn’s environmental exposure in industrialized countries with potential major influence on the microbiota are discussed in the hygiene hypothesis to contribute to the recent rise in the incidence of inflammatory diseases. Although mucosal host factors were shown to influence bacterial colonization, their nature and functional importance for the postnatal development of the microbiota are largely unknown. Using models of competitive colonization with commensal and pathogenic bacteria in both conventional and germ-free neonate mice, the proposed research project aims at the identification, characterization and functional analysis of inducible and developmental factors that drive the establishment of the enteric microbiota after birth and provide protection from pathogen colonization and infection.

The research project addresses the following three issues:

(i) The kinetic and exogenous modulators of postnatal bacterial colonization of the mouse intestine.

(ii) Inducible host factors that influence the postnatal microbiota, colonization resistance and infection susceptibility.

(iii) Developmentally regulated host factors and their effect on bacterial colonization and infection susceptibility.

The results are expected to provide a better understanding of the processes involved to establish (and maintain) the enteric microbiota and to identify new strategies to reinforce colonization resistance and intestinal host-microbial homeostasis.

Principal investigator:

Prof. Dr. Mathias Hornef, Hannover
Medizinische Hochschule Hannover

Reciprocal interactions between the intestinal microbiota and immunoglobulin A

The gut is permanently colonized with a dense and complex population of microorganisms, collectively known as microbiota. It is well know that components of the microbiota, such as commensal gut bacteria, induce IgA type antibodies. These antibodies in turn bind commensal bacteria and control their growth and expansion in the host organism. Thus the IgA system substantially contributes to control the intricate balance between protective immunity and tolerance in the gut. Disturbance of this system can result in immunopathologies such as inflammatory bowel disease and diarrhea. In this project we exploit newly available high-throughput sequencing technologies to analyse the interrelation between the IgA system and the microbiota. Specifically we aim at:

1) Studying adaptation of the IgA system in response to changes in the microbiota, e.g. after antibiotic therapy.

2) Determining the microbe-specificity of IgA antibodies and

3) Examining the impact of the IgA system on the microbiota.

These experiments will help to better understand how pathological misbalances between immunity and tolerance can develop and trigger disturbance of the microbiota. Moreover, these experiments might have profound effects on the development of oral vaccines.

Principal investigator:

Prof. Dr. Oliver Pabst, Aachen
Universitätsklinikum Aachen

Characterization of the dysregulated networks between the intestinal microbiota and the immune system in NLRP6 inflammasome-deficient mice

The intestinal microbiota is composed of hundreds of microorganisms and has important functions in the regulation of metabolism and the immune system. New scientific studies suggest that the intestinal microbiota contributes to the development of common disease such as auto inflammation and diabetes. These disease associations correlate with altered composition of the microbiota, a so-called dysbiosis.

In mice lacking a part of the innate immune system, we discovered a dysbiotic microbiota that is directly responsible for the development of intestinal inflammation. We propose to use this model system to study the mechanisms the microbiota employs to modulate the immune system and to identify which specific microorganisms are responsible for exacerbating disease severity. These experiments explore new functions of the intestinal microbiota that may play important roles for human health and may enable new therapeutic approaches in the future.

Principal investigator:

Privatdozent Dr. Till Strowig, Braunschweig
Helmholtz-Zentrum für Infektionsforschung GmbH

Microbes and chronic inflammation

Projects aim at understanding microbe-host interactions in the pathologic transition from immune homeostasis to chronic inflammatory disorders. Crohn’s disease and ulcerative colitis serve as hallmark pathologies to define the role of the intestinal microbiota in the disease susceptible host.

Necroptosis Regulation in Paneth Cells and its Role for the Intestinal Microflora

Previous studies identified a caspase-independent mode of programmed cell death, denoted necroptosis, which is mediated by the ripoptosome protein complex. In a very recent study we could demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells and intestinal immune homeostasis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8ΔIEC) spontaneously develop inflammatory lesions in the terminal ileum and are highly susceptible to experimentally induced colitis. Casp8ΔIEC mice lack Paneth cells due to necroptosis and demonstrate decreased expression of antimicrobial peptides, suggesting dysregulated anti-microbial immune response of the intestinal epithelium. How necroptosis of Paneth cells is triggered, whether Paneth cell necroptosis induces barrier dysfunction and is causative of inflammation remains an open question and is the subject of the current proposal.
As preliminary data for this proposal, we demonstrate alterations in the composition and distribution of the microbial flora. Moreover we found a direct attachment of microbes to intestinal epithelial cells in Casp8ΔIEC mice and a profound systemic spread of bacteria upon challenge of these mice with DSS. Thus, our data so far implicate a critical role of caspase-8 and Paneth cell necroptosis in regulating intestinal immune homeostasis and antimicrobial defence.
The overall goal of the project is to investigate the relationship between Paneth cell necroptosis, the microbial flora of the gut and intestinal inflammation. Accordingly, we will determine the role of the intestinal microbiota on necroptosis, ripoptosome complex formation and necroptosis-induced intestinal inflammation in different in vitro and in vivo systems. Our analyses will include conventional and gnotobiotic mouse strains that will be left untreated or will be treated in experimental disease models, including models of intestinal infection and inflammation. Moreover, we will study how Paneth cell necroptosis affects the composition of the microbial flora and barrier function in the gut.
The project described in this proposal will provide novel insights into the regulation of necroptosis in the intestinal epithelium and its consequences for intestinal immune homeostasis. A better understanding of these pathways might uncover new therapeutic options for the treatment of intestinal infection and inflammation.

Principal investigators:

Prof. Dr. Christoph Becker, Erlangen
Universitätsklinikum Erlangen AöR

Dr. Claudia Günther, Erlangen
Universitätsklinikum Erlangen AöR

Therapy of IBD: Identification of bacteria, bacterial components and the host intestinal immune mechanisms

The intestinal microbiota is a complex community of microorganisms that colonizes the gastrointestinal tract. Composition of the intestinal microbiota and number of microorganisms differs in dependency of the local environmental conditions. In previous work we showed that the commensal strain B. vulgatus is able to prevent and even more strikingly to heal IBD and that the bacterial factor promoting healing, prevention and even induction of IBD seems to be Lipid A, the bioactive part of LPS. The acylation and phosphorylation pattern of Lipid A seems to decide on an anti-inflammatory or pro-inflammatory effect. Therefore we will focus on two aspects, first the prerequisites of the host immune system and the microbes in healing IBD and second the interaction of therapeutic LPS with TLR4. We will analyze whether (i) the effect of B. vulgatus is a species or a strain specific effect, (ii) which cell type mediates healing of IBD by Bacteroides vulgatus/LPSBV, (iii) whether administration of LPSBV alters the composition of intestinal microbiota, (iv) the mechanisms of therapeutic LPS/TLR4 interaction, and (v) the intracellular processes mediating the antiinflammatory effect of B. vulgatus/LPSBV. The project will result in a deepened understanding of LPS/TLR4 interaction in IBD, the contribution to intestinal immune homeostasis and the potential of this interaction in restoring immune homoeostasis. Understanding the mechanisms and players influencing balance or imbalance of intestinal immune homoeostasis is essential for the development of novel therapeutics and therefore for new strategies in treatment of intestinal inflammatory disorders.

Principal investigator:

Prof. Dr. Julia-Stefanie Frick, Tübingen
Eberhard-Karls-Universität Tübingen

Functional analysis of the intestinal microbiota in a mouse model for Crohn’s disease-like ileitis using a gnotobiotic approach

The increasing incidence of inflammatory bowel diseases (IBD) is considered to be the consequence of environmental and individual risk factors. A major focus of research into disease mechanisms underlying chronic inflammation is the gut microbial ecosystem and its interaction with the intestinal mucosa. Crohn’s disease is one of the two major IBD phenotypes mostly affecting the terminal ileum. Despite the fact that a variety of susceptibility genes suggest a role for microbial triggers in the pathogenesis of Crohn’s disease, numerous IBD-related mouse models target chronic inflammation only in the colon and functional evidence for the intestinal microbiota in shaping a spontaneously developing, chronic inflammatory process of the small intestine is still lacking. The aim of the present project is to identify and mechanistically characterize a disease-relevant microbiota in a unique murine model for Crohn’s disease-like ileitis (heterozygous Tnf ΔARE/+ mice). First experiments showed that antibiotic treatment (vancomycin/metronidazole) of Tnf ΔARE/+ mice inhibited ileal inflammation, supporting the hypothesis that microbial factors play an essential role in the disease pathogenesis of the small intestine. Improvement of ileal pathology was associated with marked changes in mucosa-associated and luminal bacterial diversity, but not cell density, as measured by high-throughput sequence analysis of 16S rRNA gene amplicons. Based on these findings, we will use isolated bacterial strains of the endogenous microbiota to initiate (Objective 1) or prevent (Objective 2) recurrent pathology in germ-free and antibiotic-treated Tnf ΔARE/+ mice. The interplay of selected microbiota with the disease-susceptible host will be analyzed at the epithelial (ileum) and T cell level targeting innate and antigen-specific mechanisms. In summary, we propose to use one of the few model systems for Crohn’s disease-like ileitis to study the functional role of the non-infectious intestinal microbiota in initiating chronic inflammation of the small intestine.

Principal investigator:

Prof. Dr. Dirk Haller, Freising-Weihenstephan
Technische Universität München

Control of intestinal inflammation through CD101 expression

Dysregulated host-microbe interactions in genetically susceptible individuals have been implicated in the pathogenesis of inflammatory bowel diseases (IBDs). However, molecules and signalling pathways controlling the interactions of the intestinal microflora with the immune system of the patient and their role in the suppression of intestinal inflammation have been rarely identified.
We have recently characterized CD101, a negative costimulatory molecule shared by myeloid and lymphoid cell subsets, as critical factor for the control of T cell proliferation and Th17 differentiation in a chronic T cell transfer colitis model. Recipients of T cells from wild-type mice exhibited less severe intestinal pathology than CD101-/- littermates correlating with an acquisition of CD101-expression on T cells and decreased bacterial translocation. Conversely, human IBD patients exhibited lower numbers of CD101-expressing T cells correlating with increased systemic immune responses to enteric bacterial antigens. Based on these findings we propose that CD101 is a novel clinical marker for tissue-specific inflammation in IBD. Thus, the overall goal of the present proposal is to determine the role of CD101 in directing tissue-specific immune protection. Although the mechanisms by which CD101 interferes with T cell activation are unknown, our preliminary data support the hypotheses that a lack of CD101 expression on T cells inhibits the generation of regulatory T cells, triggers T cell proliferation and Th17 responses and subsequently promotes intestinal inflammation and bacterial translocation. Thus, we aim to: 1) explore the regulation of CD101-expression under physiologic and inflammatory conditions in response to intestinal commensal bacteria; 2) determine the impact of CD101-expression on the protection from colitis; define the role of CD101 on 3) IL-2R
a- and FoxP3-expression and 4) the generation of regulatory T cell and Th17 responses; 5) specify the interaction(s) of T cells with myeloid cells in the presence and absence of CD101; and 6) correlate the expression of CD101 on T cells in IBD patients to the clinical disease score, the reactivity of serum samples to oligosaccharides of intestinal bacteria and shifts in the balance of Treg-Th17 homeostasis.

Principal investigator:

Prof. Dr. Jochen Mattner, Erlangen
Universitätsklinikum Erlangen AöR

Analyse mikrobiota-abhängiger IL-23/Th17 Immunantworten bei chronischer Colitis

Complex regulatory mechanisms work together to maintain intestinal homeostasis, and a breakdown in these pathways may finally result in inflammatory bowel disease (IBD). Much of our present understanding of intestinal homeostasis is built around the concept that the microbiota continuously interacts with the intestinal immune system and that the microbiota composition regulates the fine equilibrium between pro-inflammatory and tolerogenic immune responses. However, whether disease related shifts in microbiota composition that were shown to occur in IBD directly cause disease or reflect the adaptation of bacterial communities to inflammation or host genotype related changes in the intestinal environment is still incompletely understood. Genetic polymorphisms in the IL-23R gene and other regions that encode proteins implicated in the IL-23/TH17 pathway in IBD as well as murine studies suggest that appropriate regulation of IL-23 dependent immune responses is critical for intestinal immune homeostasis. On the other hand the IL-23/TH17 axis may play a central role in protective immunity to gastrointestinal infections.  In this project, we will mechanistically investigate the crosstalk between IL-23 producing cells, IL-23R+ immune cell populations, their effector cytokines and the commensal flora. An IL-23 reporter mouse strain will be used to analyze the spatiotemporal expression of IL-23 under germfree conditions and after re-colonization with conventional or defined microflora. Using a mouse model of IL-23 dependent spontaneous colitis, we will characterize the intestinal T cell and innate lymphoid cell (ILC) pool in intestinal tissues before and after onset of colitis; these results will be compared to the situation in IBD patients. In parallel, the compositional analysis of the intestinal microbiota by 16S rRNA metagenomic studies will help to characterize the microbiota/immune system interactions in the transition phase from healthy to inflammatory settings. Moreover, as the cytokines IL-17 and IL-22 that are particularly produced by TH17 cells and RORγt+ ILC in an IL-23 dependent manner are important regulators of antimicrobial peptide production in intestinal epithelial cells (IEC), we will determine how cytokine induced antimicrobial peptide production affects microbial composition and diversity in the gut. In line with this, we will analyze in models of infectious colitis, how IL-23 dependent production of antimicrobial factors in IEC contribute to physiological host responses to intestinal pathogens.

Principal investigators:

Prof. Dr. Markus Neurath, Erlangen
Universitätsklinikum Erlangen AöR

Dr. Stefan Wirtz, Erlangen
Universitätsklinikum Erlangen AöR

TLR-mediated microbiota–host interaction in the regulation of intestinal homeostasis and inflammation

The intestinal lumen is colonised by trillions of commensal bacteria that provide essential digestive support but also influence the regulation of mucosal and systemic immune responses. The cross talk between host cells and the microbiota is now believed to be the major determinant of health and disease in the gastrointestinal tract. Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory conditions of the intestine with unclear aetiology. Deregulation of the cross talk between the intestinal microbiota and the host immune system is currently believed to be the main factor contributing to IBD. The single-layered intestinal epithelium forms a mechanical barrier separating the luminal contents from the mucosa but also provides an active immunological barrier directly regulating and interacting with both the microbiota and the host immune system. The cross-talk of intestinal epithelial cells with the microbiota and with the host immune system is believed to be critical for the regulation of intestinal homeostasis, however the mechanisms regulating epithelial responses to intestinal bacteria and mucosal immune cells remain poorly understood. We showed previously that inhibition of NF-κB signalling in the intestinal epithelium by epithelial specific knockout of NEMO/IKKgamma triggered the spontaneous development of severe chronic colon inflammation. The development of colitis in these epithelial-specific NEMO knockout mice depends on MyD88-mediated TLR signalling and on the presence of intestinal bacteria. In this project we aim to address the role of the TLR-mediated cross talk between the host and the microbiota in the regulation of intestinal homeostasis and inflammation. In the first part of the project we will use genetic mouse models to address the mechanisms by which TLR signalling induces the pathogenesis of chronic colon inflammation in mice with epithelial-specific NEMO knockout. In the second part of the project we will use mouse models allowing the inducible inhibition of TLR signalling specifically in the intestinal epithelium in order to address the role of epithelial TLR signalling in the regulation of the composition of the intestinal microbial communities. Together, these studies will provide important information that will advance the current state of the art in understanding the TLR-mediated mechanisms controlling the cross-talk between the host and the microbiota and their impact in the regulation of epithelial homeostasis and inflammation.

Principal investigator:

Prof. Dr. Manolis Pasparakis, Ph.D., Köln
Universität zu Köln

Interaction between microbiota, antimicrobial defense and differentiation factors in Inflammatory Bowel Disease

Inflammatory bowel diseases (IBD) are to date incurable and characterized be recurring and severe inflammations of the intestinal tract. Even though there is an ongoing dispute about basic and general disease mechanisms, a major role of an imbalanced microbial-epithelial interaction at the intestinal barrier is commonly accepted among clinicians and researchers. In this regard, our group focusses on a potentially disturbed epithelial differentiation. In our research approaches we and consecutively also others have found disturbances in an important Wnt pathway transcription factors (TCF7L2 also known as TCF4) in patients with small intestinal Crohn’s Disease (CD), a subgroup of IBD. Following studies also pinpointed a Wnt coreceptor (LRP6) in a similar context. The Wnt pathway is of major importance in maintaining proliferation, regeneration and paradoxically also directed differentiation of epithelia in the gastrointestinal tract. In particular, Paneth cell maturation and function is controlled by the β-catenin dependent branch of the pathway. Paneth cells are normally specifically found on the bottom of crypts right next to the stem cells in the small intestine. They are specialized producers of antimicrobial peptides which they store in granules and release upon stimulation of pattern recognition receptors. Their most abundant products, HD5 and HD6, are transcriptionally controlled by the Wnt pathway and exhibit a primary decrease in ileal CD. HD5 and HD6 as well as other Paneth cell AMPs are crucial in fending off pathogenic threats but also in controlling the relationship towards intestinal commensals. Their reduced expression in patients represents an interesting possibility for the development of targeted therapy approaches, but further studies are indispensable to gain a better understanding of the complicated system and the involved networks. We therefore plan to analyze the role of microbiota in the context of Wnt controlled antimicrobial defense, as well as to identify potential additional factors which might underlie the imbalanced host-microbiota interactions at the epithelial barrier in patients.

Principal investigator:

Privatdozent Dr. Jan Wehkamp, Stuttgart
Robert Bosch Gesellschaft für medizinische Forschung mbH

 

 

X-linked inhibitor of apoptosis protein in intestinal homeostasis and the pathogenesis of inflammatory bowel disease

Inflammatory bowel diseases (IBD) are a group of diseases that affect an estimated two million people in Europe and that are characterized by chronic intestinal inflammation and an increased risk of colorectal cancer. While the precise mechanisms of IBD pathogenesis are not known, it is believed that inflammation results from an exaggerated host immune response to environmental factors including the intestinal microbiota. Current treatment is therefore based on immunosuppression, targets final steps in immune pathways common to pathogenic and protective immunity, and is associated with susceptibility to infection and malignancy. Moreover, the efficacy of current treatment is limited and at any time half of people with IBD are not in remission.

Recent studies suggest that a subset of IBD cases might be related to congenital or acquired immunodeficiency and that chronic inflammation in these patients may result from an inability to control microbial expansion and invasion at the intestinal mucosal surface thus explaining the lack of efficacy of immunosuppression in these patients.

In studies aiming to identify the genetic contributions to early onset IBD, we have identified mutations in the X-linked inhibitor of apoptosis protein (XIAP), a gene associated with a primary immunodeficiency called X-linked lymphoproliferative syndrome. These results are in accordance with the concept of immune defects underyling intestinal inflammation in IBD. Using primary human cells from these patients, we could demonstrate a selective and severe defect in NOD2-dependent bacterial recognition. Moreover, Xiap-/- mice showed pronounced defects in the expression of NOD2- and Wnt-dependent antimicrobial peptides in Paneth cells. These results suggest that XIAP acts as a master regulator of two pathways central to intestinal homeostasis and control of the intestinal microbiota.

Here, we propose to study the role of XIAP in the regulation of the intestinal microbiota and the pathogenesis of intestinal inflammation. Specifically, we propose to investigate (a) whether Xiap-/- mice exhibit impaired microbial control upon challenge in vitro and in vivo, (b) whether Xiapdeficiency in mice and humans is associated with alterations in the intestinal microbiota that might predispose to intestinal inflammation, (c) whether Xiap-deficiency in mice is associated with increased susceptibility to intestinal inflammation and (d) whether susceptibility to inflammation is dependent on the intestinal microbiota.

Together, these studies will provide unique insight into the mechanisms that control intestinal homeostasis and will reveal whether primary immunodeficiency associated with defects in XIAPdependent microbial control contribute to the pathogenesis of intestinal inflammation. These studies have major implications for IBD and might force a reconsideration of the mechanisms underlying intestinal inflammation and the strategies required for efficacious targeting of inflammation in IBD.

Principal investigator:

Prof. Dr. Sebastian Zeißig, Kiel
Universitäts-Klinikum Schleswig-Holstein

Microbes and infectious inflammation

Projects aim at understanding the role of the intestinal microbiota in creating a disease-conditioning milieu for enteric infections. Establishing colonization resistance and regulating virulence of infectious organisms are key questions to define health-related functions of the microbial ecosystem in the intestine.

Interaction between Yersinia enterocolitica, the intestinal microbiota, and the host: From molecular analysis to therapeutic intervention

Co-evolution of the intestinal microbiota with its host has resulted in a state of mutual benefit. Besides contributing to host nutrition, physiology and mucosal immunity, the intestinal microbiota protects the host from enteric infections, a function designated as colonization resistance. By expression of virulence and fitness factors, enteric pathogens may exploit structures and signaling pathways of the host in order to subvert specific functions of the immune system. While hostpathogen interactions have been studied in detail during the past decades, the role of the microbiota in this interaction is largely elusive, and the trilateral interaction between enteric pathogens, the intestinal microbiota and the host is not well understood. Herein we address whether and how virulence factors of enteric Yersinia enterocolitica (Ye) affect this interrelationship, and whether and how specific components of the microbiota might be used to interfere with Ye pathogenicity. To this end, we will be using Ye wildtype and isogenic mutant strains, an experimental mouse Yersinia infection model including germ-free (GF) and gnotobiotic mice as well as metagenomic analyses to address the specific hypotheses. The results of these investigations will provide new insights into the intricate interaction between Ye, the intestinal microbiota and the mucosal immune system, and might result in novel strategies for treatment of enteric infections.

Principal investigators:

Prof. Dr. Ingo Autenrieth, Tübingen
Eberhard-Karls-Universität Tübingen

Prof. Dr. Daniel Huson, Tübingen
Eberhard-Karls-Universität Tübingen

 

 

The role of blood group-related glycosyltransferases in shaping diversity of the intestinal microbiota and susceptibility to inflammation

Glycan metabolism is an important factor contributing to the composition and physiology of the intestinal microbiota, and variation in the glycosylation profile of the GI tract is often mediated by blood group-related glycosyltransferases. This class of genes is known to influence both symbiotic and pathogenic bacteria, and accordingly displays clear signatures of pathogen-driven selection among populations. This proposal will focus on the interplay between host glycosylation, the intestinal microbiota and susceptibility to GI pathogens and inflammation by investigating two blood group-related glycosyltransferases that are conserved between mice and humans, namely B4galnt2 and Fut2. Their role in the development of intestinal inflammation will be investigated using the pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium, as well as a dextran sodium sulfate (DSS)-induced colitis model. To relate changes in host glycosylation and inflammatory response to changes in the intestinal microbiota, microbial ecological analyses will be performed at the level of 16S rRNA gene- and shotgun metagenomic sequencing. Together, these experiments will elucidate to what degree changes in microbial communities, host glycosylation or their interaction contribute to differences in susceptibility to enteric bacterial pathogens and inflammation.

Principal investigators:

Prof. Dr. John Baines, Kiel
Christian-Albrechts-Universität zu Kiel

Prof. Dr. Guntram Grassl, Kiel
Christian-Albrechts-Universität zu Kiel

Generation of gnotobiotic mice to investigate the role of the intestinal microbiota in Salmonella enterica spp. I serovar Typhimurium colitis in AGR2-deficient mice

The intestinal microbiota plays a pivotal role in protection against the enteric Salmonella serovar Typhi-murium (S. Tm) infection. In mice, treatment with the antibiotic streptomycin transiently disrupts the microbiota and enables S. Tm colonization of the gut, tissue invasion and induction of intestinal inflam-mation. Besides the microbiota, the mucosal barrier significantly contributes to immediate immune de-fence against S. Tm. So far, the interplay of the microbiota and the innate immune system is poorly un-derstood. We analyzed S. Tm infection in mice deficient in anterior gradient 2 (AGR2), which have se-vere mucosal barrier defects. Yet, in opposition to our prediction, AGR2-/- mice were protected against S. Tm induced gut inflammation, in contrast to AGR2+/- littermate controls. AGR2-/- mice exhibited altered microbiota composition and increased microbiota density in response to streptomycin. Thus, we hypothe-size that imbalanced mucosal homeostasis in AGR2-/- mice induced microbiota alterations which provided increased resistance against S. Tm infection. In our case, the intestinal microbiota could compensate for mucosal barrier defects. In this proposal we aim at further characterizing the influence of altered mucosal homeostasis on the intestinal microbiota and how this is related to increased resistance against S. Tm in-fection. To dissect the effect of mucosal homeostasis and the microbiota on the outcome of S. Tm infec-tion, we aim to generate germfree and gnotobiotic AGR2-/- mice associated with a defined gut microbiota. To this end, we will apply a novel “Oligo-Mouse-Microbiota” developed in our laboratory.

Principal investigator:

Prof. Dr. Barbara Stecher, München
Ludwig-Maximilians-Universität München

Mutual interactions between the pathobiont Helicobacter hepaticus and the mouse intestinal microbiota: Ecology, mechanisms and relevance to the induction of IBD

The prototype pathobiont, Helicobacter hepaticus, induces inflammatory bowel disease in susceptible immunocompromised mouse strains, and H. hepaticus infection has become a widely used model to investigate bacterial factors involved in IBD pathogenesis. Available data indicate that the pathology induced by H. hepaticus infection depends on both specific pathogenic mechanisms of the bacterium, as well as the composition of the intestinal microbiota. We showed that C57BL/6 IL-10 ko mice reared at two different institutions (MHH and MIT) display very different susceptibilities to IBD after H. hepaticus infection. Microbiota analyses of these mice by deep sequencing of 16S rDNA amplicons (454 FLX technology) have identified multiple culturable bacterial species that are only present in one group of mice and therefore might be involved in augmenting or suppressing inflammation. In a first part of the project, we will test the hypothesis that the presence of these single bacterial species affects susceptibility to H. hepaticus-induced colitis. Candidate species will be added to the microbiota, followed by H. hepaticus infection, and the resulting pathology and shifts in microbiota composition will be analyzed. In a second part, we will investigate the influence of H. hepaticus colonization on the composition of the intestinal microbiota in both inflamed and non-inflamed conditions.

The H. hepaticus genome contains a pathogenicity island termed HHGI1. This island encodes a type 6 secretion system (T6SS) capable of transporting multiple proteins out of the bacterial cell. Loss of a part of the island, or inactivation of one single gene, vgrG1, was previously shown to strongly attenuate the ability of H. hepaticus to induce colitis. A further objective of this project is to understand how loss of the T6SS function affects the interaction of H. hepaticus with the microbiota. Finally, we will study the genomic adaptation of H. hepaticus to mice under conditions where H. hepaticus is part of a complex microbiota in comparison with mice that are monoassociated with H. hepaticus. In summary, to better understand the complex interactions between a model pathobiont, the gut microbiota and the host, this project combines an experimentally versatile persistent infection model with state -of-the-art microbiota analysis and whole genome comparison technology. We hope that findings obtained during this project will be transferable to interactions between other epsilon proteobacteria (e.g. Campylobacter species) and the physiological microbiota in mice and humans, and to contribute to understanding their complex interplay within the intestinal host ecosystem, and to positively influence it.

Principal investigator:

Prof. Dr. Sebastian Suerbaum, Hannover
Medizinische Hochschule Hannover

Proteomics, Metabolomics and Gnotobiology

Functional analysis of the microbiota and the metabolic synapse in colites by metaproteomics

The maintenance of the stable and healthy symbiosis between microbiota and the host depends on the composition ofthe microbiota which In turn Is controlled by immunological and metabolic factors. Both the Immunological as well as the metabolic synapse are located In the contact zone between microbiota and the host's tissue, the mucus layer. Hence we will focus on the analysis of this specific habitat. It Is reported that the phylogeny and the metabolic functionality are significantly different between the mucus layer and the gut content. The production and maintenance ofthe mucus layer Is known to be strongly affected by Inflammatory diseases like chemically Induces colitis. The Immune response ofthe host Influences directly the composition of the microbiota which then affects metabolic processes. The latter are resulting from the complex metabolic Interdependencles within the microbiota and demand thereby for the analysis of the functional rather than for an assessment of the mere phylogenle diversity. This can be achieved by applying metaproteomlcs and especially protein-stable Isotope probing as a well established variation of metaproteomlcs, In this technique 13C and 15N labelled substrates will be applied and the Incorporation of these Isotopes into proteins determined by high resolution mass spectrometry. The identification of peptides and the according proteins on the basis of metagenome data provides simultaneously phylogenle and functional Information. The degree of Incorporation Is used as a measure of metabolic activity. Furthermore the shape of the isotopologlc distribution allows distinguishing between direct and Indirect metabollsation and hence supports the information on the carbon and nitrogen flux through the microbiota. In addition also the flux from the microbiota Into the host tissue can be assessed. The effect on the metabolic synapse will be also followed by metabolomic profiling ofthe host serum. Since also the endothelium Is crucially Involved in the metabolic synapse and It Is affected by the Inflammatory response we will also analyze the proteomic effects In these cells In colitis In comparison to healthy tissue. The global quantitative proteomics will Indicate the general pathways of the molecular response whereas the phospoproteomlcs approach will lead to Insights Into the cellular signaling Induced by colitis. In summary, we will Identify metabolic pathways and interactions within the mlcroblata and the effect on the metabolic synapse caused by colitis and will combine this with insights Into the response of endothelial cells. Furthermore we will offer proteomic and metaproteomlc support to other subprojects within the SPP.

Principal investigator:

Prof. Dr. Martin von Bergen, Leipzig
Helmholtz-Zentrum für Umweltforschung - UFZ

In depth characterization of Gut microbial Sulfometabolome in a mouse model of Crohn´s Disease

Inflammatory bowel diseases (IBD) refer to intestinal chronic diseases and main forms are Crohn´s disease and ulcerative colitis in which lot of research is invested to reveal the genetic and environmental risk factors. Recently, our group showed the importance of applying metabolomics to describe biomarkers in fecal samples associated with Crohn´s disease in humans using high resolution mass spectrometry (FT-ICR-MS) (J. Jansson et al. 2009). Our group pinpointed different pathways that are regulated between healthy and Crohn´s disease humans from which some involve many on-identified sulfur containing metabolites. Several other studies were performing a metabolite profiling approach to study the impact of the metabolome in IBD. Our aim of the present project is to investigate this specific role of sulfur and its involvement in Crohn´s disease etiology. Thereby, we want to crystallize the central role of bacterial metabolism in the intestine in Crohn´s disease mouse model to elucidate microbial metabolome between healthy and inflamed mice. Moreover, we are concerned about the functional role of sulfur and sulfur containing metabolites and the impact on intestinal microbiota, which are assumed to be one of the important environmental factors which are involved in the pathogenesis of IBD. Furthermore, we want to point out the linkage between sulfur metabolism and the host – gut microbiome axis between healthy and inflamed mice by using specific mouse colonization studies. The implementation of high resolution mass spectrometry and NMR techniques by applying a metabolomics approach will support us to understand the sulfur involvement in an intestinal inflammation in a complex mammalian organism.

Principal investigator:

Privatdozent Dr. Philippe Schmitt-Kopplin, Oberschleißheim
Helmholtz Zentrum München, GmbH

A gnotobiology unit within the SPP 1656

Axenic and defined colonized animals provide unique opportunities to model and study physiological and pathophysiological microbiome-host interactions. They can be utilized to investigate the “naive” mucosa as well as the impact of specific germs or microbial products on a host otherwise devoid of microorganisms. The Institute for Laboratory Animal Science and Central Animal Facility of the Hannover Medical School (Ztm) has a long tradition of gnotobiotic work, including generation and maintenance of germ-free models and their utilization in experimental settings. This expertise has been successfully incorporated in various collaborations and cooperative research projects, e.g., the SFB 621. The aim of this proposal is to make this techniques and experience available to members of the SPP 1656. Axenic and defined colonised animals will be accessible, models will be derived into germ-free state and maintained over defined periods of time, and collaborative gnotobiotic experiments can be conducted at Ztm. In addition, a network of gnotobiology units shall be established to best support the members of the SPP 1656. Thereby, this project is intended to contribute to unravel physiological mechanisms of colonization and development of immune responses as well as to identify determinants of the microbiome-host interplay that contribute to development of pathological conditions in a variety of disease models or resistance against pathogens.

Principal investigators:

Prof. Dr. Ingo Autenrieth, Tübingen
Eberhard-Karls-Universität Tübingen

Prof. Dr. André Bleich, Hannover
Medizinische Hochschule Hannover

Prof. Dr. Dirk Haller, Freising-Weihenstephan
Technische Universität München


FUNDING PHASE 2


Z-Projects

Z-Project: Gnotobiology unit within the SPP 1656

Z-Project: MIMIC - Culture-based and molecular study of the mouse gut microbiome Towards Minimal Microbial Consortia

The gut microbiota is very important for mammalian host physiology. It is thus crucial to put effort into understanding its structure and functions. Omics technologies have generated major breakthroughs in microbiome research, but they only show correlations. To test causal relationships and to study molecular mechanisms, mouse models, have been extensively used in many research projects worldwide and within SPP 1656. However, there is an obvious deficit of bacterial strains and genomes from the mouse gut microbiome, because emphasis has been put on molecular studies over the last decade. This is a major limitation for annotation of omics datasets and for colonization studies in germfree mice to test causal effects of microbiomes known to have specific features depending on their host species of origin.

To overcome these limitations, the present central project MIMIC aims at improving preliminary work performed during phase 1 of SPP 1656 on the isolation and description of mouse gut bacteria to deliver a unique and well-curated strain collection. The collection will give researchers easy access to bacterial strains and corresponding genomic information, but most of all will serve as a foundation to implement a new bioinformatics tool (MIMIC) for automated design of minimal microbial consortia adapted to individual experimental designs in mouse models of interest. The design of minimal consortia will be based on metagenomic coverage strategies and will be embedded into the newly established bioinformatic platform IMNGS for dissemination (www.imngs.org). Considering known phenotype variability between mouse facilities, an innovative approach that allows generation of customized minimal consortia of bacteria based on the cultivable fraction of native ecosystems is highly relevant regarding standardization of mouse models and mechanistic understanding of bacteria-bacteria and bacteria-host interactions. The MIMIC project is a perfect match to current needs in the field and within SPP 1656. It is embedded in the consortium activities via multiple interactions with partners, e.g., to obtain materials from different facilities for isolation of bacteria, to gain access to already isolated strains that require proper description, and to generate minimal consortia adapted to current needs within SPP 1656.

In summary, the present MIMIC proposal is a unique opportunity to use complementarity culture-based and molecular approaches to discover novel bacterial diversity and to create and deliver useful resources to the priority program and more generally to the scientific community. Funding by the DFG will allow putting preliminary activities on a solid basis and integrating an internationally recognized bioresource center (the DSMZ) to SPP activities on microbe-host interactions.

Principal investigator:

Dr. habil. Thomas Clavel
Technical University of Munich

PHYSIOLOGY

Characterizing the interaction between blood group-related glycosyltransferases and the intestinal microbiota using multi-omic approaches

Characterization of mononuclear phagocyte populations that mediate microbiota-ILC3 Interactions

 

 

Spatial Organization of the Microbiota - Impact of Direct Bacterial Interaction with the Colonic Crypt Epithelium and Long-lived Stem Cells

Inflammatory, dietary and malignant conditions are associated with quantitative changes of the microbial composition in the gut. While these quantitative differences are well established, little is known about the exact localization and changes of the spatial organization of the microbiota. Recent work has demonstrated that dietary and inflammatory changes might be associated in translocation of luminal bacteria towards the gut epithelium but the exact conditions and the impact of this translocation are still not known.

We hypothesize that development of chronic pathological conditions in the gut are promoted by pathological translocation of luminal bacteria towards the gastrointestinal crypts. Direct attachment of luminal bacteria to specialized crypt cells and in particular to stem cells in the crypt base may result in direct epithelial damage as well as indirect injuries through induction of severe immunological responses.

Principal investigator:

Dr. med. Michael Sigal
Charité University Medicine Berlin

Role of bactericidal permeability increasing protein (BPI) in bidirectional hostmicrobiota communication in the gut

Bactericidal/permeability-increasing protein (BPI) has been characterized as high-affinity LPS-binding protein displaying strong antimicrobial activity against Gram-negative bacteria. Expression of BPI is not only found in myeloid hematopoietic cells such as neutrophils but also in epithelial cells including those of the gastrointestinal tract. Besides its features as very potent endogenous antibiotic, recent studies have shown that BPI- neutralizing autoantibodies as well as BPI gene polymorphisms (Lys216Glu) are associated with inflammatory bowel diseases (IBD, Crohn´s disease and ulcerative colitis). We aim to further investigate the role of BPI for stabilizing/restoring the interrelationship of gut microbiota and the gastrointestinal mucosal surface in homoeostatic and inflammatory conditions. Applying newly generated BPI gene-deficient and BPI-reporter mice as well as BPI-humanized BAC-transgenic mice we will address I) the influence of the microbiota on the mucosal expression of BPI as well as II) the role of BPI for shaping the microbiota composition, III) their spatial segregation from the host mucosa and IV) the control of bacteria-driven inflammatory processes in the gut. A better understanding of the role of BPI in the bidirectional microbiota-host interaction will provide the basis for future therapeutic strategies in inflammatory bowel diseases. 

Principal investigator:

Prof. Dr. Dr. André Gessner
Institut f. Med. Mikrobiologie u. Hygiene Regensburg

Host control of the enteric microbiota during the postnatal period

Prior to birth, the intestinal tract is devoid of viable bacteria. With rupture of the membranes, the neonates body surfaces become exposed to environmental bacteria and are rapidly colonized. The early postnatal microbiota exhibits a high interindividual variation and low diversity and is strongly influenced by the maternal contact and environment. With ongoing environmental exposure, the diversity steadily increases ultimately leading to a mature and beneficial microbiota that remains relatively stable throughout life. The host-microbial interaction during the postnatal period was shown to play a critical role for the establishment of life-long immune homeostasis and disease susceptibility in the adult host. Given the non-redundant contribution of the postnatal period, mechanisms must exist that shape the bacterial composition during the neonatal period. Indeed, our work during the last funding period identified a host mechanism of microbial selection that acts solely during the postnatal period but influences the life-long microbiota composition and disease susceptibility. The proposed research project extends on these findings and characterizes the kinetic of bacterial colonization during early development and analyzes host factors that determine the postnatal microbiota development using genetic tools, immune modulation and competitive colonization with bacterial sentinel mixtures. Also we identify mechanisms to manipulate the emerging microbiota and evaluate possible functional consequences.

Principal investigator:

Prof. Dr. med. Mathias W. Hornef
Institut für Medizinische Mikrobiologie

Neonatal imprinting of tolerogenic properties in stromal cells from gut-draining lymph nodes by commensal microbiota

Foxp3+ regulatory T cells (Tregs) play a central role for the maintenance of immune homeostasis, self-tolerance and particularly mucosal tolerance. The vast majority of Foxp3+ Tregs is generated already in the thymus, however, a subset of Foxp3+ Tregs can be converted from conventional CD4+ T cells in the periphery. This peripheral de novo generation of Foxp3+ Tregs very efficiently takes place within gut-draining lymph nodes (LNs), including mesenteric LNs (mLNs). Recently, we studied cellular and microenvironmental factors that contribute to the high Treg-inducing capacity of gut-draining LNs. Using LN transplantations, we could not only demonstrate that LN stromal cells critically contribute to the high Treg-inducing capacity of gut-draining LNs, but also that these tolerogenic properties of gut-draining LNs are stably imprinted within LN stromal cells and cannot be substantially influenced either by the local microenvironment of the skin or by inflammatory perturbations such as gastrointestinal infection or chronic colitis. Importantly, contact with commensals in the neonatal phase could be identified as the microenvironmental factor being responsible for the stable imprinting of tolerogenic properties in mLN stromal cells, further emphasizing the critical role of the neonatal period for the development of a fully functional immune system.

In the present project, we aim to identify the molecular mechanisms by which intestinal microbiota stably imprint tolerogenic properties within mLN stromal cells in the neonatal period. Utilizing LN transplantation experiments in conjunction with mono-associated mice, neonatal infection models and low-input transcriptomics and epigenomics, we aim to i) identify bacterial species and commensal microbiota-derived molecular mediators that contribute to tolerogenic imprinting, ii) study the impact of commensals on the formation of a Treg-specific epigenetic and transcriptional signature during peripheral de novo generation of Foxp3+ Tregs iii) investigate the long-lasting consequences of neonatal infections and subsequent changes in intestinal microbiota composition on the imprinting of tolerogenic properties within mLN stromal cells. Viewed as a whole, the insights from this project will shed further light into one of the layers of the complex and intimate connection of Foxp3+ Tregs with commensals.

Principal investigator:

Prof. Dr. Jochen Hühn
Helmholtz Centre for Infection Research

Aging-associated modification of intestinal homeostasis and barrier function: Role of microbiota, iNOS and innate immunity

Results of several recent studies suggest that not only the development of diseases like inflammatory bowel disease and non-alcoholic fatty liver disease are associated with alterations of phylogenetic composition of gut microbiota but also age-associated degeneration and decline may be at least in part a result of changes of intestinal microbiota and barrier function. Indeed, it has been suggested that intestinal microbiota and barrier function may impact health maintenance and longevity contributing to an extension of a healthy life-span. However, intestinal barrier function and microbiota composition may also be responsible for the low-grade inflammation typically found during aging. It has been suggested that these changes are also associated with an increased number of activated TH1 cells, a TH1/TH2 imbalance but also an altered redox status. However, the interaction between gut microbiota, intestinal barrier function, immune system and aging-associated degeneration and decline is only partially understood.

The objective of our present project is to investigate mechanisms involved in aging-associated modifications of intestinal homeostasis and barrier function. Herein, a particular focus will be on alterations of intestinal microbiota and NO generated through inducible nitric oxide synthase (iNOS) as well as TH1 cells in the upper part of the small intestine as well as in colon. Furthermore, the role of dietary composition and energy bioavailability as well as novel targeting strategies to prevent these changes will be determined. 

Principal investigator:

Prof. Dr. Ina Bergheim
Universität Wien

Dr. rer. nat. Amélia Camarinha Silva
University of Hohenheim

Genetic circuits underlying fungal-bacterial interactions in the mammalian intestine

The ~100 trillion microorganisms that inhabit the human body comprise members of all three domains of life, i.e. bacteria, archaea and eukaryotes. Little is known, however, about how non-bacterial members of the microbiota interact with the host and with other cohabiting microbes. The project seeks to define functional mechanisms that underlie the in vivo interplay between one of the most prevalent fungi in the human gut, Candida albicans, and other members of the microbiota as well as the host.

Preliminary work conducted in my laboratory indicates that several genetic determinants of in vivo fitness in this fungus are influenced by the gut microbiota. Based on these observations, the project is specifically aimed at: (1) Determining whether C. albicans relies on a microbiota-derived molecule as a key source of nitrogen to proliferate in the mammalian gut. The in vivo and in vitro characterization of C. albicans strains harboring deletions in putative allantoate transporters will allow me to accomplish this goal. (2) Establishing how the microbiota shapes the interface between C. albicans and the mammalian intestinal lining. Immunohistochemistry of intestinal sections of germ-free and conventionally raised mice inoculated with C. albicans wild-type or adherence-impaired mutants will be used to achieve this aim. (3) Establishing whether in vivo fitness determinants in the human gut bacterial commensal Bacteroides thetaiotaomicron are influenced by C. albicans. We will evaluate the patterns of colonization in gnotobiotic mice mono- or co-associated with these species to attain this goal.

The research project is expected to reveal basic principles underlying in vivo interactions among C. albicans, the gut microbiota and the host. Furthermore, since C. albicans’ overgrowth in the gut is a major source of life-threatening infections, our findings will allow us to devise potential interventions that target the gut flora to prevent these infections.

Principal investigator:

Dr. J. Christian Pérez
Universitätsklinikum Würzburg

Healing Mucosa, interaction of microbial ecology and the immune system

Immune modulation by metagenomic DNA from the gastrointestinal microbiota of newborn infants

Maintaining a healthy equilibrium between pro- and anti-inflammatory microbial triggers and host immune responses is crucial for gastrointestinal homeostasis, in order to avoid both increased susceptibility to infection and overly excessive inflammation. This balance appears to be of special importance in neonates that, after leaving the sterile environment of the womb, need to establish a healthy gut microbiota while both controlling colonization with infiltrating (opportunistic) pathogens and avoiding excessive inflammation in response to these bacteria.

Our goal is to study host-microbiota interactions of the neonatal gastrointestinal tract via the innate immune receptor Toll-like receptor (TLR) 9, which recognizes specific short oligomers of microbial DNA and is involved both in the pro-inflammatory activation and anti-inflammatory modulation of host immune responses. We propose to use fecal microbiota characterization by metagenomic sequence analysis to quantify TLR9-stimulating and TLR9-regulating sequence motifs, in combination with TLR9-dependent experimental immune assays, to model the dynamics of host-microbe interactions and to potentially identify sequence-based diagnostic biomarkers for gastrointestinal dysbiosis.

Principal investigator:

Professor W. Florian Fricke, Stuttgart
University of Hohenheim

INFECTION and IBD

Generation of gnotobiotic mice to investigate the role of the intestinal microbiota in Salmonella enterica spp. I serovar Typhimurium colitis in AGR2-deficient mice

The intestinal microbiota plays a pivotal role in protection against the enteric Salmonella serovar Typhimurium (S. Typhimurium) infection. Besides the microbiota, the mucosal barrier (e.g. mucins) significantly contributes to the innate immune defense against S. Typhimurium. So far, the interplay of the microbiota and the mucus layer is poorly understood. We have analyzed S. Typhimurium infection in mice with a defective mucosal barrier function (AGR2KO) in a colitis model. Interestingly, in opposition to our prediction, AGR2KO mice are protected against S. Typhimurium colitis. In addition, they exhibit an altered gut microbiota composition. We identified commensal bacteria correlating with protection against Salmonella in AGR2KO. The same bacteria also conferred protection in a gnotobiotic mouse model. Our further aims are to characterize these organisms with respect to their role in gut microbial ecology as well as interactions with the host and with S. Typhimurium. We will employ a gnotobiotic mouse model, Salmonella mutants and mucosal- and bacterial transcriptome analysis to elucidate the mechanisms by which they confer protection.

Principal investigator:

Prof. Dr. rer. nat. Barbara Stecher
Max von Pettenkofer-Institut

Impact of Prevotellaceae on the interplay between host and microbiota during health and disease

Impact of the intestinal microbiota and microbiota mediated immune responses on the host cell death machinery in the intestinal epithelium

In healthy individuals, intestinal homeostasis mainly depends on the diverse functions of intestinal epithelial cells, which form a physical barrier to separate the commensal and pathogenic microorganisms from the underlying immune system. In addition, specialized intestinal epithelial cells, such as Paneth cells and Goblet cells provide innate immune function by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria directly adjacent to the intestinal epithelium. Given these fundamental and diverse functions of intestinal epithelial cells, it is obvious that proliferation, differentiation and cell death of these cells needs to be tightly controlled in order to maintain intestinal homeostasis. The association between increased bacterial translocation and the risk of developing inflammatory bowel diseases (IBD) suggests a central role of dysregulated epithelial barrier function. Previous studies have demonstrated that intestinal barrier dysfunction is strongly associated with a dysregulation of intestinal epithelial cell death. Our own studies already identified a critical role of necroptosis (a novel form of programmed necrotic cell death) in the regulation of intestinal homeostasis. Although our previous studies demonstrated that necroptosis strongly contributes to intestinal inflammation and barrier dysfunction, little is known about the impact of the intestinal microbiota on this particular form of cell death and the effect of epithelial necroptosis on the gut microbiota. The aim of the proposed project is to define the bi-directional cross-talk between the intestinal microbiota and the host cell death machinery under physiological and pathophysiological conditions. The research project addresses the following issues: (1) Contribution of the microbial environment on intestinal inflammation in a mouse model for Crohn´s disease like ileitis and colitis (Casp8deltaIECmice). (2) Impact of the intestinal microbiota on the host cell death machinery. (3) Impact of bacterial driven Stat1-signaling on (a) Paneth cell death, (b) small intestinal inflammation and (c) gastrointestinal infection. (4) Role of microbiota induced immune response on Paneth cell homeostasis. A better understanding of the host-microbial interaction in the context of establishing and maintaining intestinal barrier function is essential for the development of novel strategies for the management of intestinal inflammatory disorders.

Principal investigator:

Dr. Claudia Günther, Erlangen
Universitätsklinikum Erlangen AöR

Molecular analysis of the immune-modulatory properties of colitogenic intestinal Microbiota

Previously published studies imply that Dendritic cells (DCs) play a complex and subset-dependent role both during the maintenance of intestinal homeostasis and prior and upon intestinal inflammation. Therefore, experimental model systems are in need to specifically assess the spatio-temporal contribution of individual DC subsets to intestinal homeostasis in the steady state and during initiation, promotion and resolution of colitis. Given the intimate relationship of microbiota, microbiota-sensing cell types and intestinal homeostasis on the one hand and the frequent finding that IBD patients display an intestinal dysbiosis on the other hand, studies are needed to assess the DC subset specific role to the composition and functionality of microbiota in the steady state and upon colitis induction. The emergence of novel DC deficiency models allows the field to conduct such studies. For example, we have recently described that the AP-1 transcription factor Batf3 controls the formation of related CD8a+ and CD103+CD11b- DCs in vivo while T cell-intrinsic differentiation programs appear virtually unaffected. Utilizing novel genetic model systems we aim at I. identification and functional validation of colitis-promoting microbial entities in the context of altered DC homeostasis; II. molecular characterization of DC-dependent microbiota- vs. host-employed, colitis-promoting mechanisms and III. translational studies to assess the intestinal DC-microbiota-axis in patients suffering from inflammatory bowel diseases (IBD). 

Principal investigator:

Prof. Dr. med. Kai Hildner
University Hospital Erlangen

X-linked inhibitor of apoptosis protein in intestinal homeostasis and the pathogenesis of inflammatory bowel disease

NUTRITION

Functional dynamics of dysbiosis in a gnotobiotic transfer model of Crohn`s disease-like ileitis: Impact of diet and inflammation

Compositional and functional changes in the intestinal microbial ecosystem are considered to be key mechanisms in the pathogenesis of inflammatory bowel diseases (IBD). High-throughput sequencing analysis of microbial communities from patients with Crohn’s disease and ulcerative colitis identified risk patterns associated with these distinct IBD phenotypes, however functional evidence for the existence of disease-conditioning ecosystems (dysbiosis) or the selection of single taxa (pathobionts) as underlying cause of chronic inflammation is still lacking. Until now mechanistic studies about the role of bacteria in the pathogenesis of Crohn’s disease (CD) with predominant ileal inflammation were hampered by the lack of adequate germfree mouse models. In the first funding phase, we demonstrated that germfree Tnf ΔARE/+ mice do not develop CD-like ileitis, supporting our initial hypothesis that bacterial triggers are an essential part of this pathogenesis. Most importantly and for the first time, we showed that inflammation-associated changes in the microbiota caused transmissible disease in the genetically susceptible but not wildtype host. Despite these breakthrough findings, we still have no understanding about the characteristic features of disease-conditioning (dysbiotic) microbial communities. In the second funding phase, we aim to better understand the functional plasticity of these complex microbial ecosystems towards changes in the intestinal milieu including inflammation and exposure to diet. We will address the host specificity of a complex microbiota to induce inflammation in two IBD-relevant mouse models including germfree Tnf ΔARE/+ (ileitis phenotype) and germfree IL10 deficient (colitis phenotype) as well as the possibility of the host milieu to reprogram the disease-conditioning nature of the microbiota. In addition, we will functionally substantiate our previous observations that diet is an efficient modulator of microbiota composition and disease activity. Metagenomic analyses will complement 16S-based compositional characterization of the microbiota. In summary, the combination of sequence-based analyses and transfer of complex and minimal microbial consortia into germfree mouse models will help to understand the functional role of the non-infectious intestinal microbiota in initiating chronic inflammation of the small intestine. 

Principal investigator:

Prof. Dirk Haller
Technische Universität München

Role of dietary sulfonates in the stimulation of intestinal bacteria promoting gut Inflammation

The intestinal microbiome is crucial for the maturation and proper functioning of the immune system. Disturbances in the interaction between the intestinal microbiota and the mucosal immune system may lead to inflammatory bowel diseases. In which way a disturbed microbiota may contribute to an inflamed gut is not completely understood, but it has been demonstrated that the proliferation of pro-inflammatory intestinal bacteria may lead to gut inflammation in a susceptible host. Diet is the main source of bacterial substrates in the digestive tract and therefore influences the composition and activity of the intestinal microbiota. A diet rich in saturated fats has been demonstrated in mice to stimulate the growth of pro-inflammatory Bilophila wadsworthia by shifting the bile-acid spectrum toward a higher proportion of taurine conjugates. The sulfonyl group of taurine provides sulfite, which is used by this organism as an electron acceptor and becomes reduced to sulfide. Host enzymes oxidize sulfide to thiosulfate, which in turn stimulates the growth of pathogenic Salmonella. With the present project we aim to investigate whether dietary sulfonated compounds such as sulfoquinovosyldiacylglycerol (SQDG), which occurs in the membranes of chloroplasts and is ingested with green fruits and leafy vegetables, may contribute to the pool of sulfonates, lead to intestinal sulfide production and stimulate the growth of colitogenic bacteria such as B. wadsworthia. In view of the potential stimulation of enteropathogenic bacteria by thiosulfate, we aim to clarify, to which extent dietary sulfonates, in particular SQDG and sulfoquinovose (SQ), undergo further microbial degradation. Escherichia coli has recently been demonstrated to harbor a set of enzymes that catalyze the breakdown of SQ to 2,3-dihydroxy-1-sulfonate, which may contribute to the sulfonate pool in the intestinal tract. We will examine whether intestinal bacteria other than B. wadsworthia contribute to the formation of sulfide from sulfonates. Such bacteria will be isolated and the biochemical pathways involved in sulfonate utilization will be identified. We will test whether sulfonate utilizers have pro-inflammatory properties like B. wadsworthia. Using mouse models we will investigate how these dietary sulfonates affect the intestinal microbiome and examine whether these effects promote gut inflammation. 

Principal investigators:

Prof. Dr. Michael Blaut, Nuthetal
Deutsches Institut für Ernährungsforschung Potsdam-Rehbrücke (DIfE)

Dr. Annett Braune, Nuthetal
Deutsches Institut für Ernährungsforschung Potsdam-Rehbrücke (DIfE)

The impact of dietary proteins for mucosal homeostasis and inflammation

Functional consequences of intestinal microbiota altered by ingestion of gluten and wheat alpha-amylase-trypsin inhibitors

Chronic inflammation in the gastrointestinal tract, as seen in inflammatory bowel disease (IBD) and celiac disease (CD), arises from a failure to maintain tolerance to certain bacterial and/or food antigens. Yet ill-defined nutritional or microbial triggers, combined with a proinflammatory genetic predisposition, can switch the intestinal immune system from a tolerogenic to a proinflammatory state. We could show that IL-22 produced by intestinal dendritic cells through bacterial stimulation regulates intestinal epithelial STAT-3 activity, promoting mucosal wound healing and contributing to intestinal homeostasis. We also identified a highly relevant nutritional trigger of innate immune activation, wheat amylase trypsin inhibitors (ATIs), which activate TLR4 on intestinal macrophages and dendritic cells and which can exacerbate intestinal and extraintestinal inflammation. ATIs are therefore the long sought for cause of non-celiac (nonallergy) wheat sensitivity, which is characterized by worsening of pre-existent inflammatory disease with wheat consumption. ATIs resist intestinal degradation and trigger innate immune activation after oral ingestion in vivo. Mice exposed to a diet that is devoid of ATIs are less susceptible to experimental colitis compared to mice exposed to a diet containing wheat/ATIs. Mice ingesting ATIs exhibit enhanced activation of intestinal myeloid cells, which appear to promote expansion of inflammatory T cells in the gut. ATIs induce intestinal dysbiosis. We plan to explore the contribution of nutritional ATIs to intestinal inflammation and the mechanisms by which they induce intestinal dysbiosis. A special focus will be the direct and indirect effects of luminal ATIs on the pro- vs- anti-inflammatory activities of the intestinal microbiota in connection with disease severity. We will also explore how far the IBD promoting activities involve migration and/or activation of myeloid cells to the mesenteric lymph nodes where major T cell activation may occur.

Principal investigators:

Prof. Dr. Dr. Detlef Schuppan
Institute of Translational Immunology, Mainz 

Dr. Geethanjali Pickert
Institute of Translational Immunology, Mainz

The influence of gut microbiota composition and diversity on the resorption and de novo synthesis of fatty acids in the small intestine

The mammalian gut microbiota is a complex ecosystem, its composition and diversity depends on various factors including diet, environment, health and disease. Our preliminary data strongly indicate that gut microbiota influence absorption of dietary lipids, intestinal fatty acid trafficking, de novo synthesis and secretion into the circulation. A dysregulation of the intestinal lipid metabolism may be associated with metabolic overload. Moreover, intestinal FA synthesis may be involved in the formation of the intestinal barrier function and associated inflammatory pathologies. Gut microbiota might represent attractive targets for modulating dietary lipid resorption and intestinal lipid synthesis. Therefore, the primary objectives of this project are to investigate if and how gut microbiota composition and diversity affects the resorption of fatty acids, intestinal lipid synthesis and storage. This includes its effects on lipid composition of the intestinal barrier as well as the role of microbiota derived short chain fatty acids.

Principal Investigators:

Dr. Josef Ecker
Technische Universität München (TUM) 

PD Dr. Gerhard Liebisch
Universitätsklinikum Regensburg