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Microbial Community in Familial Mediterranean Fever

#A-1727


Microbial Community as a Pathogenic Factor in Autoinflammatory Disorder-Familial Mediterranean Fever

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-CHM/Biochemistry/Biotechnology
  • BIO-DIV/Biodiversity/Biotechnology
  • BIO-INF/Bioinformatics/Biotechnology
  • BIO-MIB/Microbiology/Biotechnology
  • MED-DIS/Disease Surveillance/Medicine

Status
3 Approved without Funding

Registration date
18.02.2009

Leading Institute
Institute of Molecular Biology, Armenia, Yerevan

Collaborators

  • Kyoto Prefectural University / Graduate School of Life and Environmental Sciences, Japan, Kyoto\nNational Institute of Livestock and Grassland Science, Japan, Ibaraki\nIowa State University of Science and Technology / College of Veterinary Medicine, USA, IA, Ames\nUniversity of Aberdeen / Rowett Institute of Nutrition and Health, UK, Aberdeen\nUniversity of Leicester / Department of Biochemistry, UK, Leicester\nUniversity of Dundee, UK, Dundee\nCalifornia Institute of Technology / Division of Biology, USA, CA, Pasadena\nUniversity of British Columbia / Michael Smith Laboratories, Canada, BC, Vancouver

Project summary

The goal of the project. The main goal of this project is to investigate the nature of gut microbiome transformation that makes it pathogenic and identify the novel pathways controlling inflammation in the autoinflammatory condition such as FMF. The study will reveal the fundamental aspects of the complex interplay between the host and gut microbiome and will identify molecular mechanisms behind this interaction. The findings will serve as a basis for designation of novel therapeutic strategies for FMF and related autoinflammatory diseases through the manipulation of the gut ecosystem.

The state of the art in the field. Familial Mediterranean fever (FMF, MIM249100) is the most commonly encountered member of hereditary autoinflammatory diseases. FMF is characterised by recurrent self-resolving attacks of fever and polyserositis, and is often complicated by amyloidosis, which may become a life-threatening condition. Genetically, FMF is caused by mutations in the MEFV gene, which encodes the protein called pyrin. The functional role of this protein is regulation of innate immunity, inflammation and apoptosis. Experimental data describing its regulatory role remain contradictory and support both possibilities, as of an anti-inflammatory as well as of a pro-inflammatory agent, and pyrin may have unique signaling and microbial sensing functions. However, the exact molecular mechanisms contributing to the autoinflammatory nature of this disease remain obscure.

This proposal is the continuation of the previous project that was supported by the International Scientific-Technical Centre (ISTC). The main goal of the preceding project A-1055 was to reveal the role of host genetics on intestinal microbiota. Briefly, it had been proposed to investigate the gut microbial community structure in FMF and healthy subjects to reveal a possible contribution of the host on the composition of gut bacteria and clarify the contribution of gut bacteria in the induction and modulation of immune responses in FMF. All the specific tasks within the project were completely accomplished and the main findings have been published in national and international peer-reviewed journals. More specifically: (i) we found, for the first time, that FMF affects bacterial persity in the gut resulting in specific restructuring of its composition and metabolic imbalance both in remission and acute stages of the disease; (ii) our investigation of cytokine profile in FMF revealed T cell differentiation into the previously unrecognized Th17 lineage and confirmed subclinical chronic inflammation in attack-free periods of the disease; (iii) our results demonstrated the increased systemic reactivity against the commensal gut microbiota in FMF. Nevertheless, no particular species, metabolites or antigens were specifically linked to FMF, thus leading us to the conclusion that the restructured community as a whole may contribute to the disease state rather than a single bacterium or bacterial species.

These findings raised an important question: if the structural changes in microbiota are indicative of its transformation into a “pathobiota” in FMF? Thus we propose to explore further the mechanisms contributing to the disease and test the hypothesis that the gut microbiota in an autoinflammatory condition is restructured in a way that it acquires pathogenic properties, possibly through the production of the unusual range of bacterial metabolites and antigen repertoire. Presently, it is not clear how the composition of the normal microbiota is restructured to become a “pathobiota” and which properties make it pathogenic. To address this question, in the present proposal we aim to investigate the mechanisms of aberrant host-microbe interaction leading to disease state with the use of metagenomic, metabolomic and proteomic approaches and in in vitro experiments.

The impact of the proposed project on the progress in this field. Continuing to advance our knowledge of host-microbe signaling and gain insights into their molecular mechanisms may enable us to explain many aspects of pathogenesis in FMF and related autoinflammatory disorders. Knowledge of these mechanisms is of extreme importance for the development of strategies to enhance the natural disease resistance. It may also permit the development of treatments that suppress undesirable immune responses such as those associated with autoinflammatory diseases. As far as the growing incidence of autoinflammatory syndromes such as FMF is concerned, the results of this project will be applicable worldwide to understand the general mechanisms of autoinflammatory conditions with the aim of developing strategies to control disease activity, progression and outcome.

The competence of the project team in the specified area. The research activities of our scientific group focus on the microbiological and immunological aspects of FMF. Our relevant findings, as reflected in our publications, have made a significant contribution to the study of the problem. The scientific leader of the project – Dr. Ktsoyan Zh.A., is a leading scientist with a wide range of professional interests including various aspects of medical and biological sciences. She has more than 45 years of experience, with more than 100 scientific publications. Dr. Ktsoyan developed the approach to elucidate the role of microorganisms in FMF pathogenesis that became the cornerstone of ISTC A-1055 project that she managed. The said project was successfully accomplished, and the findings of this pioneering study have been presented in relevant publications. The manager of the project Khachatryan Z.A. – Ph.D., is a research scientist whose scientific interest is focused on microbial molecular persity of gut microorganisms, human gut microbiota in health and disease (FMF), host-microbe interaction in FMF. She has extensive research experience in various modern microbial ecology techniques and bioinformatic tools. She is one of the core participants of the previous ISTC A-1055 project and is (co)author of more than 20 scientific publications. Our team also includes investigators from the Institute of Cytology of the Russian Academy of Sciences in Saint Petersburg (Russia), Prof. V. N. Parfenov and Dr. A. Mittenberg.

Expected results. Comparative metagenomic sequencing using the 454 technology will produce a comprehensive view of the phylogenetic structure and functional persity of the gut microbial community in FMF and in the healthy state. We will then be able to correlate the phylogenetic and functional (metabolic pathways) profiling of the microbiomes in order to reveal changes in the functional potential of the microbiome in FMF (how the gut microbiota in FMF is assembled and how it operates in comparison with the healthy microbiota?). Simultaneous direct monitoring of bacterial product shifts will confirm and complement the metabolic breakage in FMF. Further testing these metabolic products with colonocytes will define if they possess inflammatory properties and induce enhanced permeability and which metabolites are most important in this process. Testing the effect of bacterial metabolites and products on neutrophil function will provide the background information for therapeutic intervention through the adequate immunomodulation in FMF. Continuing to improve our knowledge of host-microbe interactions and gain insights into their molecular mechanisms may enable us to explain many aspects of pathogenesis in FMF and related autoinflammatory disorders. Such a multifaceted approach will allow to understand what are the selective factors governing the “pathogenic” shape of the microbiota in FMF, and to develop more effective therapies by administration of microbe-derived agents (reshaping the microbiota as a therapeutic strategy).

Meeting ISTC Goals and Objectives.

  • The project is of great value and importance from the point of view that it presents a unique opportunity for the scientists of the Institute of Molecular Biology NAS RA, which were earlier involved in the defensive activity of the former USSR, to be involved in health-oriented research. In this project, about half of these studies will be carried out by these scientists.
  • The project also promotes the integration of scientists of CIS states into the international scientific cooperation. The project includes investigators from the Institute of Cytology, Russian Academy of Sciences, Saint Petersburg, Russia, Prof. V. N. Parfenov and Dr. A. G. Mittenberg.
  • The project will contribute to the investigation and designation of technologies in the field of human autoinflammatory diseases, gut microbial ecology, and gut immunology. In academic terms, the goal is to reveal the mechanisms of molecular cross-talk between the host and gut microbiota to understand its role in normal human physiology and in pathological condition with the aim of the elaboration of recommendations for solving health-related problems.

Scope of activities. The project is planned for three years. The basic research will be carried out on blood and fecal samples of FMF patients (in attack and remission periods of the disease) and healthy control group. At the same time data set of clinical manifestation will be collected. The specific tasks of this proposal are: 1. Investigation of the complex gut microbiome to monitor the changes in the functional gene repertoire in FMF condition; 2. Screening of bacterial metabolites to reveal the metabolic activity features in FMF; 3. In vitro evaluation of impaired metabolic consequences on the proinflammatory responsiveness and gut permeability; 4. Investigation of the susceptibility of systemic polymorphonuclear neutrophils towards bacterial metabolites. This multidisciplinary project is the first of this kind and will study the complex interplay of the gut microbiome, host genetics and environmental exposures, which will enable us to guide therapeutic and preventive strategies through the gut microbiota modulations.

The role of foreign collaborators. The role of foreign collaborators will be in assessment of the project quality, coordination of efforts, help with technical challenges, monitoring the progress of the project. Routine communication will be primarily through the electronic mail. The cooperation will be coordinated also through the reciprocal visits, which will allow discussions, and preparation of results for publications. The collaborators in this project are Dr. R. Aminov (UK), Prof. G. Macfarlane (UK), Dr. N. Barlev (UK), Dr. R. Singh (USA), Prof. K. Ushida (Japan), and Dr. A. Takenaka (Japan).

Technical approach and methodology. The methodology proposed for the present work, namely metagenomics, metabolomics, proteomics and in vitro experiments, has the potentials to uncover mutual relationships and molecular ways of the complex host-microbe signaling, the aim of achieving the conclusions that are rigorously cross-validated. Comparative metagenomic sequencing using the 454 technology will produce a comprehensive view of the phylogenetic structure and functional persity of the gut microbial community in FMF vs. the healthy state. This may help to elucidate the pathogenic role of FMF microbiome. The metabolomic approach will be applied to characterize metabolic activity of gut community in FMF. Short-chain fatty acids and phenolic compounds of microbial origin will be identified and quantified in fecal content of FMF patients and healthy controls using GLC. Direct monitoring of bacterial product shifts will confirm and complement the metabolic breakage in FMF. In vitro experiments will be performed with Caco-2 and HT29 cells followed by analysis of the transcriptome for inflammatory gene expression by real-time PCR technique to uncover the role of FMF gut community and bacterial metabolites in induction of proinflammatory response. The changes in the intestinal proteome will be monitored by the proteomic analyses that will identify the host protein responses through peptide mass fingerprinting by MALDItof and nano-flow HPLC combined with ESI mass spectrometry. The effect of metabolites and microbiota on gut permeability will be assessed in model epithelial cell culture. Tolerance of the whole blood neutrophils in response to exposure to repeated doses of bacterial metabolites and agents will be monitored by measuring the intracellular production of proinflammatory cytokines using FACS. Such molecular based and multifaceted approach has the potential to uncover the mechanisms responsible for the initiation and resolution of inflammation in FMF. Thorough understanding of these processes will help to develop new therapeutic approaches with the aim of resolving inflammation in a directed and controlled way as well as to extend the remission periods in this and other autoinflammatory diseases.


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The International Science and Technology Center (ISTC) is an intergovernmental organization connecting scientists from Kazakhstan, Armenia, Tajikistan, Kyrgyzstan, and Georgia with their peers and research organizations in the EU, Japan, Republic of Korea, Norway and the United States.

 

ISTC facilitates international science projects and assists the global scientific and business community to source and engage with CIS and Georgian institutes that develop or possess an excellence of scientific know-how.

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