Project structure


WPs schema

 

WP1 Fungus-driven allergy and inflammation

Partners involved:
N° 1 Luigina Romani(WP Leader), N° 3 Jagadeesh Bayry, , N° 4 Thierry Jouault, N°6 Mihai Netea, N°7 David Denning, N° 9 Thierry Calandra, N° 10 Claudia Covaciu, N° 11 Maria Diaz-Torres

Objectives

The main objective of WP1 will be the study of the specific cellular and molecular mechanisms by which ubiquitous airborne or commensal fungi contribute to immune homeostasis and its deregulation leading to inflammatory diseases.
The study will be centred on yeasts, such as C. albicans and Malassezia spp, and filamentous fungi, such as A. fumigatus and other filamentous fungi, known to be associated with a number of inflammatory, autoimmune and allergic diseases.
By the completion of WP1 and with the contribution of WP3 and WP4, through iterative wet-lab and data exchange, the main objectives of the present WP will:
(i) define the molecular and cellular pathways leading to resistance or tolerance to inhaled Aspergillus spp, or commensal Candida and Malassezia yeasts;
(ii) identify the molecular connection between failure to resolve inflammation and lack of antifungal immune resistance
(iii) define a predictive model that will incorporate information from the fungal-mouse system to clarify the predictive nature of mouse studies in the human context
(iv) produce a systems biology analysis of fungal/immune system interaction in different clinical settings.
We will take advantage of the mouse models of inflammatory fungal diseases, such as lung allergy to airborne fungi and mucosal (gut and vagina) and skin inflammatory responses to yeasts commensals. We aim to exploit epithelial cells (ECs) and dendritic cells (DCs) through a systems biology approach for high-throughput screening of molecular signatures and pathways in response to fungi and/or fungal components.
We want to study the signalling pathways activated in ECs and DCs at the different mucosal sites with the ultimate goal to determine how ECs and DCs drive mucosal immune responses to fungi. This will allow not only the dissection of inflammatory/anti-inflammatory pathways that distinguishes pathogenic vs non-pathogenic fungi but also the identification of fungal molecules putatively promoting allergy and inflammation. A comparative analysis will be performed between the different mouse models and PBMC or mucosa-associated cells from patients with pulmonary allergy, Crohn’s disease, vaginal candidiasis and atopic dermatitis in response to different fungal morphotypes or fungal PAMPs.
The ultimate discovery of common or unique signalling pathways and effectors mechanisms that direct protection against medically important fungi will open up novel areas of research in the fungal field but also across a whole spectrum of epithelial immunity field. Likewise, determining how epithelial cells discriminate between the commensal and pathogenic states of a microorganism will benefit the whole immunology field.


WP2 Common immunogenic molecules and auto-antigens: towards an universal Diagnosis

Partners involved:
N°8 Reto Crameri(WP Leader), N°2 Anne Beauvais, , N° 4 Thierry Jouault, N°5 Neil Gow, N°7 David Denning, N°10 Claudia Covaciu, N°11 Maria Diaz-Torres, N°13 Claudia Alcaro

Objectives
The overall objective of WP2 is the description of the fungal microbiome affecting lung and skin followed by the identification, purification and characterization of common immunogenic fungal molecules including proteins, cell wall polysaccharides and lipids. These molecules will serve as a basis to study their immunogenicity in serology and to develop diagnostic kits based on multiplexed highly sensitive label-free biosensor technology. These molecules will be the foundation of the studies of WP3 centred on the analysis of PRRs and their interactions with PAMPs to clarify the role of these molecules during the time course of exposure or infections.
The airways and the skin are not sterile and many fungal species can be isolated from BAL of the lung of asthmatic patients and from skin biopsies of atopic eczema patients after cultivation. In a clinical setting a microbe is seldom found isolated but is part of a complex microbial environment and many fungi adapted to the host are unable to grow in culture media. These organisms represent a major gap in our understanding of the groups of potential pathogens with unknown influence on immunologic responses. Therefore, the spectrum of the fungal microbiome affecting lung and skin is largely unknown. Because culture-based analysis of mixed populations introduces bias in the species observed due to the preferential recovery of fast growing fungi, new methods for the identification of the whole fungal microbiome associated with severe asthma and atopic eczema based on whole microbiome sequencing will be developed.
Fungal allergens can be subdivided in species-specific and cross-reactive allergens. Species specific allergens which are of outmost diagnostic values will be cloned out of fungi with sequenced genome derived from the lung and skin microbiome by selective enrichment of cDNA libraries displayed on phage surface, sequenced, characterized, and produced in large amounts as recombinant allergens.
Cross-reactive fungal allergens have been shown to be widely distributed among the fungal kingdom, however, the true extent of allergen cross-reactivity and its clinical relevance is far to be complete. Sets of orthologous allergens from the fungal genomes of the identified microbiomes associated with severe asthma and atopic eczema will be cloned, produced and characterized in parallel with the homologous structures of fungi already known to be associated with these diseases. Potentially cross-reactive homologous human self antigens will be searched since IgE-mediated cross-reactivity to self antigens is an important component contributing to the exacerbation of severe chronic atopic diseases.
Fungal cell wall components are the first structures coming in contact with the immune system during the time course of exposure and would be expected to elicit strong immune responses. In spite of this fact only two fungal cell wall allergens, a minor allergen of C. herbarum and a major allergen of M. sympodialis have been described.
The spectrum of allergenic cell wall proteins including the neglected family of glycosylphosphatidylinositol anchored proteins (GPI) will be re-evaluated. Most immunological research to date is based on proteins and DNA and has totally underscored two classes of molecules that in fungi account for the majority of the cell wall: polysaccharides and lipids. Oligo- and polysaccharided are well known in allergy as -cross reactive carbohydrate determinants (CCD). The role of these sugar structures in fungal allergy, which might strongly contribute to the extended cross-reactivity observed among phylogenetically unrelated fungal species is, however, fully unknown and needs to be investigated in detail.
Fungal lipids are indeed sensed by the host immune system. However, there is an almost complete lack of investigations about the role of lipids in immune reactions mainly due to the lack of purified fungal lipids. Major aim of this sub-project is to provide biochemically purified fungal lipid fractions to allow basic studies on their influence of inflammation and immunity.


WP3 PRR/PAMP interaction network

Partners involved
N°5 Neil Gow (WP Leader), N°2 Anne Beauvais, N°3 Jagadeesh Bayry, N° 4 Thierry Jouault, N°6 Mihai Netea, N°9 Thierry Calandra

Objectives
The collective objective of WP3 is to study the complex interaction between Pattern Recognition Receptors
(PRRs) and Pathogen Associated Molecular Patterns (PAMPs) for a range of common fungal pathogens in a
variety of human and murine cell types in order to characterise the recognition phenomena leading to innate and adaptive immunity of fungi. This work will be based in the setting of fungal allergic and inflammatory diseases.
Filamentous fungi in the atmosphere, such as the genus Aspergillus, are very different morphologically and have very different temperature as well as aw optima. For yeasts, different species colonize/infect the human body surfaces, but the specific immunological alterations underlying the switch from symbionts to pathobionts is not known. We want to correlate the immune response to one species to its phenotype, genotype and metabolic activities by using cutting-edge technologies developed in the labs of the WPs 1 and 2. The main focus of attention will be on yeasts such as Candida and Malassezia spp. and A. fumigatus.
Execution of this WP (combined with deliverables within WPs 1 and 2) will:
(i) Define the shared and unique PAMP repertoires and PRRs involved in innate immune recognition of the major fungal pathogen using combinations of fungal cell wall mutants and purified PAMPs obtained through classical methods of molecular biology and modern high-throughput technologies, creating a comprehensive genome-wide examination of the interaction of fungi with host PRRs.
(ii) Investigate signal transduction pathways downstream PRRs activation by genetically-modified fungi and
purified fungal PAMPs.
(iii) Define the role of the PRR/PAMP interaction network on immunity, tolerance and immune
deregulation in murine models of infections and immunopathology associated with fungi (partly in conjunction
with WP1).
(iv) Investigate the specific PRR/PAMP interaction network operating in immunity and its
deregulation in human fungal allergy and inflammatory diseases.
We want to discover which fungal components are endowed with the capacity to activate distinct innate PRR responses in mice and humans. This is a feasible approach based on findings showing that distinct fungal components are endowed with the capacity to activate specific PRRs (55) and the ensuing pathogenic or protective Th immune responses in mice and humans (56). Thus, the ultimate goal of this WP3 is to identify immunoactive host/fungal antigens to be used as natural or bio-engineered antigens to exploit the broadness and specificity of human T cell repertoire against fungi in the different clinical settings.
This WP will take advantage of the specific expertise of WP2 participants in the isolation and characterisation of fungal PAMPs, generation of fungal cell wall mutants through high-throughput technologies as well as collective experience in innate and adaptive immunity in mice and humans.
Results gathered in WP3, in conjunction with those of WP1, will be entered into the knowledge base in WP2 and used in the construction of predictive systems biology models for interventional strategies in WP4.


WP4 Intervention strategies in fungal allergy and inflammation

Partners involved
N°3 Jagadeesh Bayry (WP leader), N°1 Luigina Romani, N° 4 Thierry Jouault, N°6 Mihai Netea, N°7 David Denning, N°9 Thierry Calandra, N°10 Claudia Covaciu, N°11 Maria Diaz-Torres, N°13 Claudia Alcaro

Objectives
The main goal of WP4 is to design and validate Intervention strategies in fungal allergy and inflammation. Thus, WP is a ‘Translational Research’ part based on the knowledge gained in WP1 and WP3. Therefore WP4 is a clear indication that all the beneficiaries of ALL FUN consortium have a clear vision of translational research in order to translate basic research findings into appropriate interventional tools. Thus WP4 represents a perfect platform for thorough blending of ideas and cooperation among all the beneficiaries of ALLFUN.
We divide the intervention strategies in fungal allergy and inflammation into two broad objectives: prophylaxis and therapeutic strategies.
Prophylaxis strategies
i. to decipher the gene polymorphisms in immune genes of patients with fungal mediated allergy and inflammatory diseases
Therapeutic strategies
ii. to target the PRR (host)-PAMPs (fungi) interface
iii. to target inflammatory signalling pathways of innate cells
iv. to validate therapeutic strategies based on Treg-based molecules
v. to target migration of Th2 cells
vi. to explore therapeutic utility of polyclonal intravenous immunoglobulin (IVIg) in fungi-mediated allergy and inflammatory conditions
Prophylaxis strategies are aimed at identifying the risk factors that predispose the patients and population for fungi-mediated inflammatory diseases. For clinical samples, we will take advantage of cohort of patients we establish for WP1. It is anticipated that implementation of this program with other groups and sharing fungi-derived molecules as well as clinical materials will shed new insights on the molecular mechanisms governed by fungi and human genome leading to diseases due to breach in tolerance. The discovery of SNPs has an important impact on our capacity to predict, prevent and treat fungal opportunistic infections. If verified, our observation will have tremendous implications. SNPs will be exploited as a prognostic marker for the risk of developing inflammation and autoimmune diseases, and will help clinicians to choose appropriate therapeutic strategies so as to reduce the risk of developing a disease. Alternatively prescreening of the donors for such SNP will aid the clinicians to choose appropriate donors for the transplantation purposes.
For therapeutic intervention strategies we will take advantage of the murine models of inflammatory fungal diseases established by the beneficiaries Based on the results we obtain, we can envisage phase I clinical trials as a follow-up of ALLFUN.


WP5 Management, Review and Assessment

Partners involved
N°1 UNIPG (WP Leader), N°14 ALTA

Objectives
The aim of the management Work Package is to ensure full synergy and integration among ALLFUN beneficiaries to provide added European value and extend the state of the art in research on fungus-related
diseases. This will facilitate the following outcomes:
- Maintenance of a defined time-schedule of the research
- Achievement of the planned objectives
- Immediate recognition of problems, their evaluation and the adoption of actions to solve them.
- Integrate and expand european state-of-the-art in research on yeasts and filamentous fungi known to be
associated with a number of inflammatory, autoimmune and allergic diseases.
Expected outputs. An efficient management will guarantee the fruitful collaboration among ALLFUN beneficiaries
and the successful progress of the project within the agreed time, cost and quality limits as defined by the project contract signed with the EU.


WP6 Training, Dissemination and Diffusion of project results

Partners involved
N°1 UNIPG (WP Leader), N°14 ALTA

Objectives
The general objectives of this Workpackage are to disseminate results, to increase general awareness of issues addressed by ALLFUN and to sustain or create occasion for young scientists to improve their skills
For this purpose a dissemination and a training plan will be carefully designed at the beginning of the project and regularly updated in order to guarantee an effective communication of ALLFUN results to the scientific and non scientific community as well as among ALLFUN Beneficiaries and to ensure an efficient use of the resources to train young scientists in order to encourage their active participation in research activities and to improve their professional development. The training activities promoted by ALLFUN will concern on fungal biology, immunopathology and fungal diseases as well as on additional skills such as scientific writing, technology transfer, intellectual property issues, ethical issues etc.