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Microbes in Allergy and Autoimmunity Related to the Skin

Final Report Summary - MAARS (Microbes in Allergy and Autoimmunity Related to the Skin)

Executive Summary:
Evidence is accumulating that infections and dysbiosis of the human microbiota may critically modulate the development of allergy and autoimmunity. Although the concepts are intriguing, the underlying mechanisms remain largely elusive. Our overall goal has been to unravel the inflammatory pathways during host-pathogen interactions which may trigger allergic or autoimmune inflammation using AD (surrogate for allergic diseases) and PSO (surrogate for autoimmune diseases) as disease models. We have identified key microbes and molecular targets to develop novel intervention strategies with the expected impact of delaying the onset of these diseases and improving quality of life.
The collection of clinical samples has been an instrumental part of the project. Study subjects have been stratified and samples collected within work package 1 (WP1) "Clinical studies".

Characterization of the skin microbiome has been performed in WP2 (Microbiome analysis) and transcriptional profiling of skin samples has been performed in WP3 (Transcriptomics). All the resulting data has been collected, analyzed and integrated in WP4 (Bioinformatics and systems biology) and the first set of results from WP1-WP4 are currently under review at the journal Science. Based on data from WP1-WP4, WP5 (Molecular and cellular networks) and WP6 (Animal models) have performed target validation and functional studies.

Project Context and Objectives:
Adequate immune responses control infections, eliminate pathogens and ensure the integrity of the host. In contrast, inadequate immune responses against foreign antigens may lead to the development of allergy while inadequate immune responses against self-antigens result into the onset of autoimmunity. Accumulating evidence suggests that infections and dysbiosis of the human microbiota may critically modulate the development of allergy and autoimmunity. Although the concepts are intriguing, the underlying mechanisms remain largely elusive.
The skin provides a first line of defense against microbial pathogens and represents a frequent site for the development of allergy and autoimmunity. Its easy accessibility makes the skin an ideal organ system for studies aiming to unravel pathogen-host interactions in the establishment and persistence of allergic and autoimmune disorders.
Genetic studies for susceptibility genes in AD and PSO have identified several disease-associated loci and immunological mechanisms of AD and PSO have been studied extensively over the last decades. However, responsible molecular networks in the context of microbial exposure still remain elusive. We know very little how host-pathogen interactions trigger and regulate the inflammatory cascade leading to allergic or autoimmune skin inflammation, and in the MAARS project aims to take the next step to fill in the open gaps. We propose that microbiomes and genetic factors, characteristic to particular disease, initiate a cascade of inflammatory events through the modulation of anti-microbial defense. The dysregulation of innate as well as adaptive immune pathways leads to inappropriate responses to physical, microbial or allergen challenge, finally manifesting in the clinical symptoms of skin-related allergy (AD) or autoimmunity (PSO). We also propose that the nature and duration of microbial stimuli and associated changes in the epidermal barrier will also give grounds for the development autoimmunity.
Our overall goal has been to unravel the inflammatory pathways during host-pathogen interactions which may trigger allergic or autoimmune inflammation using AD (surrogate for allergic diseases) and PSO (surrogate for autoimmune diseases) as disease models. We have aimed at identifying key microbes and molecular targets to develop novel intervention strategies with the expected impact of delaying the onset of these diseases and improving quality of life.
The specific objectives have been:
• In clinical studies we will generate highly standardized and genetically stratified clinical sample material to unravel the role of microbiomes in the pathogenesis of AD and PSO. Samples from three European countries will be studied to reduce the possibility of population-based systematic errors in results. (WP1)
• We will characterize the skin microbiome from patients and healthy subjects using state-of-the-art high-throughput sequencing techniques. We will also sequence virus enriched serum samples from the same individuals to identify potentially relevant viruses. (WP2)
• We will perform gene expression profiling using DNA microarrays and high-throughput DNA sequencing to investigate microbe-regulated pathways in AD and PSO in genetically susceptible versus non-susceptible backgrounds. We will investigate expression profiles both from skin biopsies as well as from microbe stimulated skin explants from patients and from controls. Finally, we will explore kinetics of microbial-induced transcriptional network in primary keratinocytes by deepCAGE. (WP3)
• We will use advanced bioinformatics tools and systems biology methods to connect the different datasets from different WPs to identify key disease associated microbe as well as key gene targets. Reverse engineering methods will be also used in order to infer pathogenic stimuli from transcriptional profiles generated from lesional skin samples. (WP4)
• Identified molecular and microbial targets will be validated in vitro in cellular models relevant for AD and PSO. We will investigate the activation and regulation of antimicrobial defense molecules and cytokine networks in AD and PSO. Moreover, cellular interactions in the pathopysiology of AD and PSO will be investigated. (WP5)
• We will utilize established animal models of AD and PSO to unravel and validate molecular mechanisms and to test therapeutic targets to interfere with diseases processes of AD and PSO. Gene-targeted as well as transgenic mice, xenotransplant models and neutralizing antibodies will be used. (WP6)
For this purpose we have gathered a multidisciplinary research consortium with members that are at the forefront of biomedical research and have made significant contributions in the areas of epidemiology/risk factors of AD and PSO, genetics, innate and adaptive immune regulation, and systems biology. In addition, state-of-the-art research techniques, comprehensive patient materials and top level animal models utilizing gene-targeted mice as well as humanized mouse models are basis of our research proposal.

Project Results:
1. Work progress and achievements

In WP1 tasks 1 and 2 (collection of patient swab samples in skin biopsies) were finalized during the 2nd reporting period. A total of 134 psoriasis (PSO) patients, 91 atopic dermatitis (AD) patients and 126 healthy volunteers were recruited, samples were collected and submitted to analysis in WP2 and WP3. Task 3 (Patient stratification) was a central part of WP1 activities during the 2nd and 3rd reporting period, providing additional clinical information to aid data analysis, and patient genotypes will soon be available for genetic stratification. For Task 4, (Skin explants) WP1 has established the production of skin equivalents and an an ex vivo skin organ culture model, for further analysis and validation of the cutaneous response to microbes. In WP2 the MAARS patient skin microbiome has been characterized by 16S rRNA sequencing and metagenomics shotgun sequencing (Task 1), and patient blood samples have been subjected to the analysis of virus content (Task 2), in addition to more in depth analysis of the microbiome data (Task 3). In WP3 RNA has been extracted from all patient skin biopsies (Task 1) and the gene expression has been profiled by using DNA microarrays and high-throughput sequencing (Task 2). Task 3 was modified into combining data from microbiome and transcriptome analyses. In WP4 emphasis has been on data management (task 1) and analysis strategies (task 2-4) and patient stratification. We have set up a pipe-line that involves pre-processing steps and more advanced statistical approaches, and introduced models of data integration, and we have developed an information system KDS2 to centralize and retrieve all the data from the MAARS project. During the 3rd period of the project emphasis has been on data integration, and biological interpretation in order to draw important biomedical and conceptual conclusions from the large amounts of high quality data generated throughout the project, and the first set of results from WP1-WP4 are currently under review at the journal Science. WP5 objectives included the production of protein, antibody and cell-based tools (Task 1) and in vitro investigation of microbial targets identified by WP4 (Tasks 2-5), and WP6 objectives included validation of microbial targets in experimental AD and PSO (Tasks 1-3). According to the colonization pattern of microbes identified by WP1-WP4, C. simulans and C. kroppenstedtii are thought to be pro-inflammatory in PSO, whereas L. crispatus might have an anti-inflammatory role in both, AD and PSO. Indeed our in vitro and in vivo data confirm immune-modulatory, protective roles of lactobacilli and a pro-inflammatory function of Corynebacteria spp, supporting WP1-4 conclusions.

1.1. WP1 Clinical studies

To evaluate differences in the cutaneous microbial colonization and transcriptional profile in atopy- and autoimmune-type skin diseases, adult patients (18-70 years) with mild-to-severe chronic AD (SCORAD score > 25, n=91) and plaque-type PSO (PASI score >7, n=134) as well as healthy volunteers (n=126) were recruited from three Depts. of Dermatology, at University Hospitals located in Duesseldorf (HHU, Germany), London (KINGS, Great Britain) and Helsinki (UH, Finland) (table 1). Each subject underwent a physical examination by a dermatologist and the medical history was recorded. The diagnoses were made by a dermatologist based on clinical presentation, personal history, laboratory findings and the criteria of Hanifin and Rajka. The exclusion criteria included concomitant autoimmune diseases (e.g. rheumatoid arthritis, diabetes, alopecia areata, etc.) the use of systemic antibiotics within 2 weeks and systemic immunosuppressive therapy or phototherapy or systemic biologic agents within the previous 12 weeks prior to screening.

Before skin sampling, the biopsy sites were left untreated for at least 2 weeks and cleansing with only the non-antibacterial Dove soap was allowed and washing was avoided for 24 hours prior to sampling. The patients or healthy volunteers who did not match these clinical exclusion criteria were removed from the study. The following biological samples were then obtained and submitted to analysis: 1) microbiome samples from upper/lower back, posterior thigh or buttocks (PSO, AD, healthy volunteers) with no prior cleaning or preparation of the skin surface using sterile gloves to prevent cross-contamination, were obtained placing a sterile ring (2.5 cm diameter) onto the appropriate skin area, 1.5 ml PBS was supplemented into the ring and the area sampled scraping a glass rod in a circular motion 10 times to the left and to the right. Subsequently, the microbiome-enriched PBS was harvested and stored. 2) 6 mm punch biopsies from skin at the “microbiome” sites were taken in local anaesthesia. Subsequently, samples were stored in RNAlater (Sigma-Aldrich) and subjected to further analyses (Fig. 1). The study was approved by the appropriate local Institutional Review Boards, and all subjects provided written informed consent before participation.

Figure 1. Flow chart of study design and samples included in the study.
Punch biopsies (6 mm) were taken after obtaining microbiome samples, from the same skin sites as the swab samples, from lesional and non-lesional skin of AD patients (n=91, 2 biopsies per patient), PSO patients (n=134, 2 biopsies per patient) or from normal healthy individuals (n=126, 1-3 biopsies per patient). Samples were stored in RNAlater (Sigma-Aldrich) and subjected to further analyses, as described above.
In addition, heparinized blood was drawn from each patient (in CPT tubes), as well as from the healthy donors, and PBMC were isolated locally using standard protocols and either used fresh or frozen for later studies (for details, see WPs 3, 4 and 5). The plasma collected from these blood samples has been shipped to Partner 8 for viral DNA analyses.
10 ml of EDTA blood was drawn from each participant for genomic analyses (for details, see WPs 3, 4 and 5). The samples have been shipped to Partner 8 for standardized DNA extraction.

Related to milestone 4 (MS4) Stratification of patients for high-throughput transcriptomics and microbiome studies, WP1 provided clinical stratifiers for psoriasis and atopic dermatitis. For genetic stratification of the patients, central extraction of the patient DNA at KINGS (P8) is under progress. The three most common filaggrin mutations (in AD) are being analyzed in Stephan Weidinger’s laboratory in Kiel and HLA Cw6 variations will be detected by DNA microarrays (Affymetrix, Illumina chip version II).

Related to Task 4, (Skin explants), HHU (P5) has established the production of skin equivalents and ex vivo skin organ culture models, setting up important tools for further analysis and validation of the cutaneous response to microbes in vitro and ex vivo in genetically-defined and disease-related settings.

1.2. WP2 Microbiome analysis

The microbiome sequencing of patient samples has proceeded at a satisfactory pace and, as previously reported, we have extracted DNA from all samples. PCR amplification has been carried out and 16S sequence data has been obtained for all samples. The analysis of these sequences showed that clear identification of bacterial taxa has been achieved (Task 1) (Fig.2).

Figure 2. Characterization of the skin microbiome in patients with atopic dermatitis (AD), psoriasis (PSO) and healthy volunteers (HV) by 16S rRNA sequencing.

The data have been analyzed further and followed up with data integration with other datasets. The results will be submitted for publication shortly.
We have also completed the shotgun sequencing task. The setting up of the methodology for this task was time consuming, but successful. Thanks to lower sequencing costs and efficient novel library preparation protocols, we managed to produce shotgun data from all samples instead of a subset, which has resulted in data of tremendous importance for the study (Task 1). A bioinformatics pipeline has been established for primary analysis of the shotgun data and the analysis is ongoing. The initial results indicate a deep sampling of the bacteria, fungal and viral skin microbiomes.

For the identification of virus in serum samples, a complete set of protocols for virus enrichment, library preparation and sequencing were established and tested extensively using different types of samples. In addition an efficient bioinformatics pipeline for the subsequent analysis was established. Illumina Miseq data was produced for all MAARS patient samples, as pools representing the two diseases and the healthy subjects. The resulting data have been analyzed and multiple viral species were identified (Task 2).
The identification of interesting candidates has progressed along with the data analysis. Bacteria selected for further study include Lactobacillus and Corynebacterium, and fungal and viral species will also be studied further (Task 3).

1.3. WP3 Transcriptomics

In WP3 task 1, biopsy samples from 91 AD patients, 134 PSO patients, and 126 healthy volunteers were used for total RNA extraction. At least 250 ng of high quality total RNA per sample was sent to Institute Curie (P6) Affymetrix platform for generation of transcriptional profiles by usage of Affymetrix Chip Human Gene ST2.1 array. Quality controls at the cRNA and cDNA levels were satisfactory, except for 3 samples out of 600 for which no amplification product was detected. This corresponds to a 0.5% failure, which is very satisfactory for such a large number of samples. There was no outlier chip after Gene PLIER normalization, confirming a good quality in the hybridization procedure. After preprocessing and normalization of the raw data by WP4, a PCA on the 1000 most variant genes revealed good separation between the groups (Fig. 3).

Figure 3. Transcriptomics analysis of the three patient groups. (A) Projection of AD (red), PSO (orange) and HV (green) transcriptome profiles in the subspace spanned by the 2 first components of the Principal Component Analysis (PCA) performed on the 1.000 most variant genes.

In task 2, 60 ng of the same samples were delivered to KI (P2) sequencing facility for generation of transcriptional profile by high-throughput sequencing analysis by STRT method. STRT sequencing libraries were prepared and a commercial control RNA (10 ng) that was received from Curie (P6) was added to each batch to control the variation between the libraries. The quality and amount of libraries were checked before sequencing and when not adequate, the library was re-prepared. After the raw data was received from the sequencing facility, it was transferred to Curie for storage, and can be accessed by MAARS members.

1.4. WP4 Bioinformatics and systems biology

WP4 is a central work package which includes partners of diverse and complementary expertise in data management, bioinformatics and systems biology. Connections with other WPs are crucial to ensure a smooth and efficient processing and analysis of the data in order to move towards the global objectives of the MAARS project. During the first reporting period, emphasis was given to the data management, in order to implement the most appropriate solutions to address data storage and handling and ensure success of research tasks as well as clinical practice. Frequent interactions with WP1 and WP2 were important to ensure that clinical information/annotation was made available in the right format for subsequent biostatistics/bioinformatics analysis. An electronic CRF was built in accordance with the paper CRF and clinical input from WP2 partners. Downstream interactions with WP5 were established in order to prepare the biological database (BIRD). Interactions with WP3 were critical to link RNA samples to data generation. Several important issues were also addressed regarding the choice of transcriptomics methods, future analysis strategies, and task-sharing among partners. In particular, a meta-analysis of public transcriptomic data was performed (FIOS GENOMICS (P10)) in order to establish some biological benchmarks for comparison with our own data. By the end of the first reporting period, everything was in place to store, annotate and analyze data according to state of the art bioinformatics methods.

During the second reporting period, emphasis was maintained on the data management, in order to ensure continued quality of the data, manage transfer of new large-scale data, and perform all required annotations and quality controls. In parallel, data analysis gradually increased as the data became available. Frequent interactions with WP1 and WP2 were important to ensure that clinical information/annotation was made available in the right format for subsequent biostatistics/bioinformatics analysis. Downstream interactions with WP5 were continued as in first reporting period, in order to finalize the biological result database (BIRD), and to discuss molecular targets to focus on within WP4. Interactions with WP3 were critical to link RNA samples to data generation for the final large transcriptome dataset. By the end of the 2nd reporting period, transcriptomics and microbiome data on the final large dataset of 600 samples had been generated, and were available to all consortium members.

During the third reporting period emphasis was on data integration, and biological interpretation in order to draw important biomedical and conceptual conclusions from the large amounts of high quality data. We found that AD (n=88) and PSO (PSO, n=129) are classified by distinct microbes, which differ from healthy volunteers (n=117) microbiome in composition and relative abundance. AD is dominated by a single microbe (S. aureus), associated to a specific disease relevant transcriptomic signature (Fig. 4). In contrast, PSO is characterized by co-occurring communities of microbes impacting psoriasis core gene transcripts. Our work underscores the importance of distinct microbial classes in tissue inflammation and provides a basis for discovery of biomarkers and targeted therapies in skin dysbiosis. These results are currently under review at the journal Science.

Figure 4. Differential analysis between healthy and AD samples revealed 16,716 genes (FDR < 0.05) for which we created an AD gene co-expression network by Pearson correlation analysis, followed by partitioning of the network into modules using network community detection (network on the left). Hypergeometric tests revealed significant enrichment in S. aureus-associated genes (coloured red) that exclusively mapped to module 4 (FDR<0.05) (network on the right). The functional characterization of this module using Ingenuity Pathway Analysis identified 78 significantly enriched pathways (FDR < 0.05) (Fig. 3D). Top enriched pathways were predominantly related to cytokines/cytokine signaling and antimicrobial peptides.

1.5. WP5 Molecular and cellular networks

Cutaneous surfaces are the first sites that encounter microbial threats and therefore may initiate and shape the innate and adaptive immune response to invading pathogens through activation of pathogen recognition receptors ultimately leading to diseases, like atopic dermatitis (AD) and psoriasis (PSO). Conversely, commensals colonize the skin and critically contribute to tissue homeostasis. Indeed, the MAARS consortium (WP1-WP4) identified a specific colonization pattern in healthy skin that differs significantly from that observed in AD and PSO (as described above). Among those, L. crispatus, C. simulans and C. kroppenstedtii are of special interest and, therefore, have been cultured in vitro. According to the colonization pattern C. simulans and C. kroppenstedtii are thought to be pro-inflammatory in PSO, whereas L. crispatus might have an anti-inflammatory role in both, AD and PSO.

In WP5 we have focused on five different aspects: 1) production of relevant protein, antibody and cell-based tools, 2) dysregulated responses at cutaneous surfaces, 3) the micromilieu and its effect on DC function and T cell priming, 4) novel effector pathways of memory T cells within the skin, and 5) the role of the allergic effector unit. Concerning task 1, we have successfully produced the following tools: human 2B4 rabbit polyclonal antibodies, human TSLP rabbit polyclonal antibodies, ELISA tests for detection of human 2B4, mouse 2B4, human CD48 and mouse CD68, and ELISA tests for human RNase7.

In work related to tasks 2-5, we observed that L. crispatus extracts induce regulatory cytokines and decrease Th2 type responses in human PBMCs and primary keratinocytes (Fig. 5), and diminish IgE mediated degranulation of mast cells, but have no effect on Th1 type inflammation. The data indicate that L. crispatus enhances the immune-modulatory and protective function of immune cells, antagonizes Th2 type inflammatory responses, and may be involved in the maintenance of skin homeostasis.

Figure 5. L. crispatus culture supernatant decreases TH2-, but not TH1-inflammation. Primary human keratinocytes were pre-treated with L. crispatus culture supernatant (A-579-1 in a 1:10 dilution) for 24 hrs followed by a stimulation with IL-4 or IFN-gamma for 24 hrs. Cells were harvested, RNA was prepared and subjected to qPCR analysis (n=8). **, p<0.01; ***, p<0.001.

Lactobacilli and other skin commensals produce short chain fatty acids (SCFAs) such as butyrate and propionate, which might harbor immune-modulatory properties. We found that human primary keratinocytes express SCFA receptors GPR109A and 109B, indicating that microbes that produce these compounds have direct effects on keratinocytes. We observed that sodium butyrate (BA) enhances Th2 responses, downregulates FceRI expression on mast cells, and interferes with Th1 responses in human primary keratinocytes. Further, BA interferes with Th1 responses in dendritic cells (DCs), and reduces the expression of MHC-II, suggesting that BA down-regulates antigen-specific activation of T-cells. BA also differentially regulates psoriasis-related chemokines in DCs, suggesting a role for BA in the regulation of leukocyte trafficking towards the inflammatory site.

C. simulans and C. kroppenstedtii are over-represented in psoriasis. We found that sera from both psoriatic and healthy subjects contain specific IgG against C. simulans, but the magnitude and pattern of antigen recognition differ between psoriasis patients and healthy subjects. A psoriasis specific band was identified, cut out and sequenced, and several peptide fragments of hornerin and filaggrin were identified. The current hypothesis is that C. simulans might contribute to the generation of autoantigens in psoriasis, via triggering of supramolecular forms of hornerin. Moreover, C. simulans significantly up-regulates the expression of PSO-related genes (CCL20, IL36G, S100A7) in human primary keratinocytes, and synergizes with IL-17.

Finally, the fungus Malassezia which is known from the literature to play a role in both AD and PSO, induces the expression of pro-inflammatory cytokines and chemokines in human primary keratinocytes, and synergizes with psoriasis related cytokines IFN-gamma and IL-17 to further drive the expression of pro-inflammatory genes. To conclude, our data suggest immune-modulatory, protective roles of lactobacilli and disease promoting roles of Corynebacteria spp, supporting WP1-4 conclusions.
1.6. WP6 Animal models

Cutaneous surfaces are the first sites that encounter microbial threats and therefore may initiate and shape the innate and adaptive immune response to invading pathogens through activation of pathogen recognition receptors ultimately leading to diseases, like atopic dermatitis (AD) and psoriasis (PSO). Conversely, commensals colonize the skin and critically contribute to tissue homeostasis. The MAARS consortium (WP1-4) identified specific colonization pattern in healthy skin that differs significantly from that observed in AD and PSO. Among those, Lactobacillus crispatus, Acinetobacter lwoffii, Candida albicans and Staphylococcus aureus are of special interest and, therefore, have been cultured in vitro. L. crispatus and A. lwoffii were abundant on the skin in healthy volunteers and lost in AD and PSO lesions, and L. crispatus is suggested to have an anti-inflammatory role in both, AD and PSO. In contrast, according to the colonization pattern seen in patient samples C. albicans and S. aureus are thought to be pro-inflammatory in AD.

In the mouse model of AD (FIOH, P1), L. crispatus antagonized Th2 type allergic responses by reducing the expression of IL-4 and IL-13 mRNA in the skin and by lowering the level of OVA-specific IgE in the serum (Fig. 6). Similarly in the IMQ-model (KINGS, P8) pretreatment of the skin with L. crispatus extracts reduced skin thickness and macrophage infiltration.

Figure 5. a) Exposure to L. crispatus during the AD model protocol resulted in reduced b) inflammation in the skin, including c) lower numbers of eosinophils in the skin, reduced expression of Th2 type cytokines and lower levels of OVA specific IgE in the serum.

A. lwoffii is also present at a higher abundance in the skin in healthy individuals compared with AD patients, and is associated with protection against atopy and allergies in previous studies. Topical exposure of the mouse AD model to A lwoffii extracts significantly diminished the inflammatory response in wild type mice, including decreased numbers of eosinophils and lower expression of Th1, Th2 and Th17 related genes in the skin. In reverse, the yeast C. albicans, which is more abundantly present in AD lesions compared with healthy skin, substantially exacerbated skin inflammation in the mouse AD model through the amplification of Th2 components in addition to Th1 and Th17 type responses.

To validate the role of microbial targets in driving disease pathways in PSO and skin inflammation, KINGS (P8) optimized two in vivo models, human skin transplantion model and psoriasiform with imiquimod, and showed that L. crispatus ameliorated local skin inflammation associated with decreased skin thickness and macrophage infiltration.

To conclude, our results suggest that microbes identified by the MAARS study have a strong impact on the cutaneous inflammatory responses in vivo. Understanding of these processes and interactions will open new avenues to treat and even prohibit

Potential Impact:
Impact of allergies. Atopic dermatitis (AD) has become an epidemic, now more than 15% of European children suffering from the disease. The prevalence of AD keeps on increasing both in developed and developing countries. Asthma develops in approximately 30 percent of children with atopic dermatitis, and allergic rhinitis in 35 percent, causing additional costs and burden. Thus, AD acts as a model disease to unravel common pathways relevant for allergic rhinitis and asthma. The total costs of AD for EU member states has been estimated at 12 Billion euros. (Williams HC, N Engl J Med 2005; 352:2314-2324). Psychosocial abnormalities in preschool children with AD include behavioral problems, excessive dependency, clinginess, and fearfulness. In addition to daytime scratching, sleep in children with AD is marked by overall reduced sleep efficiency with frequent awakenings, which for many children persist even during disease remission. Difficulty falling asleep and night awakenings in children with AD correlate with behavior and discipline problems. Coincident with its impact on the child, childhood AD has a social, emotional, and financial impact on families. AD is associated with increased incidence of occupational hand dermatitis, causing work disability.

Impact of autoimmunity. Psoriasis (PSO) represents a chronic inflammatory and autoimmune skin disorder characterized by epidermal hyperproliferation and dermal inflammation that vary in severity, from minor, localized patches to complete body coverage. It affects 2–3% of the population, making it one of the most prevalent autoimmune diseases worldwide, and can be associated with other inflammatory conditions, such as inflammatory bowel disease and coronary artery disease. In the USA, total annual direct and indirect costs of PSO over 11 billion US $, with missed work days accounting for 40% of the cost burden. In comparison, the costs are estimated to be similar or higher in the EU. Psoriasis has been shown to affect health-related quality of life to an extent similar to the effects of other chronic diseases such as depression, myocardial infarction, hypertension, congestive heart failure or type 2 diabetes. Depending on the severity and location of outbreaks, individuals may experience significant physical discomfort and disability. Itching and pain can interfere with basic functions, such as self-care, walking, and sleep. Plaques on hands and feet can prevent individuals from working at certain occupations, playing sports, and caring for family members or a home. Medical care can be costly and time-consuming and can interfere with an employment or school schedule. Up to 30 percent of people with PSO also develop psoriatic arthritis, which has symptoms similar to rheumatoid arthristis, including pain, stiffness and swelling in and around the joints. Psoriatic arthritis may progress to a severe, deforming and destructive arthritis possibly causing permanent damage to joints. Inflammatory processes in PSO are associated with metabolic syndrome, involving diabetes, hypertension and atherosclerosis.

The global market to the pharmaceutical industry for psoriasis medication is $3.5 billion per annum, while the cost of treating AD is $6 billion dollars per annum in the US, and in EU a similar amount is estimated. AD and psoriasis therefore impose an enormous societal and financial burden on the European Community and other developed, as well as increasingly also developing, nations worldwide. MAARS aims to utilize the skin as a model organ to identify novel targets from microbiome-host-interaction for prevention, early diagnostics and therapy of allergy and autoimmunity. Resulting findings are likely to hold promise to open opportunities for European industry in global markets.

Contribution towards the expected impacts listed in the work programme

MAARS contributes towards the expected impacts in call HEALTH.2010.2.4.5-2: Infection and dysbiosis as the triggers of the development of inflammatory processes in allergies and autoimmune diseases. It is translational research which obtains mechanistic insights into the early processes underlying chronic inflammatory allergic or autoimmune diseases, such as AD and PSO, respectively. Emphasis is directed to on identifying and validating microbe-host-interaction networks involved in the establishment development and persistence of the chronic inflammatory reaction. MAARS aims utilize the skin as a model organ to identify novel targets for the prevention (pre- and probiotics), early diagnostics and therapy of allergy and autoimmunity. The consortium has a multidisciplinary approach involving microbiologists, immunologists, pharmacologists, molecular and cellular biologists, molecular biologists, geneticians, bioinformaticians, systems biology researchers, and dermatologists. MAARS uses existing genetic information, as well as immunological response markers, to stratify patient collectives. It employs state-of-the-art techniques to assess microbe-host interactions, validates findings in relevant in vitro systems and finally performs proof of concept studies using animal models for its research, which are amenable to genetic testing and manipulation. MAARS integrates vast amounts of data spreading from microbiology, genetics, transcriptomics, immunology systems biology and immunology, to the clinical phenotype, in a systems biology approach for understanding and modeling of the underlying biological processes of allergy and autoimmunity within the model organ skin. This is possible by bringing together of critical masses of various expertise and resources in MAARS, not achievable at a national level. The scientists in consortium represent the forefront of biomedical research in their disciplines.

MAARS fully responds also to the general objectives and rationale of Framework 7 “HEALTH” area. Its aim is to improve the health of European citizens through development of prevention measures as well as treatments and prevention measures for allergy and autoimmunity. Its aim is also to increase the competitiveness and innovative capacity of European health-related industries and businesses, in the field of allergy and autoimmunity, through IPR acquisitions and collaboration with SMEs and European drug industry. MAARS addresses global health issues, allergy and autoimmunity, which are also an emerging epidemic in developing countries. MAARS has an emphasis on translational research, i.e. translation of basic discoveries into clinical applications, including scientific validation of experimental results obtained from large-scale microbiome and transcriptome studies. The project aims at the development and validation of new therapies Findings in AD are highly relevant for other allergic diseases, especially allergic rhinitis and asthma. Conversely, findings in psoriasis are likely relevant for other autoimmune diseases including arthritis and inflammatory bowel disease. AD is a major disease of childhood and risk factor for childhood asthma and MAARS promotes child health in unraveling the mechanisms of early processes of allergic sensitization. AD is also increasingly a disease of aging people and prevalence of PSO gets higher with aging. Therefore, the strategic issues of child health and pediatric diseases as well as of the health of the ageing are taken into account in this project. The project also aims at providing diagnostic tools for early detection of AD and PSO for a more efficient preventive intervention.

Steps needed to bring about impacts in the work programme
MAARS will obtain mechanistic insights to early processes of inflammatory disorders featuring the continuum from allergy to autoimmunity. For this purpose we have gathered a multidisciplinary research consortium with members that are at the forefront of biomedical research and have made significant contributions in the areas of epidemiology/risk factors of AD and PSO, microbiology, genetics, innate and adaptive immune regulation, and systems biology. In addition, state-of-the-art research techniques, comprehensive patient materials and top level animal models utilizing gene-targeted mice as wells as human xenotransplant mouse models are basis of our research proposal.

MAARS will identify novel targets for the therapy of different types of AD and PSO, and will also produce a substantial body of information about the molecular underpinnings of AD and PSO. The information will be integrated to ensure that maximum added value will come from interpretation of the study results. The project is designed to identify and test genes, proteins, antibodies, cells and model systems that will be the basis for the identification of novel small-molecule and biologics reagents for AD and PSO.

A large number of small molecule receptor antagonists and agonists for these targets are currently identified and under investigation in research institutes and pharmaceutical companies worldwide. Some of them have already reached phase one and two clinical studies in the treatment of diseases involving the immune system, e.g. AIDS and myeloproliferative disorders, such as lymphomas. Hence, results of the proposed study will directly identify promising candidates for the treatment and prevention of AD and PSO will lead to the initiation of evidence-based clinical trials using those specific small molecule antagonists and agonists. MAARS will therefore reinforce the competitiveness of the European Biotechnology and Pharmaceutical sectors in a major global therapeutic market.

MAARS will provide a large body of unique diagnostic targets from studies on pathomechanisms and systems biology. Use of these agents will allow prediction of the onset of AD at early time-pointpoints in susceptible individuals, which will then enable prevention of the disease processes at early stage. The agents will also be of use in the early identification of individuals who will have a difficult or severe disease. Diagnostic information that predicts the response to particular therapeutic agents will also be generated. This type of testing is likely to have a major impact on effective clinical management of AD and PSO. Partner 8 has already a large experience in developing and marketing diagnostic tools for use.

Rationale for a European (rather than a national or local) approach.

The MAARS consortium includes scientists from fields of immunology, genetics, systems biology, medicine, and molecular biology, who are in the forefront of their research. A comprehensive project at the forefront of science to identify the potential targets for AD and PSO allergy and autoimmunity treatment would not be possible at a national scale, as the best expertise is distributed across Europe. The only possibility for this would be a pan-European effort with the best laboratories and research groups on the continent in their areas of expertise (microbiology, genetics, transcriptomics, immunology, systems biology, immunology, clinical research), which concentrates on this aim. A national or local approach would not be able compete with the consortiums and research organizations of the world, including the U.S. A pan-European approach is also needed to ensure that the patient material studied is not genetically area-specific and biased.

How other national or international research activities are taken to account.

It should, however, be noted that most of the targets studied in MAARS will actually come from within its own translational research, as well as research done in other laboratories globally. MAARS will also collaborate with other groups and consortiums from Europe and elsewhere when win-win situations emerge for further research.

Assumptions and external factors that may determine whether the impacts will be achieved.

MAARS is a prospective research project and the general external factors, i.e. the yet unsolved host-pathogen-interaction in AD and PSO determine whether targets obtained from microbiome, transcriptomics and bioinformatics will be identified for diagnostics and possible treatment and prevention. Finding such targets is, however, very likely, as the approach in MAARS is truly comprehensive.

3.2 Dissemination and/or exploitation of project results, and management of intellectual property

The research done in MAARS program will provide an immense amount of new information on the pathomechanistic processes in immunopathogenesis of skin-related allergy and autoimmunity. These results will first be disseminated within the consortium to improve the knowledge base of its members. The program also provides new reagents, produced by Partner 8, an SME, which will be available for consortium members. Know-how and knowledge will also be disseminated through human mobility program of the project (WP0).

Scientific dissemination and exploitation

Scientific dissemination of the results will primarily be through the publication of research papers in leading biomedical journals. In case a potential IPR, scientific publication will be done after IPR panel has decided whether patent announcement should be done prior to scientific publication or presentation.

All scientists who have contributed to the innovation, planning, performing scientific laboratory work or data handling, interpretation of data, and who have participated in the process of writing, will be co-authors in those parts of the project in which they have collaborated. The results of the project are published in scientific congresses, i.e. Annual Meetings of the European Society for Dermatological Research, European Academy of Dermatology and Venereology and European Academy of Allergology and Clinical Immunology. The results of research are published as original research reports in top peer-review journals in the fields of microbiology, immunology, autoimmunity, allergology, dermatology, cell biology, general medicine and general science. However, when possible, journals with Impact Factor of at least 20.0 will used for publication. Additionally, review articles on the research will be written, as well as book chapters for books on the area of research. A textbook from the information based on final workshop and final report on the topic may be suggested to major science book publishing houses and, if accepted, thereafter written and published. A website will be created that has open part for public, including patient and other education material, closed parts for registered collaborators, as well as fully secure internal parts with extranet features, video conference applications and social networks (see also 3.2.4. Human mobility).

Commercial dissemination and exploitation

The call HEALTH.2010.2.4.5-2: "Infection and dysbiosis as the triggers of the development of inflammatory processes in allergies and autoimmune" diseases aims at identification of novel pathways in skin-related allergy and autoimmunity, to target and create new treatments to these chronic inflammatory diseases within spectrum of allergy and autoimmunity. Therefore, IPR matters are of paramount importance in MAARS. For this, special attention is devoted during this project on IPR matters.

The consortium will stimulate patenting of all discoveries made in the research laboratories, where compensation is given to the inventors and at the same time the findings are also made accessible to other partners. Patenting should not, however, prevent publication in scientific journals, once IPR matters have been considered and, when decided feasible, secured.

The consortium will make a binding contract on the intellectual rights of the results, including matters related to transparency inside the consortium and the necessary secrecy during patent applications prior to publications at the kick-off meeting.

A separate IPR panel will be established for IPR matters (WP1). The panel will consist of five scientists from consortium who also have experience on IPR matters, Project manager, Coordinator, patent expert, as well as two outside experts. The panel will also invite, on case basis, more outside experts from industry and/or academia to facilitate decisions on whether an innovation is worth pursuing for a patent. Coordinator has a separate budget for IPR panel (WP 1). Additionally, each Partner has a separate budget for arising IPR procedures.

The Inventor will report to Coordinator about a new invention. Coordinator will convene the IPR panel. After review of the IPR panel, Inventor will either (1) patent the finding, after which it can be published, or (2) if there are no IPR matters worth for pursuing in terms of commercial dissemination, the results are published. The other Partners will immediately be informed via e-mail of the process after patent has been filed or a decision not to patent has been reached. The project will also create and produce cutting-edge new reagents for study of skin-related
allergy and autoimmunity and other allergy and autoimmune disorders beyond the skin. These reagents will be commercialized by Partner 8, which is a SME specialized in this type of production and commerce. The economic advantages from this activity will take into account the respective IPR matters in discoveries from which creation of new reagents is enabled. Partner 8 has all facilities for larger scale production of fine-tuned biologicals, as well as a marketing department for all merchandise in products and/or intellectual properties. The projects other responsible scientists from academic centers have also diverse contacts to pharmaceutical industry, especially related to the development of novel therapeutics through biotechnology.

Expertise dissemination and exploitation: Human mobility

Coordinator establishes a fund which is used to finance Visiting Fellowships to participating laboratories. These fellowships will cover costs of travel and accommodation during 2-3 month visits. Approximate cost for these is approximately 6000 euros. Coordination places 0.8 % of total budget, i.e. 48 000 euros, for this purpose over 4 years. This will allow 2 fellowships per year for the consortium, i.e a total of 8 fellowships. The salary of the visiting scientist will be paid by the Partner employing the scientist and the laboratory costs will be paid by the hosting Partner. These fellowships will enable establishment of true pan-European network for scientific community. Additionally, Marie Curie fellowships will be applied to enable longer (1-3 years) fellowships between participating partners. The project will also provide training for PhD students in the project in virtually all participating centres, as well as postdoctoral training.

Human mobility is today not only physical mobility, but also virtual human mobility in the form of social internet and messaging. MAARS will form a Social Internet Networks for the whole Consortium, as well as subgroups of the Consortium. The social networks will be secure and closed LinkedIn/Facebook-like networks allowing discussion and sharing of research experience and data. Individual or identifiable group patient information or information about patient visits will not be shared. Subgroups will be e.g. those working in same WP. Secure Twitter, Jaiku, Qaiku etc type instant messaging systems may be incorporated. The junior scientists would have their own secure social network with monitors chosen amongst junior scientists themselves, to ensure a more relaxed virtual group to discuss everyday matters at ease, to promote cohesiveness among scientists across borders. The social networking and messaging will create a virtual pan-European lab where information and working together will occur without restricting distances. Virtual mobility is ecological and cost-effective at the same time. The virtual mobility and networking will act, in addition to scientific excellence and aiming to lessen the burden of disease, as one more key attractor for highest talent in Europe and other world for MAARS in competition for this scarce resource. Especially, in coming years of projects time-table the talented graduate and postgraduate student generations of 1980's and even 1990's would demand this kind of environment for their workplaces and would choose consortiums such as MAARS that have it.

Public dissemination and exploitation

Allergic and autoimmune skin disorders, such as AD and PSO, are multifactorial diseases and therefore their treatments can be very complex and potentially confusing to patients and their families. This is especially true in case of children suffering from AD. Both lack of proper diagnostics and insufficient compliance to treatments cause unnecessary worsening of disease, both in children and adults. Even more confusing is existence of alternative theories and treatments for AD and PSO, which may drive patients away from medical treatments with proven efficacy. For this reason, one of the missions of MAARS is to strengthen the position of science among public in Europe. The project's public dissemination program will enhance perceptions of scientific research in its true context, i.e. an activity to promote health and well-being in society. For this purpose, several activities are taken.

The use of internet is of paramount importance during the course of MAARS project. The project will launch two major websites during its first year. The first, an official website, will be for scientific community, health professionals, public institutions and organizations, as well as patient organizations.

The website will:

• Describe the aims of MAARS
• Describe the scientific strategy of the project
• Describe the benefits to European Community
• Report on scientific progress of the project

A second website will be a popular science website, aimed at the general public. The awareness and understanding of scientific mechanisms behind AD and PSO will result in better treatment and quality of life among patients suffering from AD and/or PSO and their families. Scientists in MAARS will provide material to this website. This website will have special attention to provide understandable information, in addition to public in general, also to young people.

The website will contain:

• Descriptions of microbiome, atopic dermatitis, psoriasis, immune system, genetics and research methods
• Information on the benefits of scientific research, including treatments
• Individual descriptions of scientific research in project, including
- web videos on live-microscopy (cellular interactions)
- animations
- interactive simulations of scientific research processes, on a how-to-do approach
- success stories in AD and PSO research
• Links to and from patient organizations and popular science websites

Both websites will be built and managed by Partner 1 (FIOH), where Project Manager will have experience and know-how on internet website managing. Professional website service providers will be used for creation of multimedia applications. The websites are linked to each other. Success of the websites will be monitored according to visit numbers and usage of multimedia in the websites.

The consortium will also initiate contact with the popular media through a press release announcing the launch of MAARS, that will coincide with the Kick-Off meeting. Regular press releases will be published to announce the achievement of scientific milestones by MAARS members.

Patient organizations and special interest groups will be invited to attend selected training activities. The consortium actively promotes the participation of MAARS members in public dissemination activities, including interviews and articles in internet, newspapers and magazines, as well as cooperative public actions through national research councils or private foundations.

List of Websites:

P1 FIOH, Coordinator Professor Harri Alenius, TYOETERVEYSLAITOS, Topeliuksenkatu 41b, 00250 Helsinki, Finland, Tel. +358304742175, Fax +358304742116, E-mail:

P2 KI Professor Juha Kere, Group Leader, KAROLINSKA INSTITUTET, Department of Biosciences and Nutrition, Hälsovägen 7, SE-14183 Huddinge, Sweden, Tel. +46-8-524 81057, Fax +46-8-774 5538, E-mail:

P2 KI Professor Björn Andersson, Group Leader, KAROLINSKA INSTITUTET, Institutionen för cell- och molekylärbiologi (CMB), Berzelius Väg 35, SE-171 77 Stockholm, Sweden, Tel. +46-8-524 839987, E-mail:

P3 HUJI Professor Francesca Levi-Schaffer, Group Leader, School of Pharmacy, Faculty of Medicine, THE HEBREW UNIVERSITY OF JERUSALEM , Ein Kerem Campus, Ein Kerem Street, POB 12065, Jerusalem 91120, Israel, Tel. +972 2 6757512, Fax +972 2 6758144, E-mail:

P5 HHU Professor Bernhard Homey, Chairman, HEINRICH-HEINE-UNIVERSITAET DUESSELDORF, Moorenstr 5, 40225 Düsseldorf, Germany, Tel. +492118117600, +492118117853, Fax +492118117049, E-mail:

P6 CURIE, Dr. Vassili Soumelis, Principal Investigator, Senior staff physician, INSTITUT CURIE
rue d'Ulm 26, 75248 Paris, France, Tel. +33144324227, Fax +33153104025, E-mail:

P7 CAU, Professor Jens-Michael Schröder, Head of the Clinical Research Unit, CHRISTIAN-ALBRECHTS-UNIVERSITAET ZU KIEL, Schittenhelmstr. 7, 24105 Kiel, Germany, Tel. +49 431 597 1536, Fax +49 431 597 1592, E-mail:

P8 KINGS, Professor Frank Nestle, Director of Clinical Research Facilities, Professor Jonathan Barker, Head of the Department, Dr. Sophia Tsoka, Lecturer of Bioninformatics KING'S COLLEGE LONDON, 9th Floor, Tower Wing, Guy's Hospital, Kings College
SE1 9RT London, United Kingdom Tel. + 44 207 188 8086, Fax + 44 207 188 8050, E-mail: E-mail: E-mail:

P9 ICOS Professor Mart Ustav, CEO, Dr. Andres Tover, CSO ICOSAGEN AS, Nooruse 9, 50411 Tartu, Estonia , Tel. +3725225418, Fax +3727377077, E-mail: E-mail:

P10 FIOS Dr. Max Bylesjö, Head of Data Analysis, Fios Genomics Limited, ETTC, King's Buildings, Mayfield Road, EH9 3JL Edinburgh, United Kingdom, Tel. +441314724800, Fax +441316624678, E-mail:

P11 UH Professor Annamari Ranki, Principal Investigator, Professor Antti Lauerma
HELSINGIN YLIOPISTO Meilahdentie (Po Box 22), 00014 University of Helsinki, Helsinki, Finland, Tel. +358 9 471 86300, Fax +358 9 471 86561, E-mail: E-mail: