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Since the end of World War II there has been a dramatic increase in the incidence of chronic inflammatory diseases that appears to follow a geographical pattern of industrialization and urban living1 This includes allergic conditions (asthma, allergic rhino-conjunctivitis, food allergies and eczema) as well as autoimmune disorders such as type I diabetes (T1D), chronic inflammatory bowl disease (IBD) and neuro-degenerative disease. Genetics can not account for the increase in prevalence over time, but epidemiological evidence clearly supports a combination of environmental and genetic risk factors -The nature of these risk factors and the pathogenic mechanisms involved, however, are only just beginning to be understood. In October, the European Science Foundation (ESF) brought together an international group of experts on subjects ranging from epidemiology to epigenetics for a 5-day meeting in Barcelona  , Spain, intended to help define strategic directions for research into this increasing public health concern. In this first meeting of the "ESF Forward Look initiative", discussions focused on the grand challenges that face research into gene-environment interactions in metabolic and immune-mediated pathologies with inflammatory processes as the key driver of disease initiation and/or perpetuation. Surprisingly, diseases as diverse as obesity and asthma at the phenotype level share similarities at the molecular and cellular levels. Such striking findings shed new light on this area and triggered the organisation of an interdisciplinary ESF initiative. In order to structure this process, the Barcelona meeting covered intrinsic and extrinsic mechanisms of various chronic diseases together with emerging concepts in disease therapy and prevention.
Intrinsic mechanisms in chronic inflammatory disease
The increasing incidence of chronic inflammatory disease such as diabetes, asthma, allergy, and inflammatory bowel disease (IBD) are widely considered to be due to a combination of environmental and individual risk factors. Recent scientific findings highlight similarities among these conditions at both the genetic and mechanistic level.
The advent of genome-wide association studies (GWAS) raised the hope that disease risk could be clearly stratified according to individual susceptibility alleles. These efforts resulted in the identification of several disease-associated polymorphisms , which clearly shows that there are overlapping susceptibility genes in these inflammatory disorders 2. A prototypic example is the discovery of ormdl3 polymorphisms in both asthma and IBD3, 4.
Despite the recent identification of numerous potential susceptibility genes 5, 6, their individual effects appear to be quite modest. Thus, in many diseases the alleles that have been identified by GWAS only explain a very small proportion of disease heritability both in IBD and asthma. Similar observations have been made in type-1 diabetes (T1D) where the most common polymorphisms explain less than 30% of the heritable risk7, 8, and even in identical twins a concordance of only about 50% has been observed.. The situation in type-2 diabetes (T2D) is even more striking9. Having a first-degree relative with T2D is associated with a hazard ratio of between 3 and 4 for development of the disease compared with individuals who do not have affected relatives. Despite clear heritability, the susceptibility genes identified by GWAS only account for approximately 1% of T2D heredity, making them essentially useless for risk prediction10, 11. Similar findings have been also observed for other chronic inflammatory disease including asthma and allergic eczema, indicating the multigenicity and complexicity of these disorders. Thus, simple genetic explanations for individual susceptibility to chronic inflammatory disease have not been forthcoming.
These findings highlight a key challenge in defining the genetic factors that determine disease susceptibility in chronic inflammatory disease, namely the failure of single alleles or even allele combinations so far to facilitate clear risk prediction. It is important to note, however, that the lack of clear associations with common single-nucleotide polymorphisms (SNPs) does not mean that these genetic differences do not underlie susceptibility. Individual SNPs with small effects may together have metabolic consequences that can be used as biomarkers in risk stratification. Sequencing technology has advanced to such an extent that it can now be considered trivial to analyze 1500 SNPs at a time. The real challenge is the ability to discern relevant patterns from the data obtained. Furthermore, genetic variation is not only about SNPs. Other factors such as repetitive DNA elements or copy-number variants could also be involved, and these may be much more difficult to address.
Since risk alleles are very frequent in the healthy population, epidemiologic evidence supports the role of environmental triggers in the initiation and/or perpetuation of chronic diseases12. Epigenetic variables have been recognised as a possible missing piece in the puzzle linking the human genome, the environment and phenotype development. Comparison of methylation patterns between individuals exposed to severe prenatal famine and their unexposed siblings, for instance, has shown that transient prenatal exposure to a specific environmental stimulus leads to persistent and heritable epigenetic changes13.
A crucial element of epigenetic studies will be the generation of tissue- and cell-type specific data. One example in which evidence has been gained for the epigenetic regulation of specific cell types is in the differentiation of T-helper (TH) cells from naÃ¯ve CD4+ lymphocytes14, 15. These immune cell types are linked to autoimmunity and allergy, and they may be central to the development of chronicity by generating a long-lasting cellular memory of earlier environmental exposures. The level of methylation of the IFNG promoter has been identified as a marker of TH1 lineage commitment16. Thus, in a specific immune cell type, epigenetic changes can determine important cell fate decisions that are likely to influence disease17. In the light of these findings, epigenetic signatures might serve as biomarkers for specific prenatal environments, while GWAS studies may reveal biologically significant changes18.
In order to achieve these goals, however, future studies will need to obtain a reliable catalogue of prenatally induced epigenetic signatures associated with adverse events or environmental exposures. Determining how to sample the environmental impact is perhaps the single greatest challenge for research of environment-gene interactions. It will also be important to obtain robust evidence that these signatures have a causal relationship with the pathogenesis of the disease. As in genomic analyses, obtaining an overview of epigenetic associations will require more detailed information on disease phenotype, including temporal changes from initiation through to chronicity.
A major focus for research into the cellular mechanisms underlying chronic inflammatory disease is the tissue-environment interface19. It is here, particularly in the mucosa lining the gut and airways, that a careful balance must be maintained between host defense and uncontrolled non-resolving inflammation. One area in which substantial advances have been made is in understanding the role of endoplasmic reticulum (ER) stress, autophagy, and microbial sensing in the pathogenesis of IBD20. The unfolded protein response is a pathway from the endoplasmic reticulum (ER) to the nucleus that protects cells from stress caused by unfolded or misfolded proteins. Interference with this pathway in mice, for instance by knocking out the transcription factor Xbp1, results in an IBD-like phenotype21. Closer analysis reveals that the gut epithelium lacks Paneth cells and, to a lesser extent, goblet cells. These are the most secretory cell types, and therefore most susceptible to ER stress. One of the biological functions of the Paneth cells is to secrete anti-microbial peptides. Thus, when ER stress cannot be controlled in Paneth cells, they are unable to respond appropriately to their environment nor can they fulfil their role in managing the microbial milieu. Most recently, Toll-like receptor (TLR)2- and TLR4-mediated activation of xbp1 links ER stress signalling to microbial sensing, supporting a role for cellular stress mechanisms in intestinal inflammation22, 23. A number of genetic risk factors for IBD converge on the Paneth cell. Interestingly, one such risk allele of the Atg16l1 gene, which is involved in autophagy and is linked to Crohn's disease, requires an environmental insult in order for the disease phenotype to be expressed24. When susceptible cells are infected with norovirus, they become abnormal and hyperinflammatory, but this is not converted into chronic inflammation. Thus, at least two gene-environment interactions appear to be necessary to initiate the disease that then progresses to a chronic inflammatory phenotype. In addition, nutritional factors such as luminal iron, have recently been identified as risk factors in experimental ileitis; targets are inherent ER stress mechanisms in the epithelium and also the microbial milieu25. This study indicates that luminal iron may directly trigger epithelial cell stress-associated apoptosis through changes in microbial homeostasis. This links ER-stress, microbial composition and development of inflammation.
The transcription factor NF-ÎºB is linked to a wide range of disease processes26. Tissue-specific activation of NF-ÎºB in the gut epithelium of mice leads to spontaneous colitis27. However, this genetic effect depends on the gut microbiota because mice reared under germ-free conditions remain healthy. Thus, expression of the disease phenotype is clearly determined by gene-environment interactions, most likely involving a response of the epithelium to the microbial milieu in the gut.
Unequivocal experimental and clinical evidence have linked inflammation, or the inflammatory signalling response in various tissues, to the development of obesity-related alterations such as insulin resistance, glucose intolerance and endothelial dysfunction28. Some fatty acids activate inflammatory pathways directly via pattern recognition receptors of the innate immune system and consistently, Tlr4-/- mice are substantially protected from the ability of systemic lipid infusion to suppress insulin signalling29. ER stress-related mechanisms have emerged as a novel mechanistic link between inflammation and the development of insulin resistance and glucose intolerance, providing evidence that common cellular mechanisms are shared between metabolic and immune-mediated diseases30.
Extrinsic mechanisms in chronic disease
The prevalence of inflammatory disorders such as IBD, T1D and asthma has risen sharply and the spread of these diseases seems to follow lifestyle factors such as nutrition, hygiene, and exposure to environmental microbial components. Exogenous variables have a clear impact on the intrinsic mechanisms of chronic inflammatory disorders 31. For instance, in the non-obese diabetic (NOD) mouse model of T1D, the cumulative incidence of diabetes is higher in female than in male animals when reared under specific pathogen free (SPF) conditions32. When NOD mice are reared in fully germ-free conditions, however, this sex bias disappears. Furthermore, early exposure to a single commensal bacterial species in NOD mice reared under germ-free conditions confers protection against T1D. Thus, differences in microbiota exposure interacts with intrinsic factors such as sex to determine the expression of a pathologic phenotype. Similar evidence emerges from IBD-related animal models, clearly indicating that disease initiation depends on the presence of commensals33. Interestingly, the influence of the gut microbiota on the development of disease is, however, not unidirectional; a mutualistic cross-talk has been established between the immune system and the microbiome to maintain intestinal homeostasis34. Evidence suggests that the microbiome of NOD mice differs from that found in diabetes resistant (NOR) mice35. Exactly how these genotype differences influence the microbial environment remains to be determined but similar findings have been reported recently for the development of metabolic syndrome in mice lacking TLR536. A new classification of "commensal" and "facultative pathogenic" microbes of the gut microbiota needs invented as it appears to be variable in the context of genetic host factors.
Sequencing technology has been instrumental in opening up a whole new avenue of research into environment-gene interactions, namely the role of the microbiota living on various body surfaces in promoting and protecting against chronic diseases. The reference gene sets for the human gut microbiome that have been generated by collaborative networks such as the MetaHIT Consortium are already being used to identify associations between individual bacterial species and diseases including IBD and obesity37. Looking to the future, however, it is clear that a number of challenges have to be overcome. The identification of organ-specific core microbiomes (e.g. respiratory and gastro-intestinal tracts) and the possibility to identify disease-relevant alterations seems essential in order to address therapeutic options with respect to the inherent microbial ecosystem. Additional key issues include the ability to determine functionality when most mucosal commensals cannot be cultured, and to mechanistically dissect causality in animal models. Defined microbial ecosystems in disease-relevant animal model systems including gnotobiology will be essential in future efforts to understand microbe-host interactions on normal and susceptible genetic backgrounds. Furthermore, the necessary bioinformatic analyses for such studies will be at least as complex as they are for genome wide data.
Despite the common perception that the respiratory tract is largely a sterile body compartment, microbial sampling of the airways, including the lungs, reveals that the number of bacteria is similar to that in the upper intestine. The most prevalent species differ according to the anatomical level of the airway (e.g. oropharynx and upper lobes of the lungs). Comparison of the microbiota in the same region of the airway between healthy individuals and patients with respiratory inflammatory disorders such as asthma or chronic obstructive pulmonary disease also reveals clear differences, both in adults and children. For instance, patients with respiratory disease have larger numbers of known respiratory pathogens than healthy individuals. In contrast, anaerobic species appear to be much more common in healthy airways (unpublished, Cookson). Interestingly, host-associated microbial communities such as those of the human gut, skin, and vagina, show a very strong phylogenetic clustering, much stronger than that of samples from different terrestrial or marine environments38. Thus, each individual area of the body appears to represent a discrete ecological niche that determines which bacteria are selected from an apparently more phylogenetically homogeneous environment. Obtaining a detailed spatial description of the human microbiome presents a number of important challenges for future research.
Significant advances have nevertheless been made in understanding the influence of early-life microbial exposure on the subsequent risk of chronic disease. Children born in families living on farms with animals have a substantially reduced risk of allergy and asthma, and this effect appears to be due to the diversity of environmental microbes and fodder components to which they are exposed39. Interestingly, protection appears to begin prenatally, with maternal exposure to multiple species of farm animal seemingly having a cumulative effect. A birth cohort (the EFRAIM cohort) to examine the effect of prenatal microbial exposure is currently running.
Assessment of environmental exposure is limited by the extent to which we are able to measure its impact. There is a considerable lack of validated methods for more detailed determination of the variables. The sequencing of the human gut microbiome has raised hopes of microbiome-wide association studies, but no such approach can be taken in relation to the environment as a whole. Even in something as apparently straightforward as nutrition, experience has shown that accurate, reliable information on individual food consumption is hard to obtain. The challenge, then, must be to develop paradigms for accurate sampling of the environment. Efforts must be made to determine which environmental factors to sample, when, and how.
By combining genetic and epigenetic data with careful collection of environmental and biological samples, well designed cohort studies are expected to provide important clues as to the relationship between intrinsic and extrinsic mechanisms of disease. It may be time, however, to consider extending these approaches to include an embedded mechanistic component. As samples and data are generated, close collaboration with experimental research groups could allow hypotheses to be tested at an earlier stage while retaining the open-minded approach characteristic of epidemiological studies.
Emerging concepts in therapy and prevention
The emerging knowledge about intrinsic and extrinsic mechanisms underlying chronic inflammatory disease should be translated to the benefit of human and animal health. Although this represents a great challenge, insight into the response of the host to the environmental impact that determines the transition to disease chronicity will help identify novel therapeutic targets. Furthermore, identification of the exogenous variables that promote or protect against chronic inflammatory disease later on in life could raise realistic hopes of prevention.
A knowledge gap between research and clinical development has been identified in several disease areas. In Crohn's disease, for instance, many of the recent drug developments target TH1-mediated immunity which was thought until recently to play a central role in pathogenesis. However, clarification of the role of the NOD2 pathogen receptor in the gut highlights the importance of defects in innate recognition and handling of bacterial components and how this can impact on Crohn's disease. In addition, TH17 cells have been also implied recently as important mediators of inflammation in Crohn's disease. Their development is also linked to both, genetic variants as well as bacterial derived triggers40. This situation demonstrates the need to streamline the translation of research for clinical development41.
Target identification and development lead to the important question of whether our current knowledge of disease phenotypes and individual susceptibility supports the search for a single effective pharmacological target42. One such example is the development of novel drugs for the treatment of asthma. IL-13 was identified as a key therapeutic target in asthma, based on its ability to influence TH2 cell differentiation, airway inflammation and hyper-responsiveness. The importance of IL-13 was shown by several approaches with allergen-induced TH2-driven models of airway inflammation and remodelling. However, the results in human clinical trials were disappointing. Further subset analysis revealed several major sub-phenotypes of asthma: TH2 high-responders with up-regulation of IL-13 targets, increased bronchial hyper-responsiveness and a good response to corticosteroid treatment, and finally TH2 low individuals. These novel data clearly indicate that cellular and molecular characterisation and definition of phenotypes is necessary in order to identify target-responsive patient subsets43.
In the future much more information is therefore required on the range of phenotypes in disease classification. This will be achieved by "deep phenotyping" of well-defined patient groups. In parallel, experimental models used to identify putative cellular and molecular signalling cascades amenable to therapeutic intervention must adequately reflect the disease phenotype. Thus, both patient selection criteria and choice of animal models will depend on an improved classification of chronic inflammatory disease44.
The concept of probiotics-viable bacterial strains with a health-promoting activity has gained recently great attention as a possible mode for prevention of chronic inflammatory disease. In contrast to substantial data supporting the concept and efficacy of treatment e.g. viral intestinal infections with specific probiotic strains, there is inconsistent support for the efficacy of probiotics in preventing chronic inflammatory disease such as allergies45, 46. As with clinical trials of new asthma therapies, the challenge may be to define the correct biological variables. Furthermore it is now well-appreciated that all probiotic strains even of a given species are unique, while most of the results to datehave been obtained using distinct strains. Currently, a large number of bacteria are marketed without any clear demonstration of beneficial health effects. Hopefully, new regulations will require demonstration of such effects as well as the proven safety of probiotic strains. A major challenge will be to identify changes in the microbiota associated with disease. The relationship between different strains of bacteria and interactions with the gut epithelium is important in this area47. A further approach represents the development of recombinant therapeutics such as genetically engineered Lactococcus lactis expressing the tolerogenic cytokine IL-10. Experiments in murine models of ulcerative colitis have shown down-regulation of ongoing inflammation with this approach. Another potential therapeutic target represents T1D. Preliminary evidence from recent-onset diabetes in NOD mice suggests that the combination of this recombinant microbial strain and pro-insulin mediates effective reduction of the dose of anti-CD3 treatment, thereby limiting the potential toxicity associated with anti-CD348. A further challenge represents consistent dosing. Carefully controlled studies are required to identify the biological variables that determine viability and, therefore, dosage control in both genetically modified and naturally-occurring probiotics.
Breast feeding represents a natural way of modulating offsprings' cellular and molecular homeostasis through maternal environment. Many cellular and molecular components have been recently identified which do not only provide "passive protection", but impact immunological and metabolic maturation of the infant in a direct fashion. Evidence from several clinical studies shows that breast-feeding is associated with a reduced risk of obesity later in life. The risk of obesity continues to decline with longer periods of breastfeeding up to around seven months. Breastfeeding is also associated with slower weight gain. In contrast, rapid weight gain carries a 2-3 fold grater risk of obesity at school age and in adulthood. There are preliminary indications that this protective effect of breast-feeding is linked to the profile of the gut microbiota, perhaps influenced by maternal transfer of bacteria. Novel data suggest that probiotics given to infants can have a preventative effect on the development of obesity. In addition to the microbiota, protein intake might also play an important role because it is markedly higher in formula-fed than breast-fed infants. Reduced protein intake in the first twelve months of life is significantly correlated with reduced body mass index over a twenty-four month follow-up period. This is an important example indicating the relevance of the nutritional composition of breast milk49, 50.
Future research will determine the extent to which dietary modification can directly or indirectly influence the risk of developing diseases related to obesity, such as T2D, cardiovascular disease and also TH2-mediated allergy51. It will be important to better understand the impact nutritional and environmental variables exert on the development of the gut microbiota and the immune system. Furthermore, taking into account the role of genetic susceptibility, it will be important to determine who is likely to benefit from specific intervention in these disease areas.
Effective translational research
The hypothesis-generating research characterised by genome- and metagenome-wide association studies yields large amounts of data that must be validated in hypothesis-testing experimental animal models. Such animal model systems should consider the above mentioned intrinsic and extrinsic mechanism of disease development. This could be achieved by assembling the various modules of intrinsic and extrinsic disease modifiers, leading to well-defined (sub)phenotypes. This approach could be termed "synthetic macrobiology". A key challenge for the future, then, will be to obtain maximum benefit from the approaches and increase the likelihood of identifying effective treatment and prevention strategies. This calls for unbiased, high-throughput translational research in which the right disease targets are analysed in the most appropriate model and potential treatments tested in the right patients by clinical trials.