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OUTLOOK ALLERGIES

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Seeing beyond the hand we’re dealt


OUTLOOK ALLERGIES 24 November 2011 / Vol 479 / Issue No. 7374

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Magazine Editor Tim Appenzeller

ur picture of allergy is changing. For one thing there is a dramatic rise in prevalence. Eczema, hay fever, asthma, and potentially life-threatening allergies to certain foods, drugs or other substances, have become alarmingly common: the World Allergy Organization White Book on Allergy 2011–2012 estimates that about 30–40% of the world’s population is affected by one or more allergic conditions. As allergies spread, our understanding of them is changing. For 40 years, from its first identification in the 1960s, immunoglobulin E (IgE) was the focus of the field. As the physiological processes of hypersensitive reaction were described, chemical mediators identified and drivers of those mechanisms pinned down, it began to look as though control of the IgE pathways would open the way to allergy prevention and effective therapies. Epidemiology could also expect more evidence from geneticists equipped with new tools for hypothesis-free association studies thanks to advances in genome sequencing (page S10). This Outlook, however, is not a triumphant account of strategies that overcame allergy. Recent exploration of the complexities of the adaptive immune system has turned some assumptions about allergies upside down, and researchers are swimming in unfamiliar waters. Asthma, for instance, develops and responds in many different ways, not all triggered by allergy. It is better understood not as one disease, but many (page S20). Defects in skin and other epithelial barriers, once considered as symptoms of allergy, are being re-evaluated as potential primary causes (page S12). And, while environmental and lifestyle changes have been linked to the rising prevalence of allergy (page S2), attention to the role of gut-dwelling microbes has given a new dimension to the concept of a crucial balance toppled, with the human body seen not as a single organism but a complex ecosystem (page S5). We are pleased to acknowledge the financial support of Nestlé Research Center in producing this Outlook. As always, Nature retains sole responsibility for all editorial content.

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Editorial Herb Brody, Michelle Grayson, Roger East, Tony Scully Art & Design Wes Fernandes, Alisdair Macdonald, Kate Duncan Production Donald McDonald, Emilia Orviss, Leonora Dawson-Bowling, Stephen Russell Sponsorship Reya Silao, Yvette Smith, Gerard Preston Marketing Elena Woodstock, Hannah Phipps Project Managers Helen Anthony, Claudia Deasy Art Director Kelly Buckheit Krause

Nature Outlooks are sponsored supplements that aim to stimulate interest and debate around a subject of interest to the sponsor, while satisfying the editorial values of Nature and our readers’ expectations. The boundaries of sponsor involvement are clearly delineated in the Nature Outlook editorial guidelines available at http://www. nature.com/advertising/resources/pdf/outlook_guidelines.pdf. CITING THE OUTLOOK Cite as a supplement to Nature, for example, Nature Vol XXX, No. XXXX Suppl, Sxx–Sxx (2010). VISIT THE OUTLOOK ONLINE The Nature Outlook Allergies supplement can be found at http:// www.nature.com/nature/outlook/allergies. It features all newly commissioned content as well as a selection of relevant previously published material.

CONTENTS S2 LIFESTYLE When allergies go west Modernity nurtures these afflictions

S5 MICROBIOME Gut reaction Our friendly bacteria might be of help

S8 FOOD Picky eaters Taking the sting out of risky morsels

S10 GENETICS Seeking a gene genie Clues to who is prone are painting a bigger picture than anybody envisaged

S12 SKIN Into the breach A radical new theory of allergic disease

S14 ATOPY Marching with allergies Living from one disorder to another

S16 PERSPECTIVE Acting on the evidence Hywel Williams

S17 TREATMENT In search of a booster shot From symptoms to the root of the problem

S20 ASTHMA Breathing new life into research Recent insights are redefining the condition

S22 PERSPECTIVE A human touch Stephen Holgate

All featured articles will be freely available for 6 months. SUBSCRIPTIONS AND CUSTOMER SERVICES For UK/Europe (excluding Japan): Nature Publishing Group, Subscriptions, Brunel Road, Basingstoke, Hants, RG21 6XS, UK. Tel: +44 (0) 1256 329242.Subscriptions and customer services for Americas – including Canada, Latin America and the Caribbean: Nature Publishing Group, 75 Varick St, 9th floor, New York, NY 10013-1917, USA. Tel: +1 866 363 7860 (US/Canada) or +1 212 726 9223 (outside US/Canada). Japan/China/Korea:Nature Publishing Group — Asia-Pacific, Chiyoda Building 5-6th Floor, 2-37 Ichigaya Tamachi, Shinjuku-ku, Tokyo, 162-0843, Japan. Tel: +81 3 3267 8751. CUSTOMER SERVICES Feedback@nature.com Copyright © 2010 Nature Publishing Group S1


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When allergies go west Modern living seems somehow to make our immune systems overly sensitive. Is cleanliness at fault — or something else? BY DUNCAN GRAHAM-ROWE S 2 | NAT U R E | VO L 4 7 9 | 2 4 NOV E M B E R 2 0 1 1

erms can be good for us. This ‘hygiene hypothesis’ claims the urban ‘Western’ lifestyle, with its relatively limited exposure to infectious agents during childhood, might be behind the post-war epidemics of asthma, eczema and food allergies. But can the explosion in allergies in the developed world really be explained so simply? A more complicated theory is emerging. For, while it is apparent that there is something about Western living that increases the risk of developing allergies, research has shown that it’s not just a culture of antibacterial soaps, antibiotics and excessive cleanliness that’s to blame. There is evidence to suggest there is more about the way children are raised — where they are raised, what they are fed and how many siblings they have — that influences their burgeoning immune systems. It’s not just that their exposure to microbes or potential allergens seems to be limited. Sanitary living conditions disrupt the delicate balance between our bodies and a complex ecology of microbes and parasites with which we coevolved, and through which our immune systems are balanced and regulated (see ‘Gut reaction’, page S5). In 1989, epidemiologist David Strachan proposed the theory that a reduced exposure to dirt could render a person prone to allergy. The idea originally had a far narrower scope and, as Strachan, then at the London School of Hygiene and Tropical Medicine, but now at St George’s University of London, is at pains to point out, he didn’t actually use the term ‘hygiene hypothesis’. Strachan, like others, was trying to understand a perplexing public health paradox. With clean drinking water, sanitation, vaccinations and advances in medicine, many diseases were on the wane. So why was it that allergic diseases were becoming more prevalent, particularly in industrialized and urban parts of the world? The prevalence of allergies varies considerably, but many Western countries experienced a twentyfold increase in incidence. Asthma affects as much as 40% of the population in regions of New Zealand, Australia and the United States1,2. Cases of eczema identified as atopic, meaning that they are associated with a propensity for allergies, doubled and even tripled in some industrialized countries. While these increases appeared to plateau, allergies rose rapidly in developing nations where living conditions and hygiene standards were becoming more like those in the West. It was starting to look as though the causes of allergies had something to do with the nature of Western lifestyles. Strachan’s insight was to link increases in allergic disease to the declining size of families. NATURE.COM In his landmark study, For some of the which involved following latest research on more than 17,000 British allergies: children born in 1958, go.nature.com/fa6vfc

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OUTLOOK ALLERGIES


ALLERGIES OUTLOOK he found that there was an inverse correlation between allergic rhinitis, or hay fever, and the number of older siblings. This, he suggested, “could be explained if allergic diseases were prevented by infection in early childhood, transmitted by unhygienic contact with older siblings or acquired prenatally”. Older siblings appeared to increase the range of bugs that either a pregnant mother or a younger sibling would be exposed to, boosting a younger sibling’s protection either indirectly or directly.

A QUESTION OF BALANCE “The initial response from the immunologist community was highly sceptical,” recalls Strachan. The consensus was infections acted as triggers of allergic sensitization rather than as offering some form of protective influence. Strachan’s theory went against the grain. A few years later, in 1992, a plausible mechanism came along and people started taking Strachan’s idea seriously. This new explanation hinged on the upsetting of the delicate balance between populations of white blood cells, specifically two types of helper T cells — Th1 and Th2. Bacteria and viruses tend to elicit an immune response mediated by Th1 cells in which immune cytokines, such as interleukin-2 and interferon-gamma, are released. However, in animal models it was found that this Th1 response also serves to downregulate the Th2-mediated response, which can produce immunoglobulin E (IgE), the class of antibody that reacts to common allergens. This discovery supported Strachan’s theory — in developed countries, where the microbial burden might be low during childhood, insufficient stimulation The hygiene of the Th1 response hypothesis would fail to dampen gained ground, Th2 leading to overacand was tive Th2 responses. applied to other Evidence for the autoimmune hygiene hypothesis, diseases. as it quickly became known, started to pile up. Anne Wright and colleagues at the University of Arizona in Tucson found that children attending day care during the first six months of life were less likely to develop both eczema and asthma. Researchers started to study why farmers’ children suffer so much less from allergies (see ‘Benign exposure’). The hygiene hypothesis gained ground, and was applied to other autoimmune diseases, such as multiple sclerosis, inflammatory bowel disease, Crohn’s disease and type 1 diabetes. Meanwhile, the fall of the Berlin Wall in 1989 had opened up a unique opportunity to compare the effects of lifestyles of the East and West. Throughout the 1990s, Erika von Mutius, an allergist at Munich University Children’s Hospital in Germany, carried out a series of studies that found substantially lower incidence

of asthma and atopy among East German children compared to those growing up in the more developed West Germany, despite the fact that those living in East Germany were exposed to far higher levels of pollution. These results were echoed by other studies of children from Poland or Estonia and Sweden — relatively similar cohorts in terms of their genetic make-up, but which for 40 years had lived under very different economic and environmental circumstances. Much research has focused on asthma and eczema, although it remains unclear how the hygiene hypothesis explains food allergies. It is clear, however, that they too are on the rise. This has directed attention to the link between food allergies and other allergic diseases. While genetics definitely play a role, so too do environmental factors such as diet and antibiotics, says Clare Mills at the Institute for Food Research in Norwich, UK. “Environment affects the kinds of microflora in your gut. So if you live in a clean environment you may end up with a different group of bugs that promote allergy.” Little is known about the prevalence of food-specific allergies, particularly in developing countries, but efforts are underway to understand more. Mills led a project called EuroPrevall, which set out to study the allergy problem in Europe. This project also involves other studies in India and China, trying to understand how socioeconomic factors, diet, lifestyle and geographic variation can influence food allergies. The results of this work in Asia, which have yet to be published, were very revealing, says Mills. “What we found, particularly in India, was that they had virtually no food allergies.” The exception was highly developed Hong Kong, she says, where allergy incidences differed from the rest of China and were more like the West. Other longitudinal studies in developing countries are now recording increases in immunological disorders as countries become more affluent. One such study led by Emmanuel Addo-Yobo at the Komfo Anokye Teaching Hospital in Kumasi, Ghana, consisted of a 10-year study, comparing atopy, an inherited propensity to allergic hypersensitivity, and exercise-induced asthma in urban-rich, urban-poor and rural children. In partnership with Strachan and Adnan Custovic, at University of Manchester, UK, Addo-Yobo’s work shows that it was the urban and richer middle class Ghanaian children who were most likely to have developed both allergies. Other evidence does not always fit so well with the hygiene hypothesis. In highly urbanised Japan, where hygiene standards are high, asthma levels are much lower than in the United States or Australia. In the United States, asthma is increasing among children who live in very poor housing. And people in Barbados who live close to main roads and have high levels of endotoxins in their homes appear to

B ENIG N EXP OS U RE Help on the farm In 1999 Charlotte Braun-Fahrländer, an epidemiologist at the University of Basel, Switzerland, confirmed what rural doctors had observed for years, but which surprised most others: children who grow up on farms tend to have a reduced risk of developing hay fever and other allergies. This was backed up by work by Erika von Mutius, head of the Asthma and Allergy Group at the Munich University Children’s Hospital in Germany, who says that such children could be as much as three times less likely to develop allergies, compared with both city children and those from rural, but non-farming households. In 2002 the farm studies produced a fresh insight. Roger Lauener, head of allergy research at Zurich University Children’s Hospital in Switzerland, published research with von Mutius that showed that children growing up on farms had blood cells with significantly higher levels of toll-like receptor 2 (TLR2), which binds to microbes known to trigger innate immune responses. Yet none of them had any signs of infection and their white blood cell counts were normal. “How could it be that the environment was having an effect on the immune system without triggering an inflammatory response?” asks Lauener. The answer appears to be genetics: microbial exposure modulates the immune system at a genetic level by triggering the expression of these toll-like receptors, all without infecting the child. In farmers’ children, says von Mutius, the trigger has to do with contact with animals, and also the consumption of unpasteurized milk. Several studies have now shown a protective effect from unpasteurized cows’ milk against asthma, eczema, hay fever and allergic sensitization. It has even been found that both animal exposure and unpasteurized milk consumption during pregnancy can pass on protection to the unborn child for the first two years of a child’s life. It is not exposure to any single microbe that seems to count, but their diversity. In early 2011, von Mutius reported that children growing up on farms are exposed to a significantly wider range of microbes than other children, and she described an inverse correlation between the diversity of microbial exposure and the risk of asthma. Later in 2011 she published findings on the influence of unpasteurized milk consumption, which indicate that whey protein is implicated, although the mechanism has yet to be explained.

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ASTHMA AROUND THE WORLD UK

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Prevalence is the highest in the world, reaching 18.4% in Scotland and 15.3% in England.

7.6–10 5.1–7.5 2.5–5.0

CHINA Asthma prevalence is 2.1%, slightly below Russia (2.2%) but higher than Indonesia (1.1%).

0–2.5 No standardised data available

AUSTRALASIA New Zeland and Australia, at 15.1% and 14.7%, respectively, have higher prevalence of asthma than outside the UK.

AMERICAS Canada (14.1%) and the US (10.9%) have typically high asthma rates; Brazil and Peru also have high rates, wheras Mexico’s rate is only 3.3%.

receive no allergenic benefit, and instead have high levels of asthma.

TV, CARPETS AND CULTURE Thomas Platts-Mills, at the University of Virginia in Charlottesville and a former president of the American Academy of Allergy, Asthma and Immunology, says that the problem may lie with the word hygiene’. He believes in the hypothesis, he says, but not that hygiene alone is responsible; other aspects of lifestyle must play a part too. Platts-Mills sees a clue in the timeframe of these allergy epidemics. It’s no coincidence, he says, that asthma rates in the United States started to rise after the advent of

popular children’s TV shows such as the Mickey Mouse Club. “Prior to 1955 children came home from school and went outdoors to play. We now have a population that sits around the house and sits still in ways that children have never sat still before,” he says. This is important because research by his group has shown that people tend to sigh fewer times when watching TV, compared with when reading. Without these periodic expansions of the lung, the bronchial smooth muscle suffers, leading to non-specific bronchial reactivity, he says. Combine this with less exercise (which also reduces the amount of deep breathing and thereby stretching of

LOST PROTECTION The helminth connection Another, perhaps unlikely, source of protection against allergies might come from parasites. Many parasitic diseases that persist in developing countries, such as filariasis and schistosomiasis, have been almost eradicated in developed countries. While commendable from a public health standpoint, a perverse effect of this eradication might be a loss of protection from allergy. Parasitic worms, or helminths, were implicated in providing protection against allergies even before the hygiene hypothesis was proposed, says Maria Yazdanbakhsh at the Leiden University Medical Center in the Netherlands. Her research in Ghana has shown that catching schistosomiasis disease can modulate the expression of toll like receptor2 (TLR2), a similar effect to that found by Roger

Lauener and Erika von Mutius among children growing up on farms in Europe. This supports the idea that the organisms with which we co-evolved are important in helping to prop up our immune systems. Other studies in Gabon and Venezuela, and work in Vietnam by Carsten Flohr, have also shown that helminth eradication increases atopic skin sensitization. What’s more, research in 2011 by Harriet Mpairwe of the Uganda Research Unit on AIDS, established by the UK’s Medical Research Council and the Uganda Virus Research Institute in Entebbe, found that treatment against helminths during pregnancy is associated with an increased risk of infantile eczema — suggesting that, conversely, any protection against allergens afforded by helminths could be passed down to the as yet unborn child.

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the lungs), and dust mites lurking in wall-towall carpeting, and the conditions are ripe for asthma. Carsten Flohr, a paediatric dermatologist at St Thomas’s Hospital, London, makes another point about the influence of lifestyle factors. Despite evidence that suggests introducing foods early may reduce the risk of developing food specific allergies, concerns about food allergies have made parents in developed countries more cautious. As a result, we are introducing foods to children later in life, a behaviour which may inadvertently be making the problem worse, Flohr says (see ‘Picky eaters’, page S8). Strachan agrees that the term hygiene hypothesis has become a little tired. Microbial deprivation still seems the most promising candidate to explain the rise in allergies, he says, and the evidence remains solid that sibling numbers are an influence, but attributing it to Westernization rather than hygiene is a better way of capturing the multifaceted nature of the phenomenon. Regardless of what the theory is termed, no one is advocating that the developed world abandon modern urban lifestyles and move en masse to the countryside to work on farms. Nor is it reasonable to have more children or deliberately catch a bad case of worms (see ‘Lost protection’) just to develop better protection against allergies. Yet the evidence continues to mount that there is something amiss about modern urban life in the developed world, and something about the West cleaning up its act that is exacerbating the burden of allergies. ■ Duncan Graham-Rowe is a science writer based in Brighton, UK. 1. Peat, J. K. et al. The Med. J. Austria 163, 22–26 (1995). 2. ISAAC Steering Committee. Lancet 351,1225–1232 (1998).

MASOLI, M. ET AL. THE GLOBAL BURDEN OF ASTHMA (2004).

Prevalence of clinical asthma as proportion of total of population (%)

Global studies confirm an association with westernized lifestyles


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University of Gothenburg in Sweden, brought gut microbes into the equation. Wold and her colleagues observed that typical gut bacteria colonize infants in Pakistan earlier than they colonize infants in Sweden. This delay, Wold suggested, could compromise immune tolerance — affecting the ability to cope with harmless antigens such as food and pollen. Gary Huffnagle, a microbiologist at the University of Michigan in Ann Arbor, has built on this concept of the hygiene hypothesis. He proposes that the Western lifestyle can dramatically alter our gut microflora leading to allergies and other inflammatory diseases, an idea he calls the ‘microflora hypothesis’. The theory is supported by observational evidence. City dwellers, increasingly the predominant demographic, are exposed to a narrower range of microbes than people in rural areas — and they get more allergies. Children in rural Burkina Faso, where allergies are rare and the typical diet is high in fibre, have a different profile of microbes in their faeces than children living in Europe. The rapid increase in allergic diseases in the West has coincided with widespread use of antibiotics, especially broad-spectrum drugs. Antibiotics can profoundly alter the microbial composition of the gut, and studies show that children who are given antibiotics in their first year are more susceptible to allergies. “More and more of these smoking guns point to the role of the microbiota affecting immune development,” says Brett Finlay, a microbiologist at the Michael Smith Laboratories, University of British Colombia in Vancouver.

BUGGING THE IMMUNE SYSTEM

MICROB I OME

Gut reaction Microbes are under the spotlight in efforts to unravel — and combat — allergies. B Y C A S S A N D R A W I L LYA R D

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he twists and turns of the human gut support an active and diverse microbial ecosystem. The tens of trillions of bacteria aren’t just hitchhikers; they interact intimately with the immune system, and are so integral to our health that some scientists have deemed them the “forgotten organ”. Today scientists are trying to unravel the relationship between changes in lifestyles in recent decades, changes in our microbiota, and the skyrocketing prevalence of allergies in the

developed world. Establishing a link between these phenomena could lead to treatments for allergies and asthma. It was the ‘hygiene hypothesis’ (see ‘When allergies goes west’, page S2) that first posited a causal link between Western lifestyles and allergy. Scientists found that zealous use of antibacterials, from cleaning products to antibiotics, had limited exposure to pathogens in early childhood. They suggested that the regulation of immune responses was compromised by this limited exposure. In the late 1990s, Agnes Wold, a bacteriologist at the

The immune cells in the gut are in constant contact with a diverse microbial milieu, and the human gut “has more immune cells than the rest of the body put together,” says David Artis, a microbiologist at the University of Pennsylvania in Philadelphia. To an immune cell, beneficial or harmless bacteria (known as commensals) look much like harmful ones, but the beneficial bugs have developed methods of shaping the function of the immune system, so that their presence doesn’t provoke an immune attack. “These bugs are flipping switches,” says Sarkis Mazmanian, a microbiologist at the California Institute of Technology in Pasadena. If these beneficial microbes fail to colonize our guts early in life, or if they succumb to a course of antibiotics, then switches don’t get flipped and the immune system can become hypersensitive, attacking harmless microbes and other substances such as pollen, pet dander or shellfish — or so the thinking goes. NATURE.COM Scientists are still tryJust how friendly ing to figure out which is our gut switches are flipped, microbiota? how the commensal go.nature.com/loqptl bacteria flip them, and

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what the consequences are. “I think there will probably be multiple pathways through which commensals can influence allergic disease,” Artis says. Several of these pathways appear to involve regulatory T cells: immune cells that suppress inflammation by keeping the immune system in check. “Our immune system is sort of like a loaded gun, and as soon as there’s a microbe, it wants to fire,” says Mazmanian. Mice lacking regulatory T cells develop allergies or autoimmune diseases, and research suggests that some microbes can increase their abundance or boost their activity. Kenya Honda, an immunologist at the University of Tokyo, has been investigating this link. Honda’s research focuses on bacteria in the Clostridium genus, many species of which live symbiotically in the intestines of mice and humans (although others, like C. dificile, are highly pathogenic). His team took mice that had been bred to be free of microbes, and inoculated them with a mixture of 46 different Clostridium strains. Sure enough, this was a catalyst for the production of regulatory T cells in the colon; inoculation with other types of bacteria had little or no such impact2. The team then used the same 46 Clostridium strains to boost the microbiota of standard laboratory mice, which typically already have Clostridium bacteria at a low level, and subjected them to tests that would ordinarily provoke an allergic response. They found that the Clostridium-boosted mice exhibited much more muted allergic responses than a control group, suggesting that microflora rich in Clostridium can provide at least partial protection against allergies. Another mouse study, yet to be published, reinforces Honda’s findings. A team led by Cathryn Nagler, an immunologist at the University of Chicago in Illinois, found that mice treated with antibiotics designed to eliminate Clostridium species had more food allergies than untreated mice. Nagler’s team also found fewer regulatory T cells in the lining of the colons of these mice. “One of our challenges now is to see how those regulatory cells get out of the colon to mediate protection against allergic disease,” Nagler says.

SEEKING OTHER SWITCHES Mazmanian has focused his attention on an entirely different member of the gut microbiome, Bacteroides fragilis, which produces a molecule called polysaccharide A (PSA). Mazmanian and his colleagues have Mice treated already shown that with antibiotics PSA can prevent and designed to treat inflammatory eliminate bowel disease and Clostridium multiple sclerosis in species had more mice, and speculates food allergies that it might work than untreated against allergies too. “It directly activates mice.

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OUTLOOK ALLERGIES

Scanning electron micrograph of bacteria (coloured rods and spheres) among the milk solids in yoghurt.

those regulatory T cells,” he says, by signalling through Toll-like receptor 2 (TLR2), a protein found on T cells and other immune cells. Tolllike receptors bind to microbial molecules, and activation of this receptor typically ramps up immune activity. But Mazmanian found that PSA instead enhanced the function of immunesuppressing regulatory T cells3. Nagler’s work also implicates a Toll-like receptor, TLR4. In 2004, Mazmanian and her colleagues reported that a mutation in TLR4 made mice particularly susceptible to food allergies, and that administration of antibiotics to mice with normal TLR4s made them as susceptible to food allergies as their counterparts with defective receptors. TLRs appear to be one of the switches that commensals use to flip immune activity, although others are likely to exist, Mazmanian says. “There is indirect evidence that other commensal bacteria do not use TLRs to coordinate immune responses.”

BEYOND THE YOGHURT CURE As bench scientists work to unravel exactly how commensals interact with the immune

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system to curb allergies, clinicians and the food industry are searching for ways to tweak our microflora to prevent and treat allergic diseases. Yoghurt and other cultured food products labelled ‘probiotic’ contain bacteria said to be effective against a variety of ailments. More than 25 randomized clinical trials have examined the use of probiotics to treat or prevent allergies. Many of these studies have focused on strains of Lactobacillus, a genus of bacteria long used to manufacture cheese, yoghurt and other cultured foods. In a 2001 study of newborns in Finland, one group of pregnant women with a family history of allergies were given a strain of Lactobacillus casei known as ‘GG’ during the last weeks of gestation, and their babies were given GG for the next six months. This appeared to reduce the children’s susceptibility to eczema, which often occurs in children who later develop asthma. At the age of two, the frequency of eczema in the probiotic group was half that of a placebo group. A follow-up study published in 2007 suggested that the beneficial effects


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ALLERGIES OUTLOOK were still evident at age seven. However, a more recent study in Germany with a similar protocol failed to find any effect of Lactobacillus GG on childhood eczema4. Why the contradictory results? Marko Kalliomäki, a paediatrician at Turku University Hospital and author of the Finnish study, notes that the studies used different methods for diagnosing eczema. Genetics may have played a role as well, he says. Michael Cabana, chief of paediatrics at the University of California, San Francisco, who is testing the effects of the same Lactobacillus strain on eczema in 270 infants, speculates that dietary differences could be relevant too. “If you have a diet that’s already high in fermented foods and probiotics, you might not see the effect of a probiotic supplement,” he says. Another study has found that 6-month-old infants who received supplements of Lactobacillus GG had other beneficial bacteria too, and that the microbial communities in their guts appeared to be resistant to perturbations and the growth of pathogens. The probiotic appears to be “affecting the whole community structure,” Cabana says. He likens it to baseball. “Sometimes mid-season a team will add one player, and that one player just changes the whole dynamic, and they become a championship team,” he says. Research looking at using probiotics as a treatment for allergies is equally mixed. A 2008 review by the Cochrane Collaboration, based in Oxford, UK, included 12 randomized controlled trials. It concluded that the studies, taken together, “do not suggest that probiotics are an effective treatment for eczema”. It’s possible that probiotics researchers have simply not yet found the correct bacterial strain or combination of strains. Clinical trials tend to examine strains with good safety profiles, so those with the longest safety record, such as Lactobacillus, “are the ones that get studied more”, Cabana says. Honda and his colleagues are now working to isolate Clostridium from the human gut, and intend to test whether these strains induce a regulatory T-cell response in humans like that observed in mice. “And perhaps those Clostridium species can be applied to treat allergies,” Honda says.

FORTIFYING THE BENEFICIAL BACTERIA Another approach to enriching the microbiota is nourishing bacteria in the gut with substances called prebiotics. One such prebiotic is inulin — a compound produced by plants that humans cannot digest. Inulin travels through the digestive tract to the colon. Once there, it provides food for bacteria, especially bifidobacteria — a genus found in breast-fed infants and also used as a probiotic. Today’s prebiotics are what David Mills, a molecular biologist at the University of California, Davis, calls first generation and broad acting. As yet, he says, “there are no prebiotics that are tailored to enrich a specific population”.

TH E LU NG L INK Bacteria’s role in asthma It’s not just microbes in the gut that shape our susceptibility to allergies. Bacteria in the lungs, once thought to be a sterile environment, might also play a role. A study by Hans Bisgaard at the University of Copenhagen found that infants who had harmful bacteria in their lungs soon after birth were more likely to develop asthma than babies that didn’t have those strains. And in 2011, researchers at the University of California, San Francisco, led by Homer Boushey, reported that the lungs of asthmatic adults contained far more bacteria than the lungs of people without asthma. Furthermore, individuals with more severe cases of asthma had a greater diversity of bacteria than patients with less acute disease. A stomach-dwelling bacterium long associated with ulcers and stomach cancer, Helicobacter pylori, has also been linked with asthma, but in this case the link appears to be beneficial. Studies by Martin Blaser, a microbiologist at New York University, show that children infected with H. pylori were 40% to 60% less likely to have asthma than children who weren’t infected. A study, published in August 2011, supports Blaser’s finding5. Researchers in Switzerland and Germany infected 6-day-old mice with H. pylori and then attempted to induce an asthma-like disease by exposing them to allergens. The lungs of uninfected mice became inflamed, while mice infected with H. pylori didn’t experience any such allergic

In theory, probiotics and prebiotics could be combined to increase the propensity of particular bacteria to take up residence in the gut and thrive. Huffnagle predicts that, some day, patients will go to the doctor and be prescribed a combination of probiotics and prebiotics designed according to their individual floral profile. But this generation of probiotics may not be bacteria you’d want to eat in a cup of yoghurt. “Some of the stuff they make smells nasty,” Huffnagle says. “They’ll be capsuled.” Another approach could be to isolate and administer only the beneficial molecules produced by the microbes. Mazmanian, for example, speculates that “some day PSA could be a drug, just like insulin”. There are many obstacles to overcome. Researchers don’t yet know what a healthy gut looks like, nor have they uncovered a profile for the ‘allergic’ gut. Although the evidence suggests that our microflora play a role in

Helicobacter pylori bacteria, coloured scanning electron micrograph

symptoms. Their lungs did become inflamed, however, after researchers gave them a dose of antibiotics to eradicate the bacteria. The researchers also noticed that regulatory T cells accumulated in the lungs of infected mice, and they speculate that these cells keep the allergic response in check. Anne Müller, a cancer researcher at the University of Zurich and author on the study, points out that regulatory T cells can travel from one mucosal surface, such as the stomach, to another, like the lung, and in this way “a stomachdwelling bacterium can influence systemic immune responses”.

the development of allergies, Artis points to a host of other factors. “It’s host genetics coupled with lifestyle coupled with potential effects of commensal bacteria, all operating simultaneously to influence disease susceptibility,” he says. The complex origins of a disease may help explain why some people appear to respond to probiotics while others do not. Therapies that work for one person may not work for another, Huffnagle says. “Fixing the ‘broken’ microflora — that’s still a shot in the dark.” ■ Cassandra Willyard is a freelance science writer based in Brooklyn, New York. 1. Bisgaard, H. et al. J. Allergy Clin. Immunol. 128, 646–652 (2011). 2. Atarashi, K. et al. Science 331, 337–341 (2011). 3. Round, J. L. et al.Science 332, 974–977 (2011). 4. Kopp, M. V. et al. Pediatrics 121, 850–856 ( 2008). 5. Arnold, I. C. et al. J. Clin. Invest. 121, 3088–3093 (2011).

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once only common in children younger than five or six years of age, now often endure into adolescence. The reasons remain obscure, but the costs are clear: in the United States, where food allergies are the leading cause of anaphylaxis, the cost of healthcare and lost productivity amounted to US$510 million in 2007, according to estimates in a Virginia Commonwealth University study published in April 2011.

ADVICE IN FLUX

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Picky eaters Clinical trials are testing how careful exposure could protect people with potentially lethal allergies to everyday fare. BY REBECCA KESSLER

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spring roll may seem to be an innocent appetizer, but the shadow of death lurks between its crispy golden layers for the peanut allergy sufferer. The peanut butter sometimes used as spring roll sealant can send unwitting victims into the throes of anaphylaxis. At first, their skin may break out in hives. Their lips, tongue, and throat may start to tingle or itch. Then, in the worst cases, blood pressure drops, breathing becomes

difficult, consciousness fades, and death ensues. The prevalence of food allergies in developed countries has escalated to the point where 8% of children in the United States have one or more allergy, according to a study led by researchers at Northwestern University in Chicago, Illinois, published in June 2011. Similar rates are reported or estimated for Canada, the United Kingdom, France and Norway. Allergists have observed that individual patients tend to react to more types of foods than was typically the case a few decades ago. And allergies to dairy and egg,

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Babies with a known allergy-related disease, including eczema, or who have either a sibling or parent afflicted, are considered at high risk for developing food allergies. Until recently, it was standard practice in several developed countries to advise mothers of such children to avoid eating potential allergens during pregnancy and while breastfeeding; breastfeed infants exclusively for 4–6 months; and introduce potential allergens to a baby’s diet only after 2–3 years of age. The idea was to “wrap the infant up in a sort of immunological cocoon and not expose them to proteins that could launch allergic reactions,” says Gideon Lack, professor of paediatric allergy at King’s College London. However, research suggests this could be the entirely wrong approach. “There is a possibility that we were achieving the reverse of our intentions through this avoidance policy,” says Lack. A 2008 study that Lack co-authored showed that the incidence of peanut allergies among Jewish children in Israel is tenfold lower than among Jewish children in the United Kingdom, although young Israelis eat a great deal more food containing peanuts. This and other research led the American Academy of Pediatrics to revise its recommendations to its members in 2008, to state that there is insufficient evidence to support restricting either the mother’s diet during pregnancy and lactation or the infant’s diet after 4–6 months. The UK Department of Health followed suit in 2009. “Some mothers, when they have a peanutallergic child, say: ‘I don’t want to expose my [other] child to peanuts.’ We can cite studies that suggest that may be the way to go. If they say ‘no, I want to eat everything’, we can cite studies that say that may be the way to go,” says Hugh Sampson, director of Mount Sinai School of Medicine’s Jaffe Food Allergy Institute in New York and a specialist in paediatric allergy. “The biggest thing is not to let mothers feel guilty about whatever choice they make, because at this point we really don’t know the best answer.” Three large studies currently underway aim to resolve the lingering uncertainty about when, and whether, to expose children to potential allergens. Lack leads the Learning Early About Peanut Allergy (LEAP) study, which began in 2006 and is due to publish results in 2013. It follows 640 babies, half of whom are considered high risk for NATURE.COM food allergies, to find out For some of the whether eating peanut- latest research on containing foods during allergies to food: infancy leads to peanut go.nature.com/2qzyoz

JONATHAN BURTON

OUTLOOK ALLERGIES


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RANCÉ, F, GRANDMOTTET, X. & GRANDJEAN H. CLIN. EXP. ALLERGY 35,167–172 (2005). GUPTA, R. S. ET AL. PEDIATRICS 128, 9–17 (2011).

ALLERGIES OUTLOOK allergies. Lack is also leading the Enquiring About Tolerance (EAT) study, due to report in 2015, probing the best time for infants to begin eating solid foods in general. EAT is comparing two strategies to see whether exclusive breastfeeding up to 6 months of age better prevents allergies than breastfeeding but also introducing 6 potentially allergenic foods at 3 months of age. A third study is tracking more than 500 babies who tested positive for allergies to milk or egg, which puts them at high risk for developing peanut allergy. It aims to illuminate the role of maternal diet by seeing which babies go on to develop peanut allergies. Interim results published in 2010 seem to support the “cocoon hypothesis” that it is best to minimize exposure to allergens, since infants whose mothers ate more peanuts during pregnancy had higher levels of the peanut antibody in their blood, but it remains to be seen whether more of them will develop full-blown peanut allergies, says Sampson, the study’s principle investigator.

REDUCING SENSITIVITY, CHASING TOLERANCE Attempts to treat allergy sufferers by exposing them to incremental doses of the substance that torments them date back a century. Allergy shots, which physicians administer to treat allergies to dust, pollen, pet dander and other irritants, are a prime example of the technique, called desensitization or immunotherapy. But researchers have trodden warily in applying it to food allergies, because the reactions can be so severe. The death by anaphylaxis of a 15-yearold volunteer in a study on peanut injection immunotherapy in 1991 spread a pall over the field. John Oppenheimer, an allergist at the University of Medicine and Dentistry of New Jersey, led that ill-fated study. Twenty years later, he is sad that patients are still languishing in a “timebomb-like existence” where a mouthful of the wrong food could mean death, though he is heartened by recent research progress. “I tell this to all my young patients,” Oppenheimer says. “I’m hopeful that by the time they go to college I’ll have something to offer them that’s better than avoidance, which is all I have right now.” If injecting food allergens is too risky for therapy, what about administering a tiny controlled dose with food? This seemingly more obvious avenue of delivery has yielded some positive results. In March 2011, researchers at the University of Cambridge, United Kingdom, reported that an oral immunotherapy trial of 22 peanut-allergic children fed incremental doses of peanut flour each day over the course of 30 weeks had raised their median tolerance by a factor of 1,000 — enough to abate trouble after any accidental ingestion. After treatment, 14 of the 22 children had no allergic reaction to eating the equivalent of about 32 peanuts, and another 4 children had only mild or moderate reactions. Also in March 2011, researchers at Duke University Medical Center in Durham,

PREVALENCE OF FOOD ALLERGIES AMONG CHILDREN USA Reporting one or more food allergy (%) Reporting specific allergy to (%):

Peanut

France

8.00

6.70

2.02

0.55

Milk

1.69

0.80

Shellfish

1.38

0.36

Other foods

2.92

5.00*

*includes eggs (0.63%), kiwis (0.6%), tree nuts (0.52%), fish — unspecified (0.52%)

North Carolina, reported early results from an ongoing oral immunotherapy study involving 25 peanut-allergic children. After a year, the 16 children in the test group all ate the equivalent of 20 peanuts, with only one suffering a mild reaction, whereas five of the nine taking placebos could still tolerate little or none, and the median tolerance of the placebo group was just one peanut. In May 2011, Sampson’s group reported the results of a study on a similar approach sometimes referred to as ‘natural immunotherapy’. Over a 5-year period, 70 children allergic to milk were fed muffins and waffles, and later cheese pizzas, containing milk baked at progressively lower temperatures. Children in the treated group were 16-times more likely to tolerate uncooked milk than those in the control group.

Spring rolls: not always a treat for the allergic.

Oral immunotherapy seems to help about 80% of people tested so far, according to Sampson. Adjusting the treatment regime could help the remainder. In clinical trials, Sampson, Lack and others have shown that the safety and efficacy of this therapy improve when subjects take the drug omalizumab, a treatment developed for allergic asthma. Widespread use of omalizumab for this purpose, however, would be hugely expensive; its average wholesale cost in the United States works out at roughly US$20,000 per patient per year. Researchers are also investigating the delivery of allergens in liquid form under the tongue or through a skin patch. Both approaches seem to reduce adverse reactions, Sampson says. However, despite encouraging results, immunotherapy studies have so far tended to be small, lack sufficient controls, and be of too limited duration to reveal any long-term effects of treatment. There’s also little evidence of any improved tolerance persisting if the regular allergen dose is discontinued; typically the effects wear off after

anything from 1 week to 2 months, according to Sampson. “That’s what people don’t really understand,” says Wesley Burks, director of the Duke University group. “They see the desensitization effect and they think it’s a more permanent cure.”

INTO THE CLINIC In an effort to hasten the arrival of a safe treatment for food allergies, the US not-for-profit group Food Allergy Initiative (FAI), based in New York, gathered about 40 allergists to debate the best way forward. The group’s consensus, according to FAI’s executive director Mary Jane Marchisotto, was that out of several experimental treatments — including a Chinese herbal formula, a parasitic worm, and vaccines — oral immunotherapy has the best prospects. Subsequently, FAI has undertaken a campaign to orchestrate and raise funding for a phase III clinical trial in hundreds of subjects to lay the scientific groundwork for approval of an oral immunotherapy regimen by the US Food and Drug Administration (FDA). Sampson, who participated in the FAI gathering and receives FAI funding, estimates that this trial might yield an approved treatment within seven years. He notes, however, that the Chinese herbal formula developed, patented, and tested by his team, has promise and might bear fruit sooner. His colleagues are currently enrolling subjects to test it against several food allergies. If the herbal formula passes trials, it could be brought to market as early as 2013. As a herbal supplement, unlike a drug, it doesn’t need to go through the lengthy and stringent FDA approval process, although the team does intend to pursue FDA approval in the long run. Meanwhile, Burks says more than 100 families are lined up to enrol in his oral immunotherapy studies. At this stage, however, he is concerned when he hears of allergists using oral immunotherapy on patients outside the watchful supervision of controlled trials at major medical centres, and even of desperate parents attempting to treat their food-allergic kids at home. This is a bad idea, he warns, since serious adverse reactions can occur. “The use of oral immunotherapy should be still investigational,” Burks says. “It’s really not ready for practice.” ■ Rebecca Kessler is a freelance journalist in Providence, Rhode Island.

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OUTLOOK ALLERGIES

G ENETICS

Seeking a gene genie Rare gene variants could be key to unlocking the underlying genetics of allergy, now that whole genome sequencing and other technologies have sharpened the focus of epidemiology. B Y E R I C A W E S T LY

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o to a doctor with allergy-related symptoms and it’s a safe bet you’ll be asked about family history. Clearly there is a heritable component to allergies. The genetic elements, however, have proved fiendishly hard to pinpoint. Since a working draft of the the human genome was published in 2001, the focus of research has shifted from trying to link specific genes and allergies, to genome-wide association studies of increasing sensitivity. Evidence of ethnic propensity, and an interplay between environmental factors and genetic predisposition, are starting to acuminate this more subtle search for the heritability factors in allergy and asthma. The potential rewards are huge. For a start, allergic diseases are heterogeneous in terms of symptom profile and symptom severity. Deciphering the underlying genetics would help researchers make connections between the various allergy types and subtypes, which could aid both diagnosis and treatment. Clinicians are also interested in using genetics to clarify the relationship between allergy and asthma;

epidemiological reports indicate that about 80% of childhood asthma patients also suffer from allergic rhinitis, or hay fever, but there are still many questions about how, why and when this association develops (see ‘Breathing new life into research’, page S20).

NO SINGLE CULPRIT When geneticists started studying allergy 40 years ago they followed the functional candidate gene approach, using symptom profiles to identify genes likely to be of interest. Most of the early gene associations in this field involved the immune system. A particular favourite for investigation was the human leukocyte antigen (HLA) gene family, on chromosome 6, which encodes proteins that help the body recognize allergens and other invaders. The problem was that few of these gene associations reached statistical significance. By 2010, nearly 1,000 studies had been published proposing various candidate genes for allergic disease, but there was still no clear answer. Some intriguing candidates have emerged, however, particularly in the past few years. One contribution came when researchers at the

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University of Dundee in the UK linked a skin disorder called ichthyosis vulgaris to a relatively rare mutation in FLG, the gene for filaggrin, a key epidermal protein. Subsequent studies tied FLG mutations to several allergic conditions, including eczema, asthma and, most recently, peanut allergy. This genetic data, combined with biochemical evidence, led researchers to the hypothesis that allergen susceptibility may be because of an impaired epithelium rather than further downstream in the immune system. Genome-wide association studies are one of the strongest methods for finding new candidate genes because they use larger sample sizes and they allow researchers to scan for thousands of variants at once. “One of the benefits is that you do genome-wide association studies without a hypothesis,” explains Michael Kabesch, an asthma genetics researcher at Hannover Medical School in Germany. “So you can find connections you hadn’t thought NATURE.COM about before.” For exam- Research the ple, in 2007, a European genetics behind genome-wide association allergies: study linked a mutation go.nature.com/e5keru


CORBIS

ALLERGIES OUTLOOK on chromosome 17 to asthma but found no link to atopy, which was unexpected given the high incidence of overlap between the two conditions. This finding, which has since been replicated by groups in the United States, Japan and elsewhere, suggests that asthma is not necessarily as tied to allergen exposure as previously believed and may involve novel pathways. Even more surprisingly, there seemed to be an association with the digestive disorder Crohn’s disease, a link that Kabesch is investigating further.

of California, San Francisco. When it comes to developing allergies and asthma, “it shows that ancestry matters,” he adds. For example, a variant of the DENND1B gene has been associated with asthma in children of European ancestry, but it appears to offer a protective effect in African American children. Geneticists refer to this as the ‘flip-flop phenomenon’. The epigenetics approach, which seeks to understand how a phenotype may result from the interaction of genes and environmental factors, has been paired with ethnicity-based perspectives through research by Burchard’s group. In a recent study they showed a correlation between a mutation in CD14, an immune system gene, and asthma severity in Mexican children who had been exposed to tobacco smoke. Providing more evidence of the relevance of epigenetics in understanding allergy, recent European studies suggest that microbes found on pets and farm animals can cause alterations in CD14 and in genes that control the Toll-like receptors, which are also involved with the immune response and confer a protective effect against asthma and allergies. In order to translate these findings into treatments or preventive strategies, however, researchers need to figure out when and how these epigenetic interactions occur.

ALLERGEN-SPECIFIC PROFILES The evidence is equivocal when it comes to determining correspondence between genetic profile and specific allergies. One school of thought suggests that although genes control a person’s susceptibility to develop allergies, the actual allergens to which the person reacts are determined by the environment. This theory is supported by anecdotal reports of patients trading one allergy for another, say pollen for mould, after moving to a new climate. On the other hand, there is evidence connecting particular mutations to specific allergens: a 2003 study, for instance, found a link between a specific HLA variant and sensitivity to rat allergens. “There is a lot of conflicting evidence,” says Rana Tawil Misiak, a clinician who studies allergy at the Henry Ford Health System in Detroit, Michigan, and the lead author of a recent review of allergen specificity 1. Misiak says that the best prospect of clarification lies in studies that are large enough to include genetic variants that have been associated with different allergies. Indeed, some researchers, including Misiak, have proposed that allergy may only be associated with rare genetic variants, such as the FLG mutation, which would explain the absence of positive data from genome-wide association studies that look for links with common gene variants. The trouble is that genome-wide analyses simply haven’t been sensitive enough to detect rare variants. That is changing, however, as whole genome sequencing technologies become more affordable. Genome-wide association studies normally just scan for single DNA mutations (single nucleotide polymorphisms, or SNPs), but incorporating whole genome sequencing would allow researchers to analyse a larger set of genetic variations. The multi-institute 1000 Genomes Project, the largest whole genome-sequencing effort to date, has already sequenced the genomes of more than 2,000 people and identified millions of previously unknown gene variants. It’s still unclear whether any will relate to asthma and allergies, but Carole Ober, a geneticist at the University of Chicago, Illinois, who specializes in asthma and allergy, is optimistic. “My guess is that there are at least a few important rare variants that haven’t yet been discovered,” she says. Expanding the population genetics studies to include subjects of non-European ancestry

What role does ethnicity play in allergy risk?

should also help identify new candidates. Epidemiological evidence suggests there is a connection between ethnicity and risk of developing allergy and asthma. According to the Centers for Disease Control and Prevention, children of Puerto Rican descent have the highest risk for developing asthma of any group in the United States, with prevalence rates as high as one in six; 125% higher than Caucasians and 80% higher than African Americans. Another study in 2009 found that eczema is significantly more prevalent in African and Latin American countries than in Asian nations, such as China and India. Yet non-European ethnic groups have been severely under-represented in the population genetics studies of allergy-related disease. This is starting to change. In 2010, researchers from Johns Hopkins University in Baltimore, Maryland, published the first genome-wide analysis of asthma and allergy to focus exclusively on people of African American and African Caribbean descent2. The chromosome 17 mutation associated with asthma in Asian and European populations did not prove significant in this study, but the researchers did find an association between four other genes, including one on a different part of chromosome 17. Ethnicity could also play a role in a person’s reaction to an allergen. Researchers are using ethnicity-specific gene chips with single DNA mutations to study asthma and allergy genetics in Latin American and African American populations. “We’re finding that the same variant can confer either risk or protection depending on ancestry,” says Esteban González Burchard, an asthma genetics researcher at the University

LOOKING BEYOND BLOOD Methodological issues may explain why it is so difficult to replicate findings like this in largescale studies. “The tricky part when you start to get into epigenetics is you have to be looking at DNA from the appropriate cell type, and we don’t always know what that’s going to be,” says Ober. “It’s a challenging area because we’re really in the dark about where to look. You may need to have a specific lung or immune cell type to see the relevant genetic modification.” Collecting the tissue samples for this is a lot harder than obtaining blood, the traditional biologic material used for genetic studies. As a result, these studies are in their infancy, and the sample sizes will be quite small compared to genome-wide association studies, which typically include thousands of subjects. “Many people are willing to give blood for science, but bronchoscopy (the procedure used for taking lung tissue) is much more invasive,” says Kabesch. Fortunately, advances in stem cell technology might offer a non-invasive alternative. What’s more, epigenetics allergy studies might do more than help scientists understand the development of allergies. Their findings could also inform research into other disorders that involve gene-environment interactions such as autism and schizophrenia, which is a rich incentive indeed. ■ Erica Westly is a freelance science writer based in Brooklyn, New York. 1. Misiak, R. T. et al. Curr. Allergy Asthma Rep. 10, 336–339 (2010). 2. Mathias, R. A. et al. J. Allergy Clin. Immunol. 125, 336–346 (2010).

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STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY

OUTLOOK ALLERGIES

The orderly layers of healthy skin contrast with the eruptions of eczema (right image).

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Into the breach A focus on skin barrier disorders has opened up new thinking about how allergies kick in. BY CLAIRE AINSWORTH

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n the autumn of 2005, Alan Irvine, a dermatologist at Trinity College Dublin, noticed something unusual about a cohort of patients with ichthyosis vulgaris — a dry, scaly skin disease. Irvine had been working with geneticist Irwin McLean at University of Dundee, UK, whose team had recently identified two mutations in the gene behind the inherited disorder. But Irvine’s new observation pointed to something much bigger: a radical new explanation of what causes allergic diseases. Irvine had spotted that far more ichthyosis vulgaris patients also had atopic eczema compared to the general population. Could their skin be at fault? Indeed, could epithelium defects be responsible for other allergies too, such as asthma, allergic rhinitis and food allergies, rather than a misfiring adaptive immune system as was widely believed? While the two hypotheses are not mutually exclusive — faults in both could combine to

cause allergies — the idea that the epithelium is a major player could explain several phenomena, including why people with atopic eczema often go on to get a set of other allergies. The epithelium is being targeted in new approaches to allergy treatments. Stephen Holgate, a clinician and asthma researcher at Southampton University, UK, describes it as “the new boy on the block”.

REVEALING MUTATIONS The conventional view of allergies is that some people have an inherited tendency to develop multiple allergies — a condition known as atopy — because of a faulty immune response, dominated by the activity of Th2 lymphocytes and a type of antibody called immunoglobulin E (IgE). A decade ago, however, hints started emerging that this wasn’t the entire story. One clue came from research into a rare skin disease called Netherton syndrome. Netherton sufferers have fragile, scaldedlooking skin prone to cracking, and they develop rampant atopy. Netherton syndrome

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is a recessive monogenic condition, caused by mutations to the SPINK5 gene. The normal version of SPINK5 encodes a protein that inhibits the activity of certain skin proteases (enzymes that cut up proteins). These serine proteases are needed for everyday skin renewal, but if they become active too early, as happens with Netherton syndrome, they destroy the younger layers of skin before they mature. Bacteria and other organisms present in the environment also produce proteases, such as those found in pollen and in the droppings of that bane of asthma sufferers, the house dust mite, whose proteases Der p I and Der p II damage epithelia. In 2001, a team at the Wellcome Trust Centre for Human Genetics in Oxford, UK, showed that a particular variant of SPINK5 was strongly associated with eczema and asthma in families with atopy, but who did not have Netherton syndrome. This suggested to the researchers that lesions in the epithelial barrier might be predisposing people to eczema, and that the atopy might be secondary. Three years later came the breakthrough involving McLean’s team in Dundee and Irvine’s team in Dublin. Their work focused on the f ilaggrin gene FLG, which This theory shifts is needed to help the blame from keratinocytes form the adaptive and moisturise the immune response skin’s outer imperto the innate one. meable layer, the


STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY

ALLERGIES OUTLOOK

stratum corneum. Only about 1 in 100 northern Europeans has ichthyosis vulgaris resulting from mutations in the FLG gene, but 1 in 10 northern Europeans is heterozygous, carrying one mutated copy and one normal copy. Irvine noted that the heterozygous carriers, although free of full-blown ichthyosis, nonetheless had overly creased palms and unusually dry skin, suggesting that it was more fragile than normal. They compared 50 children with ichthyosis vulgaris with 200 unaffected individuals and found a remarkably strong association between FLG mutations and eczema.

MORE THAN SKIN DEEP Subsequent work, says Irvine, showed that eczema patients who carry FLG mutations tend to have a more severe form of eczema, and are more disposed to develop a series of other allergies too, a phenomenon known as the ‘atopic march’ . The finding that filaggrin mutations are strongly associated with asthma was a particular surprise, since filaggrin is not expressed in the lung. Furthermore, McLean’s dermatologist colleague Sara Brown, also at Dundee, found a strong association between filaggrin mutations and peanut allergy. Filaggrin has not been detected in the gut, but is found in the lining of the mouth as well as in the skin. These and other findings, including the link between atopy and another rare fragile skin disorder called peeling skin syndrome, are consistent with the idea that allergies are triggered by a leaky epidermis. Although the precise mechanism is poorly understood, it

is thought that leaky skin allows allergens to penetrate the body and prime the immune system for an allergic reaction. Recent work in a murine model tested the concept. Mice lacking filaggrin have dry, flaky skin that is abnormally thin and porous; if such mice have their skin smeared with ovalbumin, a protein abundant in egg white, they develop a systemic IgE allergic response to it, while normal mice do not. “Dermatologists had always said they thought that eczema was primarily a skin disease, and that the effects throughout the body were secondary to that,” says Brown. In the past few years, the field has exploded, extending the faulty epithelium idea to allergic rhinitis (porous nasal epithelium) and food allergies (leaky gut epithelium). The intriguing thing about this theory is that it shifts the blame from the adaptive immune response to the innate one. Epidermal cells, long thought to form only a passive barrier to invaders, are now emerging as players. Keratinocytes not only present antigens to immune cells, they also secrete cytokines such as thymic stromal lymphopoietin (TSLP), which drives atopic march (see ‘Marching with allergies’, page S14) as well as other cytokines that fan the Th2dominated immune response. “The epidermis is quite immunologically active,” says Irvine. “It isn’t just inert bricks and mortar.”

ASTHMATIC COMPLICATIONS Meanwhile, asthma researchers are questioning the idea that the adaptive immune system is the sole trigger of allergy. Over the past 20 years or so, a growing body of work has shown that the airway epithelium seems to be more fragile in asthma patients. For example, Holgate’s team in Southampton has shown that tight junctions, the massive protein complexes that help hold cells together in epithelia, do not assemble properly in the airways of asthmatic patients. Other teams have found that faults in different protein complexes also involved in epithelial cell junctions are linked to asthma and that the airway epithelium in asthmatics is poorly repaired following injury. What’s more, studies of cultured lung epithelia show that several common triggers of asthma also disrupt cell junctions, particularly in epithelia from asthmatic patients. And some allergens compound their effect by stimulating the immune system. The house dust mite protease allergen Der p II, for instance, interacts with innate immune cell receptors called Toll-like receptors, triggering a ‘danger signal’ that attracts the attention of the adaptive immune response. “If you add that to the epithelial weakness,” says Holgate, “you have a perfect storm.” ■ Claire Ainsworth is a freelance science writer based in Southampton, UK.

S COP E F OR TREATMENT Fixing the barriers Academic researchers and pharmaceutical companies are already investigating therapies that bolster the integrity of epithelia as a treatment for allergies. For example, Irwin McLean and colleagues at University of Dundee, UK, are searching for small molecules that could boost the production of filaggrin and perhaps other components of the skin barrier machinery in eczema patients. They are also investigating drugs that could help epithelial cells ‘ignore’ so-called nonsense mutations to the FLG gene — nonsense mutations are the most common mutations in the filaggrin gene in northern Europeans, and cause the cell’s protein synthesis machinery to halt part way through translating the genetic code into the protein. Certain kinds of antibiotics called aminoglycosides are already known to let cells read-through nonsense mutations, and drugs based on the read-though approach are in clinical trials for other genetic diseases such as cystic fibrosis. McLean and his colleagues have been awarded a patent covering the use of readthrough drugs, including aminoglycosides such as the off-patent antibiotic gentamicin, for the treatment of ichthyosis vulgaris and other atopic conditions. Such drugs would enable cells to bypass nonsense mutations and thereby restore filaggrin production. The treatment could help the skin develop a more natural, stronger structure. Gentamicin, however, has harmful side effects, so the team is screening for alternative read-though drugs and is following a number of leads in cell and animal models. Another way of strengthening the epithelium is to help it repair itself. The airway epithelium of asthma patients is known to be poor at self-repair, but experiments in tissue culture suggest that its ability to regenerate improves in response to growth factors. Holgate’s team at Southampton University, UK, is currently testing the safety and efficacy of keratinocyte growth factor (KGF) on asthma patients. KGF is present in the skin and the lining of the gut, where it promotes the growth of cells that help repair damage and maintain tissue strength. Work by other researchers in Lille and Paris, France, on a rat model of asthma suggests that KGF can reduce inflammation and leakiness in the airway epithelium, as well as making it more resistant to being damaged by allergens.

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Marching with allergies They come not single spies, but in battalions. The latest research helps explain why an individual may experience the ‘atopic march’ from one allergic disorder to another. B Y PA I G E B R O W N S 1 4 | NAT U R E | VO L 4 7 9 | 2 4 NOV E M B E R 2 0 1 1

nimal dander was the culprit in my first asthmatic trauma. On a childhood sleepover, I was sharing my friend’s spare bed with her cat. Its hair was all over my pillow. Far too shy to complain, I remember trying to breathe shallowly, in a vain effort to avoid inhaling the essence of cat from the bed. Not until three in the morning, face swollen and airways inflamed to the point that I could hardly breathe, did I finally gather the courage to wake my friend’s parents. That night ended with hospital-administered inhalant bronchodilators and a good dose of antihistamines. But my lifelong struggle with allergic disorders and hyper-reactivity continues. My mother, herself allergy-prone, remembers my inflamed and itchy skin lesions in infancy. These are classic symptoms of atopic eczema, better known in the United States as atopic dermatitis. Then, in my toddler years, as my father recounts, I started to show “this peculiar reaction” around animals, scratching at my throat as my eyes went red, watery and swollen. These typical symptoms of allergic rhinitis, or hay fever, owe their origins to human immunesystem responses to specific protein allergens, for example those found in animal dander. An allergy-prone child is likely to demonstrate sensitization to various allergens within the first few years of life. The sensitization phase takes place as the body gradually mounts an immune response to foreign substances introduced through the respiratory tract or through skin that lacks natural barrier functions, although the response can become systemic. In my case, for example, it may have been sensitization by way of my skin that led to my lungs becoming sensitized to inhaled allergens. The mechanism of sensitization is dominated by Th2-type inflammation, in which T-helper type-2 (Th2) cells, a subgroup of white blood cells, produce cell-signalling proteins called cytokines that drive the allergic response (see ‘Breathing new life into research’, page S20). Exposure to an allergen causes another subgroup of white blood cells, activated by these Th2 cytokines, to produce specific immunoglobulin E (IgE) antibodies. These antibodies then bind to circulating white blood cells known as basophils, and to mast cells (similar to basophils but found mainly in connective tissue), priming them to release inflammatory mediators such as histamine upon re-exposure to the original allergen or structurally similar proteins.

WATERMELONS AND ECZEMA For the past decade, from my teens to midtwenties, my life has been plagued by allergic asthma. Perhaps more frustrating and stressful has been an explosion in my food allergies and chronic allergy-related atopic dermatitis, causing discomfort, sleeplessness caused by scratching, and skin abnormalities. “Make a note on her chart, displays symptoms

ILLUSTRATION BY JONATHAN BURTON

OUTLOOK ALLERGIES


ALLERGIES OUTLOOK characteristic of an atopic individual,” said my dermatologist, Mary Dobson, when I visited her clinic in Baton Rouge, Louisiana, for a particularly severe flare-up. Atopy is the tendency to be hyperallergic, the genetic propensity to mount an IgE response to triggers including pollen, animal dander and food-based allergens. Thomas Platts-Mills, head of asthma and allergic disease at the University of Virginia in Charlottesville, describes the atopic individual of today as one who is ‘better’ at recognizing small quantities of foreign protein and reacting to it: “allergies are the price we pay for an immune system that protected us against big killers in the past”, he says. Sometimes I feel like a walking illustration of the atopic march. This term describes a progression of allergic diseases through childhood into adulthood, and it involves a diverse interplay of factors. Some of these are genetic: variations or mutations in the genes controlling important constituents of the immune system or skin integrity. Others are environmental: allergen exposure, early-life infections, pollution and chemical irritants that break down the skin barrier and promote allergen sensitization1. Atopic diseases now affect up to 20% of the population in developed countries2. Individuals with one atopic disease are more likely to develop a second or third atopic disorder, with many suffering from the ‘triad’ of atopic dermatitis, allergic rhinitis and asthma: a 2004 study by Donald Leung, head of paediatric allergy and immunology at National Jewish Health in Denver, Colorado, and colleagues, showed progression occurring from atopic dermatitis to allergic rhinitis in up to 75% of patients, and to asthma in 50%. And, although food allergies might seem to be completely different phenomena from this triad, many of the same mechanisms mediate these disorders as well. When I started looking into my own food allergies, for instance, which involve strong inflammatory reactions to a range of fruits and vegetables including apples, cherries, kiwis, bananas, carrots, pineapples, tomatoes and even watermelon, I found out that they are probably due to a cross-reactivity phenomenon known as the pollen–food allergy syndrome. Certain pollen proteins, including ragweed allergens and the birch pollen allergen Bet v1, are common causes of allergic rhinitis and asthma. They are also common inducers of allergic sensitization to particular fruits and vegetables, in those of us with pollen–food acquired immunity dominated by the IgE antibody response (rather than the IgG4 response, which favours food tolerance)3. SensitizaNATURE.COM read the latest tion occurs because of research on immunological crossatopic march: reactivity between the go.nature.com/g7xczl pollen allergen and

structurally related food proteins, such as the apple protein Mad d1. The recognition of food proteins by pollen-specific IgE antibodies can then lead to IgE-mediated reactions including inflammation and itching of the mouth, as well as late-phase Th2 inflammatory responses such as atopic dermatitis. The pollen–food allergy syndrome provides one link between the various manifestations of allergy. Another lies in the condition of the skin, the first line of defence against infections and allergens. It has long been hypothesized that skin barrier abnormalities and hydration impairments, such as various dry skin conditions, might have systemic consequences for allergen sensitization and inflammation (see ‘Into the breach’, page S12). The inflamed and broken skin characteristic of atopic dermatitis aids contact between environmental allergens and the immune cells that initiate and sustain Th2 pathway responses.

LEAKY SKIN It sometimes seems that my skin is providing less of a barrier to the outside world than it should. I tend to have dry skin, and even tiny nicks on my legs, hands and feet can turn into chronic lesions. My more severe atopic dermatitis flare-ups seem to be closely linked to my food allergy symptoms, and to subsequent bouts of worsened asthma. But what molecular mechanisms underlie my symptoms? “The common disease features of the allergic diseases that comprise the ‘atopic march’ strongly suggest a common underlyThe pollen– ing mechanism,” says food allergy Steven Ziegler, head syndrome of immunology at provides one Benaroya Research link between Institute in Seattle, the various Washington. Ziegler’s manifestations group, as well as other of allergy. groups, has identiAnother lies in fied high levels of one the condition of important protein of the skin. the cytokine family, called thymic stromal lymphopoietin (TSLP), as a likely common factor. “TSLP expression is elevated in humans with these diseases, as well as in mouse models, and blocking TSLP can prevent disease in these mouse models,” explains Ziegler. TSLP is highly expressed by the outermost skin cells in atopic dermatitis lesions, especially chronic lesions. When expressed in high quantities, it has been shown to promote local environments dominated by Th2-type inflammation. A 2007 study also showed that TSLP is released from human epithelial cells in response to infections and physical trauma4. So the explanation for accidental breaks in my skin turning to atopic dermatitis may be that the TSLP protein is mediating a Th2 inflammatory response initiated by the mechanical injury.

Studies using mouse models have also shown an association between TSLP expression by cells at barrier surfaces, including the skin, gut and lung, and more basophils in the spleen, blood, lung and bone marrow where bloodcirculating basophils mediate systemic Th2type allergic responses. Earlier this year, Mark Siracusa, an immunologist at the University of Pennsylvania in Philadelphia and colleagues showed that such TSLP-mediated increases in basophil activity led to a body-wide elevation of IgE levels and allergic inflammation in mice5. Moreover, they showed that TSLP-elicited human basophils differ from healthy basophil populations in the ability to create a state of systemic susceptibility to Th2 inflammation. What these results indicate is that abnormal TSLP production at one barrier surface can trigger the proliferation, mobilization and activation of immune system cells that promote allergies throughout the body. This might help to explain why the exposure of cells lining my gut (a barrier surface) to a food allergen such as watermelon might be linked to worsening of my eczema symptoms, and why a flare-up of my eczema (at another barrier surface) might aggravate my asthma symptoms. TSLP is now a therapeutic target. Michishige Harada, of the laboratory for respiratory diseases at Institute of Physical and Chemical Research in Japan, and colleagues had already revealed in 2009 that a particular genetic variant of TSLP leads to increased protein expression, for example in response to viral respiratory infection. This discovery, pointing to TSLP as a genetic orchestrator of the atopic march, was reinforced by further studies from the same group, published in 2011, which established long-form TSLP gene variants as factors of susceptibility to atopic asthma6. Based on these studies and others, establishing TSLP as a major causative factor of atopic march disease progression, Ziegler says there is now an anti-TSLP drug in phase II clinical trials. “With no pharmacologic cure for asthma in sight, there is a need for drugs which can halt the atopic march,” says Leung. He believes that the current scientific data strongly support the use of systemic anti-TSLP drugs as an intervention strategy. Although it is too late to save me from my sleepover trauma, the parents of children with early signs of atopy should be watching with the keenest of interest. ■ Paige Brown is a freelance science writer based in Baton Rouge, Louisiana. 1. Platts-Mills, T. A. & Woodfolk, J. A. Immunol. Rev. 242, 51–68 (2011). 2. Zheng, T. et al. Allergy Asthma Immunol. Res. 3, 67–73 (2011). 3. Geroldinger-Simic, M. et al. J. Allergy Clin. Immunol. 127, 616–622 (2011). 4. Allakhverdi, Z. et al. J. Exp. Med. 204, 253–258 (2007). 5. Siracusa, M. C. et al. Nature 477, 229–233 (2011). 6. Harada, M. et al. Am. J. Respir. Cell Mol. Biol. 44, 787–793(2011).

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OUTLOOK ALLERGIES

PERSPECTIVE Acting on the evidence Allergy isn’t the whole story on atopic eczema, says Hywel Williams.

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IMAGE COURTESY OF HYWEL WILLIAMS

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topic eczema, the largest group among nine types of eczema, Prevention is nevertheless a real possibility. Early life (in utero and has become so widespread that it now affects approximately soon after birth) is a critical period to evaluate strategies, including one in five children living in cities across the world, according modifying bacterial gut flora with probiotics, or exposure, rather than to estimates by the International Study of Asthma and Allergies allergen avoidance, to induce immune tolerance. It is also possible in Childhood (ISAAC)1. The name ‘atopic eczema’, however, is a that barrier enhancement with emollients in newborns onwards could prevent, delay or reduce the severity and progression of allergic disease misnomer. Diagnosis is based on itching, visible signs of eczema affecting the skin folds, and family history, but ISAAC studies show in those so predisposed. that around half of those so described are not, in fact, atopic (predisTreating eczema remains a challenge3. Whilst atopic eczema is posed to allergic hypersensitivity) — they do not have elevated IgE not a killer, witnessing children scratch themselves until their skin antibodies in their blood against common environmental allergens. bleeds is heartbreaking, and the associated sleep deprivation has an Most children with severe eczema attendimpact on the family as a whole. Quality of ing hospital clinics are atopic, often with life impairment and costs associated with either asthma or food allergies, or both. atopic eczema are often greater than with Identifying a potential culprit allergen and other conditions such as childhood diabethen limiting exposure to it, however, can tes. Complications such as skin infection rarely control their condition. Even if they with Staphylococcus aureus, herpes simplex are truly allergic to something like cat danvirus and associated asthma, hay fever and food allergies pose additional problems. der or house dust mites, immune tolerance can occur with repeated exposure — in The biggest problem is undertreatment — not because the treatments don’t work, but which case reducing exposure by frantic cleaning, or intermittent exposure, could because of a lack of education and an excesmake things worse (and could also explain sive fear of side effects of topical corticowhy atopic eczema is more likely to flare steroids reported in the 1960s. Just as inhaled steroids are needed to deal with the when near an unfamiliar cat). Moreover, non-allergic factors such as: temperature inflammation of asthma, topical steroids extremes; dry wintry or conditioned air; are needed to treat skin inflammation. A soaps and shampoos; sweating; abrasive potent topical corticosteroid plus a soothgarments; Staphylococcus aureus infection; ing emollient is usually enough to treat psychological stress; and habitual scratchatopic eczema in children, inducing remising might be more provocative to their sion with a 2–3 week blast of corticosteroid eczema than allergens2. treatment to gain control over the condition Genes responsible for the integrity of and then maintain remission through weekthe skin barrier determine much of the dry end corticosteroid therapy4. Other creams skin associated with atopic eczema. This or ointments such as pimecrolimus and tacrolimus are also useful for treating sensitive might contribute to an enhanced reactivity to environmental irritants, such as soap, Telling this child it’s only eczema and you will grow out of sites such as the face. or to allergens. Contact with irritants could it is not the way forwards. Drugs that calm the immunological storm trigger low-grade eczematous inflammaneed to be developed to improve quality of tion, which then leads to atopic eczema, possible followed by asthma life and disease progression for those with severe atopic eczema — new, smarter and more specific agents, with fewer adverse effects than and hay fever, and so proceeds the ‘allergic march’. Genes that govern aberrant inflammatory responses might also play a role. Yet, genetics the older systemic agents. ■ cannot account for the findings that atopic eczema is more common in wealthier, smaller families, or that children migrating from lowHywel Williams is Director of the Centre of Evidence-Based to high-prevalence countries assume higher rates in their adopted Dermatology, Nottingham University Hospitals NHS Trust, and a countries. Nor can geneticists explain the rapid increase in eczema steering group member of the International Study of Asthma and Allergies in Childhood. symptom prevalence reported in the ISAAC study. The interaction between genetic and environmental factors may be complex. Just as e-mail: hywel.williams@nottingham.ac.uk a fruit machine only pays out when all three cherries align, flares of 1. Odhiambo, J. et al. J Allergy Clin. Immunol. 124, 1251–1258. (2009). eczema might only occur when multiple components such as genes, 2. Williams, H. C. N. Eng. J. Med. 352, 2314–2324 (2005). allergens and low humidity coincide. 3. Langan, S., Silcocks, P. & Williams, H. C. Br. J Dermatol. 161, 640–646. (2009). 4. Nankervis, H., Maplethorpe, A. & Williams, H. C. BMC Dermatology 11, 10 For all these reasons, it is important not to over-emphasize allergic (2011). factors in understanding atopic eczema, or in strategies for prevention and treatment. The author declares competing financial interests: go.nature.com/xwss7n


ALLERGIES OUTLOOK

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n 1911, a British researcher named Leonard Noon attempted to do for hay fever what his predecessors had done for smallpox and rabies. Using small amounts of grass pollen, Noon injected ‘pollen vaccines’ into people suffering from grass allergy, gradually increasing the amounts to help build up a tolerance to the irritant. A century later, Noon’s immunotolerance therapy has matured into a technique widely used by allergists, one that can be tailored for dozens of airborne allergens. At their worst, such allergens — which cause the sniffing, sneezing and sinus misery also known as rhinitis — rarely cause more than severe annoyance. These days, people who suffer from allergies like pollen, mould, or dust mites have a few more choices than Noon’s patients. Over-the-counter antihistamines provide relief for mild cases, while people with more persistent allergies can use prescription corticosteroids to decrease inflammation and keep their sinuses clear. Those with the most severe cases, however, still benefit from allergy shots or a combination of all of the above. The current protocol for allergic rhinitis shots requires injections as often as twice a week, and a course of treatment takes between three to five years to yield a long-lasting effect. But this approach is futile in as many as 25% of sufferers and many hope that a more effective treatment must be possible. The same airborne allergens which cause rhinitis can also cause a more serious problem: allergic asthma. It’s not clear how the conditions are related, but during the past 20 years researchers have shown that serious allergies can evolve into acute asthma, which can require sufferers to have hospital care and is very occasionally fatal. As with rhinitis, most sufferers can control their asthma with existing drugs. The remaining 25%, however, have drawn the eye of drug developers.

WHEN GOOD IMMUNE SYSTEMS GO BAD

TREAT M ENT

In search of a booster shot A plethora of therapies can keep the symptoms of allergy under control, but they can’t cure. New research aims to prevent allergies from developing in the first place. BY LAUREN GRAVITZ

Pollen and other airborne allergens should be harmless. But in 20% or more of the world’s population, something triggers an immune response. In these over-reactive immune systems, allergens prompt the activation and proliferation of a group of white blood cells known as T helper 2 (Th2) cells, triggering a cascade of events that causes overproduction of a class of antibody called immunoglobulin E (IgE) and leads to inflammation and irritation (see ‘Targeting Th2 activity’). In rhinitis, such inflammation causes the classic range of hay fever symptoms. In asthma, inflammation and other unknown culprits — perhaps coupled with a genetic predisposition (see ‘Seeking a gene genie’, page S10) — cause the airway to remodel and become prone to constriction upon exposure to an allergen. The more researchers learn about the cellular chain of events, the closer they are to determining which links are most vulnerable to intervention1.

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OUTLOOK ALLERGIES BUILDING TOLERANCE Most rhinitis treatments focus on variations of the same immunotolerance on which Noon capitalized a century ago. The method exploits the very response that causes allergies in the first place: the body’s adaptive immune system, which evolved to remember and attack foreign bodies or antigens after first encounter. With airborne allergies, the adaptive immune system reacts each time it sees an antigen to which it is sensitized (such as mould, cat dander or ragweed pollen). A course of shots, injecting increasing quantities of allergen extracts over many years, can gradually build tolerance in the immune system and prevent it from launching an offensive every time it meets the offending molecules. This approach is, however, fraught with problems. Shots don’t always work, they require a substantial time commitment from a patient, and they bring a rare but inherent danger — after all, people are being injected with the substance to which they are allergic. “It’s medieval stuff, in a way,” says Mark Larché, an allergist at McMaster University in Hamilton, Ontario. “Allergists still mix up cocktails of extracts in their offices to give to people. There’s a weird juxtaposition of a disease-modifying approach with this dark-arts business of using extracts, and people having terrible allergic reactions.” One reason these extract-based cocktails may fail is the omission of particular antigens. “People aren’t just allergic to one allergen of dust mites. There can be as many as twenty that are important,” Larché says. Although extract makers try to standardize their products, it’s a difficult goal. “Studies have shown that some of the most important antigens can be missing,” adds Larché, who co-founded the UK-based biotech company Circassia in 2006 to try to improve that hit rate. Circassia is one of a few companies working to create a second generation of allergy shots and, from a regulatory standpoint, is perhaps the furthest along. The company’s approach is based on identifying specific parts of the allergen molecules, called epitopes, that T cells recognize and interact with. Rather than extracting the whole allergen from pollen or the faeces of dust mites, the company is building synthetic versions of the relevant epitopes that can be put together into a dose that is quantifiable and replicable. “We can ensure our synthetic epitopes are consistent from batch to batch,” says Rod Hafner, Circassia’s senior vice president of research and development. “And because we’re using epitopes that don’t interact with [the antibody] IgE, they don’t have a risk of anaphylaxis. We get a faster onset of efficacy and it persists much longer.” Circassia is developing products for four of the most common allergens: cat, ragweed, dust mite and grass pollen. Early data suggests that, at least for cat allergies, 4 shots given 4-weeks apart are enough to prevent symptoms for at

TARGETING Th2 ACTIVITY How the cytokines produced by Th2 cells stimulate inflammation – and hence offer theraputic targets Allergen Naïve T-helper cell activated by allergen IL-5 Antigen presenting cell

Eosinophil IL-4 Releases soluble pro-inflammatory mediators

IL-13 B cell

Th2

lgE

Th17 IL-9

Th1

TReg

Epithelial cells

IL-13

Mucus

Mast cell

Increased production of mucus

Connective tissue

Smooth muscle cells

Releases histamine, leukotrienes and cytokines

Contraction of smooth muscles

Increased vascular permeability Blood vessel

Target

Mode of action

Example (In development at)

Immunoglobulin E

IgE increases allergen uptake by antigen-presenting cells, priming the immune system for attack against the allergen. Anti-IgE therapies aim to prevent it from binding to the antigen-presenting cells.

Lumiliximab (Biogen Idec) Anti-IgE vaccine (Pfizer, United Biomedical)

Mast cells

Mast cells release histamine, cytokines and other immune-system mediators that promote vascular and smooth-muscle changes, increase mucus, and recruit additional inflammatory cells. Blocking their activation could prevent downstream effects.

R343 (Rigel)

Th2 Cytokines (e.g. IL-4, IL-5, IL-13)

Cytokines produced by Th2 cells orchestrate allergic inflammatory responses. Numerous approaches take aim at specific cytokines in order to improve the Th1 to Th2 ratio.

Mepolizumab (GlaxoSmithKline) MEDI-528 (Medimmune) IL-13 MAB (GlaxoSmithKline)

Toll-like receptors (e.g. TLR9)

TLRs are immune-cell specific receptors. Different ones regulate different aspects of the immune response, and the hope is that stimulating the appropriate TLR will promote a more appropriate balance of Th1 to Th2 cells.

CYT003-QbG10 (Cytos vaccine) IMO-2134 (Idera Pharmaceuticals)

least a year. The company is moving the catallergy product into phase III trials, aiming for regulatory approval by 2015, and expects that the ragweed trials could come a year later. Rudolf Valenta, an allergist at the Medical University of Vienna, is approaching the allergic rhinitis problem from a different angle. Valenta is also interested in creating homogenous synthetic vaccines, but he wants

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to take it a step further into diagnostics. He and his colleagues developed an allergen library, with DNA fragments from different antigens that could be placed on a protein chip. Then, from just a single drop of a patient’s blood, the chip can rapidly detect which molecules a patient is allergic to. “The beauty is that in just a few minutes you can record the complete reactivity profile of a


ALLERGIES OUTLOOK patient against hundreds of allergy components,” Valenta says. Valenta and his colleagues are using the data to build molecules called recombinant hypoallergens. They take an allergen, pluck out the peptides that don’t induce an immune attack and attach them to carrier molecules from the hepatitis B virus. When injected, instead of triggering production of IgE, these hypoallergens stimulate production of another antibody, called IgG, that prevents IgE from binding and thereby keeps the patient safe2. “In a study where we applied the vaccine components to the patients’ skin, we saw no reaction at all,” Valenta says. These vaccines have the potential to induce no side effects. Ultimately Valenta envisions an entire system in which a protein chip determines each patient’s allergy profile, and then a series of vaccines is tailored to match these specific sensitivities. For now, though, his team is still working one allergy at a time. In collaboration with the Vienna-based biotech company, Biomay, they are about to begin a 3-year phase II trial, testing their approach with a vaccine made of 4 different grass–pollen antigens.

STAYING A STEP AHEAD A growing number of studies indicate that children with allergic rhinitis are at higher risk of developing allergic asthma. No one is precisely sure how allergic rhinitis can be a catalyst for the development of asthma. One theory gaining credibility suggests that overactive immune cells, triggered by a response to airborne allergens, damage the epithelial and smooth muscle cells that line the lungs’ airways. In regeneration, the epithelium and smooth muscle grow back abnormally, leaving the lungs increasingly vulnerable to attacks of inflammation and airway obstruction, particularly when the immune system encounters the allergens that caused the initial attack. Some scientists, therefore, wonder if it might be possible to stop allergic asthma before it starts. The key will be to identify children most at risk, and intercede before the process becomes irreversible. One study, led by Christian Möller at Umeå University in Sweden, observed 117 children with pollen allergies for over 10 years. Half of the children were 6–14 years old when the trial began, received a 3-year course of allergy shots for grass and/or birch pollen; the other group received a placebo. Seven years later, the researchers found that the risk of developing asthma was nearly halved for the children receiving the allergy shots, confirming the hypothesis that early treatment of rhinitis could very well stop the onset NATURE.COM of asthma3. For some of the A related, 4-year latest research on study, set to conclude allergy therapy: at the end of 2011, aims go.nature.com/das6s9 to determine if allergy

prevention, of both rhinitis and asthma, can start even earlier. Led by Patrick Holt, an immunologist at the University of Western Australia in Perth, it follows young children — between 18 months and 30 months old when the study began — who were beginning to show signs of allergic disease. Each day for one year, the children received just a few drops of a mixture of dust mite, cat and grass pollen extracts under their tongue. “It’s not immunotherapy, because they weren’t yet sensitized,” says Holt, who leads the cell biology division at the university’s Telethon Institute for Child Health Research. “We wanted to expose their mucosa to allergens that are important in their environment in order to drive the process of mucosal tolerance, a natural process through which individuals can escape sensitization4.”

The more researchers understand about immune cascades, the more they can hone their attack.

SCULPTING THE IMMUNE RESPONSE Once allergic asthma develops, it can prove a difficult beast to tame. When long-acting corticosteroids fail — as they do in about 25% of allergic asthma cases — there are not many alternative treatments. Part of the reason these cases are so intractable is that researchers still don’t fully understand the precise cause and effect of the disease. In fact, most researchers are coming around to the belief that allergic asthma is not a single disease but a cluster of similar symptoms generated by a variety of underlying causes. “Asthma is a garbagecan term,” says Sally Wenzel, director of the Asthma Institute at the University of Pittsburgh Medical Center. “The definition of asthma is incredibly broad.” Unlike the allergic rhinitis approach of immunotolerance, most allergic asthma approaches use a tactic known as immunomodulation, which aims to interrupt the cellular cascade that leads to the overabundance of IgE. “Immunology is maturing to the point where we have a much bigger, more complete picture. And it could be a false dawn, but it’s an exciting period in this area of science,” says Roberto Solari, head of respiratory biology at GlaxoSmithKline (GSK), based in London. The more researchers understand about immune cascades, and which cells are involved, the more they can hone their attack. Some approaches aim for the top of the chain, altering the proportion of the various T helper cells among the white blood cells. Since different types of white blood cells drive different immune-response pathways, the hope is that stimulating Th1 immunity could muzzle the allergy-prone Th2 activity. But this approach has its drawbacks. “The problem with targeting something that high up,” says Thomas Casale, chief of allergy and immunology at Creighton

University in Omaha, Nebraska, “is that you’d likely affect other processes that might be important, increasing susceptibility to infections or lowering your ability to fight infections.” With that concern in mind, other researchers are aiming further down the cascade, trying to eliminate the Th2 products that stimulate inflammation, such as particular interleukins (IL-4, IL-5, IL-13). Some are even attempting to eliminate what those products target, such as the white blood cells called eosinophils or IgE itself5. Not only does asthma range in severity, even patients with similar disease severity can have very different characteristics or phenotypes. And different phenotypes will respond better to some interventions than others, depending on which point in the immune cascade a drug is targeting. The only anti-IgE drug to hit the market so far is a monoclonal antibody called omalizumab, which was approved in the United States in 2003, but only works for the roughly one-third of intractable allergic asthma cases in which IgE levels fall within a narrow range. (Omalizumab appears to be effective for allergic rhinitis, but the cost, which can surpass US$20,000 per year, is beyond the means of most sufferers. “Although allergic rhinitis has a high prevalence, it’s not going to kill you,” says Casale.) Omalizumab is likely to be the first of many drugs that will be aimed at specific subsets of allergic asthma. Multiple therapeutics, which takes aim at a variety of molecules in the Th2 cascade, are in various stages of clinical trials. The interleukins produced by Th2 cells drive different elements of the inflammation process (see ‘Targeting Th2 activity’) and two in particular, IL-5 and IL-13, have proven particularly attractive targets; GSK completed a phase III trial of anti-IL-5 mepolizumab in 2010. But these, too, are showing promise only in very small subsets of patients. People with high levels of eosinophils in the mucus of their lower airways appear to benefit from antiIL-5 therapy, whereas people with high levels of a blood protein called periostin seem to be helped most by anti-IL-13. “In the past, we’ve treated asthma with the medicines we have. But in the future, we need to define those diseases better, define the patients better, and target therapies to those stratified patients,” says GSK’s Solari. “This is where 21st century medicine needs to go.” ■ Lauren Gravitz is a freelance science writer based in Los Angeles, California. 1. Holgate, S.T. & Polosa, R. Nature Reviews Immunology 8, 218–230 (2008). 2. Niespodziana, K. et al. J. Allergy and Clin. Immunol. 127,1562–1570 (2011). 3. Jacobsen, L. et al. Allergy 62, 943–948 (2007). 4. Holt, P. et al. Nature Immunology 5, 695–698 (2004). 5. Casale, T. B. & Stokes, J. R. J. Allergy and Clin. Immunol. 127, 8–15 (2010).

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might gain an insight into why the prevalence of asthma and other IL-17-driven diseases — such as inflammatory bowel disease and multiple sclerosis — has increased in the industrialized world.

ROUNDING UP THE SUSPECTS

AST H MA

Breathing new life into research Asthma was once thought to be a uniform disease triggered by one type of immune cell. Researchers are now revealing the complexity of the condition and hope to hasten new drugs for forms unresponsive to steroids. BY AMY MAXMEN

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ccording to textbooks, type 2 helper T cells (Th2 cells) preside over asthma. T cells are a subset of the white blood cells known as lymphocytes, and the conventional view is that an asthma attack occurs when Th2 cells secrete a certain set of immunesignalling proteins called cytokines that inflame the lungs, irritate the chest and cause asthma’s characteristic wheezing. However, another cytokine, interleukin-17 NATURE.COM (IL-17), which does not For some of the belong to Th2’s signalatest research on ture set, has been caught asthma: lurking in lung tissue, go.nature.com/4Itbg5 sputum and the blood of

asthmatic patients (see ‘New partner in crime?’.) “We thought we understood asthma, but now we know it’s much more complex,” reflects immunologist Manfred Kopf at the Swiss Federal Institute of Technology in Zurich. Recent research is reframing our picture of asthma. Numerous molecular, immune-system pathways are now being implicated in different manifestations of a disease once considered uniform. By mapping these networks, scientists plan to identify new treatment targets for each form of asthma. The discovery of IL-17 in the lungs of some asthmatics has drawn attention to IL-17-producing cells previously thought unrelated to asthma. What’s more, if researchers discover what spurs the anomalous and harmful surge in IL-17 in the first place, they

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Implicating IL-17 in asthma and proposing new disease subtypes has solved several puzzles. For example, in many asthmatic patients, other types of white blood cell called eosinophils accumulate in the lungs, but individuals with the most severe form of asthma harbour neutrophils, yet another type of immune cell. Normally, these neutrophils surround the site of an acute infection or injury, lured there by IL-17, to protect the body. It appears that in asthma, IL-17 is drawing neutrophils into the lung — but they are not there to protect. Instead, they make asthma attacks worse. Furthermore, compared to people with mild asthma, individuals with severe asthma not only have more IL-17, they tend to have more of the cells known to secrete IL-17, namely Th17 cells. Research has also shown that mice with more Th17 cells get little breathing relief from steroids, a common treatment for asthma but one to which severe asthma patients rarely respond. “Patients who come into the hospital needing help are usually those with severe asthma who are resistant to steroids,” explains Bart Lambrecht, at Ghent University in Belgium. “For a long time we knew these patients had neutrophils in their sputum, but the link to Th17 cells wasn’t made until recently.” By secreting IL-17 and other cytokines, Th17 cells constrict the lungs’ airways. What triggers Th17 cells to churn out these lung-damaging molecules, however, is less well understood. Viruses, allergens, cigarette smoke and airborne pollutants have all been fingered as potential culprits, but how they initiate Th17 cells in asthma remains elusive. For example, cigarette smoke triggers the proliferation of Th17 cells, and smoking is a risk factor for asthma, but the steps between these two observations are obscure. “Perhaps you first have a predisposition to drive a Th2 response, and IL-17 comes on board later,” says Marsha Wills-Karp, an immunologist at the Cincinnati Children’s Hospital Research Foundation in Ohio. “It may have something to do with the exposures you have early in life, such as infections, cigarette smoke or air pollutants.” In 2010, Wills-Karp and her colleagues discovered a way for allergens to provoke Th17 cells, and thereby exacerbate asthma. Their discovery concerned the complement systempart of the immune system that connects innate and adaptive T cells, and Th17 in particular. After an extract consisting of the allergy-inducing portion of house dust mites activated the C3 system, a molecular cascade caused the number of Th17 cells to rise. And IL-17A, a cytokine produced by Th17 cells, reactivated the C3 system and perpetuated the response. Wills-Karp says that abnormally bad, and

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OUTLOOK ALLERGIES


ALLERGIES OUTLOOK EDITORIAL ADVISOR: CLARE LLOYD.

sometimes lethal, infections of the common respiratory syncytial virus (RSV) might cause asthma through the C3 complement system as well. “Almost 100% of kids have been infected by RSV by the time they are two years old, and besides a cold, they’re just fine,” she explains, “but a subset get hospitalized, and if they survive they almost always go on to have severe asthma.” Wills-Karp had previously led a study that found postmortem signs of complement activation in children who died during an RSV vaccine trial in the 1960s — these children had experienced breathing problems. This study was conducted before we knew about Th17 cells, she says, but she now speculates that, when the RSV activated the complement system, a Th17 cascade was set in motion leading to severe asthma. “If we went back and looked at those children’s lungs, we’d probably find Th17 cells”, she says. Another possible factor in the role of Th17 in asthma is vitamin D. This vitamin seems to slow production of cytokines by Th17 cells, so a vitamin D deficiency might lead to the overproduction of cytokines by Th17 cells, which could be a problem if the triggering agents were benign. In a study of asthmatic children in Costa Rica, those with lower levels of vitamin D circulating in their blood were more likely to be hospitalized for severe asthma than those with higher levels of vitamin D.

A THICKENING PLOT The story does not end there: Th17 cells might not be the sole producers of IL-17 in people with asthma. In a murine model of asthma, Dale Umetsu, a paediatrician at Harvard Medical School and Children’s Hospital in Boston, Massachusetts, reported that natural killer T (NKT) cells produce IL-17 and cause asthmalike symptoms. Umetsu’s mice had difficulty breathing after inhaling ozone at concentrations equivalent to ozone-polluted air. Although the mice produced more IL-17, Th2 and Th17 cells were not to blame. “You can take mice that don’t have helper T cells, expose them to ozone, and they still have trouble breathing because they have plenty of NKT cells,” explains Umetsu. “This was at odds with the prevailing wisdom,” he says, “but there is a growing feeling that Th2 cells are not the only way to get asthma.” Another unusual suspect is a T helper cell going against the grain. In a study reported in 2010, Th2 cells from asthmatic patients were found to produce IL-17. Although surprising, this wasn’t the first report of T helper cells secreting uncharacteristic proteins. Immunologists are unsure of how this plasticity arises in a small percentage of T cells. It is also uncertain whether a T cell producing a new cytokine or combination of cytokines should be assigned to a new T-cell subset. “People are slowly acknowledging that there are more T-cell subsets than we had imagined,” says immunologist Carsten Schmidt-Weber, at the Technical University and

NEW PARTNER IN CRIME? Allergens, infections, or pollution might trigger T cells involved in asthma to produce IL-17. This cytokine seems to make asthma worse (although sometimes it may bring relief).

Mediate steroid resistance

IL-17 Airway protection?

!

Th2

NKT

IL-4

Airway constriction

Th17 IL-5

IL-4 IL-13

IL-13

IFN Airway constriction Airway constriction

Mucus Airway constriction

Eosinophils

Helmholtz Center Munich, Germany. “But if we want to design new treatments for asthma, what matters is their function, not their name.”

PERSONALIZING ASTHMA TREATMENT Figuring out what cells produce IL-17 in response to certain stimuli should help the design of new medicines to treat asthma in patients unresponsive to current steroid treatments. In order to test any new drug, patients must be clearly categorized to define the underlying pathology. Otherwise, a drug tailored for one form of asthma could be ineffective in treating a different form of the disease. “There is a lot to learn before we start to use highly specific therapies,” says Schmidt-Weber. Phenotyping asthmatic patients is one aim of the German Center for Lung Research, a virtual centre that links asthma research from across five cities in Germany. Meanwhile, in the United States, the multicentre Severe Asthma Research Program (SARP) has thus far assessed about 1,600 patients and outlined three main types of severe asthma. SARP investigators are analysing sputum and blood samples from patients in each of these groups to determine the immune response associated with symptoms. Blood and sputum, however, might not reflect what occurs in the lungs. And there are limits to what tests you can do in people in the name of research. Clare Lloyd at Imperial College London cuts tiny tissue samples from the airways of consenting asthmatic patients in order to analyze the protein expression of immune cells. Yet even this invasive method permits only a snapshot of

Mast cells

Neutrophils

the lungs. “We hope the sample represents what’s going on in the lungs, but really it is only a small slice,” she says. “In an ideal world, we could see which cytokines are being secreted in real time within a patient’s lungs — but we are far from having the technology to do that in a way that’s safe and ethical.” Such issues help account for asthma researchers’ heavy reliance on mice. Dependence on a single murine model of asthma originally misled researchers into believing Th2 was the primary immune cell at fault, some immunologists argue that new models are needed to help scientists understand other immune pathways. “People have said that the old mouse model of asthma didn’t explain how a viral infection or ozone exposure caused asthma,” Umetsu says. “The key is to develop new models to explore different types of asthma.” Recent findings on IL-17 and NKT cells have raised more questions than answers, but Imperial’s Lloyd finds the challenge invigorating. “People are starting to work towards a greater understanding of all kinds of asthma,” says Lloyd. “If we can identify the cytokines involved in each case, we can develop better, more personalized treatments.” ■ Amy Maxmen is a freelance writer based in New York City. 1. Brehm, J. et al. Am. J. Res Crit. Care Med. 179, 765–771 (2009). 2. Pichavant, M. et al J. Exp. Med. 205, No. 2 (2008) 3. Wang, Y. et al JEM 207, 2479–2491 (2010). 4. Lajoie, S. et al Nature Immunol. 11 (10) (2010).

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OUTLOOK ALLERGIES

PERSPECTIVE A human touch Stephen Holgate argues for a return to more human-centred studies of allergy and asthma.

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ince the discovery of immunoglobulin E (IgE) almost half a century ago, there has been a massive expansion in knowledge about how IgE antibodies work. Research has unravelled IgE’s role in a myriad of cellular and molecular targets driving inflammatory responses and underlying complex allergic disorders. This knowledge might have been expected to lead to novel preventative and therapeutic pathways — unfortunately, this has not been the case. The dramatic rise in allergy and asthma worldwide has increased the clinical need for treatment, but research focusing heavily on IgE as the main malefactor in allergies has not been translated into widespread patient benefit. Part of the reason lies in inherent limitations of the animal models on which researchers have so heavily depended. Most pharma effort in drug discovery for allergy has been directed at asthma. Almost all novel therapeutics for asthma are developed using mice and, to a lesser extent, non-human primates, to investigate whether they inhibit antigen-driven models of lung inflammation. While such acute or chronic models result in a strong immune response by the T helper 2 lymphocytes (Th2) in the lungs, they fail to take account of the many other exposures that are now known to cause human asthma: genetic susceptibility; viral infection; air pollutants; and drugs such as aspirin and paracetamol. A compounding problem with animal models is the widespread use of ovalbumen as a sensitizing antigen in trials on rodents. Ovalbumen is not a natural inhalant allergen and does not prime airway dendritic cells the same way as dust mites, pollens and other allergens do in human asthma. Traditional therapy of allergic disease has in large part relied on the abatement of symptoms with H1-antihistamines (rhinoconjunctivitis, food allergy, urticaria), adrenaline (anaphylaxis) or β2–adrenoceptor agonists (asthma), and the suppression of inflammation with corticosteroids. Besides improving the pharmacology of known drugs, the only novel asthma therapies to emerge are leukotriene inhibitors (for example, montelukast) and the non-anaphylactogenic anti-IgE, omalizumab, both of which are directed at targets identified well over 40 years ago. There have been disappointments with a wide range of biologics targeting activating receptors on T cells, cytokines, chemokines, adhesion molecules and inflammatory mediators. Having shown convincing efficacy in in-vitro cell systems and animal models, and possibly some level of efficacy in acute allergen challenge in mild asthma, all of these have fallen short of expectations when trialled in human asthma. In moderate–severe asthma, where the unmet therapeutic need is greatest, trials of novel biologics have revealed only small subgroups in which efficacy has been shown or is suggestive.

Most of the potential new therapeutics have been directed towards aspects of the Th2 pathway, and yet gene profiling of epithelial cells of asthmatic patients indicates that in only half of cases could they be classified as Th2 predominant. Indeed, the premise that asthma is primarily an allergic condition is a concept now being challenged, and attention is shifting to impaired innate immunity sensitizing a person to allergy. In the future, it is essential that asthma is not treated as a single disorder, but rather defined by causative pathways. We need new diagnostic biomarkers to identify patients most likely to respond to highly selective biologics, such as anti-IL-5 biologic (mepolizumab) and anti-IL-13 (lebrikizumab). These therapies are only active in particular subtypes of asthma, when the molecules they target lie on a causative disease pathway. Forms of allergic disease affecting organs other than the lungs have taken second place to asthma in therapeutic research, despite the large and increasing unmet clinical need. Repositioning the biologics for use in diseases other than asthma, however, is leading to some therapeutic success. Omalizumab has shown benefits in a range of diseases, including: IgE autoimmune urticaria; recalcitrant atopic dermatitis; allergic bronchopulmonary aspergillosis; and therapy-resistant systemic mast cell activation disease. It can also provide protection against anaphylaxis during food allergen immunotherapy. There have been successes, too, with mepolizumab for eosinophilic oesophagitis, ChurgStrauss syndrome and other hypereosinophilic disorders. The World Allergy Organization White Book on Allergy stresses that allergy and asthma have not only increased in prevalence, but also in severity and complexity — with attendant health costs. Recognizing that they are reaching worrying proportions, it is necessary to focus more on studying all these diseases as they occur in humans, using well-phenotyped patients, biomarkers and experimental medicine approaches. Understanding the pathobiology of the disease in wellphenotyped humans will then enable a direct assessment of clinical efficacy, using relevant disease-related stressors besides allergen challenge, such as viruses and pollutants. A different type of more open and trusting relationship is also needed between academia and industry, where greater collaboration is encouraged in the precompetitive space. The current model of blockbuster drug discovery is unsustainable. ■

RESEARCH FOCUSING HEAVILY ON IgE HAS NOT BEEN TRANSLATED INTO WIDESPREAD PATIENT BENEFIT – PARTLY DUE TO THE LIMITATIONS OF ANIMAL MODELS.

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Stephen Holgate is UK Medical Research Council Clinical Professor at the University of Southampton, Honorary Consultant Physician at the Southampton University Hospital NHS Trust and Chairman of the UK Respiratory Research Collaborative. e-mail: s.holgate@soton.ac.uk


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Nutrition and the Immune System Authors Stephanie Blum, Sami Damak, Sophie Nutten, Jalil Benyacoub, Jennifer Dean, Annick Mercenier, Peter van Bladeren & Johannes le Coutre NestlĂŠ Research Center, 1000 Lausanne 26, SWITZERLAND

The immune system is a fascinatingly complex and dynamic system protecting the body against challenges such as pathogens, foreign substances and tissue damage. It must be able to distinguish pathogens from innocuous self or food antigens, to maintain homeostasis and optimal health. The immune response comprises two complementary components: innate and adaptive immunity. At the innate level, the immune response to invading pathogens or toxin is immediate, but non-specific. The complementary adaptive immune system provides a more targeted and specific reaction to pathogens- and antigens for protection which is more robust and long lasting. The 2011 Nobel Prize in Physiology or Medicine was awarded to Bruce Beutler, Jules Hofmann and Ralph Steinman for their pioneering characterization of innate immune cells (dendritic cells), pathways (Toll-like receptor signalling) and mechanisms that instruct adaptive immunity. Their findings acknowledge the integral role of innate immunity in the onset, course and control of adaptive immune responses.

Chronic inflammatory disorders Inflammation is the culmination of physiological responses by the immune system acting to protect the body from infections, irritants and tissue injury. In normal conditions, the interplay of innate and adaptive immune responses leads to the cessation of inflammation, a return to homeostasis and initiation of tissue repair (Fig 1). If environmental factors and/or genetic susceptibilities impair control of inflammation, it may become chronic and lead to disease conditions. Persistent inflammation of which the cause is ill defined, and does not result from infection or tissue damage, is implicated in a variety of diseases such as: inflammatory bowel disease (IBD), allergy, obesity, diabetes, cardiovascular disease and a variety of autoimmune disorders which predominantly affect people in developed countries1. The prevalence of such chronic diseases has rapidly increased in the past decades, linked with the adoption of a sedentary lifestyle and Western diet2-3. Although the phenotype of these chronic diseases may be diverse, there are

Lower immune responses to avoid exacerbation of inflammation & immune disorders

Figure 1 | Potential influence of nutrition to enhance immunity and reduce the risk of chronic inflammation. SPONSOR RETAINS SOLE RESPONSIBILITY FOR CONTENT


Incidence

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Age in Years

World Allergy Association data

Figure 2 | The atopic march.

similarities at the molecular and cellular levels. Because nutrition may influence various mechanisms of inflammation, specific food components and nutrients have therapeutic potential to ameliorate some conditions of chronic inflammation. Inflammatory bowel disease is manifested in the two main forms of Crohn’s disease and Ulcerative Colitis, which are deemed acute and chronic inflammatory diseases. IBD is characterized by an aberrant immune response to normal bacteria in the gut microbiota in individuals with a weakened epithelial barrier. Though the etiology is not fully known, research indicates that IBD involves numerous factors such as: genetics and environment, microbial influences, reduced barrier function and a loss of mucosal homeostasis, and metabolic stress1. Obesity is characterized by the storage of triglycerides in adipose tissue, but frequently also in skeletal muscle and the liver. This brings metabolic changes and insulin resistance, which may promote inflammation. Hotamisligil et al first illustrated the link between obesity and inflammation through the positive correlation of adipose tissue and expression of TNFα1,2. It was further established that the adipocyte has an impact on the inflammatory response through the release of a cocktail of inflammatory mediators and signalling molecules. It is speculated that obesity is a state of low-grade inflammation which is stimulated by adipocytes1,4. A major cause of cardiovascular disease is atherosclerosis, or the hardening of the arteries. The incremental adhesion and accumulation of atherosclerotic plaque is complex, and its evolution is marked by the inflammatory response of monocyte/macrophage and T-lymphocyte permeation1. Allergy – like other chronic inflammatory diseases – has been on the rise in Western countries over the past decades. Statistics show that the recent incidence of allergic reactions is higher than reported cardiovascular diseases

and diabetes. Figures from 2008 provided by the National Center for Health Statistics in the U.S. indicate that four out of every 100 US children have a food allergy. And it is predicted that the incidence of allergic rhinitis will continue to increase. Similar to inflammatory bowel disease, allergy is a multifactorial disease which can stem from genetic, environmental and immune factors. Other contributors can include diet and living conditions, family size, contact with pets and farm animals, exposure to airborne allergens and pollution, and characteristics of the host itself including the gut microbiota3. Allergic reaction is the heightened response of the immune system (often in predisposed individuals) to ordinarily harmless antigens. Food (e.g. milk, egg, peanuts/nuts, soy, wheat/cereals, fish, shellfish), airborne (e.g. pollen, dust mites, pet dander) and contact allergens (e.g. Nickel) can all cause sensitization in the sufferer, which upon re-exposure to the allergen will lead to different symptoms at the level of the gastrointestinal tract, the respiratory tract or the skin. With the exception of anaphylactic shock, allergic diseases are not life threatening but can drastically reduce quality of life. Atopic eczema (dermatitis) affects 10-20% of young children and 1-3% of adults, while diagnosed food allergies occur in 5-6% of young children and 2-4% of adults. Globally, the prevalence of respiratory allergies is much higher, exceeding 30% in some countries3. Treatment for food allergy typically means avoiding foods which contain the offending allergen(s)5. When patients are allergic to wheat or egg, it may be difficult to find appropriate foods that do not exacerbate the allergy. Children who do not spontaneously outgrow their food allergy are at a much higher risk (a three-fold increase) of subsequently developing allergic rhinitis. This predisposition to allergy development, known as the “atopic march,” (Fig 2) therefore reinforces the importance of developing preventive strategies.

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New insights in the development of inflammatory disease There has been recent focus on new features of the physiopathology of inflammation including: microbiota dysbiosis, defects in T cell homeostasis and differentiation, authophagy and endoplasmic reticulum stress. Endoplasmic reticulum (ER) stress response Multiple cellular stress responses are involved in chronic disease. Different exogenous, but also endogenous insults can lead to cellular stress (Fig 3). Beyond its initial role for membrane protein synthesis, folding and transport, the ER also serves as a cellular stress integrating site6. Accumulation of misfolded proteins during protein synthesis leads to so-called ‘ER stress.’ In antagonistic response to this ER stress, eukaryotic cells develop a tightly controlled mechanism, the unfolded-protein response (UPR), enabling elimination of un- and misfolded proteins. ER stress and inflammatory pathways were shown to intersect at several stages and in experiments, has been shown as a potential mechanism implicated in IBD. Moreover, genetic polymorphism studies reported that UPR molecules are associated with IBD in human patients7. Increasing evidence suggests that energy metabolism and the UPR are connected, and the ER acts as a sensor for cellular lipid status and exposure6,7. It therefore presents an exciting avenue to explore the possible use of nutrition to modulate ER stress response, and further links to chronic inflammatory diseases. Th17 cells – new players in the initiation of inflammation Besides the differentiation of naïve CD4+ T cells into Th1 and Th2 helper cells, a new T helper subpopulation, Th17 cells, has been implicated in the onset and cause of antigen-specific


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Figure 3 | Genetic and environmental factors drive ER stress in the intestinal epithelium.

auto-immunity and tissue inflammation. Activated Th17 cells produce inflammatory cytokines, with a primary role of clearing pathogens. In line with this protective function, Th17 cells are potent inducers of inflammation in a variety of tissues. The molecular mechanism by which an adaptive immune response is skewed towards Th17 seems to rely, at least partially, on the specific pathogen sensed by innate cells. The role of Th17 pathogenicity has been highlighted in the context of several chronic inflammatory disorders including Crohn’s disease, rheumatoid arthritis and multiple sclerosis8,9. Better understanding of Th17 cell differentiation determinants could open new possibilities for therapeutic and nutritional intervention strategies. Both ER stress response and T cell homeostasis are new aspects under investigation in Nestlé research programmes on inflammation and metabolic diseases. Interactions of the gut microbiota and the host immune system The gastrointestinal tract is a multifaceted system integral in educating the early immune system and modulating host responses. It is populated by diverse microorganisms (>1,000 bacterial species) who vary in number and type along the intestine according to local biochemical conditions and nutrient availability. This complex microbiota interacts with the host, impacting its metabolic and immune functions, and is also able to send signals to the gut and enteric nervous system. Numerous studies have demonstrated the importance of the microbiota in mucosa barrier function, immune modulation and metabolism1,10-11.

A specific characteristic of the gut-associated immune system is to mount a protective inflammatory response against invading pathogens while developing homeostatic/immune tolerance towards commensal bacteria. Most commensal bacteria are coated with secretory IgA antibodies (SIgA). The SIgA interaction is not exclusively antigen specific, but can be glycan-mediated, suggesting an important role for polyreactive SIgA in controlling microbiota composition. SIgA, as well as its primary role in immune exclusion and prevention of pathogen translocation, has the capacity to selectively retro-transport bound bacteria into Peyer’s patches and initiate an immune response without harmful inflammatory reaction. This process is thought to be important in developing tolerance to endogenous microbiota12. Dysbiosis of the microbiota is a perturbation in the microbiota composition, resulting in changes in host metabolic and immune activity. Evidence suggests that dysbiosis may be associated with chronic diseases such as IBD, allergy, obesity and type 2 diabetes10-11. For example, it has been observed that allergic patients have an altered gut microbiota, as well as IBD patients; however, it has not been established whether changes in the gut microbial composition are a cause or a consequence of the aberrant immune response11. Nestlé’s interest in the role of nutrition and inflammation Although persistent inflammation and related tissue damage occurs in an organ-specific manner (gut, joints, skin, adipose tissues, etc), they share similarities at the molecular and cellular levels.

This presents a variety of possibilities for nutrition interventions common across inflammatory pathologies. In addition, due to the significant role of the gut and associated immune system, certain foods and ingredients may help ameliorate or prevent chronic inflammation and related disorders. Potential nutrients that may have antiinflammatory benefits include; omega-3 fatty acids, polyphenols, antioxidants, vitamins and pre-and probiotics1,2. Nestlé researchers are working to further understand the complexity of the gut ecosystem and its implications for health and disease prevention – in addition to exploring potential nutrients that may exert beneficial effects. Using an integrative, multidisciplinary approach and modern molecular technologies, Nestlé is investigating the link between genetic and metabolic signatures of the host, as well as the impact of environmental factors, to gain deeper insight into the pathophysiology of chronic inflammatory disorders. These studies are accompanied by analyses of the fecal microbiota and in-depth study of how the microbiota can be influenced by diet and functional food ingredients. The future challenge will be to decipher the role and impact of diet or single nutrients to prevent or delay the onset of disease, to alleviate symptoms or to prevent related mortality. Polyphenol antioxidants are keenly relevant for Nestlé, due to their presence in many foods and beverages and the possible benefits for health. Polyphenols are being investigated by Nestlé for their anti-inflammatory profile, especially for their possible impact on allergic manifestations and the ageing process.

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Cocoa is also being studied by Nestlé researchers for its associated health benefits, some of which could indirectly impact inflammation including lipid lowering, endothelial function, antioxidant activity, suppression of platelet activation, etc2. More recent studies are targeting the direct effects of cocoa on inflammatory markers. Another key area of interest for Nestlé Research is the role of lipids to modulate inflammatory disorders. Essential fatty acids, specifically fish oils rich in long-chain polyunsaturated fatty acids (LC-PUFA), have been extensively studied. Dietary intervention with omega-3 (N-3) fatty acids such as eicosapentataenoic acid (EPA) and docosahexaenoic acid (DHA) proved both experimentally and clinically to promote brain development and to reduce the risk of cardiovascular disease. It is thought that EPA and DHA interfere with pro-inflammatory pathways such as cyclooxigenases (COX) and lipoxygenases (LOX), activating the formation of anti-inflammatory metabolites1. Modulation of microbiota composition and metabolic activity Given the vital role of the microbiota in inflammatory and metabolic diseases, nutritional strategies to maintain and/or restore microbial balance are of great interest. Prebiotics, non-digestible carbohydrates that promote the growth and/or metabolic activity of selected colonic bacteria, have been thoroughly investigated10. Several studies have reported their positive impact on microbiota composition, particularly in the case of inulin-type fructans and galactooligosaccharides, for promoting immune defence and reducing infection in infants and the elderly. An improvement in clinical outcomes was reported for inulin-type fructans when consumed by IBD patients1. Another approach to balance the microbiota and influence its metabolic activity is through the use of probiotics – live microorganisms which improve health when consumed in sufficient quantities. Interest in probiotics among nutrition and health researchers has intensified in recent years. Myriad studies have examined the effects of various probiotic strains in ameliorating symptoms of diarrhea, constipation, chronic intestinal inflammation, irritable bowel syndrome, atopic dermatitis and allergy1,5,10-11. The anti-inflammatory actions of certain probiotic strains, including lactobacilli and bifidobacteria is said be a result of their ability to interact directly with intestinal epithelial cells and the gut-associated immune system10-11. For probiotics to be effective in disease treatment, finding the correct strain for the right outcome is necessary10. Nestlé is extremely active in the field of probiotics research, aiming to gain deeper knowledge about the effects of probiotics on inflammation and allergy. In the context of inflammatory conditions such as IBD, some specific strains of probiotics were experimentally shown to activate the innate immune system, support maintenance of mucosal homeostasis and interfere with inflammatory reactions, typically by promoting

maturation of regulatory dendritic cells, induction of regulatory T cells, and by producing immune modulatory molecules such as TGF-β and altering the NF-Kb cascade1,10-11. More research is needed to fully elucidate the benefits of probiotics and their impact on chronic inflammation. Nestlé’s nutrition strategies to mitigate food allergies Food can be a valuable intervention in the management of allergy. Nestlé has broadened its interest in food allergies to skin and respiratory manifestations of allergy. Current research covers two main approaches to mitigate the development of food allergy: Reducing ingredient allergenicity Lowering the allergenic potential of raw materials can be achieved through different processes, including a combination of heat treatment and hydrolysis, as pursued by Nestlé. These processes break down allergenic proteins into lessallergenic components, such as peptides. For example, extensively hydrolyzed infant formula contain mostly very small peptides of cow’s milk proteins (CMP) and thus offer a safe alternative for infants with established cow’s milk allergy. When hydrolysis of CMP is partial, the reduced allergenicity of the formula is accompanied by a capacity to induce oral immune tolerance to allergens5,13. This concept of less allergenic, tolerogenic foods has been extended beyond milk to wheat and egg (typically consumed by children at weaning), as proven by preclinical data presently consolidated in small pilot human trials. Preventing or reducing symptoms of allergic manifestations Functional ingredients may help prevent the development of allergies in susceptible consumers through modulation of their immune system towards a non-allergic state, thereby minimizing digestive, respiratory or skin allergy symptoms. Probiotics are at the forefront of investigation as health-preserving and/or health promoting food ingredients. Monitoring of allergy management with probiotics has shown encouraging results worldwide. Nestlé has identified and selected a proprietary probiotic strain (Lactobacillus paracasei) to alleviate allergic rhinitis symptoms in adults14. Other probiotic strains are currently being studied for their potential in preventing or reducing symptoms of skin and food allergies. In addition to probiotics, specific food ingredients are being tested in vitro, in vivo and in clinical trials for their efficacy in prevention of allergy and/or symptom reduction. It was recently shown by Nestlé researchers that a polyphenolenriched apple extract can attenuate food allergy symptoms in experimental models15. More research is needed to elucidate the mechanisms of action of these candidates and significantly, the optimal window of intervention should be adapted to the respective type of allergic manifestation (skin, respiratory, digestive) and the targeted effect, i.e. prevention or symptom alleviation.

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Taste ‘interventions’ against inflammation Certain inflammatory signal transduction mechanisms seem to interfere with the chemical senses of smell and taste, which have notable implications for nutrition. Among groups such as the elderly or cancer patients undergoing chemotherapy, in which low grade inflammation is common, maintaining adequate nutrition can be difficult due to reduced appetite. Various studies with Crohn’s disease patients showed increased taste thresholds for all tastes, or only for salt or sweet compounds16. Subjective taste disorders is a common complaint in cancer patients, though changes in taste thresholds are minimal, which may actually be related to decreased or modified olfactory function17. Taste disturbances have also been described in people with rheumatoid arthritis; however, data is not sufficient to confirm this as a direct consequence of inflammation. Nestlé Research considers the interaction between taste and inflammation as important in understanding the nature of changes which may occur during inflammation at both sensory and molecular levels. This knowledge will guide the development of foods and nutritional supplements to be adapted to the specific sensory profiles of patients – palatable food which provides optimal nutrition. Work by Nestlé Research to decipher the interaction between molecular signal transduction pathways of taste and inflammation was rewarded through the discovery of intestinal “taste cells.” It was observed that populations of solitary cells throughout the gastrointestinal tract express taste receptors and taste signalling elements such as umami and sweet receptors T1rs, the G-protein gustducin, and the cation channel Trpm518. Interestingly, those cells also express inflammatory markers, including COX-1, COX-2, cytokines and chemokines, suggesting an interaction between inflammation and taste signalling. Preliminary data suggests that mice lacking Trpm5 have lower expression of inflammatory markers in the colon. Similar cells are found in the nasal epithelium and express bitter taste receptors (T2rs), gustducin and Trpm5. Recently, it was shown that stimulation of these bitter taste receptors with denatonium benzoate induces leakage of plasma albumin into the nasal epithelium and activates dendritic cells, therefore inducing an inflammatory and immune response in the nasal cavity. This effect disappears in mice lacking Trpm5 or gustducin19. Taste tissue expresses several inflammatory molecules and receptors such as Toll-like receptors, interleukins, and interferon receptors. Using a rodent model, Wang et al showed that inflammation activates the interferon signalling pathways in taste tissue, shortening the lifespan of taste bud cells20. Work by Nestlé researchers discovered that CCR6, the receptor for the cytokine CCL20 is expressed in type II taste cells, the subset of cells involved in the transduction of sweet, bitter and umami tastes and Nestlé is continuing the investigation of its physiological significance.

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Future Considerations Much remains to be discovered about the intricacies of the immune system, especially pertaining to chronic inflammation, disease development and the effect of nutrition on these conditions. Nestlé is committed to the pursuit of scientific knowledge and technological advancements to leverage the benefits of food and nutrition for health, wellness and improved quality of life for people at all ages and stages of life. References 1. Calder, P. C. et al. Inflammatory disease processes and interactions with nutrition. British Journal of Nutrition 101 (Suppl 1), S1–S45 (2009). 2. Egger, G. & Dixon, J. Inflammatory effects of nutritional stimul: further support for the need for a big picture approach to tackling obesity and chronic disease. Obesity Prevention 11, 137–149 (2010). 3. Bach, J. F. The effect of infections on susceptibility to autoimmune and allergic diseases. N. Engl. J. Med. 347 (12), 911–920 (2002). 4. Stienstra, R., Duval, C., Müller, M. & Kersten, S. PPARs, obesity, and inflammation. PPAR Research 2007, 1–10 (2006).

5. Zuercher, A. W., Fritsché, R., Corthésy, B. & Mercenier, A. Food products and allergy development, prevention and treatment. Curr. Opin. Biotech. 17, 198–203 (2006). 6. Todd, D. J., Lee, A. H. & Glimcher, L. H. The endoplasmic reticulum stress response in immunity and autoimmunity. Nat. Rev. Immunol. 8, 663–674 (2008). 7. Kaser, A. et al. XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell 134, 743–756 (2008). 8. Korn, T., Bettelli, E., Oukka, M., & Kuchroo, V. K. IL-17 and Th17 Cells. Annu. Rev. Immunol. 27, 485–517 (2009). 9. Peters, A., Lee, Y. & Kuchroo, V. K. The many faces of Th17 cells. Curr. Opin. Immunol. Epub of print. doi:10.1016/j.coi.2011.08.007 (2011). 10. Wallace, T. C. et al. Human gut microbiota and its relationship to health and disease. Nutr. Reviews 69(7), 392–103 (2011). 11. Nagalingam, N. A. & Lynch, S. V. Role of the microbiota in inflammatory bowel diseases. Inflamm. Bowel Dis. epub ahead of print. doi: 10.1002/ibd.21866 (2011). 12. Kadaoui, K. A. & Corthésy, B. Secretory IgA mediates bacterial translocation to dendritic cells in mouse peyer’s patches with restriction to musocal compartment. J. Immunol. 179, 7751–7757 (2007).

13. Szajewska, H. & Horvath, A. Meta-analysis of the evidence for a partially hydrolyzed 100% whey formula for the prevention of allergic diseases. Curr. Med. Res. Opin. 26, 423–437 (2010). 14. Wassenberg, J. et al. Effect of Lactobacillus paracasei ST11 on a nasal provocation test with grass pollen in allergic rhinitis. Clin. Exp. Allergy 41, 565–573 (2011). 15. Zuercher, A. W., Holvoet, S., Weiss, M. & Mercenier, A. Polyphenol-enriched apple extract attenuates food allergy in mice. Clin. Exp. Allergy 40, 942–950 (2010). 16. Zopf, Y. et al. Alterations of taste perception in Crohn’s disease and their dependency on disease activity and nutritional behavior. J. Clin. Gastroenterol. 43, 617–621 (2009). 17. Sherry, V. W. Taste alterations among patients with cancer. Clin. J. Oncol. Nurs. 6, 73–77 (2002). 18. Bezençon, C., le Coutre, J. & Damak, S. Taste-signaling proteins are coexpressed in solitary intestinal epithelial cells. Chem. Senses 32, 41–49 (2006). 19. Tizzano, M. & Finger, T. E. Nasal solitary chemosensory cells link irritation to inflammation, ACHEMS 2011 Annual Meeting (2011). 20. Wang, H., Zhou, M., Brand, J. & Huang, L. Inflammation activates the interferon signaling pathways in taste bud cells. J. Neurosci. 27, 10703–10713 (2007).

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