Asthma's Inner World
Clues to the three-decade surge in asthma rates may be found not in cells of the lung, but among the universe of bacteria in the gut.
It began as a mystery.
For years, Marsha Wills-Karp, PhD, had used the same strains of laboratory mice to study the molecular mechanisms of asthma. And for years standard tests had shown that one commonly used strain, A/J mice, was susceptible to asthma, while another standard strain, C3H mice, was resistant to the disease.
That changed when Wills-Karp moved her lab from the Bloomberg School to the Cincinnati Children’s Hospital Medical Center 13 years ago. Suddenly, the A/J mice were less asthmatic, while the C3H mice were more susceptible to the disease.
“We were baffled,” says Wills-Karp.
Other than geography, nothing had changed. The mice were the same genetic strains she had always used, ordered from the same company she had always patronized. Even the scientists handling the animals were the same—graduate students who had accompanied Wills-Karp to Cincinnati.
“When you want wisdom and insight as badly as you want to breathe, it is then you shall have it.” —Socrates
The mystery renewed in 2012 when Wills-Karp returned to the Bloomberg School to chair the Department of Environmental Health Sciences and continue her asthma research. And the mice have resumed their old patterns: A/J’s are susceptible to asthma and C3H’s are resistant.
Wills-Karp’s group spent hours brainstorming what might account for the differences, and they painstakingly devised new protocols to adjust for the changes.
The mercurial mice had the potential to turn into “a big headache,” says Wills-Karp during a June interview in her seventh-floor office on Wolfe Street. “Sometimes, however, a big headache can turn out to be exciting because we can use it for our own devices.”
In this case, Wills-Karp says, the headache has helped her recognize a new paradigm for asthma, a model that could help explain some of the disease’s unsolved puzzles.
New ideas, new insights are welcome in a field that has seen asthma rates skyrocket over the past 30 years. Worldwide prevalence is now 300 million, with cases projected to reach 400 million by 2025. Genes and the environment clearly factor into asthma’s development, but cannot explain everything about the disease’s development or its rise. Severe asthma—the source for most asthma-associated hospitalizations, deaths and health costs—presents another conundrum. According to the CDC, asthma in the U.S. is responsible for 1.9 million emergency department visits per year and $56 billion in health costs and lost productivity. Why some patients develop severe forms of the disease while others experience only mild cases is not known.
Its symptoms—inflamed bronchi, wheezing, coughing, labored breathing—clearly mark asthma as a lung disease. Yet the mystery presented by the furry A/J’s and C3H’s has led Wills-Karp to focus on a different organ system: the gut. Specifically, she’s targeting the horde of bacteria that reside there. Known collectively as the intestinal microbiota, these microbes help us digest our food, metabolize certain vitamins and keep disease-causing bacteria in check. A growing body of evidence also links disruptions in the microbiota to a host of diseases. Asthma, says Wills-Karp, especially severe asthma, may be one of those diseases.
She now believes that different intestinal microbiota accounted for the discrepancies in asthma between the Baltimore and Cincinnati mouse colonies. Different types of feed in the two animal houses may have facilitated the growth of different microbiota, says Wills-Karp, a hypothesis she will examine in future studies. In the meantime, the unexpected discrepancy has given Wills-Karp the opportunity to understand the microbiota’s possible role in asthma.
The Disease Connection
Research on the microbiota and microbiome (all the genes of the microbiota) has grown exponentially in recent years. Scientists funded through NIH’s Human Microbiome Project are studying everything from the urethral microbiome of adolescent males, to the role of the gut microbiota in obesity in the Amish and the skin microbiome associated with acne. And at Hopkins, Wills-Karp has organized a Microbiome Interest Group, which includes more than 100 scientists from diverse disciplines (see sidebar).
“We’re born alone, we live alone, we die alone,” Orson Welles once lamented. Not from a biological perspective. Microbiome studies make it increasingly clear that we move through this world in congress with trillions of microbial companions—in our intestines, on our skin, in our eyes, and on every surface of the human body.
And some studies are beginning to produce tantalizing results showing microbial patterns that correlate with certain diseases. For instance, Cynthia Sears, MD, a Microbiome Interest Group member and professor of Medicine, has shown in mice that certain toxin-producing microbes are associated with colon cancer. When these microbes colonize the gut, they may induce conditions that cause or exacerbate colon cancer. Her findings and those from other labs are early but tantalizing, says Sears. “They raise real hope there is a bigger story and also hope that the microbiome will be manipulable in ways that help treat or diagnose disease,” she says.
The big question, though, is what the findings mean. To date, researchers have been charting the organisms that make up the microbiota, says Jonathan Braun, MD, PhD, a professor of Pathology at the UCLA School of Medicine whose research involves the microbiome. Now, he says, “we’re moving from cartography of the microbiome, finding out what [microbes] are in there, to finding out what do they do and what to do about it, what parts are useful and what parts are scary.”
Scientists might exploit such knowledge to develop microbiome-based diagnostic tools or microbiome-targeted therapies. Braun envisions, for instance, a home test kit that measures the metabolic products of a person’s gut microbiota. The results might be used to help consumers adjust their diet to reduce their risk of certain diseases.
But some scientists caution that we are not there yet and that it’s important not to oversell the microbiome. “I think it is very likely that microbiomes are involved in an incredible diversity of host phenotypes—including health, disease, etc.,” says Jonathan Eisen, PhD, a professor of Medical Microbiology and Immunology at UC Davis. “I also think largely because microbiomes are the hot thing, that there is a massive amount of overselling.”
Seeking Asthma’s Switch
Wills-Karp’s interest in asthma grew out of research she conducted in 1986 as a postdoc at Yale, where she studied the effects of aging on the muscles that control the lung’s blood vessels. A prominent theory at the time held that children with asthma outgrew the disease as their lungs matured. Wills-Karp and her colleagues set out to look for an “aging component” responsible for that effect. They never found one, but their studies piqued her interest in the immune system’s role in asthma.
That work led her to take a close look at the hygiene hypothesis, which posits that being exposed to a wide variety of microorganisms in childhood helps program the immune system, and that the hygienic Western lifestyle deprives children of this important driver of immune development.
“In the developed world, we’ve reduced microbial exposure in early life in children,” says Wills-Karp. The widespread use of antibiotics, a high rate of Caesarean section deliveries (which prevents the newborn from being exposed to the vaginal canal’s microbiota), a low rate of breast feeding, migration from rural areas to cities, and other factors that go along with economic development reduce the variety of microorganisms children encounter. Without a rich microbial “education,” the regulation of the immune system becomes skewed in a manner that makes it more sensitive to certain antigens, according to the hygiene hypothesis. This imbalance leads to a heightened risk for asthma and allergies, as well as autoimmune disease.
Indeed, as childhood has become less germy in the last 30 years, rates of asthma, type I diabetes, Crohn’s disease and certain other chronic illnesses have climbed. Zeroing in on asthma, Wills-Karp calls up on her computer screen a color-coded world map showing asthma prevalence. As the map’s colors reveal, rates vary dramatically from country to country. For instance, the United States, United Kingdom and Australia are colored fire red, the shade for countries in which more than 10 percent of the population has asthma. Russia and China, on the other hand, appear as pale green, signifying asthma prevalence of 2.5 percent or less.
Other observational evidence supports the hygiene hypothesis as well, says Wills-Karp. Scientists have shown, for example, that people in developing countries have a more diverse assortment of microbiota in their GI tracts than people in developed countries. Other studies show that children with a lot of siblings, or people who live on farms, have lower rates of asthma, endorsing the view that encountering a panoply of germs in early life reduces asthma risk.
One piece missing from the hygiene hypothesis, though, has been a plausible biological mechanism. Which microbes and which immune pathways incline a child toward developing asthma? Scientists have proposed many theories, says Wills-Karp, but none has held up to close scrutiny. “It’s still an open question.”
In 2001, Wills-Karp published a review of the hygiene hypothesis in Nature Reviews Immunology. The prevailing model for asthma focused on two subclasses of immune cells, T-helper cell 1 (Th1) and T-helper cell 2 (Th2). Reduced exposure to germs early in life skews the immune balance toward producing more Th2 and less Th1. But Wills-Karp suggested that there might be more to the story and proposed that immunologists consider a broader model.
“Everything you’ll ever need to know is within you; the secrets of the universe are imprinted on the cells of your body.” —Dan Millman
A breakthrough on this idea began in 2008, when Wills-Karp read a journal article by immunology researchers at New York University. The scientists had noticed something strange. They had purchased the same strain of mice from two different popular vendors, Taconic Farms and Jackson Laboratory. But even though the mice were genetically identical, their small intestines contained dramatically different levels of a recently discovered immune cell called T helper cell 17. Taconic mice had a high level of Th17, while the Jackson mice had a low level—a difference reminiscent of Wills-Karp’s Baltimore and Cincinnati mice. In both cases, genetically identical groups showed surprisingly dissimilar results in a health parameter. Some difference in the facilities (Baltimore vs. Cincinnati; Taconic vs. Jackson) must underlie the inconsistent results.
As it happened, Wills-Karp had recently published the results of her own study in mice related to Th17 cells. “We found that Th17 had some connection with severe forms of asthma,” she says. “The more Th17, the more severe the asthma.” She suspected that the Taconic mice would have more severe levels of asthma; studies in her lab confirmed this hypothesis.
Research in people echoed the mouse results, further evidence that an overzealous Th17 response might underlie or augment severe asthma. But that still left a question: What would cause Th17 cells to spike in one mouse and not in its genetically identical cousin?
Around the same time, several studies began to suggest that the answer had something to do with the microbiome. Further studies by the NYU group pointed to one microbe in particular, an elusive bacterium that has not been definitively classified but may belong to the Clostridium genus.
These clostridia-related bacteria live in the small intestine of mice and various other species where they burrow deep inside the epithelial lining. Scientists have not succeeded in replicating this specialized environment in a culture dish, so these bacteria, like many other microbiota members, can’t yet be cultured.
In recent studies, Wills-Karp examined whether the clostridia-related bacteria could indeed be driving the immune change behind severe asthma. Because of the bacteria’s growing constraints, for these experiments she had to devise strategies.
In one experiment, for instance, her team called upon a Japanese company that had developed special mice that have absolutely no germs in their bodies except for the clostridia-related bacteria. They collected fecal material from the mice and shipped it to Wills-Karp’s lab. Presumably, the fecal material would contain the clostridia-related bacteria. Wills-Karp’s team transplanted the fecal material into a common breed of laboratory mice that scientists had shown were free of the clostridia-related bacteria. They then compared the rate of asthma symptoms in those mice to a comparable group of mice that had not received the fecal transplants.
“I know it sounds strange,” says Wills-Karp. “The things scientists do.”
Strange or not, the experiment proved useful. The mice receiving the fecal transplants went on to develop severe asthma, a finding that, along with other results, provided strong evidence that this member of the microbiome may drive severe asthma.
Further studies revealed another interesting aspect of this process. “In mice, this bacteria is cleared during weaning or maturation,” says Wills-Karp. Her initial experiments involved young mice, those still colonized by the clostridia-related bacteria. So her group did a series of experiments to determine whether the immune changes brought about by the bacteria endure.
Indeed they do, her studies showed. “We see that past the time of clearance of that bacteria, this Th17 response persists,” says Wills-Karp. “Whatever happens is changing the immune response indefinitely.” Her studies suggest that the presence of the bacteria in the guts of young mice gets communicated to the bone marrow, the site of immune cell production. This information skews the immune system’s normal balance, biasing it in favor of the production of Th17 cells.
It’s easy to see how the same scenario might occur in people, says Wills-Karp.
“The going assumption with asthma is that the first year of life is critical to the establishment of the disease,” she says. Certain events early in life can disrupt the gut’s normal microbial balance. If a child receives repeated courses of antibiotics, for example, those medicines might skew the balance in a way that allows a clostridia-related species to flourish. As in the mouse, that information would get telegraphed to the immune system, and so on.
A Proof of Concept
Wills-Karp’s findings demonstrate something that no scientist has shown before, says Richard Markham, MD, a Bloomberg School professor of Molecular Microbiology and Immunology and an expert in sequencing technology essential to much microbiome research.
“They suggest for the first time that the presence of a single species of bacteria has influence on whether an individual can develop asthma,” he says.
Markham and Wills-Karp add that asthma may not be the only disease that follows this pattern. Th17 cells have also been associated with arthritis, multiple sclerosis, Crohn’s disease and other autoimmune conditions. The microbiota may underlie those diseases as well.
Her results, says Wills-Karp, “are proof of concept.” One big unknown is whether the human disease truly does parallel the mouse pattern. Patients with severe asthma do have elevated levels of Th17 cells, scientists have found. But no one has shown that the clostridia-related bacteria underlie those cases.
“our purest, sweet necessity: the air.” —Mary Oliver
Wills-Karp is starting to address this question in a study with Stacey Burgess, PhD, a former student and now an infectious disease researcher at the University of Virginia. The pair will examine whether the clostridia-related bacteria are more prevalent in children with asthma. This and Wills-Karp’s other studies could help guide the way toward new asthma treatments—perhaps a drug that dampens or eliminates the clostridia-related bacteria or a new and improved probiotic.
Of course, preventing asthma in the first place would be even better. Here, too, growing knowledge about the microbiome might offer some guidance. Research implies that microbially rich environments reduce asthma risk. So does it make sense to raise your kids on a farm or simply not wash off the pacifier after it falls on the floor, five-second rule or no? The New York Times Magazine recently ran a story on the microbiome; the opening photo showed a baby slathered in mud, mouthing a grimy toy car that was clenched in a dirt-encrusted fist.
Wills-Karp won’t go so far as to endorse muddy playtime, although she observes that there is an evolutionary argument to be made for this practice: “Kids when they are young touch everything and put it all in their mouths. Maybe there’s a reason for that.” She would, however, advise germ-phobic parents to temper their fear, saying, “I would tell parents not to go overboard with the hand sanitizers. Babies do need some kind of exposure to the environment.’”
Greater understanding of the microbiome’s role in asthma may help scientists refine such prevention strategies, as well as develop new treatments for those already afflicted. The microbiota may contribute a little or a lot to asthma, says Wills-Karp. “We don’t yet know. Microbiome research is in the early stages.”
The microbiome mystery continues.