As pediatrician Kari Nadeau and I leave California’s Pacheco Pass and head east, the Bay Area foothills give way to acres of orchards and level farmland. After three hours in the car, the jagged contours of the Fresno metropolis appear stark against a clear sky — but only because the cold air had pushed the hazy, gray smog below the horizon, Nadeau explains. She stops midsentence as the truck ahead of us coughs a cloud of black smoke. “Did you see that?” she says, eyes widening, her voice rising with a blend of awe and disgust.
Nadeau, who specializes in asthma, is heading to Fresno to meet with other researchers working on the San Joaquin Children’s Health and Air Pollution Study. It’s a trip she makes about every three months. In Palo Alto, where she lives in an airy, two-story home with her husband and five kids, she sees asthma patients a few times a week at Stanford’s Lucile Packard Children’s Hospital. Most of the children live in Palo Alto, but a few journey from Fresno for care they can’t get back home.
As we near the outskirts of the city early that afternoon, caravans of big rigs with soot-streaked trailers groan past. Not far away, dusty-faced migrant workers and their families, as well as homeless people, live in vast shantytowns — rows of tents, and shopping carts, sofas and bicycles strewn about. Although it is the most profitable agricultural area in the nation, Fresno County had the highest poverty rate in California in 2010, with 26.8 percent of its nearly 1 million residents living in poverty. Fresno-Madera ranks as the metropolitan area with the highest exposure levels to particle pollution, or soot, in the country.
Palo Alto and Fresno might as well be different worlds. And Nadeau has discovered a difference of extremes in the lungs and genes of her patients as well. She found that kids in Fresno were more likely to develop asthma not due to lung damage, but because changes on the surfaces of just two genes — and likely more — altered the way their lungs worked.
These two genes are crucial for tightening the reins on the immune system to prevent it from reacting to benign agents and triggering asthma symptoms. Unlike mutations, these changes to the surfaces of genes — part of what’s called epigenetics — alter how those genes behave without rewriting the information they encode. It’s like tagging them with Post-It notes that tell a cell to switch a gene on or off. Nadeau has discovered that in the Fresno children, long-term exposure to air pollution and secondhand smoke switched off two specific genes. Similar changes happened in the Palo Alto children, but at significantly lower rates.
Nadeau believes heavy pollution causes asthma-inducing epigenetic changes that can last a lifetime — and even transcend generations. That connection took years for Nadeau to make. The question now is whether this insight might someday lead to treatment of the disease.
A Right to Breathe
Nadeau, 49, knows asthma especially well — she’s been living with it since about age 3. Back then, she dreaded bedtime. Each night, she’d sit upright for as long as possible, a nebulizer with asthma medication strapped to her face, on guard for an asthma attack: the heavy, searing pain; the panic of gasping for air. Living for a couple of years on a houseboat off the smog-laden Newark, N.J., shore didn’t help. By elementary school, Nadeau had accepted breathing problems as a reality — one that persists to this day. She knew asthma attacks could kill her.
Nadeau’s asthma inspired her to become a doctor. As an undergraduate, she helped build a medical clinic and sewage system in Nicaragua. The impact stuck with her. At Harvard Medical School, she wanted to give fellow students the same opportunity to protect people’s right to health care on a global level, launching a partnership between the university and Physicians for Human Rights.
Nadeau had gotten reports of a tuberculosis outbreak among children at a federal immigration detention center in Harlingen, Texas. It didn’t make sense that kids’ infections would spur an outbreak; they typically didn’t have the strength to cough up the contagious mucus from deep in the lungs. So in the fall of 1994, she flew to Harlingen.
When Nadeau entered the looming white building and peered into the barracks, she saw children piled onto rows of Army-style bunk beds — and among them, adults who had seeded the outbreak and were crowded out of their own quarters. The cramped conditions created the perfect breeding ground for TB-causing bacteria.
“It really hit me,” says Nadeau. “The kids were placed in this situation. They had no control whatsoever.” After she detailed her findings in the Western Journal of Medicine, the center moved the adults back to their facility. She realized then her power as a doctor to advocate for children who have no say in their environment.
After Harvard, Nadeau worked as a resident specializing in pediatric blood diseases and cancer at Children’s Hospital of Boston. Emotionally spent, she worked for a biotech company for a few years. She left the corporate world and landed a fellowship in 2008 in allergy, asthma and immunology at Packard Children’s Hospital.
Nadeau studied immune cells called regulatory T cells, or T-regs. They are what they sound like: regulators that keep another group of cells, T helper cells, from proliferating out of control. Think of T-regs as police officers, keeping a tight leash on T helper cell attack dogs. We need T helper cells; they kick-start the immune system to respond to potential invaders. But too many can move the immune system into overdrive, evoking coughing, airway constriction, mucus production and other asthma symptoms.
Suspecting that the T-regs in asthma patients didn’t function as well as they did in healthy people, Nadeau isolated T-regs from adult and pediatric patients’ blood samples and tested their ability to suppress T helper cells. The policing T-regs from most of her 200 patients kept T helper cells at bay. But 30 had poorly functioning T-regs that let T helper cells proliferate unchecked. They also had worse asthma symptoms. One girl couldn’t leave her house without triggering an attack, while a boy had allergies so severe that he had to seek disability status.
Skeptical, Nadeau repeated the experiments, yet the same 30 patients surfaced. Did they have the same ethnicity or socioeconomic status? No and no. It’s got to be some environmental exposure, she thought, perhaps something to do with where they lived in Palo Alto.
Nadeau looked up her pediatric patients’ ZIP codes. Those 15 children weren’t from Palo Alto. They lived in Fresno, a city she soon learned had high levels of air pollution — mostly diesel exhaust from trucks, cars and tractors — in contrast to Palo Alto’s clear skies. She had a strong hunch that this pollution had disabled T-reg function in her Fresno patients.
But her sample size was still small enough that she couldn’t rule out a statistical fluke. So she cold-called Ira Tager, a now-retired environmental epidemiologist at the University of California, Berkeley, with her findings in 2008. For the past decade, Tager had been running a large-scale study in Fresno looking at how pollution affects the lungs.
He found Nadeau’s results fascinating and invited her to visit him in Berkeley. The two hit it off, and he agreed to let Nadeau collect blood from his Fresno subjects to check whether pollution had worsened their T-reg function, too.
As part of Nadeau and Tager’s study, participants completed questionnaires about their exposure to pollution and secondhand smoke. Air quality monitoring and statistical modeling measured each person’s pollution exposure.
Now Nadeau had a larger patient pool that included children with and without asthma in Fresno and Palo Alto. She saw the best T-reg function in Palo Alto kids without asthma. Even Palo Alto children with asthma had better T-reg function than Fresno children without the disease. And, sure enough, Fresno children with asthma had the worst T-reg function.
To figure out the mechanism, Nadeau focused on the gene Foxp3, which spurs immature T cells to develop into those police officer cells, T-regs. Research had shown that children born without Foxp3 suffered from asthma, allergies and autoimmune diseases. Nadeau stumbled upon one study, in mice, describing how environmental factors can tag Foxp3 with chemical markers that tell T-cell precursors to switch the gene on or off. Tagging Foxp3 with a methyl group is like sticking a Post-It note on it that says “off.” An acetyl group’s Post-It note says “on.”
“That paper changed my life,” Nadeau says. “If this is happening in mice, it’s probably happening in humans.” Some studies also suggest that these epigenetic changes are heritable.
Once Nadeau understood the role of the methyl groups in gene expression, all the dots began to connect. She believed that air pollution triggered asthma in her Fresno patients by tagging Foxp3 in immature T cells with methyl groups, switching off its expression. This prevents the cells from maturing into those police officer T-regs that hold T helper cells in check. More exposure to pollution, then, would mean more methyl groups.
As it turned out, Foxp3 bore the fewest methyl groups in Palo Alto children without asthma, and more in Palo Alto children with the disease. In Fresno children without asthma — who had grown up with more pollution — the gene had still more methyl groups. Foxp3 bore the most methyl groups in Fresno kids with asthma. “It seemed amazing for just one molecule to be standing out,” Nadeau says.
Nadeau and Tager published their results in the Journal of Allergy and Clinical Immunology in 2010. Meanwhile, researchers at Columbia University, the University of Cincinnati and other institutions began publishing similar findings. But the trend was troubling: Pollution’s imprint wasn’t unique to Fresno. Scientists were seeing the same effects in polluted cities across the country.
Smoke Screen
Nadeau’s findings revealed that pollution could cause asthma by altering our biology at a fundamental level, changing how our very genes behave. After the 2010 paper was published, she wondered, could secondhand smoke have a similar effect? It also can lead to asthma, and research had shown that children, especially those living in poor communities like Fresno, are especially vulnerable to secondhand smoke. Nadeau wanted to unravel how exposure to it affected methylation and gene expression. Scientists had already found that smoking could cause epigenetic changes. But what were those changes? And how might they trigger asthma?
In a small office in Nadeau’s clinic, Arunima Kohli, an undergraduate in her lab, was sifting through the questionnaire and pollution data that Nadeau and Tager collected. Since the questionnaires asked about participants’ secondhand smoke exposure, Nadeau asked Kohli to include the responses in the analysis of their immune cells. It’s right at our fingertips, Nadeau thought.
Nadeau wanted to examine how air pollution and secondhand smoke — both linked to asthma — spurred epigenetic changes to Foxp3. And if these stressors epigenetically altered Foxp3, they probably affected other genes regulating the allergic pathway, too. Since studies had shown that children in heavily polluted areas of the Central Valley had more infections, and pollution and secondhand smoke contain similar toxic compounds, Nadeau searched for genes that played a major role in fighting infection. Understanding how they worked might help point the way to therapies that treat them. She also sought out genes that controlled the switch for maturation of T helper cells, maintaining just the right balance of T helpers — between Th1 cells that suppress allergic responses and Th2 cells that trigger them.
Nadeau finally landed on the protein-coding gene interferon gamma, important in not only fighting infection, but also maintaining the delicate balance of T helpers. When interferon gamma is covered in methyl groups — or switched off — it tips the balance, spurring the development of Th2 cells and sending the immune system into overdrive.
Nadeau suspected that Fresno kids were getting a double whammy of Th2 cells. Not only did they have low interferon gamma expression, they also had low Foxp3 expression, meaning they had fewer T-regs to police the T helpers. Nadeau suspected the bodies of Fresno children teemed with Th2 cells that triggered asthma.
It meant pollution and secondhand smoke might have a synergistic effect. To test this, Kohli plotted each child’s amount of exposure to pollution and secondhand smoke against the methylation and expression levels of Foxp3 and interferon gamma.
Gradually, a picture emerged. The highest methylation and lowest expression of these two genes were found in Fresno patients exposed to both secondhand smoke and pollution. But before Nadeau could get excited, she needed to repeat the analysis; she needed to be sure. She handed the data to Tager, a statistician and another lab. Each calculated the same results.
“Then we knew, oh my gosh, this is really real,” Nadeau says.
For years, studies had shown evidence that pollution caused asthma and that the disease tends to occur within families. Now, Nadeau and Kohli’s results, published in Clinical Epigenetics in fall 2012, suggested an underlying mechanism. They also linked two of tobacco smoke’s effects — methylation and asthma — suggesting that smoke-induced epigenetic changes could cause asthma as well.
All in the Family
But the molecular scars that pollution and secondhand smoke leave behind might not end with the person exposed to them. Research suggests that they can be passed on to children and grandchildren, meaning it may take generations to see their full toll. A 2012 Biomed Central Medicine study found that both the offspring and grand-offspring of pregnant rats exposed to nicotine developed asthma even if those descendants had no exposure to the chemical.
Nadeau gave a human analogy. Imagine a mother who smokes around her infant daughter, causing epigenetic changes in her daughter that persist into adulthood, even if she moves away. When she has her own child, “that grandchild will have the same epigenetic changes the grandma had because of smoking,” Nadeau says.
But unlike with genetic mutations, we can “undo” bad epigenetic modifications. The more we understand the mechanisms underlying what makes individuals vulnerable or resilient, the better researchers can design interventions. Nadeau’s team is working to identify signaling proteins in the T-reg pathway, as well as develop a screen to predict allergy and asthma prognosis by measuring the ratio of various biomarkers, including Foxp3 and interferon gamma expression. Preliminary data from another study of theirs suggest that Fresno youth, by moving elsewhere to attend college, might see a reversal of some epigenetic changes to their immune cells. So far, epigenetic changes to these cells have persisted for a year. “But we’re still going to test that out long term,” Nadeau says.
Some bioethicists doubt that even the most compelling research on the intergenerational impacts of pollution would persuade lawmakers to enact further reforms. “We are not particularly good stewards of the planet or for the people who come after us,” says Mark Rothstein, a bioethicist at the University of Louisville School of Medicine.
Even so, like parents who are driven to do something, anything, when their kids are suffering, Nadeau can’t sit back and relax, knowing that her work has a chance of helping kids overcome a diagnosis resulting from chance, not choice. “Children don’t deserve to suffer,” she says. “We need better drugs than when I was a kid, and I’m not going to stop until I get there. No one should watch their kid die.”
[This article originally appeared in print as "Something in the Air."]
