诱人的肠道微生物与大脑之间的联系(Nature：译文在后)

The tantalizing links between gut microbes and the brain
诱人的肠道微生物与大脑之间的联系
Neuroscientists are probing the idea that intestinal microbiota might influence brain development and behaviour.
神经科学家正在调查认为肠道菌群可能会影响大脑发育和行为。

Peter Andrey Smith
彼得·安德烈史密斯
14 October 2015 Corrected: 16 October 2015
Illustration by Serge Bloch
Nearly a year has passed since Rebecca Knickmeyer first met the participants in her latest study on brain development. Knickmeyer, a neuroscientist at the University of North Carolina School of Medicine in Chapel Hill, expects to see how 30 newborns have grown into crawling, inquisitive one-year-olds, using a battery of behavioural and temperament tests. In one test, a child's mother might disappear from the testing suite and then reappear with a stranger. Another ratchets up the weirdness with some Halloween masks. Then, if all goes well, the kids should nap peacefully as a noisy magnetic resonance imaging machine scans their brains.
“We try to be prepared for everything,” Knickmeyer says. “We know exactly what to do if kids make a break for the door.”
Knickmeyer is excited to see something else from the children — their faecal microbiota, the array of bacteria, viruses and other microbes that inhabit their guts. Her project (affectionately known as 'the poop study') is part of a small but growing effort by neuroscientists to see whether the microbes that colonize the gut in infancy can alter brain development.
The project comes at a crucial juncture. A growing body of data, mostly from animals raised in sterile, germ-free conditions, shows that microbes in the gut influence behaviour and can alter brain physiology and neurochemistry.
In humans, the data are more limited. Researchers have drawn links between gastrointestinal pathology and psychiatric neurological conditions such as anxiety, depression, autism, schizophrenia and neurodegenerative disorders — but they are just links.
“In general, the problem of causality in microbiome studies is substantial,” says Rob Knight, a microbiologist at the University of California, San Diego. “It's very difficult to tell if microbial differences you see associated with diseases are causes or consequences.” There are many outstanding questions. Clues about the mechanisms by which gut bacteria might interact with the brain are starting to emerge, but no one knows how important these processes are in human development and health.
That has not prevented some companies in the supplements industry from claiming that probiotics — bacteria that purportedly aid with digestive issues — can support emotional well-being.
Pharmaceutical firms, hungry for new leads in treating neurological disorders, are beginning to invest in research related to gut microbes and the molecules that they produce.
Scientists and funders are looking for clarity. Over the past two years, the US National Institute of Mental Health (NIMH) in Bethesda, Maryland, has funded seven pilot studies with up to US$1 million each to examine what it calls the 'microbiome–gut–brain axis' (Knickmeyer's research is one of these studies). This year, the US Office of Naval Research in Arlington, Virginia, agreed to
pump around US$14.5 million over the next 6–7 years into work examining the gut's role in cognitive function and stress responses. And the European Union has put €9 million (US$10.1 million) towards a five-year project called MyNewGut, two main objectives of which target brain development and disorders.
Nature special:
Human microbiota
The latest efforts aim to move beyond basic observations and correlations — but preliminary results hint at complex answers. Researchers are starting to uncover a vast, varied system in which gut microbes influence the brain through hormones, immune molecules and the specialized metabolites that they produce.
“There's probably more speculation than hard data now,” Knickmeyer says. “So there's a lot of open questions about the gold standard for methods you should be applying. It's very exploratory.”
Gut reactions
Microbes and the brain have rarely been thought to interact except in instances when pathogens penetrate the blood–brain barrier — the cellular fortress protecting the brain against infection and inflammation. When they do, they can have strong effects: the virus that causes rabies elicits aggression, agitation and even a fear of water. But for decades, the vast majority of the body's natural array of microbes was largely uncharacterized, and the idea that it could influence neurobiology was hardly considered mainstream. That is slowly changing.
Studies on community outbreaks were one key to illuminating the possible connections. In 2000, a flood in the Canadian town of Walkerton contaminated the town's drinking water with pathogens such as Escherichia coli and Campylobacter jejuni. About 2,300 people suffered from severe gastrointestinal infection, and many of them developed chronic irritable bowel syndrome (IBS) as a direct result.
During an eight-year study1 of Walkerton residents, led by gastroenterologist Stephen Collins at McMaster University in Hamilton, Canada, researchers noticed that psychological issues such as depression and anxiety seemed to be a risk factor for persistent IBS. Premysl Bercik, another McMaster gastroenterologist, says that this interplay triggered intriguing questions. Could
psychiatric symptoms be driven by lingering inflammation, or perhaps by a microbiome thrown out of whack by infection?
The McMaster group began to look for answers in mice. In a 2011 study2, the team transplanted gut microbiota between different strains of mice and showed that behavioural traits specific to one strain transmitted along with the microbiota. Bercik says, for example, that “relatively shy” mice would exhibit more exploratory behaviour when carrying the microbiota of more-adventurous mice. “I think it is surprising. The microbiota is really driving the behavioural phenotype of host. There's a marked difference,” Bercik says. Unpublished research suggests that taking faecal bacteria from humans with both IBS and anxiety and transplanting it into mice induces anxiety-like behaviour, whereas transplanting bacteria from healthy control humans does not.
Such results can be met with scepticism. As the field has developed, Knight says, microbiologists have had to learn from behavioural scientists that how animals are handled and caged can affect things such as social hierarchy, stress and even the microbiome.
And these experiments and others like them start with a fairly unnatural model: germ-free — or 'gnotobiotic' — mice. These animals are delivered by Caesarean section to prevent them from picking up microbes that reside in their mothers' birth canals. They are then raised inside sterile isolators, on autoclaved food and filtered air. The animals are thus detached from many of the communal microbes that their species has evolved with for aeons.
In 2011, immunologist Sven Pettersson and neuroscientist Rochellys Diaz Heijtz, both at the Karolinska Institute in Stockholm, showed that in lab tests, germ-free mice demonstrated
less-anxious behaviour than mice colonized with natural indigenous microbes3. (Less anxiety is not always a good thing, evolutionarily speaking, for a small mammal with many predators.) When the Karolinska team examined the animals' brains, they found that one region in germ-free mice, the striatum, had higher turnover of key neurochemicals that are associated with anxious behaviour, including the neurotransmitter serotonin. The study also showed that introducing adult germ-free mice to conventional, non-sterile environments failed to normalize their behaviour, but the offspring of such 'conventionalized' mice showed some return to normal behaviour, suggesting that there is a critical window during which microbes have their strongest effects.
By this time, many researchers were intrigued by the mounting evidence, but results stemmed mostly from fields other than neuroscience. “The groups working on this are primarily gut folks, with a few psychology-focused people collaborating,” says Melanie Gareau, a physiologist at the University of California, Davis. “So the findings tended to describe peripheral and behavioural changes rather than changes to the central nervous system.”
Innovations in the microbiome
But Pettersson and Diaz Heijtz's research galvanized the field, suggesting that researchers could get past observational phenomenology and into the mechanisms affecting the brain. Nancy Desmond, a programme officer involved in grant review at the NIMH, says that the paper sparked interest at the funding agency soon after its publication and, in 2013, the NIMH formed a study section devoted to neuroscience research that aims to unravel functional mechanisms and develop drugs or non-invasive treatments for psychological disorders.
Judith Eisen, a neuroscientist at the University of Oregon in Eugene, earned a grant to study germ-free zebrafish, whose transparent embryos allow researchers to easily visualize developing brains. “Of course, 'germ-free' is a completely unnatural situation,” Eisen says. “But it provides the opportunity to learn which microbial functions are important for development of any specific organ or cell type.”
Chemical exploration
Meanwhile, researchers were starting to uncover ways that bacteria in the gut might be able to get signals through to the brain. Pettersson and others revealed that in adult mice, microbial metabolites influence the basic physiology of the blood–brain barrier4. Gut microbes break down complex carbohydrates into short-chain fatty acids with an array of effects: the fatty acid butyrate, for example, fortifies the blood–brain barrier by tightening connections between cells (see 'The gut–brain axis').
Recent studies also demonstrate that gut microbes directly alter neurotransmitter levels, which may enable them to communicate with neurons. For example, Elaine Hsiao, a biologist now at the University of California, Los Angeles, published research5 this year examining how certain metabolites from gut microbes promote serotonin production in the cells lining the colon — an intriguing finding given that some antidepressant drugs work by promoting serotonin at the junctions between neurons. These cells account for 60% of peripheral serotonin in mice and more than 90% in humans.
Read next: Microbiomes raise privacy concerns
Like the Karolinska group, Hsiao found that germ-free mice have significantly less serotonin floating around in their blood, and she also showed that levels could be restored by introducing to their guts spore-forming bacteria (dominated by Clostridium, which break down short-chain fatty acids). Conversely, mice with natural microbiota, when given antibiotics, had reduced serotonin production. “At least with those manipulations, it's quite clear there's a cause–effect relationship,” Hsiao says.
But it remains unclear whether these altered serotonin levels in the gut trigger a cascade of molecular events, which in turn affect brain activity — and whether similar events take place in humans, too. “It will be important to replicate previous findings, and translate these findings into human conditions to really make it to the textbooks,” Hsiao says.
For John Cryan, a neuroscientist at University College Cork in Ireland, there is little question that they will. His lab has demonstrated6 that germ-free mice grow more neurons in a specific brain region as adults than do conventional mice. He has been promoting the gut–brain axis to neuroscientists, psychiatric-drug researchers and the public. “If you look at the hard neuroscience that has emerged in the last year alone, all the fundamental processes that neuroscientists spend their lives working on are now all shown to be regulated by microbes,” he says, pointing to research on the regulation of the blood–brain barrier, neurogenesis in mice and the activation of microglia, the immune-like cells that reside in the brain and spinal cord.
At the 2015 Society for Neuroscience meeting in Chicago, Illinois, this month, Cryan and his colleagues plan to present research showing that myelination — the formation of fatty sheathing that insulates nerve fibres — can also be influenced by gut microbes, at least in a specific part of the brain. Unrelated work7 has shown that germ-free mice are protected from an experimentally induced condition similar to multiple sclerosis, which is characterized by demyelination of nerve fibres. At least one company, Symbiotix Biotherapies in Boston, Massachusetts, is already
investigating whether a metabolite produced by certain types of gut bacterium might one day be used to stem the damage in humans with multiple sclerosis.
A move to therapy
Tracy Bale, a neuroscientist at the University of Pennsylvania in Philadelphia, suspects that simple human interventions may already be warranted. Bale heard about Cryan's work on the radio programme Radiolab three years ago. At the time, she was researching the placenta, but wondered how microbes might fit into a model of how maternal stress affects offspring.
In research published this year8, Bale subjected pregnant mice to stressful stimuli. She found that it noticeably reduced the levels of Lactobacilli present in the mice's vaginas, which are the main source of the microbes that colonize the guts of offspring. These microbial shifts carried over to pups born vaginally, and Bale detected signs that microbiota might affect neurodevelopment, especially in males.
Read next:
Gut–brain link grabs neuroscientists
In work that her group plans to present at the Society for Neuroscience meeting, Bale has shown that by feeding vaginal microbiota from stressed mice to Caesarean-born infant mice, they can recapitulate the neurodevelopmental effects of having a stressed mother. Bale and her colleagues are now wrapping up research investigating whether they can treat mice from stressed mums with the vaginal microbiota of non-stressed mice.
The work, Bale says, has “immediate translational effects”. She points to a project headed by Maria Dominguez-Bello, a microbiologist at the New York University School of Medicine, in which babies born by means of Caesarean section are swabbed on the mouth and skin with gauze taken from their mothers' vaginas. Her team wants to see whether these offspring end up with microbiota similar to babies born vaginally. “It's not standard of care,” Bale says, “but I will bet you, one day, it will be.”
Many are still sceptical about the link between microbes and behaviour and whether it will prove important in human health — but scientists seem more inclined to entertain the idea now than they have in past. In 2007, for example, Francis Collins, now director of the US National Institutes of Health, suggested that the Human Microbiome Project, a large-scale study of the microbes that colonize humans, might help to unravel mental-health disorders. “It did surprise a few people who assumed we were talking about things that are more intestinal than cerebral,” Collins says. “It was a little bit of leap, but it's been tentatively backed up.”
Funding agencies are supporting the emerging field, which spans immunology, microbiology and neuroscience, among other disciplines. The NIMH has offered seed funding for work on model systems and in humans to probe whether the area is worth more-substantial investment, a move that has already brought more researchers into the fold. The MyNewGut project in Europe has an even more optimistic view of the value of such research, specifically seeking concrete dietary recommendations that might alleviate brain-related disorders.
Today, Knickmeyer's project on infants represents what she calls “a messy take-all-comers kind of sample”. Among the brain regions that Knickmeyer is scanning, the amygdala and prefrontal cortex hold her highest interest; both have been affected by microbiota manipulations in rodent models. But putting these data together with the dozens of other infant measures that she is taking will be a challenge. “The big question is how you deal with all the confounding factors.” The children's diets, home lives and other environmental exposures can all affect their microbiota and their neurological development, and must be teased apart.
Knickmeyer speculates that tinkering with microbes in the human gut to treat mental-health disorders could fail for other reasons. Take, for instance, how microbes might interact with the human genome. Even if scientists were to find the therapeutic version of a “gold Cadillac of microbiota”, she points out, “maybe your body rejects that and goes back to baseline because your own genes promote certain types of bacteria.” There is much more to unravel, she says. “I'm always surprised. It's very open. It's a little like a Wild West out there.”
Nature 526,312–314 (15 October 2015) doi:10.1038/526312a
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Corrections
Corrected:An earlier version of this story incorrectly stated that the US Office of
Naval Research agreed to commit US$52 million into gut–brain research. In
fact, the figure is closer to $14.5 million over the next 6–7 years. The
text has now been corrected.
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Nature:肠道微生物与大脑之间的诱人关系
很多发现常常让我感到吃惊。

这个领域是开放的，它有点像“西大荒”。

自从Rebecca Knickmeyer与她最新的大脑研究项目参与者相识以来，时间已过去了1年。

Knickmeyer是美国北卡罗来纳大学教堂山分校医学院神经学家，通过一连串的行为和性格测试，她希望看到30名新生儿如何学会爬行，并成长为具有认知能力的1岁儿童。

接下来，如果一切进展顺利，当嘈杂的核磁共振对他们的大脑成像时，这些孩子应该睡得很沉稳。

“我们希望一切都万无一失。

”Knickmeyer 说，“如果有孩子想要往门外闯，我们很清楚应该做什么。

”
局面待转
Knickmeyer希望从这些儿童检查中看到其他一些令她兴奋的东西，她还希望检查这些孩子排泄物中的微生物群，即寄居在他们肠道内的一系列细菌、病毒和其他微生物。

她的项目(被亲切地称为“便便研究”)属于神经科学领域一个研究规模较小并日益壮大的分支。

神经科学家希望通过相关研究了解寄生在婴儿肠道内的微生物是否可以改变大脑发育。

这项研究启动的时刻很关键。

越来越多来自无菌条件下培育动物的数据表明，肠道微生物可以影响动物的行为，并能够改变大脑生理学和神经化学特征。

然而，在人体研究中，相关数据却极为有限。

研究人员已经在肠胃病理学和精神神经疾病学(如焦虑症、抑郁症、自闭症、精神分裂症和神经退行性疾病)之间建立了联系，然而这些联系之间因为缺乏证据而不能被接受。

“总体上说，微生物研究中的因果关系问题没搞清楚的仍然很多。

”加州大学圣迭戈分校微生物学家Rob Knight说，“很难说明你看到的与疾病相关联的微生物是导致疾病的原因还是结果。

”这中间有很多引人瞩目的问题，比如肠道细菌如何影响大脑机制的线索才刚开始萌芽，目前尚无人了解其在人类发育和健康中有多重要。

即便如此，这种现状也未能阻止膳食补充剂行业内的一些公司宣称，益生菌(据称是有助于消化系统的细菌)有助于产生正面情绪。

而那些渴望在神经紊乱领域领先一步的制药企业也开始在与肠道微生物及其产生的代谢分子等相关领域大量投资。

科学家和投资者都希望追根溯源，把问题搞清楚。

过去两年来，马里兰州国家心理卫生研究所(NIMH)已经支持了7个“微生物—肠道—大脑轴线”试点项目，每个项目支持资金达100万美元。

今年，弗吉尼亚州阿灵顿海军研究办公室同意在未来几年投资5200万美元用于支持肠胃在认知功能和压力反应中的角色。

同时，欧盟也斥资900万欧元支持一项名为“我的新肠胃”的5年计划，该计划的两个主要目标分别是大脑发育和神经紊乱。

最新的研究旨在超越基础观察和相互关系分析等问题，为揭开复杂的问题提供初步研究结果。

研究人员正在掀开一个广阔而纷繁复杂的系统的面纱，在这个系统中，肠道微生物会通过荷尔蒙、免疫分子及其新陈代谢对大脑产生影响。

“就眼下来看，各种推理和猜测远远超过了切实的数据。

”Knickmeyer说，“究竟采用何种方法才是最佳方法?这个问题的答案是开放的，所有的一切都需要探索。

”
肠胃反应
除了极少的个例之外，微生物和大脑之间很少被认为存在联系。

这里的极少数案例包括病原
体穿过血液和大脑的屏障——即防止大脑感染和发炎的细胞“堡垒”。

一旦这种情况发生，它们往往会造成强烈的反应：如导致狂犬病的病毒会引发攻击性、躁动症，甚至会产生恐水症。

但数十年来，人体内的大量微生物种类多数依然未被识别，而它们可以对神经生物学产生影响的观点，更是很难被主流科学界接受。

然而，这一局势正在发生转变。

2000年，加拿大一个名叫沃克顿的城镇发生了一场洪水，导致该镇饮用水水源被大肠杆菌和空肠弯曲杆菌污染，约有2300人发生了极为严重的肠胃感染，很多人直接产生了慢性肠易激综合征(IBS)。

在汉密尔顿麦克马斯特大学肠胃学家Stephen Collins带领的一项为期8年的研究中，研究人员注意到，IBS患者存在抑郁症和焦虑症的风险因素更高。

麦克马斯特大学另一名肠胃学家Premysl Bercik表示，这种相互作用引发了更加复杂的问题：这些心理症状是由长期炎症引发的?还是由感染导致的微生物失衡驱动的?
麦克马斯特团队开始从小鼠体内寻找答案，在2011年的一项研究中，该团队在不同品系的小鼠体内移植了肠道微生物，研究表明某一品系的小鼠独有的特征会随着微生物的移植而传播。

例如，Bercik说，在携带更富于冒险精神的小鼠肠道内的微生物后，原来“相对害羞”的小鼠表现出了更具探索性的行为。

“我觉得这令人惊奇，肠道微生物的确在驱动着宿主的行为，并表现出非常明显的差异。

”Bercik说。

目前尚未发表的研究还表明，把IBS患者排泄物中的微生物植入小鼠体内后，这些小鼠同样表现出了焦虑行为;相反，把健康人肠道内的微生物植入小鼠体内后，则没有这种反应。

然而，这样的研究结果同时也引来了怀疑。

随着该领域的发展，Knight表示，微生物学家应该从行为学家那里取经，了解动物生存环境(如关在笼子里)对其社会地位、压力甚至是体内的微生物的影响。

为此，这些实验让研究人员开始采用一种非自然的模型：无菌或限菌培养的小鼠。

2011年，瑞典斯德哥尔摩卡罗林斯卡学院免疫学家Sven Pettersson和神经学家Rochellys Diaz研究发现，在实验室测验中，无菌小鼠表现出较低的焦虑症，而植入正常肠道细菌的小鼠则表现出更强的焦躁性。

(专家表示，从进化论的角度来讲，一种天敌很多、体形却相对较小的哺乳动物具有较低的焦虑性并不总是一件好事。

)
俄勒冈大学神经学家Judith Eisen已经获得了一项无菌斑马鱼研究的经费支持，这种鱼类的透明胚胎可以让研究人员更容易看到其大脑的发育。

“当然，‘无菌’是一种非自然的环境。

”Eisen说，“但是它提供了一个机遇，可以让我们了解哪种微生物的功能对于某种具体器官或某类细胞具有多大的影响力。

”
走向治疗
同时，研究人员已开始揭示肠道细菌可能向大脑传递信号的方式。

Pettersson等科学家已经揭示出，在成年小鼠体内，微生物代谢会影响血液—大脑屏障的生理基础。

肠道微生物会把合成碳水化合物分解成可产生一系列影响的短链脂肪酸：例如丁酸盐脂肪酸可以通过强化细胞间的联系，增强血液—大脑屏障。

此外，近期的研究还表明，肠道微生物可以直接改变神经递质水平，从而使其对神经元产生影响。

在本月即将于伊利诺伊州芝加哥举行的2015年度神经学会议上，爱尔兰国立考克大学神经学家John Cryan和同事计划作一项报告，展示髓鞘形成——即隔绝神经纤维的脂肪外鞘的形成——也会受到肠道微生物的影响，至少在大脑的一些部位是如此。

其他研究也表明，无菌小鼠还免除了实验诱导的类似多发性硬化，该病主要特点之一是神经纤维髓鞘脱失。

现在，至少有一家公司——马萨诸塞州波士顿Symbiotix生物治疗公司已经开始了相关研究，如由某种肠道微生物产生的代谢物未来有一天是否会被用于治疗人类中的多发性硬化损伤。

宾夕法尼亚大学神经学家Tracy Bale怀疑，采用该疗法进行简单的人体干预已经获批。

三年前，Bale就听说过Cryan的工作。

当时，她在研究胎盘，但对于微生物如何通过母亲的压力
影响后代也非常感兴趣。

今天，Knickmeyer的胎儿研究项目代表了她所说的“来者不拒的混合样本”。

在Knickmeyer 扫描的脑区中，杏仁核和前额叶皮层是她最感兴趣的地方，在大鼠模型中，两处脑区都受到了肠道微生物的影响。

但是把这些数据和婴儿数十项其他检测汇总在一起，将是她面临的一项挑战。

“最大的问题是，要如何处理这些复杂的因素。

”她说，儿童的饮食、家庭生活以及其他环境接触都会影响他们的肠道微生物及其神经发育，因此需要把各项影响因素区分开来。

现在，还有很多谜底等着揭开，她说：“很多发现常常让我感到吃惊。

这个领域是开放的，它有点像‘西大荒’(蛮荒的美国西部)。

”
原文链接：
The tantalizing links between gut microbes and the brain
来源：Nature。