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Microbial Health Factor

By Sarkis Mazmanian as told to Sara McBride The Microbial Health Factor Just one molecule can make the difference in mediating a healthy immune response. Surprisingly, it comes from bacteria. © Gina and Matt / www.ginaandmatt.com rillions of commensal bacteria cover almost all environmentally exposed surfaces of our bodies at all times. But what are they doing? And why? If you want to understand the impact of c

By | August 1, 2009

The Microbial Health Factor

Just one molecule can make the difference in mediating a healthy immune response. Surprisingly, it comes from bacteria.

© Gina and Matt / www.ginaandmatt.com
T

rillions of commensal bacteria cover almost all environmentally exposed surfaces of our bodies at all times. But what are they doing? And why? If you want to understand the impact of commensal organisms on mammals, a good place to start is with mice that are devoid of all bacteria.

When I started working on this problem in 2002, so few people were still familiar with the germ-free mouse models that I had to persuade a retired research technician to help me set up sterile chambers and teach me the ways of “sanitary engineering.” Rather than the old steel and glass contraptions that he had used in his day (50 years ago), we were able to procure nicely modernized chambers with plastic bubbles that held up to four mouse cages. After my first few chamber contaminations, I began to understand why researchers rarely use germ-free animals.

Germ-free animals were conceived of almost a century ago, but were not successfully raised until 1945. James A. Reyniers’ group at the University of Notre Dame was the first to successfully raise and study germ-free animals. Perhaps reflecting a new fervor over hygiene, researchers concluded that wiping a mammal clean of microbes might actually be a good thing. The adult mice grew enormous bellies, stemming from digestive problems, but other than that, they seemed just as healthy and lived just as long as typical mice.

In those days, science’s relationship with bacteria was adversarial—the main purpose of a microbiologist was to study infectious disease. No one seemed too curious about what the seemingly passive commensal bacteria were doing. Indeed, 20 Nobel Prizes have been awarded for research on the immune response to harmful microbes, from tuberculosis to Helicobacter pylori, the causative agent of gastric ulcers. But in the grand scheme of things, bacterial infections are rare and opportunistic. Of the over 300,000 known bacterial species and possibly millions more, only about 170 are known to be pathogenic in mammals.

When I trained as a microbiologist around the year 2000, the focus was still on pathogenic bacteria. But I became intrigued by the potential benefits of good bacteria. After all, we’ve coevolved with symbiotic bacteria for millions of years. The hygiene hypothesis, proposed in 1989 by David Strachan1, correlated lower environmental exposure to microbes—as seen in developed countries—with higher rates of allergies. The idea made sense to me. Commensal bacteria help keep pathogenic bacteria at bay, and in the late 1990s new research was beginning to show that symbionts also contribute to the development of the intestinal architecture. If bacteria were so crucial to development, what else might they do? Could they actually make us healthier? Challenging though it was, I was convinced the best way to learn about the systemic effects of bacteria was to start with mice that lacked them entirely.

U

Upon finishing my PhD in 2002 from the University of California, Los Angeles, I went to Dennis Kasper’s lab at Harvard Medical School. He was working on a prevalent commensal bacterium, Bacteroides fragilis. His lab had worked for years on the capsular polysaccharides that cover B. fragilis like hairs on a kiwi fruit. These surface carbohydrates are chains of repeating sugar molecules that function to give the bacteria a mucous-like barrier on its surface. Kasper had discovered that two of these eight polysaccharides have a unique zwitterionic structure: the molecules have both positive and negative charges on each repeating unit. While many bacteria are covered in polysaccharides, only a handful of species exhibit zwitterionic polysaccharides.

We inoculated a wild-type mouse with the bacterium H. hepaticus to create an experimental mouse version of the autoimmune disorder inflammatory bowel disease (IBD). H. hepaticus activates Th17 cells which release cytokines asociated with inflammation, like IL-17, causing symptoms of disease. But once B. fragilis expressing the polysaccharide A (PSA) is added to the gut, dendritic cells take up and present the PSA molecule on their surface, activating CD4 T cells and regulatory T cells(Tregs). The Tregs release IL-10 which suppresses the inflammatory action of IL-17, in effect alleviating IBD in mice.

When I arrived at Kasper’s lab, I wanted to learn more about these polysaccharides and their properties. I mutated the B. fragilis genes that are involved in the production of polysaccharides to express different combinations of the eight surface polysaccharides. I was able to delete seven of the eight polysaccharides by deleting the gene’s promoter region, but despite hundreds of attempts, I never generated a viable culture of bacteria that lacked all eight. It was clear that the bacteria needed this sugar coating for normal function, but I wondered whether the polysaccharides might also be important because they offered something that the mammalian host lacked. The lab had already shown that PSA—the most prevalent polysaccharide of the eight—stimulated T cells of the immune system in the test tube. They had tested B. fragilis’s other zwitterionic polysaccharide, PSB, but had found that its stimulatory effects paled in comparison to PSA. Whether PSA influenced the entire immune system of animals was a question that could only be asked in a controlled manner in germ-free mice.

The heyday of germ-free animal work was in the 1940s and 1950s. But recently other researchers dusted off the old germ-free mouse models and found problems beyond their big bellies and digestive troubles.

The heyday of germ-free animal work was in the 1940s and 1950s. But recently, other researchers dusted off the old germ-free mouse models and found problems beyond their big bellies and digestive troubles. With new cellular and molecular tools, researchers demonstrated that these animals had serious problems with their immune systems: antibody deficiencies, higher susceptibility to infections, reduced number of Peyer’s patches and germinal centers (the locations of lymphocyte activity), less active intestinal macrophages and a reduced number and cytotoxicity of intestinal epithelial lymphocytes. In 1992, Lex Nagelkerken, at the TNO Institute of Ageing and Vascular Research in The Netherlands, had found that germ-free mice had a reduced number of CD4 T cells compared to conventionally colonized mice. CD4 T cells are critical for regulating the immune response, activating both cellular and antibody reactions. We decided to use CD4 T cell levels as a proxy marker for a healthy immune system in germ-free mice.

We colonized one group of germ-free mice with whole B. fragilis and another group with a strain of B. fragilis that lacked PSA but displayed the seven other polysaccharides. To my delight, wild-type B. fragilis restored CD4 T cell levels to those of animals with hundreds of bacteria. In mice that were colonized with the mutant bacteria lacking the zwitterionic PSA, CD4 T cell levels were no better than in germ-free mice.

This was an important result. Not only was a single strain of bacteria able to restore healthy levels of CD4 T cells, but we also identified the specific surface molecule that mediated these effects. I checked for an effect on other arms of the immune system: the CD8 T cells that can directly kill other cells, and the B cells, which produce antibodies. These cells appeared not to be affected. It looked like the bacteria with intact PSA were inducing CD4 T cells specifically.

When we looked at the histology of spleens, which, along with lymph nodes, serve as sites for the generation of immune responses, we saw that germ-free mice exposed to B. fragilis without PSA lacked the well-defined follicular structures that are a hallmark of healthy immune cell development. Mice colonized with wild-type B. fragilis contained follicles in abundance. It was the first evidence that bacteria might play a role in the development of organs other than the intestine.

To double-check the specific role of this molecule, I purified PSA from the surface of B. fragilis. When I fed the germ-free mice the polysaccharide, they developed conventional CD4 T cell levels—in the absence of all bacteria!

The next question was whether PSA was stimulating all CD4 T cells equally or if one of the two branches was activated preferentially. At the time, the “helper” CD4 T cells were divided into two classes depending on the cytokines they secreted: T-helper 1 (Th1) cells, which activate the cellular arm of the immune system, and T-helper 2 (Th2) cells, which activate the humoral or antibody-producing B-cells. The balance between Th1 and Th2 cells is important for the proper function of the immune system. When we investigated the Th profile of germ-free mice, we found that they had an abnormal balance of T-helper cytokines. Germ-free mice produce large quantities of interleukin-4 (IL-4)—a Th2 cytokine—and very little inteferon gamma (IFNγ)—aTh1 cytokine—compared to conventional mice. But germ-free mice colonized with B. fragilis restored IFNγ levels to normal and reduced Th2 cytokines. Purified PSA was able to restore Th1/Th2 balance to the entire organism. What seemed to be an intrinsic feature of a healthy immune system was in fact completely controlled by a single bacterial molecule.

S

hortly after our findings were published2 an epidemiological study that extended the hygiene hypothesis caught my eye. The new study3 suggested that nonallergic autoimmune diseases such as multiple sclerosis, type 1 diabetes, and Crohn’s disease, were also on the rise in westernized societies. It occurred to me that there might be a possible role for B. fragilis in a wide range of immunologic diseases. The immune system is supposed to recognize foreign pathogens (such as bacteria) and eliminate them, while steering clear of healthy human cells. But sometimes the immune system can’t tell the difference between self and nonself, resulting in autoimmunity. One characteristic of this class of diseases is an imbalance in Th1/Th2 ratios, resulting in the immune system attacking host tissue. So if B. fragilis could correct a Th1/Th2 imbalance, perhaps it could also improve autoimmune diseases.

I began to appreciate that B. fragilis was “shaping” a coordinated and complex immune profile to promote intestinal health.

My autoimmune disease of choice was inflammatory bowel disease (IBD), a category of autoimmune disease that includes ulcerative colitis and Crohn’s disease in humans. Patients present symptoms that include abdominal pain, diarrhea, and rectal bleeding, caused by immune cell attack on the small or large intestine. These diseases affect about 2 million people in the United States, and that number is rapidly increasing. For decades, researchers had been looking for the pathogenic strains of bacteria responsible for IBD, to no avail. But several recent studies had pointed to the role of commensal—not pathogenic—bacteria in triggering IBD. With the benefits of B. fragilis in mind, I wondered: what if it wasn’t the presence of certain commensal bacteria triggering IBD, but the absence of protective symbiotic strains?

To test his hypothesis I colonized wild-type mice with B. fragilis, then induced IBD by introducing Helicobacter hepaticus—a bacterial strain known to initiate IBD in this experimental model. B. fragilis protected mice from IBD, but mice colonized with B. fragilis lacking PSA were not protected. The B. fragilis appeared to halt the autoreactive immune cells and prevent intestinal damage.

In 2006, I took an assistant professor position at California Institute of Technology. By then, researchers had discovered a new category of CD4 T cell that acted as a critical mediator of autoimmune diseases. This class of cells soon became all the rage in immunology circles. Th17 cells produce IL-17, a potent inflammatory T-cell cytokine associated with every known autoimmune disease. It became clear that immune reactions could actually be skewed toward Th1 (cellular), Th2 (humoral), or Th17 (autoimmune) pathways. Several studies found that Th17 reactions were involved in initiating IBD in mouse models. Was PSA inducing Th1 cytokines to shift the balance away from too much Th17 (and Th2)? If true, it would support my earlier study showing that PSA initiated the production of Th1 cytokines that reduced the higher Th2 response in germ-free mice. But what was the mechanism, and did PSA also suppress proinflammatory Th17 cells? I showed that PSA was still causing a proliferation of CD4 T cells, like in the germ-free mice of my earlier research, but also that another cell type called T regulatory cells was activated, and that it dampened inflammation by producing the cytokine IL-10. This cytokine was enough to suppress the pro-inflammatory IL-17 and protect the intestines from immune attack (see graphic above). I began to appreciate that B. fragilis was not inducing discrete immune responses such as Th1 cells, but was “shaping” a coordinated and complex immune profile to promote intestinal health.4

A few labs have recently been able to sequence the microbiota of healthy humans and IBD patients and showed dramatically reduced numbers of Bacteroidetes bacteria in IBD patients compared to healthy subjects. Currently, IBD patients are prescribed anti-inflammatory medicine, but this suppresses the entire immune system and puts the patient at a high risk of other illnesses and infections. In theory, B. fragilis as a probiotic therapy, or even administration of PSA alone, would have a more localized anti-inflammatory reaction in humans while cultivating the features of a functional immune system.

B. fragilis is certainly not the only important commensal bacterium in the human gut—it is merely the first one to be discovered with an immunomodulatory molecule. The Human Microbiome Project, an undertaking funded by the National Institutes of Health (NIH) to sequence the microbiota from hundreds of humans, has challenged itself with determining the relative quantities of all bacteria present in the human gut. With a known baseline of the bacteria present in healthy individuals, it will be much easier to understand which bacteria might be missing in diseased patients. Hopefully, the Human Microbiome Project will lead to the discovery of other beneficial bacteria.

For decades, scientists have been able to colonize germ-free animals with single organisms to evaluate their contributions to health. By pairing modern immunologic tools with these models, we’re starting to uncover truly novel effects of cohabitation with bacteria on human health.

Have a comment? E-mail us at mail@the-scientist,com

Sarkis K. Mazmanian is an Assistant Professor in the Division of Biology at the California Institute of Technology. Sara W. McBride conducts research in Mazmanian’s lab.

References
1. D.P. Strachan, “Hay fever, hygiene, and household size,” BMJ, 299:1259–60, 1989.
2. S.K. Mazmanian et al., “An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system,” Cell, 122:107–18, 2005.
3. J.F. Bach, “The effect of infections on susceptibility to autoimmune and allergic diseases,” N Engl J Med, 347:911–20, 2002
4. S.K. Mazmanian et al., “A microbial symbiosis factor prevents intestinal inflammatory disease,” Nature, 453:620-25, 2008.

Comments

Avatar of: Jacob Silver

Jacob Silver

Posts: 3

August 4, 2009

Mazamian's article was very enlightening, especially how he details the scientific process of separately checking each component of a factor. On the grander scale, he is definitely moving us away from preoccupation on the minoroty of pathogenic bacteria to the important contributions of our own symbiotic bacteria. Of course we have to continue to monitor and analyze pathogenic bacteria, but the analysis of commensal/symbiotic bacteria, now that we know about them, needs to have even greater analytic effort.
Avatar of: Jeff Wine

Jeff Wine

Posts: 2

August 5, 2009

This article is a model of clarity, both in the writing, in the logic of the experiments, and in the presentation of antecedents to their work. It was pure pleasure to read it. \n\n

August 5, 2009

It would be fascinating to learn what the Mazmanian Lab has to say from the public health perspective.\n\nWhat other physiologically active polysaccharides have they looked for?\n\nIs anybody modelling the human organism covered in microbes commensal and otherwise as a controllable biosystem? \n\nHey, I loved it. Thanks Mazmanian and McBride!
Avatar of: M Aidoo

M Aidoo

Posts: 2

August 5, 2009

Its refreshing to hear alternative views of immunology. Ohad Parnes wrote a piece in 2004 (Molecular Immunology. 2004. 40:985-991) on why the immune system should be viewed as an integrative tool whose aim is to help us live with the organisms we are exposed to rather than protect us from them. Who knows what good other bacteria are doing for us.
Avatar of: Jag Rawat

Jag Rawat

Posts: 2

August 5, 2009

The changing lifestyles of the human being represented by changing diets, loss of physical activity and usage of higher levels of chemicals in agricultural systems for production of food, all have impacted the non-infectious spectrum of diseases in the post-industrial world.\n\nFor domestic animal husbandry, the industrialisation of animal production from homestead animal rearing, has resulted in changes of the similar nature in 'life-style or what we should say, their husbandry', as that of human being!\n\nThat has resulted in new type of metabolic diseases in animals as well. The broadest causes may be similar as cited above but there are further additions such as issue of 'mineral deficiency' taking centre stage, which of course, affects the human organism in similar fashion.\n\nBUT moot question in India, particularly raised in Research Review Meeting at the College of Veterinary Sciences at CCS Haryana Agri University, Hisar in India, was to classify 'digestive disorders exhibiting anorexia and tympany' into such classifications, through which researchers in physiology could conduct their research trials in more meaningful fashion. \n\nAdditional information was that in domestic buffaloes, a student's thesis results were cited, it was revealed that for some months of winter, the rumen productivity was best. That meant conditions for rumen microflora were best? \nCould it be that in other months there is a lack of symbiont(s) or commensals which could be the primary cause of anorexia or anorexia happens due to the primary diseases?\n\nThese are the questions which need rigour of research in livestock husbandry domain as well and the light thrown on role of singular molecule as keeper of rumen health and production ability could mean a breakthrough for managing the rumen system in the lean months where the food availability or utilisation, in the animals, gets affected, due to reasons, which need investigations, of similar type, as has been cited here.\n\nCONGRATULATIONS and may be collaborations could emerge in veterinary sciencs with your group?
Avatar of: LEIF JARLEBARK

LEIF JARLEBARK

Posts: 2

August 6, 2009

I'm a neuroscientist gone university administrator, so please question the validity of my comments on GI microbiology, etc below. Loved the article - excellent work and presentation, but aren't most if not all bacteria pathogens under the "right" conditions or at the very least not that good for human health?\n\nWikipedia (yes, I'm quoting that miracle of scientific accuracy... let's just assume that the people having entered info on this particular subject aren't happy amateurs) mentions that "Some species (B. fragilis, for example) are opportunistic human pathogens, causing infections of the peritoneal cavity, gastrointestinal surgery, and appendicitis via abscess formation, inhibiting phagocytosis, and inactivating beta-lactam antibiotics. ... In general, Bacteroides are resistant to a wide variety of antibiotics ? β-lactams, aminoglycosides, and recently many species have acquired resistance to erythromycin and tetracycline. This high level of antibiotic resistance has prompted concerns that Bacteroides species may become a reservoir for resistance in other, more highly-pathogenic bacterial strains."\nhttp://en.wikipedia.org/wiki/Bacteroides\n\nBacterial resistance to antibiotics is a serious problem that shouldn't be taken lightly at all - it's not unlikely that we are facing a very problematic era if or when we run out of ways to treat bacterial infections. For example, last year the Department of Health in the UK investigated the number of deaths caused by hospital bugs - 36,674 lives were cut short between 1997 and 2007. Of those, 26,208 were from Clostridium difficile and 10,466 from MRSA. And these figures are thought to be underestimated...\n\nSo, to the point... Instead of including B. fragilis in yoghurt, health industry probiotic products and the like, would it be possible to make PSA as a dietary supplement, to sprinkle on the breakfast cereal or what have you?

August 6, 2009

OK, WE ARE A SYMBIOTIC LIFE FIGHTER. \nDr. Hector Horacio Otero
Avatar of: anonymous poster

anonymous poster

Posts: 1

August 26, 2009

This was a nice confirmation of other work done in the early 1990s by the Sartor lab at UNC-CH. They showed that the peptidoglycan-polysaccharide of group A streptococci also induced helper T cells in germ-free mice.
Avatar of: Carol Mershon

Carol Mershon

Posts: 4

August 31, 2009

I know the article below deals with worms, but what if microbes behave like the worms in the following article when exposed to cell phone and wifi towers and antennas which give off electromagmetic and radiofrequency radiation 24/7?\n\nMobile Phone Emissions Increase Worm Fertility:\nhttp://www.newscientist.com/article/dn1889-mobile-phone-emissions-increase-worm-fertility.html\n\nCould this be why antibiotic-resistant bacterial strains like MRSA are on the rise in recent years? Is this why the honeybees are dying of CCD? Is this why the bats in the northeastern U.S. are dying of White Nose Syndrome?\n\nEverything I read seems to suggest that the immune system, at the very least, is adversely affected by these exposures. Are the microbes themselves behaving differently as well in response to these frequencies?\n\nI'd very much like a microbiologist to test these theories - perhaps expose MRSA to electromagnetic/microwave/radiowave radiation and see if it grows faster than normal. \n\nIf these exposures affect the growth of yeast and fruit mold, it is very possible that other microbes are also affected.\n\nDo EMFs Affect Yeast Growth?\nhttp://www.usc.edu/CSSF/History/2006/Projects/J1310.pdf\n\nThe Effect of Microwave Radiation on Fruit Mold:\nhttp://www.usc.edu/CSSF/History/2008/Projects/J1403.pdf\n\nAudio Archives - Interviews with Top Researchers:\nhttp://electromagnetichealth.org/audio-archives-and-more/#patients\n\nAttitudes to the Health Dangers of Non-Thermal EMFs:\nhttp://www.powerwatch.org.uk/news/20080117_bevington_emfs.pdf\n\nElectro Hypersensitivity - Talking to Your Doctor \nhttp://weepinitiative.org/talkingtoyourdoctor.pdf\n\nGerman Doctors Unite on RF Health Effects:\nhttp://www.powerwatch.org.uk/news/20050722_bamberg.asp\n\nBecker Interview: \nhttp://www.energyfields.org/science/becker.html\n\nBioinitiative Report:\nhttp://www.bioinitiative.org/report/index.htm\n\nMystery in the Skin:\nhttp://www.feb.se/ARTICLES/OlleJ.html\n \nAllergic Reactions Enhanced by Cell Phone Use:\nhttp://bastyrcenter.org/content/view/313/

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