Learning to live together: new research explains how bacteria's urge to survive in our gut promotes intestinal health

Get to know your insides and the benefits to having a symbiotic relationship with the microbial world! From Food Poison Journal

Public Interest Note:

The mere thought of microbes: bacteria, parasites, fungi and viruses tend to make us sick to our stomach.  We all know that when we catch a cold: a virus is to blame and when we have a nasty skin rash, we apply creams to kill off the infection-causing bacteria.  To think that our bodies are coated with an estimated 100 trillion microbial cells ¹ can be, at first, a very startling and threatening idea to believe.  100 trillion is a ridiculously large number to conceptualize, but let’s try to put this number into perspective: it is estimated that our Milky Way Galaxy contains 200-400 billion stars. ²  For those of you keeping score at home, that’s about a ten-fold difference in that for every star in our night sky, there are ten times more microbes colonizing a single person’s body.  Yes, that’s right- that estimated 100 trillion microbial cells is for ONE person.   In addition, the number of microbial cells found in and on our body outnumbers our human cells 10:1.  Needless to say, if we were to think of ourselves in terms of cellular content we would have to think of ourselves as being more microbial than human! Surprisingly, we know more about the stars millions of light years away than we do about the microbes living in and on our bodies!

from neuroanthropology.net

Whew! It can be very exhausting concentrating on such big numbers!  After all, this IS an immunology blog, not a mathematical one! Most of the microbes studied, thus being the ones that are best understood are the ones that cause severe diseases and make us sick.  However, the number of these virulent bugs doesn’t come close to the number of non-disease causing microbes that live with us everyday.  So why spend all our time and resources studying the things that we encounter rarely and that constitute a fraction of the total number of microbes we live with everyday?  One major reason is because, in order to study a microbe, it has been historically crucial to be able to isolate it and study it in the lab.  However, commensal bacteria, the microbes we live with that don’t cause disease, are very tricky bugs that don’t seem to cooperate with historical scientific practices.  They are usually very difficult to isolate and grow in the lab with usual laboratory medias.  One explanation for this is that the microbial species living inside our bodies are part of a complex ecosystem (aka microbiome/microbiota) that we do not fully understand yet, making it incredibly difficult to recreate in the lab and study.  

That is, until recently, when the National Institutes of Health launched a $140 million initiative in 2007 called “The Human Microbiome Project (HMP)”.  Through a large cooperation of the Nation’s leading microbiology, genetics, bioinformatics and immunology laboratories, the HMP mission is to generate “resources enabling comprehensive characterization of the human microbiota and analysis of its role in human health and disease”. ³   The HMP has undoubtedly spearheaded the advancement of genomic sequencing strategies to better understand what kinds of microbes live on our skin, in our nose and mouth, gastro-intenstinal and urogenital tracts.  With an enhancement of research technology advancement, and an increase in federal funding devoted to studying commensals, our understanding of our microbiome and how it relates to our overall health has greatly increased over the last couple years.

 In fact, earlier this year, fascinating research led by Peer Bork’s group at the European Molecular Biology Laboratory in Germany, discovered that humans can be classified based on three distinct gut microbiomes. ⁴ 

The research team analyzed genomic data obtained from human fecal matter (the least invasive (and least glamorous) method to analyze bacteria living in your guts) derived from people living in Denmark, France, Italy, Spain, Japan and the U.S. and discovered that three different clusters could be distinguished such that irregardless of sex, weight, height, age or geographic location, the balance of gut bacteria could be separated into 3 different groups-each differing in the bacterial contents that lived in their gut.  People who were Type 1 had a different balance of gut bacteria than people in Type 2 or 3.  Similar to bloodypes, that is that all people can be classified into one of 4 groups based on if they express A, B, AB or O antigens on their red blood cells, Bork suggests that this new biological classification, enterotypes (named for the collection of bacteria that live in the gut that distinguish the three groups) may be used to better tailor diets, drug regimines and antibiotics for an individual based on his/her microbiome. For example, someone of Enterotype 1 may respond better to particular antibiotics or diets than someone who is Enterotype 2 or 3. However, this is still a hypothesis that requires more research, funding and public interest to better understand the differences between these enterotypes and whether specific enterotypes are found in other highly colonized areas of the body such as the urogenital tract and skin, and importantly: if different microbial environments in our bodies play a role in disease susceptibility or resistance.

Probiotics: The Immunology Behind the Health Buzz

Most of the “microbial cosmos” living inside our body are bacteria, 70% of which take up residence in our digestive tract. ⁵  What are these bacteria doing there?  Perhaps the best way to answer that question is think of what happens when these bacteria aren’t there.  Researchers can discover answers to this question by using germ-free mice to model what it would be like for humans without intestinal flora.  To do this, mice are born via caesarian section and then immediately housed in a sterile environment by which all their bedding, cages, and food is autoclaved, preventing the colonization of any detectable bacteria.  Studies using such mice have revealed these germ-free mice have increased susceptibility to infections, reduced digestive enzyme activity, muscle wall thickness, and decreased vascularity and nutrient uptake.⁶  All of these observations are important for maintaining a healthy gut environment.  That’s right: bacteria can be healthy for us!  The commensal bacteria that constitute our intestinal microbiome largely include bacteria of the genera: Bacteroides, Clostridium, Escherichia and Lactobacillus.  Some of these genera might seem familiar, most likely because you might know of a common species within these genera that are pathogenic like Clostridium tetani (causes tetanus) and Escherichia coli O157:H7 (common to foodborne illness).  It’s important to re-iterate that commensal bacteria are part of your normal gut flora that you are essentially born with, and are NOT pathogenic. 

Molecules expressed by commensal bacteria that stimulate their growth are known as probiotics (as opposed to antibiotics, which kill bacteria).  Commensal bacteria express a variety of surface molecules that are an intense area of current research because of their potential to use these molecules to communicate to our cells-epithelial, neuron and immune cells to mediate their beneficial actions. The most commonly studied, and therefore advertised, are probiotics generated by Lactobacilli.  Lacto-meaning milk, is how this bacteria generally gets into your gut, therefore making Lactobacilli easy to culture and use as a medicinal tool, since it lives in fermented milk such as yogurt and cheese. Probiotics, like Lactobacilli have been clinically studied for its ability to attenuate gastrointenstinal infections, allergic symptoms, inflammatory bowel disease (IBD), decrease colon cancer severity ⁷ and alterations in behavior including autism ⁸. (Note: yogurts marketed specifically as “probiotic” contain enriched amounts of certain strains of priobiotic bacteria.) There are three main ways in which bacteria can be beneficial to the health of our guts:

1. Competing against invading pathogens by secreting antimicrobial peptides and lowering the gut pH

2. Increasing barrier protection and mucus generation to protect our guts from pathogen invasion and enhancing nutrient acquisition

3. Immunomodulation by affecting gut immune cells function and distribution.

                  For example, Lactobacillus acidophilus has been reported to inhibit pathogenic E. coli-induced inflammatory cytokine release by intestinal epithelial cells (IECs).  There is data to suggest that this happens due to a molecule expressed by common Gram-positive bacteria, called lipoteichoic acid (LTA).  It is thought that LTA, which looks structurally very similar to a molecule expressed by pathogenic bacteria, is able to outcompete and bind tighter to a receptor on IECs, thereby blocking inflammation. ⁹ Commensal bacteria have been shown to further induce an anti-inflammatory environment, by regulating the T and B cells, macrophages and dendritic cells in the intestines.  For example, Lactobacilli induces the production of the potent anti-inflammatory cytokine, IL-10, from dendritic cells and macrophages, while increases antibody secretion from B cells. ¹⁰ Although we generally know the beneficial effects of commensal bacteria, we don’t fully understand the biological mechanisms to explain how probiotic bacteria contribute to protection from such an array of diseases.

In 2005, the Nobel Prize for Physiology and Medicine was awarded to Australian Physicians/Microbiologists, Robin Warren and Barry Marshall. Warren and Marshall discovered “the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease”.¹¹  Ann O’Hara and Fergus Shanahan recall the 2005 Nobel Prize and explain one crucial key in unlocking the mystery of how priobiotics prevent disease, stating that this “is a reminder that the solution to some human diseases does not reside solely within the host but rather might be found at the interface with the microbial environment”.  ¹²

Understanding Beneficial Bacteria: Why This Research Paper Matters

Although the field of probiotic research is expanding, there remain many questions left unanswered including:

1. Which molecules involved-expressed by both the bacteria and our immune cells?

2. How are commensal bacteria beneficial to the maintenance of healthy gut?

3. If bacteria express similar molecules, how does our immune system recognize the difference between commensal and pathogenic microbes?

The answers to these questions are among the primary foci of a recent paper led by  Sarkis Mazmanian’s research group at California Institute of Technology.  One of the many reasons why Mazmanian’s research is so interesting is because he unselfishly thinks about human disease from the perspective of the microbe.  In fact it seems he tries to stand up for the little guys, explaining that, “They [the bacteria] couldn’t care less about us except that we provide them a stable and nutrient-rich habitat”.  Mazmanian, whose primary research interest is to better understand why the “good” bacteria, the commensals, are good for us, believes that commensal bacteria are just trying to live another day in our guts, skin and mouth-and they can potentially evolve new ways to do this anyway they can, weather it be by preventing cancerous tumors to form, harsh inflammatory environments or pathogenic intruders from taking over their home.  These beliefs represent a compelling juxtaposition between microbiologists and medical professionals.  Most scientists studying immunology and medicine think of particular immunological phenomena from the perspective of the host (people) such that even though we know that some bacteria are good for us, they believe that we, the host, have evolved to live symbiotically with bacteria.  But perhaps, thinking of this from an entirely human-centered perspective is not the only way to understand the fundamental question: how and why are commensal bacteria in our gut?

Why not let Mazmanian explain in his own words the importance of investigating this bacteria-focused angle to help solve some the biggest concerns in human health?:

Perhaps, it is this refreshing and novel outlook that enabled Mazmanian to be named one of Discover magazine’s “20 best brains under 40” in 2008.  In that interview, he reasons “The potential of beneficial microbes appears to be limitless” and that “this symbiotic relationship between the human body and microbes [is] a gold mine of potential therapies for a number of illnesses”. ¹³ 

What the &*%$#! Does the Title Mean?!

Round, JL., et al. “The Toll-LikeReceptor 2 Pathway Establishes Colonization by a Commensal of the HumanMicrobiota”. Science. 332:974-977. (2011).

1. Toll-Like Receptors (TLRs) are one of the most extensively studied, most important signaling molecules in immunology.  They are named “Toll-LIKE”, because the first such receptor, Toll, was discovered in Drosphilla, a common fruit fly used as a model for genetics research.  Originally identified as an important gene that regulated dorsoventral axis formation in developing embryos, a Toll-deficient fruit fly revealed that these flies were highly susceptible to fungal infections.  Since that discovery, in the early 1990’s, Toll-Like receptors have been identified in every animal with an immune system.  The signaling cascade and inflammatory output is amazingly similar between the fruit fly and humans, thus making the discovery of Toll one of the most intriguing examples of how basic research and the use of model organisms provide powerful insight into understanding human disease. 

Great cartoon illustrating the function of TLRs as receptors on the surface of cells (TLR1/2,4,5,2/6) as well as within cells (TLR3,7,9) to detect microbial patterns not expressed by humans. Immune cells are particularly good at expressing these TLRs and using them to alert the body of infection. But how do commensal microbes differ from pathogenic ones with respect to TLR activation? This is the question that drives Mazmanian's research group. Image from www.invivogen.com

In humans, there are at least 10 TLRs that are capable of recognizing unique molecules expressed exclusively by microbes, and not us.  Examples of microbial-associated molecular patterns (MAMPs) include: lipopolysaccharide (LPS), flagellin, and single stranded RNA (ssRNA) which stimulate immune cells to become highly inflammatory.  The inflammatory response is very quick and offers a way to recruit immune cells to the site of infection to destroy pathogens with speed.  Like most biological processes, if there’s an “on” switch (pro-inflammation), there’s a regulatory “off” switch.  Regulation is paramount to a functioning immune system and survival of a host, for example endotoxic shock is caused by a significant dose of LPS, which is found on the surface of Gram-negative bacteria like the pathogenic strain, Escherichia coli O157:H7.  The “shock” derives from an overproduction of inflammatory cytokines induced by TLR stimulation that mediates high fever, shortness of breath and even death.  TLR4 is the TLR that recognizes LPS. 

The focus of the paper discussed below, is TLR2, which is known to bind primarily MAMPs associated with Gram-positive bacteria and yeast.  TLR2 is expressed on the surface of nearly every immune cell and has also been detected on epithelia. ¹⁴ However, a few years ago it was discovered that a MAMP located on the Gram-negative bacteria, Bacteroides fragilis called polysaccharide A (PSA) can also stimulate TLR2.  Usually, it isn’t terribly exciting news to read: “bacteria activate TLR!”; however things began to turn into a more thrilling adventure when you learn that this is a commensal bacteria that belongs to the genera, Bacteroides, which alone makes up 30% of your gut bacterial flora. Currently, we do not fully understand why B. fragilis expresses a molecule that binds TLRs-but we do know that purified PSA can regulate the inflammatory cytokine release by T cells and dendritic cells ¹⁵ and we know that purified PSA injected into mice, can prevent the onset of inflammatory bowel disease (IBD). ¹⁶

Both of these findings are very interesting, but one of the most critical aspects to support these phenomena remains unknown: if, according to Mazmanian commensal bacteria don’t give a damn about us; that they are living with us because we let them, why does B. fragilis express PS? Does PSA/TLR2 signaling do something positive for the bacteria, in addition to comforting us with a balanced gut?

Now, with the information above, we can infer: 

This paper will provide data regarding PSA as being beneficial to, B. fragilis because of its interaction with TLR2 allowing the commensal bacteria to survive and colonize in our gut, while regulating out intestinal inflammatory environment.

Ready for an adventure? Read on for a guided-tour through the scientific data!
                In this paper, the basic question that motivates Round and colleagues’ research is this: why don’t commensal bacteria activate an inflammatory immune response, but pathogenic bacteria do, given the fact that both express MAMPs that stimulate TLRs?  Given that this research is directed by Mazmanian, the team set out to investigate this question with the hypothesis that B. fragilis, evolved ways to block our inflammatory, anti-microbial defense system so that this symbiotic bacteria can essentially go unnoticed by our gut’s immune system. 
            To begin, they looked at inflammatory T cells in the gut.  But not just any T cell (that’s right, there are at least 5 different subsets), they assessed a subset of T cells called, Th17 cells, which populate the gut in response to infection and further the inflammatory immune response by produces loads of the inflammatory cytokine, IL-17.  In the gut of wild-type (WT) mice, they found that Th17 cells constituted 7.65%. Interestingly, the germ-free mice only had about a tenth of Th17 cells compared to WT.  This result indicates that total lack of commensal bacteria (germ-free mice) results in less inflammatory Th17 cells in the gut.  But, when they mono-colonized germ-free mice with only B. fragilis, so that the only bacteria living in these mice is this particular commensal, they found that the Th17 population increases slightly to 1.46%, and that when they genetically delete the PSA gene from B. fragilis, the percent of Th17 cells in the gut increases dramatically to levels similar to that found in WT mice!  This data suggests that B. fragilis is a relatively weak inducer of Th17 development since these germ-free mice colonized with B. fragilis has nearly 7 times less inflammatory T cells in their gut and that more importantly, by deleting PSA, B. fragilis looks like a pathogenic bacteria able to greatly induce Th17 development!  Collectively, this data indicates that B. fragilis actively blocks inflammatory Th17 development in the gut by expressing PSA!  I’d say that is one pretty impressive start to a paper!  Let’s see what else they show us!

            Next, Round, et al. set out to determine the mechanism by which PSA suppresses Th17 development.  It is widely known in the T cell field, that the anti-inflammatory cytokine, IL-10, is a potent inhibitor of inflammatory T cells such as Th17.  Also, based on their previously published research, that PSA limits IBD inflammation and that PSA can bind TLR2, the team wanted to know if TLR2 stimulation by PSA results in IL-10 production from immune cells.  As mentioned before, a number of cells express TLR2, but since they are looking in the gut-where else better to start than investigating T helper cells and dendritic cells (DCs), which are among the most numbered immune cells in the gut?  In an in vitro experiment, in which purified T cells are mixed together with cultured DCs and stimulated with purified PSA, IL-10 is produced.  But where is the IL-10 coming from: the T cell or dendritic cell?  To answer this question, the researchers repeated this in vitro experiment, using DCs from WT mice and T cells (CD4+, T helper cells) from TLR2-deficient mice.  When they cultured these cells with PSA, IL-10 production was significantly reduced.  To be thorough, they also performed a co-culture experiment with WT T cells and TLR2-deficient DCs stimulated with PSA.  Under these conditions, the amount of IL-10 detected was just as high as in the presence of WT T cells and WT DCs, suggesting that PSA was selectively stimulating TLR2 on T cells, not DCs, to induce IL-10 production!  Even more interesting, is that DCs aren’t even needed to get PSA-induced IL-10 secretion; that T cells can recognize PSA via TLR2 and make IL-10 intrinsically! 

This diagram represents the "old" model of how B. fragilis regulated gut inflammation. From Mazmanian's newest research, we no know that PSA can DIRECTLY induce Treg development by stimulating TLR2 on T cells, thus by-passing the requirement for DCs to process and present antigen to T cells!!
From www.rndsystems.com

            With this data, they wanted to go back and see if TLR2 is the missing link to explain how PSA blocks Th17 development in the gut, as shown in Figure 1.  When they mono-colonize germ-free mice that lack TLR2, with either the WT B. fragilis strain or the one without PSA, Th17 development is not impaired suggesting that TLR2 is critical to the mechanism by which B. fragilis is able to prevent inflammation in the gut! 

            Now the ultimate inquiry: So what?!  It’s cool to discover the details that explain how commensal bacteria can regulate gut inflammation, but from Mazmanian’s perspective, microbes don’t care if our guts in a knot, making us want to cry in pain.  So, why does B. fragilis express a molecule that blocks inflammation?  What is the benefit of expressing PSA to the bacteria? 
             To address this important question, Round and colleagues looked at microscopic images of colon sections to look for B. fragilis.  Using confocal microscopy and 3D digital reconstruction imaging, they discovered microcolonies of the commensal closely associated with the host intestinal tissue.  There was no B. fragilis found in the germ-free mice.  Further more, when the mono-colonized mice with PSA-deficient B. fragilis, they could hardly find any microcolonies, and injecting purified PSA recovered the bacteria’s growth in the intestinal tissue!  So it appears that not only is PSA inhibits inflammation, which is good for us, but PSA is good for the bacteria, because B. fragilis needs it to survive in our guts!

             The obvious follow-up question to this extraordinary finding is: how does inflammation affect commensal bacteria survival?  With the first bits of data indicating that PSA-deficient B. fragilis can’t survive in mice and that Th17 cells increases dramatically in mice colonized with B. fragilis lacking PSA, Round, et al. wanted to know if IL-17 was the reason why B. fragilis evolved to express PSA.  To test this theory, they mono-colonized mice with PSA-deficient B. fragilis and injected a blocking antibody to inhibit IL-17.  Only in the presence of the anti-IL-17 antibody, was the PSA-deficient B. fragilis able to colonize and live in the intestines! This research team finally unveiled the reasons why and how B. fragilis live mutualistically with us!

             Of course, this still plenty of biological investigation to be done in completely understanding the purpose of commensal bacteria.  For example, is PSA a universally expressed molecule on all Gram-positive commensal bacteria, or is PSA specific to B. fragilis?  Furthermore, how do other common commensals such as Lactobacillus spp. survive in our guts and is this TLR mediated? In terms of the immunological mechanism, it is still unclear how exactly IL-17 is anti-microbial, which would complete the story of why B. fragilis wants to inhibit IL-17 function.  Lastly, it remains to be understood how TLR2, which binds pathogenic ligands to induce inflammation can also recognize PSA on B. fragilis to block inflammation.  How does TLR2 on our immune cells know the difference between MAMPs on pathogenic vs commensal bacteria? It’s always good to leave a few unsolved mysteries at the end of a paper; it really keeps the field of immunology and microbiology exciting and interesting, right?!
            Lastly, it would be interesting to see if these same findings can be observed in humans.  If this were to be true, it is clear that this novel data may result in new therapies to treat an array of gut inflammatory conditions such as IBD, Crohn’s and Celiac Disease like taking PSA-supplements purified PSA to treat a number of diseases that plague thousands of Americans! 
On a side note: if you are interested reading some of the best critical scientific writing currently available, I suggest you click here to read the paper for yourself! I was blown away by the clarity and philosophical spin in the concluding statements from Round JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA, & Mazmanian SK " The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota". Science (2011) discussion:

“It is historically believed that the micriobiota is excluded from the mucosal surface.  However, certain symbiotic bacteria tightly adhere to the intestinal mucosa and thus immunologic ignorance may not explain why inflammation is averted by the microbiota.  Our study provides new insight into the mechanims by which the immune system distinguished between pathogens and symbionts….On the basis of the importance of the microbiota to mammalian health, evolution appears to have created molecular interactions that engender host-bacterial mutualism.  In conclusion, our findings suggest that animals are not “hard-wired” to intrinsically distinguish pathogens from symbionts, and that microbital-derived mechanisms have evolved to actively promte immunologic tolerance to symbtiotic bacteria.  This concept suggests a reconsideration of how we define self versus nonself.”

This conclusion reads like an English literature paper, not a scientific one and left me wanting to push myself, when it comes time for me to write an article about my own research; to discuss it in a captivating manner that speaks to both the academic research community as well as the interested general public.

Round JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA, & Mazmanian SK (2011). The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota. Science (New York, N.Y.), 332 (6032), 974-7 PMID: 21512004




References and Further Reading: 
1: Qin, J., et al. “A human gut microbial gene catalogue established by metagenomic sequencing”. Nature. 464:59-65. (2010).

2: Grant, Jo and Ben Lin. “The stars of the Milky Way”. ChView.
3. The NIH Common Fund. “The Human Microbiome Project: Overview”.
4: Arumugam, M., et al. “Enterotypes of the human gut microbiome”. Nature. 473:174-180. (2011).
5: Tlaskalova-Hogenova, H., et al. “The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune disease and cancer: contribution of germ-free and gnotobiotic animal models of human diseases”. Cellular & Molecular Immunology. 8:110-120. (2011).
6: Hentschel, U., et al. “Commensal bacteria make a difference”. Trends in Microbiology. 11:148-150. (2003).
7: Mohamadzadeh, M. et al. “Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid”. PNAS. 108:4623-4630. (2011).
8: Heijtz, RD., et al. “Normal gut microbiota modulates brain development and behavior”. PNAS. 108:3047-3052.(2011).
9: Vidal, K., et al “Lipoteichoic acids from Lactobacillus johnsonii strain La1 and Lactobacillus acidphilus strain La10 antagonize the responsiveness of human intestinal epithelial HT29 cells to lipopolysaccharide and gram-negative bacteria”. Infect. Immun. 70: 2057-2064. (2002).
10: Ng, SC., et al “Mechanisms of action of probiotics: recent advances”. Inflamm. Bowel Dis. 15:300-310. (2009).
11: The Nobel Assembly. “Press Release: The Nobel Prize in Physiology or Medicine for 2005”. Released on-line Oct. 3, 2005.
12: O’Hara, AM and Shanahan, F. “The gut flora as a forgotten organ”. EMBO. 7:688-693. (2006).
13: Grant, A., et al. “20 Best Brains Under 40”. Discover. Published online Nov. 20, 2008.
14: Wang, Q., et al. “A bacterial carbohydrate links innate and adaptive responses through Toll-like receptor 2”. J. Exp. Med. 203:2853-2863. (2006).
15: Mazmanian, SK., et al. “A microbial symbiosis factor prevents intestinal inflammatory disease”. Nature. 453:620-625. (2008).