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).
 

How to get the most out of your next vaccine? A full night's sleep may enhance protection against viral infections

from gabby22197.glogster.com

Public Interest Note:

Let’s begin with a simple question: Why do we sleep?  To answer this, we can think of what happens when we don’t get enough or any sleep.  The day after a sleepless night, often we feel irritable, exhausted, unhappy and stressed.  If you are a parent, have a full-time job or a fellow graduate student you are acutely familiar with how precious even a few hours of sleep can be in redeeming your sense of sanity.  The side effects of sleep can become dangerous when the actions of a sleep-deprived individual affect those around him/her.  In March, an air traffic controller at Ronald Reagan Washington Airport fell asleep on the job, leaving two planes alone in the sky trying to land the planes alone.¹ Fortunately, in this case, no one was terrible injured, but the news magnified the public’s awareness and outrage of having to depend on sleep-deprived individuals for our safety.  It’s not surprising the single air traffic controller fell asleep on the job since it was reported this was “his fourth consecutive overnight shift, which runs from 10 p.m. to 6 a.m.” ¹ In fact, the American Academy of Sleep Medicine International Classification of Sleep Disorders classifies this condition as Shift Work Sleep Disorder (SWSD).  SWSD plagues nearly 70% of the 15 million Americans who work shifts between 10 p.m. and 6 a.m.²   It is further estimated that sleep deprived workers cost U.S. businesses $18 billion a year.³  

from cdc.gov

Importantly, sleepiness does not only affect the people who work the “graveyard shift”, but in an increasingly fast-paced society, coupled with a tough economy and technology-rich environment, many Americans are spending less time asleep.  The Center for Disease Control and Prevention (CDC) revealed that more than 35% of the 74,571 adult Americans surveyed get less than 7 hours of sleep a day and 37.9% reported “unintentionally falling asleep during the day at least once in the preceding month.  Alarmingly, nearly 5% of respondents admitted to nodding off while driving at least once in the past month. ⁴   Furthermore, it is estimated that sleep deprivation causes nearly 100,000 vehicle crashes and 1,500 deaths annually in the U.S.⁵ Perhaps, if people more strictly followed the CDC’s recommendation of getting 7-9 hrs of sleep (for adults), such scary statistics will improve and save our economy billions of dollars.  But why does the CDC discuss anything about sleep?  Does sleep have a role in disease prevention?

We know what happens to our bodies when we are sleep deprived: red eyes, achy joints, and sometimes we even catch a cold.  Besides behavioral problems that arise when we are sleep deprived, our immune system becomes compromised as well.  For that reason, the CDC warns, “persons experiencing sleep insufficiency are also more likely to suffer from chronic diseases such as hypertension, diabetes, depression and obesity…” The CDC refers to America’s sleep deprivation problem as an epidemic, a public health concern.  The problem of sleep deprivation in America is so great that since there is even a national organization called the National Sleep Foundation devoted to alerting the public, policy makers and healthcare providers of the consequences related to sleeping insufficiencies. Such agencies are worried that a continuously sleepy nation will result in a sick nation with increased susceptibility to infections and disease-which ultimately will cost the country billions and hamper productivity.  

Sleep Deprivation: The Immunology Behind It

EEG charts and amount of time spent in each sleep cycle, stages 3 and 4 constitue SWS which is also the time in sleep when pro-inflammatory cytokines, IL-1 and TNFalpha peak. Lange, et al. establishes that a full-night's sleep post vaccination BOOSTS your immunological memory, perhaps enhancing the vaccine's effectiveness. Image from seamist.hubpages.com

First, how does one define and measure sleep? There are two types of sleep: non-rapid-eye-movement (NREM) and rapid eye-movement (REM).    There are 5 main stages of sleep beginning with stage 1 in which you are just lying down to sleep, but still pretty conscious.  Stage 2 follows with a lower level of consciousness that develops into stages and 3 and 4.  Stages 3 and 4 are collectively called slow wave sleep (SWS).  Under SWS, the brain is at its lowest level of consciousness and is thought to be involved in restorative functions.  Lastly, REM is the final step in sleep and is when our brains are processing information and dreaming.  When asleep, we typically spend equal parts in REM and SWS.  Sleep stages can be monitored via EEG recordings as each stage has distinctly different frequencies and EEG wave amplitudes.⁶

 In the late 1980’s and early 1990’s, Toth and colleagues performed a series of infections on rabbits and monitored the animal’s sleep patterns during infections.  They tested a virus (influenza), bacteria (Staph. aureus and E. coli ), yeast (Candida albicans) and parasite (Trypansosoma brucei).  Each infection resulted in abhorrent sleep patterns, regardless of microorganism.  In general, the various infections increased the duration of slow wave sleep (SWS). ^6,7  The observation that infection increases SWS, but not other sleep stages, may mean that SWS, in particular, regulates our immune response while we are sleeping.  In humans, one of the most well studied infections to examine in regards to sleep is T. brucei, the parasite that causes Human African trypanosomiasis aka African sleeping sickness.  Infected individuals, usually by the second year of infection, lose the ability to manage their circadian rhythm resulting in the complete loss of sleep regulation and often leads to comatose.  In mice and rabbits, the release of parasites into the bloodstream is associated with acute increases in SWS, however this has not been evaluated in humans. ⁶

So what immune factors are modulated by sleep?  First, it is known that the majority of our immune cells, B and T cells and monocytes reach maximal levels in the blood during the night and are lowest when awake.⁸ In addition, the pro-inflammatory cytokines, TNFalpha and IL-1beta plasma levels also peak while we sleep, being the highest at the onset of SWS.⁸ An increase of either of these cytokines subsequently increases SWS duration, whereas decreases in TNFalpha or IL-1beta blocks SWS.⁶ This appears to be the clearest example of how our sleep patterns may regulate our immune response during infection. Both of these cytokines are critical in the clearance of infection and are some of the most abundant cytokines present during infectious diseases.  Therefore it seems to make sense why an increase in SWS during the diseases mentioned above occurs. 

Interestingly, infection is not the only thing that induces TNFa and IL-1beta release in the brain and plasma.  Sleep deprivation also promotes pro-inflammatory cytokine release, which then instructs your brain to increase the time it spends in SWS.  Alternatively, anti-inflammatory cytokines such as IL-10 is thought to inhibit SWS onset by antagonizing IL-1beta and TNFalpha.  It is also thought the induction of certain hormones such growth hormone (GH) and prolactin promote the release of IL-1beta, since inhibiting these hormones attenuates IL-1beta levels and SWS duration.  Conversely, the anti-inflammatory hormone, cortisol does the opposite, and decreases IL-1beta production.⁶ So it appears that our immune system mirrors our endocrine system in that they both regulate our sleep patterns by releasing pro- and anti- sleep mediators.  

TNFalpha and IL-1beta are generally thought of activators of the innate immune response, such as macrophages and dendritic cells.  Such cytokines act as an adjuvent, boosting the macrophage and dendritic cell's ability to be potent antigen presenting cells (APC).  The main job of an APC, is to display bits of processed pathogen (antigen) to a T cell.  Once a T cell recognizes a particular antigen, it then becomes activated and ready to fight the infection and help B cells secrete antibodies.  Realize that the next time you become ill and start to feel sleepy, your immune system is starting to work and that you should rest so that your body can adequately defend itself against infection.  Ignoring our body's demand for sleep affects the way our bodies fight diseases.  In a few human studies, it was revealed that the number of T helper cells decreased and the level circulating antibodies decreased with sleep deprivation.⁶ Not surprisingly, when the body is pushed to the extreme of total sleep deprivation, the immune system fails completely.  In one study, rats were sleep deprived for 21 days, until near death.  At the end of the 21 days, the rats had suffered from severe weight loss and septicemia with both opportunistic and pathogenic microbes in the blood.⁹ Furthermore, a recent survey of more than a million participants demonstrated a strong correlation between sleep of less than 7 hours a night and increased mortality.¹⁰

  

Sleep Deprivation and Disease: Why This Research Paper Matters

The evidence is clear: our immune system communicates with our brain while we sleep, but researchers have only begun scratching at the mechanisms behind this phenomenon.  This is very exciting and novel research that may provide not only insight into how the body operates, but also instigate new therapeutic approaches in fighting disease.  Although it is well established the role of certain cytokines in maintaining healthy sleep cycles and how the immune system responds to sleep deprivation, little is known about the effect sleep has on specific immune cell function.  The majority of what we know about the interplay between sleep and immune function is correlative.  For example, we know TNFalpha can promote APC function, and we know that TNFalpha levels increase during SWS, but it remains elusive whether sleep-induced TNFalpha promote APC function and thereby program and effective T cell response.  Sleep may represent a crucial part of the immune system that has been largely ignored by the immunological and medical fields.  Perhaps, the notion “Sick and Tired” can be immunologically explained and that greater insight into how sleep affects the body’s ability to clear infection is an underlying mechanism behind the increase of infections among infants and the elderly (acute vs chronic sleep deprivation).  How does sleep impact our ability to properly activate our immune system?  Can sleep play a role in designing better therapies and treatments for people fighting disease?   These questions motivate Jan Born’s research group at the University of Lubeck in Germany to find new ways to make more efficient vaccines…without even changing the vaccine components!  Therefore, as Lange, T. et al illustrates in their recent Journal of Immunology paper, catching a full night of ZZZ's  can make all the difference in getting the most out of your next vaccination.

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

Lange, T. et al. “Sleep after vaccination boosts immunological memory”. Journal of Immunology. 187 (2011).

There are two waves of antibody response: a slower, short-lived response during the initial infection followed by a quicker, longer-lived memory response . from click4biology.info

1) The essential point of vaccination is providing people with the antibodies needed to clear infections quickly and effectively.  Vaccines work by administering something that stimulates the adaptive immune response without causing disease.  Vaccines in the U.S. consist of purified proteins (not a whole, intact pathogen) from a pathogen that is injected or inhaled.  When APCs encounter the proteins in the vaccine, they cut the proteins into short peptide sequences.  Once processed, an APC presents the peptide on its surface to a T helper cell, which are named so because they help B cells make antibodies and help innate immune cells to destroy pathogens.  At the moment when the T helper cell recognizes a certain peptide sequence, the T cell begins to proliferate and secrete cytokines that help B cells secrete tons of antibodies- all of which can specifically target the protein that was used in the vaccine.  Vaccines help prevent infectious diseases by assisting our immune system in making the antibodies necessary to clearing an infection quickly.  When we become infected with something that we didn’t vaccinate against, our immune system has to take a lot of time (weeks) to produce the antibodies needed to fight the infection.  Some pathogens are extremely virulent and can cause a lot of harm, even death in the weeks it takes our bodies to properly protect itself against it.  Vaccines therefore help equip our immune system with the weapons (antibodies) it needs, before even heading into battle against a deadly pathogen. 
 2) Of course, this concept of a vaccine works best when the immune system can remember what the pathogen looks like when it encounters it a month, a year, or 10 years from the time of vaccination.  This is immunological memory.  An amazing aspect of your immune system is that when the adaptive immune cells, B and T cells, are activated and proliferate, some of the cells seem to last forever.  These cells are aptly named, memory B and T cells.  These memory cells will last you during your entire life time, always remembering what they are supposed to do when they encounter the specific protein or peptide that stimulated them to become active in the first place.  In order to maximize immunogenicity and achieve immunological memory you need a very good induction of the antigen-presenting cells by using adjuvants and booster shots to bolster the ability of your body to produce a reservoir of effective memory cells ready to kill a particular pathogen over the course of your life!

Compile this information together and we can infer that: Sleep following a vaccination, acts like an adjuvent promoting the development of memory cells that provides long-term immunological protection against a certain pathogen.  

Ready for an adventure? Read on for a guided-tour through the scientific data!

But first: A brief Q and A session regarding the overall experimental approach:

Q) What organism(s) was being tested?

A) All of the experiments presented here were performed on human volunteers.  The sample size consisted of 27 healthy, nonsmoking men with an average age of 26.  The group was on synchronized schedules with the same sleep-wake patterns for the 6 weeks prior to experimental testing.  All sleep-wake activity occurred in a sleep laboratory, and all participants had spent at least one night in the lab prior to experimental testing.  The group was randomly assigned to either the “sleep” or “wakefulness” group. 

Q) How did the “sleep” group differ from the “wakefulness” group?

A) The sleep patterns between the groups only differ the night following their vaccination (which occurred at 8am and occurred 3 times, one in Feb., March, and June. The “sleep” group had lights off at 11pm and lights on at 6:30am in the sleep laboratory.  During this same time, the “wakefulness” group stayed awake in bed watching TV, reading, listening to music or talking to a scientist (fun, no?)  The “wake” group was not allowed to sleep until 8pm the following day.  This may seem a bit extreme and rare that people would be awake for this long following a vaccine shot, but it does adequately distinguish the two experimental groups clearly. 

Q) What vaccine was used?

A) Hepatitis A vaccine (Twinrix, GlaxoSmithKline Biologicals)

Q) What did the researchers monitor throughout the trial period?

A) Four major conditions were tested:

1. Sleep activity via electroencephalography (EEG) to examine sleep stages.

2. Hormone analysis to investigate sleep-related hormone release via i.v. blood collection.

3. Hepatitis A Virus (HAV) - specific T helper cell response

4. HAV - specific antibody response

For immune response, peripheral blood cells (to look at T cells) and serum (where antibodies are found) was collected by drawing blood immediately before vaccination and then 1,2, and 4 weeks after each shot as well as a follow-up at 1 yr. after the first inoculation.

Now let’s dive into the results!

Because the whole basis of this paper rests on sleep activity it is first imperative that the authors of the paper show that the people in the “sleep” group slept normally.  Normal meaning that they spent time in each of the known sleep stages (see the above figure) that fit data that is already well established in the field, with nearly 50% of sleep spent in slow wave sleep (SWS; stages 3+4) and 50% in REM sleep.  There was no significant differences between the three sleep nights post vaccination. 

Over the course of the Hepatitis A vaccination period, blood was obtained to examine the T cell response. This is very important to look at because it is a standard read-out for the quality of a vaccine.  Recall that most vaccines, including the Hepatitis A vaccine, consist of purified proteins that contain immunogenic Hepatitis A viral proteins.  Once these proteins are injected, they enter the blood and tissue where they will be taken up by macrophages and dendritic cells.  Which is very convenient since these cells are antigen-presenting cells (APCs).  So then these APCs digest the viral proteins into smaller peptides and present these peptides on their surface so T cells can “see” the peptide.  If a T cell recognizes that particular peptide, the T cells become divides rapidly producing hundreds of more T cells that all are specific for that peptide.  Scientists can find out how well a vaccine is working by determining how many vaccine-specific T cells there are-the more there are, the more protection will be provided, the better the vaccine.   When a T helper cell encounters a peptide it’s specific for, it not only divides like crazy but it also upregulates an activation marker on its surface called CD40L.   For the purpose of this paper, it’s just important to understand that some proteins like CD40L can be used as a diagnostic tool to track the progression of T cell activation following vaccination.   In this experiment, Lange and colleagues, drew blood from the HAV-vaccinated men in both the “sleep” and “wakefulness” groups, then stimulated the blood cells with a pool of HAV peptides for 6 hours.  In this short amount of time, the only cells expressing CD40L are T helper cells that responded to and are therefore specific for HAV.  In both groups, the % of CD40L+ HAV-specific T helper cells increases.    It is important to note, that quickly following a shot, there is increase followed by a plateau of activated HAV-specific T helper cells and that with each vaccine shot, the T cell response to the vaccine increases; this is the purpose of booster shots.  The surprising result was that by the second HAV shot, there was a significantly lower T helper response in the “wakefulness” group than the “sleep” group.   Intriguingly, this difference lasted even 1 year after the initial vaccine shot with the “sleep” group having 2 times the number of activated, HAV-specific T helper cells than the “wakefulness” group.  Remember that the only major variable between the groups is that the “sleep” group went to sleep the night following the vaccination shots and the “wakefulness” group did not.  This one difference in sleep scheduling contributed to a two-fold difference in T cell activation and response to the HAV vaccine!

 Remember that T helper cells are called “helpers” because they produce cytokines that help other immune cells to become activated and better responsive to the vaccine leading to better protection against pathogens.  To make sure that the HAV-specific T helper cells they were detecting were in fact “helpers”, Lange, et al. measured various cytokines produced from CD40L+ cells in response to the HAV peptide pool.  In all the cytokines tested, the “sleep” group produced significantly more cytokines (Interferon(IFN)gamma, Interleukin(IL)-2, Tumor necrosis factor(TNF)alpha, and IL-4) than the group didn’t go to sleep after getting their vaccine shot.  

 In addition, T helper cells are “helpers” because they “help” B cells to produce and release antibodies (IL-4 helps this, for example).  Because antibodies are one of the major ways vaccines work to protect you against pathogens, the authors of this paper needed to assess whether antibody production is also affected by sleep.  To do this, they need to isolate the serum from the blood cells when they collect the blood samples.  Once they have the serum, which contains proteins and antibodies but not cells, they can look for HAV-specific antibodies-which is basically like searching for a needle in a haystack.  However, researchers can find HAV-specific antibodies in serum by performing an Enzyme-LinkedImmunosorbent Assay (ELISA).  The basis for an ELISA is using a culture plate coated with HAV proteins and then adding serum to the coated plate.  After a brief incubation period, the plate is washed thoroughly, and because antibodies bind extremely well to specific proteins, only the HAV-specific antibodies will stick to the plate, whereas the non-HAV antibodies floating in the serum will be washed away.  Finally, the amount of HAV-specific antibodies present (antibody titer) can be determined easily.  By this method, which is a very common, standard method used by immunologists and medical professionals, they found the people who slept the night following a vaccine shot produced significantly more HAV-specific IgG antibodies compared to those who didn’t get any sleep!

 What is very interesting is that the percentage of HAV-specific T helper cells greatly correlated with the sleep stage #4 compared to the other sleep stages, suggesting that something was present during sleep stage #4 might be regulating this phenomena.  Lange, et al. had found that hormones such as growth hormone (GH) and prolactin levels increased profoundly during SWS period.  Recall, that these hormones induce an inflammatory response, which has been studied extensively as a way the body regulates itself to become sleepy.  What is less known, is if these sleep-associated hormones have a role during the immune response as well.  What these data indicate is that GH and prolactin not only regulates the brain to induce sleep, but they also regulate the immune system to induce a highly activated immune response upon vaccination!  

Furthermore, during SWS, cortisol levels –which is immunosuppressive- is very low.  Given these data, the authors hypothesized that increase in GH and prolactin coupled with the decrease in cortisol levels during SWS provided a boost to the HAV vaccine that the “wakefulness” group did not receive.  In this way, these sleep-associated hormones behave like an adjuvant that further stimulates APCs to activate T helper cells.  By determining an “adjuvant factor” (GH x prolactin divided by cortisol levels), and correlating this “adjuvant factor” with the percent of HAV-specific T helper cells, Lange, et al. discovered the production of GH, prolactin and cortisol can eloquently predict the development of a strong HAV-specific immune response!

  As the authors admit, “the immunoregulatory functions of sleep are not well understood”.  Which makes this particular research so exciting and novel!  Of course, there are lots to expand upon and investigate to better understand how exactly these sleep-associated hormones boost the immune response to the HAV vaccine.  It would also be very interesting to know if sleep affects other vaccines besides HAV and if sleep cycles in women or people who are older or younger than the group tested in this paper provide similar results.  From an immunologist’s perspective, I think it would be valuable to know not only if the development of immune response to the HAV vaccine is significantly better, but also if the immune response is functionally improved because of sleep.  Which will be difficult to do in humans (people are ok with volunteering for a HAV vaccine, but to also volunteer to get infected with HAV? Probably not so great.) But perhaps this aspect could be assessed in mice or by looking at sleep patterns among people who have Hepatitis A and seeing if there is a smaller viral titer in people who sleep more.  It might seem ridiculous, that this paper rests all of its results on the one difference of either sleeping 7.5 hours or not sleeping at all, because it might appear that this data is only applicable if you are an insomniac.  It would be interesting to know what exactly the threshold of sleep (hours) is required to see the phenomena they illustrate in this paper.  However, what this research provides is strong evidence that little to no sleep has a profound effect on the development of your immune response during vaccination.  Interestingly, it would be important to test if sleep-associated hormones have a similar effect during infection with a live virus since this data might provide new insight into why newborns are highly susceptible to such infections (what newborn do you know that sleeps 7+ hours a night?) and why people who work 16+ hour days or graveyard shifts spend more time sick than people who work 9-5.  

Since starting graduate school, I’ve noticed that I have spent more days a year sick than I ever have, I used to think it was because of my increase use of public transportation, living in a bigger city, and interacting with more people (big lab, friends, students of mine, seminars, etc) than I had in my past.  But now, I think that I had been neglecting one of the major differences between life before graduate school and now: longer hours working in the lab generally means less sleep at home.  As if I needed another motivational reason to be done with graduate school!

Lange T, Dimitrov S, Bollinger T, Diekelmann S, & Born J (2011). Sleep after vaccination boosts immunological memory. Journal of immunology (Baltimore, Md. : 1950), 187 (1), 283-90 PMID: 21632713








References and Further Reading: 

1.    Hosford, M. et al. “Air traffic controller asleep on duty at Reagan National, NTSB says”. ABC News. (2011). 
2.    Beers, TM. “Flexible schedules and shift work: replacing the “9-to-5” workday?”. Monthly Labor Review. (2000). 
3.    Awake in Philly Community Education Group. “Fact sheet: shift work sleep disorder”. (2004). 
4.    CDC. “Insufficient sleep is a public health epidemic”. (2011). 
5.    US Department of Transportation, National Highway Traffic Safety Administration, National Center on Sleep Disorders Research, National Heart Lung and Blood Institute. “Drowsy driving and automobile crashes” (2011). 
6.    Bryant, PA., et al. “Sick and tired: does sleep have a vital role in the immune system?” Nature Reviews Immunology. 4:457-467. (2004). 
7.    Toth, LA., et al. “Alteration of sleep in rabbits by Staphylococcus aureus infection”. Infect. Immunity. 58: 1785-1791. (1988). 
8.    Born, J., et al. “Effects of sleep and circadian rhythm on human circulating immune cells”. Journal of Immunology. 158:4454-4464. (1997). 
9.    Everson, CA. “Sustained sleep deprivation impairs host defense”. Am. J. Physiol. 265:R1148-R1154. (1993). 
10. Kripke, DF., et al. “Mortality associated with sleep duration and insomnia”. Arch. Gen. Psychiatry.” 59:131-136. (2002).