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).
 

Think twice before diving into a high-fat diet: New research indicates how saturated fatty acids promote the development of Type 2 Diabetes

from ryangruss.com

Public Interest Note:

We hardly need to read or watch the news everyday to observe one of the fastest growing trends in America: obesity. Obesity is defined by the Center for Disease Control and Prevention “as a body mass index (BMI) of 30 or greater.  BMI is calculated from a person’s weight and height and provides a reasonable indicator of body fatness” According the CDC, nearly every state in the U.S. has populations containing at least 20% obese adults¹.  Of course, obesity does not merely affect Americans, as the World Health Organization estimates more than 300 million people around the world are obese².

Our friends and family and self-help books tell us that  “beauty comes in all shapes and sizes” and we want to believe this.  Hearing this makes us feel beautiful no matter what we look like, and we’re lucky to be surrounded by people that will love us unconditionally.  But at the same time it’s important to realize that it may not be so beautiful to be dependent on insulin injections to survive, suffering from chronic inflammation and increased susceptibility to infections.  The link between obesity and a variety of health concerns including heart disease and diabetes is well established.  A 2010 study spearheaded by Kaiser Permanente and published in the New England Journal of Medicine (NEJM) showed that heart disease has dropped significantly-by 24%- between 1999-2008.  Much of this decline, the research team noted, is attributable to decreases in cigarette smoking and increases in the use of cardio-protective medications during the course of those years³. Importantly, in the same issue of NEJM a perspective editorial by Drs. Jerimiah Brown and Gerald O’Conner, noted that during those same years 1999-2008, the incidence rate for Type 2 Diabetes and obesity increased.  They concluded “These trends suggest that we are succeeding some areas by reducing the burden of modifiable risk factors, such as smoking, hypertension, and high cholesterol levels, but that our society’s diabetes and obesity problems are worsening.”⁴ The data supporting a link between obesity and diabetes is clear, both of which cause detriment not only to us individuals, but also to our community with increased medical costs, unhealthy workforces and reduced life expectancies.

The role of scientists and doctors is vital and necessary in fighting this disappointing epidemic.  In the early 1950’s Dr. Hans Kraus had published a series of provoking research articles regarding the lack of physical activity of Americans, specifically the children.  In comparison to European children in a series of physical activity tests, Kraus and colleagues revealed out of 4,400 students (age 6-16) “56 percent of the U.S. students failed at least one of the test components…only about 8% of the European children failed”⁵.  These startling data quickly stirred interest among the general public, as people began to consider more the importance of diet and exercise to lead healthy, productive lives.  Not only the lives of individuals, but the life of the country as a whole and in 1955 President Eisenhower invited Dr. Kraus for a meeting to establish a course of action based on the data presented.  In less than year, the President commissioned the  “President’s Council on Youth Fitness” to organize a new initiative called the “Presidential Fitness Awards” in which for school children complete a series of physical fitness tests to receive one of these awards.  The program functions still today as a way to fight obesity and helped bolster the “Let’sMove” campaign commissioned by First Lady Michelle Obama as a way to “change the way a generation of kids thinks about food and nutrition” and reduce childhood obesity to 5% by 2030 ⁶.  The achievement lead by Kraus over 50 years ago highlights the value of cross talk between scientists and the public and set into motion America’s growing interest in fitness and healthy lifestyles. 

Type 2 Diabetes: The Immunology Behind the Disease

Eating whole foods, balanced meals and small portions play a role in maintaining our figures curvy and toned, while simultaneously regulating body’s ability to function properly.  Our bodies need to consume foods for nutritional benefit, but what happens when we eat too much, especially foods high in salt, sugar and fat?  The most witnessed observation is the accumulation of weight, in the form of fat, deposits in our bellies contributing to our subcutaneous (just beneath the skin) or visceral (covering our organs) fat tissue.  When we think of fat, most of us think of glossy white lining of our T-bone steaks.  Although it looks like it serves no better function that to moisten our steaks, our fat tissue is a quite dynamic place to be, especially for an adipocyte.  Adipocytes are the cells that largely make up our fat tissue (anatomically referred to as adipose tissue).  These cells are unique compared to all the other cells in our bodies.  Adipocytes store fats derived from food and liver metabolism.  The storage of fats by these cells are vitally important such that adipose tissue protects our organs and acts as a buffer to help ward off certain pathogens.  Moreover, because the metabolism of fat releases more energy than proteins or carbohydrates, adipose tissue represents the largest energy storage in our bodies.  When we eat too much and exercise too little, our adipose tissue increases, literally.  As our adipocytes store more and more fat they swell to accommodate the new intake.  This of course can be very bad for the rest of our body-most obviously for the organs that lie beneath this mounting fat-which often times is why obesity is linked to a variety of progressive diseases, simply because our diagnostic tools cannot access the affected organ and therefore inhibit many useful prevention strategies to combat such diseases.  

             So what does any of this have to with your immune system?  As I hope you will begin to appreciate, your immune system is an amazing, peculiar system that seems to be involved in nearly every function in your body.  Least of which includes your fat-or at least how it’s maintained.  It’s incredible that our bodies have evolved to become vastly dependent upon one, single protein, insulin, with which we wouldn’t be able to focus, our blood pressure would raise uncontrollably-it short, and our body would crash.  Insulin is a natural hormone produced by beta islet cells in the pancreas in response to increased blood glucose levels.  The insulin secreted by the pancreas signals through insulin receptors, which are expressed by the cells that regulate our blood glucose levels, largely our liver cells and adipocytes.  Once insulin has bound to its receptor it activates the adipocytes to absorb any glucose that comes its way, thereby lowering the blood glucose levels and returning the body to homeostasis.  When the body is unable to produce insulin or becomes unable to respond to insulin-diabetes develops.

            Let’s be clear about one thing: your immune system is tightly involved in the development of Diabetes-both Type 1 and Type 2, but in completely different ways.  Type 1 Diabetes (also known as juvenile diabetes and diabetes mellitus) is an autoimmune disorder.  Recall from the last post that autoimmunity is the result of self-reactive T cells circulating through the body, recognize self-antigen as foreign and trigger the death of any cell expressing that particular antigen.  In the case of Type 1 Diabetes, the self-antigen that’s being attacked is insulin-so therefore beta islet cells are under attack by the body’s own immune system.  It is for this reason that people diagnosed with this type of diabetes must take insulin shots to balance their blood-glucose levels since they cannot produce insulin independently.  Conversely, Type 2 Diabetes (T2D) is a metabolic disorder tightly associated with obesity and results in insulin insensitivity that is, although the body can make insulin just fine, the adipocytes can’t respond to it and therefore can’t remove lower glucose levels from the blood.  Essentially, the body is consuming more sugar and fat than it can regulate, and for reasons incompletely understood, the body loses the ability to recognize all the insulin being produced because of this resulting in hyperglycemia. The role of the immune system is quite evident in the case of Type 1 Diabetes, but it less obvious in the pathology of T2D. 

            In the 1990’s it was demonstrated that obese tissue released more inflammatory cytokines than lean tissue, thus beginning the investigation of the role inflammation in T2D development ⁷.  Cytokines are small molecules secreted by immune cells that activate the immune system, some cytokines promote inflammation.  Two of the most studied inflammatory cytokines are TNFalpha and IL-1beta, which are usually released during infections.  These cytokines are secreted by immune cells once the cell receives instructions to do so.  Immune cells are able to recognize invading pathogens quickly by recognizing certain pathogen-associated molecular patterns (PAMPs), which are only present on microbes and not our own cells.  PAMPs stimulate Toll-like receptors (TLRs) and NOD-like receptors (NLRs) to induce TNFalpha and IL-1beta release, respectfully.  These cytokines are inflammatory because they recruit more immune cells to the site of infection and starts the process of inflammation characterized by swelling, fever, redness, and pain-all of which is required to fight infection.  This clever detection system allows for the quick response by innate immune cells (macrophages, dendritic cells, and neutrophils) to activate the rest of the immune system to destroy the invading bug.  Our innate immune cells are the first the cells to respond to an immunological threat.   

Within the past couple decades it was discovered that fat tissue does not merely contain adipocytes, but is full of immune cells too, especially one particular immune cell-the macrophage.  The macrophage is named so because it is one of the largest immune cells and engulfs nearly everything it crosses.  You can think of it as the janitorial cell of the body, clearing tissue from cell debris and microbes.  Earlier just this year, Vishwa Deep Dixit’s laboratory based at Louisiana State University, established a role for fat-associated macrophages in controlling obesity-induced T2D.  They had established a model to study T2D by engineering mice that were deficient for a particular NLR (NLRP3), which is important for IL-1beta release.  When these mice were fed a high-fat diet, their blood-glucose levels were lower than their wild-type counterparts.  Furthermore, the fat-associated macrophages possessed anti-inflammatory characteristics whereas wild-type mice fed a high-fat diet contained highly inflammatory macrophages within their fat tissue.  Moreover, they showed that IL-1beta released by macrophages induce other immune cells to develop insulin resistance and perpetuate the inflammatory response, which ultimately leads to chronic inflammation and T2D ⁸.  Although, these authors convincingly demonstrated the role of NLRP3-induced IL-1beta in the development of T2D and chronic inflammation, what was directly activating the fat-associated macrophage to produce IL-1beta in the first place remained elusive. 

As you’ll recall, macrophages become activated via TLR or NLR stimulation.  Macrophages usually require a TLR or NLR activator in order to produce inflammatory cytokines like IL-1beta.  So, in the absence of microbial infection (which is the standard activator of these receptors) how do these inflammatory cytokines get released in the fat tissue? What is present in the adipose tissue that activates macrophages to release IL-1beta, a cytokine critically important in the progression of T2D? That is the great mystery and focus of the research discussed below that was recently published by Dr. Jenny Ting's research group at the University of North Carolina, Chapel Hill.

Treating Diabetes and Obesity: Why This Research Paper Matters:

            The best prevention is clear: exercise daily and eat balanced, small-portioned meals.  However, in some cases, people are more genetically susceptible in developing the disease or for some individuals, diet and exercise isn’t enough to control the disease.  For these people, there is little treatment options available.  Currently, anti-inflammatory diets are popping up on the scene as alternative diet plans to help combat diseases associated with chronic inflammation.  Such diets are heavy in foods that people should be eating more of regardless of their health conditions: whole vegetables and fruits rich in vitamins and anti-oxidants, which help combat inflammation.  Although the value of eating such foods is becoming increasingly acknowledged, the mechanisms by which these foods block inflammation is less known.  For example, although for decades, millions of people take omega-3 fatty acid (fish oil) supplements for its anti-inflammatory properties, it was not until 2010 that a team of researchers identified the exactly how omgea-3 fatty acids were able to dampen inflammation! Not only did these researchers discover the receptor that binds omega-3 fatty acids and that this receptor inhibits TLR-induced pro-inflammatory cytokine release from macrophages, but they also provide incredible data showing that omega-3 fatty acids that bind this receptor promotes insulin sensitivity!⁹ This new data suggests the use of omega-3 fatty acid as a possible treatment option specifically for T2D.   As it turns out, many drugs and supplements are prescribed without the knowledge of how the drug actually works.  There is no doubt that more effective drugs could be available if we better understood the underlying mechanisms behind disease.

As a testament for the work done in mice regarding the role of IL-1beta in T2D pathogenesis, recent clinical trials are underway testing a molecule that blocks IL-1beta signaling, anakinra.  A 2007 NEJM paper discussing the results of one study using anakinra showed that it improved glucose levels and reduced system inflammation in patients receiving the drug ¹⁰.  But again and importantly, this is clinical trials and the need to discover new drug targets and development of novel treatments for T2D is needed to help combat a disease that plagues millions of our parents, friends and increasingly-our children.  Dr. Jenny Ting’s UNC research group recently published in Nature Immunology compelling data indicating how saturated fats activates a newly identified pathway involved in the development of T2D.  With this latest discovery, it is likely that their data will bolster many promising new therapeutic targets to utilize in our fight against T2D.           

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

Haito Wen, et al. “Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling”. Nature Immunology. 12(5):408-415. (2011).

Diagram illustrating NALP3 inflammasome complex. Although a number of stimuli was known to induce inflammation via NALP3 activation (abestos, cholesterol, silica, etc), prior to the research discussed here, fatty acids high in diabetes patients was not known to contribute to inflammation through the same mechanism! Image from www.invivogen.com

1)    NLRP3 a type of NLR called-brace yourself-nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 or as it’s more commonly, for obvious reasons, as NLRP3.  Although the official name of this protein is a mouthful, it’s highly descriptive of what the protein looks like.  Each part of it, each domain is important for its function.  Pyrin domains are particularly important for interactions with other intracellular proteins. 

2)    ASC is a small intracellular protein that happens to also consist of pyrin domains allowing it to bind to NLRP3 very nicely.  See, NLRP3 isn’t very useful on its own; it needs ASC to carry out a function.  The value of ASC lies with what it brings to NLRP3: an enzyme.  Specifically, an enzyme called caspase-1 associates with ASC, so when NLRP3 recruits ASC, it’s recruiting caspase-1 too.  Caspase-1 is a very important enzyme for the immune response because it chops up big cytokines into a smaller, more functional fragment that can be secreted by the cell.  What kind of cytokines does caspase-1 process?  None other than the much-talked-about pro-inflammatory cytokine, IL-1beta.

3)    Inflammasome is a term to describe a group of proteins interacting with each other that promote inflammation.  The NLRP3-ASC inflammasome therefore refers to the complex containing NLRP3, ASC, and caspase-1.  It is known that a wide variety of PAMPs and danger-associated signals activate the formation of this signaling complex.  Recently, a lot of work has been done investigating the role of the NLRP3 inflammasome in a variety of infections and injuries.  Collectively, it’s now known that the NLRP3 inflammsome is activated in response to stressful conditions (stressful to a cell, that is), like when neighboring cells are dying or during infection.  It is important to recall, that in obesity these events are not happening in the adipose tissue, so what the stressful stimuli are is the point of this research paper.

     4)   Insulin signaling refers to what happens when insulin binds its specific receptor.        Upon binding to insulin, the insulin receptor recruits another protein called the insulin receptor substrate, which will instruct the cell to survive and divide, to efficiently remove glucose from the blood.  The concept with T2D progression is that too much glucose is around such that insulin is being produced by the pancreas all the time in high amounts-which desensitizes adipocytes, so that blood-glucose levels remain high.  One likely candidate that causes this effect is impaired insulin receptor signaling.

Put all these terms together and we can now infer that: Certain fats we eat cause the NLRP3 inflammasome to assemble.  The NLRP3 inflammasome contains caspase-1, which will process IL-1beta for release into tissue, which not only promotes inflammation but also blocks insulin receptor signaling, therefore instigating the onset of T2D.

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

Palmitate is an example of a saturated fatty acid, common at high levels in obese and T2D patients. Image from www.yellowtang.org

Ok, so we’re not quite ready to dive into the data.  We first need to be on the same page about fat.  Fat is essential to our well-being.  Fat provides most of our energy allowing our bodies to do all the amazing things it needs to do to live.  But if there’s one central theme about preventing T2D it’s “excess Let’s all agree that saturated fats are generally pretty bad for us, especially in excess.  Saturated fats derive mainly from meats and dairy.  Because of their compact structure, if they build up in your arteries a clog can form and increase LDL cholesterol levels.  Unsaturated fats are more flexible and come from plants and lower the “bad” LDL cholesterol.  The kind of fat used throughout the experiments discussed below is palmitate, which is an abundant saturated fatty acid in the blood of people who consume a high fat diet ¹¹, the main culprit of obesity and T2D development.  And on that note, let’s dive into the data!

The first thing Wen and colleagues logically wanted to determine was whether palmitate could in fact activate the inflammasome, as their title suggests.  To do  this, they activated macrophages in culture with lipopolysaccharide (LPS), which is required to make what is called “pro-IL-1beta”-the protein that caspase-1 cleaves into mature IL-1b for secretion.  If you want to measure IL-1beta released by macrophages stimulated with only LPS, you will hardly find a trace of it.  Wen, et al. show that LPS-primed macrophages will secrete loads of IL-1beta when there palmitate around in a time and dose-dependent manner.  Furthermore, palmitate only signals IL-1beta release from macrophages that express NLRP3 and ASC as macrophages deficient in either of these proteins significantly reduce the amount of IL-1beta produced. These cytokine effects appear to be NLRP3 inflammasome-specific since when they look at another cytokine (TNFalpha or IL-6), which don’t need caspase-1, palmitate does not affect their secretion.   Finally, the authors illustrate, biochemically, that palmitate activates caspase-1 allowing the cleavage of pro-IL-1beta into mature IL-1beta. The data is clear: high levels of the saturated fat, palmitate, promotes the release of the pro-inflammatory cytokine IL-1beta via NLRP3-inflammasome dependent mechanism.  Amazingly, these effects don’t occur when LPS-primed macrophages are stimulated by unsaturated fatty acids. But how is this happening? Is the saturated fatty acid activating the inflammasome directly or indirectly?

As mentioned, when cells are stressed out they signal the NLRP3 inflammasome to assemble and start to secrete inflammatory cytokines to warn the rest of the immune system that something isn’t right and that help is needed.  It is well established when cells are stressed, their mitochondria (the energy machine of the cell) loses membrane potential and reactive oxygen species (ROS) start to seep into the cytosol.  Furthermore, it’s also known that ROS controls NLRP3 association with ASC and is therefore crucial in inflammasome assembly.  

So it makes perfect sense for the authors to investigate whether palmitate can activate ROS.  Using a special dye that turns fluorescent in the presence of ROS, Wen, et al. show that palmitate increases ROS generation in cells and that this effect is blocked in the presence of a ROS-inhibitor.  Moreover in the presence of palmitate, they showed that IL-1beta is produced, but now-in the presence of this ROS-inhibitor, caspase-1 activity and IL-1beta production greatly diminishes.  All together, these data signify a novel finding-and the basis of how this story ended up in Nature Immunology: palmitate, a fatty acid associated with obesity, causes high levels of IL-1beta production from macrophages by stressing cells to produce ROS and activate the inflammasome.  But we have only reached the tip of this iceberg, and many more questions must still be answered to complete this story.  One of which is: how do saturated fats stress out cells?

The authors explain that “the AMP-activated protein kinase (AMPK) has emerged as an essential mediator of fatty acid metabolism and it suppresses ROS production” and its activity promotes the development of anti-inflammatory macrophages.  The researchers then set out to test their hypothesis that “AMPK plays a role during inflammasome activation by palmitate”.  Again, using that ROS-sensitive fluorescent dye, they provide data indicating that activation of AMPK inhibits palmitate-induced ROS production and IL-1beta release.  Interestingly, palmitate appears to deactivate AMPK, so that ROS can accumulate in the cell and promote NLRP3 inflammasome assembly and activity.    

These data suggest that AMPK activation plays a large role in directing inflammasome activation and that AMPK activity can be exploited by external stimuli like palmitate to promote inflammation.  Importantly, this exploitation of AMPK appears to be specific to palmitate and not PAMPs or danger signals associated with injury and infection.  This is an important distinction because they way these danger signals induce ROS production is by creating pores in the membrane and opening ion channels.  But none of these stresses were induced by palmitate, which begged the question: we now have data that palmitate inhibits an enzyme that blocks ROS, which in effect allows ROS to accumulate in the cell, but how in the world was ROS being generated in the first place by palmitate?

The answer appears to lie in process called autophagy, which cells utilize to get rid of old organelles and metabolites for energy. It’s a highly evolutionarily conserved process that is vital in cell survival. More recently, it’s been shown that AMPK positively regulates autophagy during fatty acid metabolism.  In the process of metabolizing fatty acids (aka autophagy) a little bit of ROS is generated.  Wen, et al. continue to show a number experiments to demonstrate that palmitate deregulates autophagy by inhibiting AMPK.  So not only are macrophages highly inflammatory in the presence of unsaturated fatty acids, but their autophagasomal machinery is messed up, which just further perpetuates inflammasome activation.  In short, a single stimulus, palmitate can turn a usually anti-inflammatory macrophage (expressing AMPK) to a potent pro-inflammatory macrophage.  This would be great news if the plan was to fight a bacterial infection, where inflammatory macrophages are needed to destroy the pathogen, yet in T2D, there isn’t a pathogen and these patients have chronic inflammation, which as this new data suggests is likely due to the high saturated fat diet consumed.  Things couldn’t get possibly worse, right?

Of course they can, especially with T2D since we know the disease isn’t solely an inflammatory condition, but also affects the body’s ability sense insulin.  So what does all of this palmitate data have to do with that?  Wen and colleagues proceed with a series of elegantly designed experiments to investigate the role of palmitate in insulin signaling.  First, they show that liver cells pre-treated with IL-1beta are less sensitive to insulin that unprimed cells based on insulin receptor activity.  Furthermore, they show this happens because IL-1beta blocks IRS-1 function, the protein responsible for promoting insulin sensitivity and cell survival.  Even more compelling was the data in which they illustrate that if they gave media from palmitate-activated macrophages, which would contain IL-1beta, to liver cells, the same effect was observed.    

They repeated this experiment with NLRP3 and caspase-1 deficient macrophages, which had no effect on insulin receptor activity suggesting that palmitate activates macrophages to release IL-1beta in a NLRP3 inflammasome-dependent manner, which can then go on to inhibit insulin signaling in liver cells.  If you recall, interference of insulin signaling will have detrimental effects for liver and fat cells’ ability to remove glucose from the blood, thereby promoting the development of T2D.

To study this more closely, in regards to T2D, the authors fed mice a high fat diet for 12 weeks and show that compared to mice fed on a low-fat diet, had higher blood-glucose levels.  Moreover, mice deficient in IL-1beta, ASC or NLRP3 had glucose levels similar to that of a wild-type mouse on a lean diet!  Finally, their in vivo data corroborated their in vitro findings that inflammasome activation within macrophages blocks insulin receptor signaling.  Collectively, Dr. Jenny Ting’s research group provided valuable insight how saturated fats control not only our weight, but also our immune system.   

Not only did they provide correlative evidence about palmitate, but the also went the extra mile in terms of experimental proof to identify the mechanism by which palmitate contributes to T2D disease.  This is why this particular research is regarded highly by the scientific community and published in high-profile journals.  Not only did Wen, et al. convincingly show how palmitate increased IL-1beta production, but the also revealed a promising new set of targets for T2D and chronic inflammatory diseases: autophagy.  This is very exciting research that is bound to play a significant role in supporting the development of future treatment options for the millions of people suffering from such ailments!  However, as their last figure nicely demonstrates, remember that the best prevention to T2D-even if you’re genetically susceptible- it to maintain a low-fat diet and try to fit in a bit of exercise each day.

Wen, et al. contributed significant understanding to the inflammatory mechanism behind T2D showing that the fatty acid, palmitate activates the NALP3 inflammasome, enhancing inflammation in adipose tissue and inducing insulin resistance and beta-cell dysfunction.
Image from discoverymedicine.com

Oh, and one last thing to keep in mind: mice are quite active during the day, roaming around and playing with each other, so it’s likely that the effects shown in this research paper regarding mice kept on a high-fat diet could be greatly exacerbated if the mice were sedentary! On that note, I think I’ll go for a run after work today, maybe you’ll be inspired to do something active too! 

Wen H, Gris D, Lei Y, Jha S, Zhang L, Huang MT, Brickey WJ, & Ting JP (2011). Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Nature immunology, 12 (5), 408-15 PMID: 21478880








References and Further Reading: 
1: Center for Disease Control and Prevention. “U.S. Obesity Trends: Trends by State 1985-2009” 
2: WHO. “WHO Fact Files: Ten facts on obesity”. (2010).
3: Yeh, RW., et al. “Population trends in the incidence and outcomes of acute myocardial infarction”. NEJM. 362:2155-2165. (2010). 
4: Brown, JR and O’Conner, GT. “Coronary heart disease and prevention in the United States”. NEJM. 362:2150-2153. (2010).
5:The President’s Council on Physical Fitness and Sports. “History of the President’s Council on Physical Fitness and Sports (1956-2006)”.
6: Let’s Move: America’s move to raise a healthier generation of kids. “White House task force on childhood obesity report to the president”. (2010). 
7: Hotamisligil, GS, et al. “Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance”. Journal of Clinical Investigation. 95:2409-2415. (1995).
8: Vandanmagsar, B., et al. “The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance”. Nature Medicine. 17(2): 179-188. (2011).
9: Oh, DY, et al. “GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects”. Cell. 142(5):687-698. (2010).
10:Larsen, CM, et al. “Interleukin-1-receptor antagonist in type 2 diabetes mellitus”. NEJM. 356: 1517-1526. (2007).
11: Boden, G., et al. “Interaction between free fatty acids and glucose metabolism”. Curr. Opin. Clin. Nutr. Metab. Care 5: 545-549. (2002).