Interview with David Paez-Espino (JGI): Identifying novel viruses on Earth and in the human body

June 12th, 2018 by Amy Proal

Hey readers!

Viruses are the most abundant life form on the planet

This blog and my peer-reviewed papers explore how the human microbiome can impact chronic inflammatory disease processes. It is well understood that an extensive microbiome persists in the gut (hundreds of trillions of microbial cells in total!) In addition, extensive microbiome communities have been identified in every other human body site – from the brain, to the liver, to the lungs, to the bladder, to the placenta and beyond. We now realize that no part of the human body is sterile. This includes human blood, which is now understood to contain a microbiome (even in healthy individuals).

However it’s very important to understand that even when all this new human microbiome knowledge is taken together, the global research community has still not yet identified, characterized or studied many of the microbes and viruses in the human body. In fact, almost every new human microbiome study turns up new species of bacteria, viruses and fungi that were previously unknown to science. For example, just a few months ago, Stephen Quake identified thousands of species of bacteria, viruses and fungi in human blood that were simply not known to exist before the study was performed.

Viruses are the most abundant life forms on the planet, but we know particularly little about human viruses. In other words, there are an incredible number of viruses in the human body that have not yet been identified. This is especially true of bacteriophages (viruses that infect bacteria). A recent study estimated that 31 billion bacteriophages traffic the body on a daily basis. The human body also harbors double stranded DNA viruses and RNA viruses. Most of us are familiar with double-stranded DNA viruses like the herpes viruses. We are also familiar with RNA viruses like polio, measles and influenza.

But these well-known RNA/DNA viruses comprise just a tiny fraction of all the viruses capable of persisting in the human body. Many other related viruses are still in the process of being discovered!

It follows that we cannot study human chronic inflammatory disease without understanding that viruses we have not yet identified may play a role in many human disease processes. To do so would be like going to the rainforest, studying only 2% of the animals, and coming to conclusions about how the entire rainforest functions off that information alone.

Despite this fact many doctors have been taught to test for only 10-20 well-known viruses in their human patients. If these viruses are not identified in the patient, it is assumed that a virus (or group of viruses) cannot be driving or contributing to the patient’s disease. We must work hard to change this assumption, because it greatly prevents the medical/research communities from looking at a much broader picture of what might be going on.

David Paez-Espino

In order to best make this point, I interviewed David Paez-Espino. David works at the Joint Genome Institute in Walnut Creek California, in the Department of Microbial Genomics and Metagenomics. He and his colleagues have created new, complex computational technologies that help identify both novel and known viruses. In fact, they have started a project called “Uncovering the Earth’s Virome” (viral communities). This project searches for novel viruses not just in the human body, but in ecosystems across the planet – including the ocean, the soil, the air, and in other animals. These viral sequences are stored in the world’s largest public viral database: IMG/VR.

Over just the past few years, Paez-Espino and team have identified and sequenced many, many new viruses. In a 2016 Nature paper, they unveiled more than 125,000 partial and complete viral genomes, which boosted the number of known viral genes in the world by 16-fold. By the time they published a follow-up paper in 2017, the contents of the IMG/VR database had doubled from the figure referenced in 2016. By January 2018 the database had tripled in size. At that point Paez-Espino stated the following:

“Among the million viral sequences predicted, we have now identified over 34,000 of them targeting several microbial taxa for the very first time, we’ve associated new viruses to known microbial genomes (culturable and unculturable), and the vast majority of the gene content (over 21 million genes in total) remains hypothetical or unknown, meaning that there is tremendous potential for new discoveries out of that gene pool.”

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In this interview, David and I talk more about these findings. We talk about how his team has identified all kinds of viral entities and how they focus on the identification of less-studied viral groups, e.g. giant viruses, and virophages (viruses that co-infect eukaryotic cells along with giant viruses). These giant viruses were not previously known to persist in the human body. We also talk about how the study of novel viruses holds incredible potential to improve our understanding of chronic and rare inflammatory conditions.

Here is a short video clip of part of our conversation. The full interview is below and has been edited for accuracy purposes:

Amy: Tell me about your job at JGI and what projects you’re working on now.

David: Over the past four years here at JGI I’ve started working on a project called “Uncovering the Earth’s Virome” – a very ambitious name, I know. We are trying to get a sense of what viruses are out there. To do that we are generating a comprehensive list or viral catalog of viruses from samples everywhere on Earth. I work in the Microbiome Data Science group (lead by Dr. Nikos Kyrpides), where we look for novel or known viruses in environmental samples…all kinds of samples: we have metagenomes, we have metatransciptomes. We also have some isolates. And we don’t just look for viruses – we also try to link the viruses to their specific habitats and to their specific hosts. Everything is computational. But then we try to validate our findings by working with other groups here at Berkeley or beyond.

Amy: Where do get your initial viral data from (that you then use to identify novel viruses)?

David: The JGI sequences many of these samples through collaboration projects (CSPs and FICUS programs). But we also have part of our public repository database (called IMG/M) represented by other groups’ data. We developed a virus prediction pipeline to identify viral sequences directly out of those samples, and have created IMG/VR (the largest virus repository integrated by isolates and microbiome-derived viral sequences). And everything is public. We are mining millions and millions and millions of nucleotides. So we have terabytes of sequences. And this collection of viral data in IMG/VR is very diverse – it includes marine/aquatic samples, terrestrial samples (like soil), human microbiome samples, and even microbiomes from mice, ruminants, air, arthropods etc.

Amy: So let’s say I sent you a blood sample. When it comes to identifying new viruses in this sample: What is your technology able to identify versus what a technique like PCR could identify?

David: If you’re using PCR to identify viruses you’re using an approach where you already know what you’re looking for (you are biased towards viruses that are fully characterized and have been entered in known databases). So PCR is useful if you have a well-known virus already understood to be a human health threat. For example, we could identify Zika virus that way.  

But in our case, we have different approaches. The first is a general global viral discovery pipeline. It does not only rely on genes from known viruses. Instead, we use the genes from known viruses to identify novel viruses from environmental samples: think of these novel viruses as, let’s say, the cousins of viruses already in a known database (their genomes are similar to known viruses but with a certain degree of difference). It’s a homology-based pipeline, but one that’s more sophisticated than the usual BLAST approach. 

A T4 bacteriophage

Our second approach is to target very specific groups of viruses that are unique. These include specific families from single stranded DNA viruses, double stranded DNA viruses, single stranded RNA viruses etc. In fact, we have developed specific tools that can identify hallmark genes associated with many types of viruses. These even include giant viruses. Giant viruses are eukaryotic viruses that have long genomes (sometimes 1 megabase, 2 megabases, even almost 3 megabases in size!). They also have very unique capsid proteins that can’t be found elsewhere. So we can develop and use these unique viral characteristics to create specific models for identifying similar viruses. This same process of identification goes for virophages.

Amy: What exactly is a virophage?

David: A virophages is a virus that co-infects a eukaryotic cell along with a giant virus. In the majority of cases the virophage is a parasite of a giant virus. But in some cases it seems that virophages only use part of the giant virus machinery to replicate within the final host (a eukaryotic cell that is often an algae or protozoa -as currently known). The infectious process is kind of like a Russian doll. There are many different levels of infection.

Amy: Where are you finding these giant viruses and virophages?

David: They seem to be everywhere. We are submitting a paper in which we found virophages in all kinds of environments including the human gut. Finding them in the gut was something people had speculated about but we finally proved it is true. Because most people don’t have the right tools to mine for virophages, or they don’t have the right tools to mine the database, or they don’t have samples from the right habitats or styles of life in the database. So a luxury we have here at JGI is that we can apply novel tools to a very wide variety of different samples – and we can pretty much find everything everywhere:)

Amy: Yes. It seems very important to stay up to date on the latest technologies for viral identification. Because if you don’t know HOW to look for a new virus you probably won’t find it.

David: Yes, if you only study already known viruses you may be missing part of a larger unexplored picture.

Amy: I’m interested identifying novel viruses that might play a role in the disease ME/CFS. Let’s say I had blood samples from patients with ME/CFS. Can I send them to you so that you can use your technology to search for new viruses in the samples? How would that kind of collaboration work?

David: We have two ways to collaborate. First we have official calls for research. About 40-60% of those projects get accepted. But those projects are more along the lines of the DOE (energy, carbon cycling, and environmental viruses rather than human viruses). 

But for identification of novel human viruses: We are developing new tools for the discovery of RNA and DNA viruses. And we need validation of these tools. So we want to apply them to all kinds of samples as opposed to just those from the environment. Also because everything is connected we sometimes find environmental viruses in humans and vice versa. So if you’re interested in human viral discovery we don’t have official calls for that research. But you can talk to me and say, “Hey I have these samples and I’d like to know if your RNA viral pipeline could work for my samples.” Then I can talk to my boss, and if we all have this common interest or a common outcome (a paper, a patent) we can hopefully find a way to collaborate.

Amy: What are some of the most interesting viruses you’ve identified in the human body? Do you mostly have gut microbiome samples?

David: So as I mentioned before, all the data we get is deposited in the interactive IMG/VR database. On the database website you can access the habitat of the samples to see where our human viral samples come from. At the moment IMG/VR includes viruses found in five different human body subtypes: skin, urogenital, salivary, gut, and the respiratory tract. 

One interesting thing we’ve found is that viruses are very specific to their habitats. That means if you find a virus in a marine environment it’s very unusual to find it somewhere else. You can find it across the globe in different oceans but, if it’s a marine virus, it’s a marine virus. The same is true of human viruses. If you have a human virus it’s probably going to be only found in the human body. And if it’s a human gut virus, only in the human gut. So, we saw very few connections between the different body subtypes. The findings are similar to those reported in a separate recent paper: The healthy human phageome. In that study, healthy humans were found to have a certain pattern of viruses always present in the gut. We also found that to be true – there seem to be marker viruses that could indicate the health status of a person. Probably because such viruses are maintaining a balance with the bad bacteria.

Amy: And in disease there might be shifts away from that core viral state?

David: Yes, definitely. For example, we have a very recent paper (in editorial status) where we compare the virome of IBS mice to that of healthy mice. And we found a certain core pattern in the gut virome of the healthy mice and a very stochastic pattern in the gut virome of the IBS mice. Basically, totally different virome patterns identified between the two groups. And those patterns may give us some leads as to what happens in human IBS. Hopefully the paper will be out soon.

Amy: Wow, you have some great papers coming out.

David: Yes. We are also doing work related to phage therapy. We are trying to predict certain phages and then predict their bacterial hosts. And of course, many of these bacterial hosts are harmful to humans. That gives us the opportunity to engineer the phages in ways that might allow them to best kill the bad bacteria.  

So, we predict phages in the human body and then say “This viral (phage) sequence is probably infecting this particular bacterial host.” But we are collaborating with other groups here at Berkeley on the project because, if we do identify a bacterial host, we need to know if we can culture it in the lab. Culturing the bacterium allows us to test it. We need to do that because our computer-based predictions are pretty robust – but we must be 100% sure that what we predict computationally holds up in the lab. Also, we need to confirm that the phage sequences we predict are perfect before attempting to use them as therapeutics.

Amy: That’s great. That’s what the phage therapy community needs right now. Much more information on verified phage/bacteria relationships (that can be easily accessed in a database). It’s also great for our general understanding of the microbiome. Because if we fail to account for the phages in any microbiome ecosystem, we’re unlikely to really understand the community dynamics. 

David: Yes. For example, if you study cell biology there’s another layer beneath that knowledge. You also need to understand molecular biology, because you need to understand molecules in order to understand cells. Similarly, if you want to understand bacteria in a microbiome community, it’s important to understand that the way the bacteria behave is based on their relationship to the phages. For example, there is a theory called “kill the winner.” Sometimes a bacterium grows exponentially in an environment. If its levels come back to equilibrium it’s usually because there is a phage that’s killing it. This equilibrium is built into the foundation of the ecosystem. And without understanding viruses you can’t fully understand that equilibrium. So yes, it makes sense to think that way.   

Amy: Yes that’s even true of an intervention like a probiotic. It would be helpful to understand how the bacteria in a probiotic might be modulated by neighboring phages.

So give me some numbers. I know it’s a ridiculous request. But about how many viruses are you finding in just the human samples you have so far?

David: So, starting with only DNA viruses…we have around a million sequences but some are redundant. So, if you only take the unique viral entities, we have about 400,000. Of those 400,000 about 30,000 or so are unique viral sequences coming from the human body.  

When it comes to RNA viruses, that’s a new ongoing project that we haven’t published on yet. But so far, we have around another 100,000 novel predicted RNA viral genomes. And about 20% of them are coming from human samples.

Amy: Do you see an end in sight?

David: There must be an end in sight, right? But the problem with viruses is that they also recombine. It’s complicated because it’s very hard to define a viral species. For example, there are cases where two closely related viruses are trying to avoid predation by bacteria, and to do that they recombine and create a third virus. Then…that third virus can recombine with another virus. Another example is that two viruses infecting the same host, at the same time, can also recombine. So, I think that the possibilities are countless and it’s going to be very complicated. We are definitely not even close to scratching the surface at all.

Growth rate of virus identification and microbial host prediction for the new release of IMG/VR database. Growth over time in the total and unique number of viral sequences in IMG/VR. (Image composite by David Paez-Espino).

Amy: It’s daunting but also exciting. Because we understand very little about the human microbiome and most ecosystems. So it’s great to realize there’s so much more we can learn about what might be going on.  

David: Right. We have a collection of ~21 million different genes from the viruses we’ve collected so far in IMG/VR. And 80-90% of these viral genes are hypothetical or have unknown functions. 

This same pattern is even true with E. coli. People have been studying E. coli for decades but we still don’t know the function of ~30% of E. coli’s genes. So, imagine how little we know about these novel viruses that are predicted from fragments here or there. It’s very complicated to make any kind of estimation.

Amy: It’s mind-blowing. To clarify: E. coli is one of the most well-studied bacterial species and we still don’t know a great deal about it.

David: Yes. E. coli is so well-studied that you’d think all the metabolic pathways are perfectly covered. But if you actually look at the databases, people still have no idea about what 30% of the E. coli genome is doing. So, these are exciting times, yes.

Amy: It’s clear that no matter what you’re studying related to the human body, you need to account for these viruses and other microbes. Whether you’re an immunologist, a neurologist etc. We need to account for their DNA/RNA and metabolites.

David: We have advanced a lot in science. Especially thanks to early studies of the microbiome that created reference genomes. But, for example, I recently went to a conference that discussed the drinking water microbiome. And they were not even testing for viruses (phages) in the drinking water. They are looking at 16S RNA sequences from about 20 known pathogens. And we’re are able to drink apparently safe water with just that knowledge. The basic necessities are covered. But – if we could understand even more about what microbes/viruses are in that water we could develop even better solutions.

This is especially true when it comes to human rare diseases. The majority of them could be caused by something we are not controlling. Medicine is doing a great job, but still – new knowledge about viruses can add to the picture. For example, now we understand that even some forms of cancer are caused by human microbiome dysbiosis accompanied by proliferation of a certain virus. So many of these novel or rare diseases could be better explained if we understand more about viruses and other microbes. 

This is also true for therapeutics (treatment). Now we can think about phages killing bacteria in a more precise way than antibiotics. Or we can use viruses that can be delivered to specific human tissue along with something that can access your immune system. You can design the virus to deliver something to the immune system “a la carte.” This precise medicine needs to evolve along with the new technologies.

My ideal job is applications. But I am also very pro basic science. For example – the CRISPR/Cas system. We are now engineering human cells from the use of bacterial genes that are designed to be a defense system against phage. So, the tremendous repertoire we have of novel genes can give us many new tools and technologies that we can manipulate at our will in the near future. We have been doing that for years, but the pace of discovery is now way, way faster. It’s a catalytic process: we are speeding up the different steps. 

Amy: Yes. The pace of discovery is incredible. I recently submitted a paper to a journal. They wrote back and asked me to give specific numbers for how many bacterial and viral species persist in the human body. I had to explain this wasn’t possible, and even if I made an estimate the number would change by the next month…or even the next day. 

David: Yes, for example, we have the largest viral database in this field. But even then, we’re only operating with assembled sequences larger than 5 KB. That represents less than 2% of all the sequences we have. Which is pretty much nothing. So, it’s not just the discovery pipeline that’s important. We can also go back and analyze metagenomes from 15 years ago. Because now we have different sequencing technology, we have different assembly technology, we have new binning algorithms, we have more robust models for gene annotation and functional predictions. And everything is still getting improved and improved and improved. So yes, as you said, there are no magic numbers. 

Amy: Definitely no magic numbers:) David thanks for speaking with me. I’m fascinated by your research and very excited to see where your findings lead. 

 

 

 

 

 

 

 

 

 

 

 

 

 

Re-thinking the theory of autoimmunity in the era of the microbiome

April 16th, 2018 by Amy Proal

Antibodies are Y-shaped molecules that target pathogen antigens

Hey there! Last podcast I mentioned the “theory of autoimmunity” and the range of immunosuppressive treatments that have stemmed from its promotion. I stated that the theory needs to be re-evaluated to account for the discovery of the human microbiome. Today I will go into even more detail on why that’s the case.

First, let’s talk about how the theory of autoimmunity gained popularity and widespread acceptance in the first place. Around the turn of the century, scientists began detecting Y-shaped molecules called antibodies in patients with a range of “acute” infectious diseases: diphtheria, tuberculosis, polio and pneumonia among others. Researchers (like the famous immunologist Paul Ehrlich) soon realized that these antibodies play a key role in helping the human immune system correctly target the bacterial and viral pathogens driving these and related diseases. They came up with a model of antibody activity that still largely holds up today. It contends that:

  1. Antibodies are released by B cells and T cells of the human immune system in response to an infectious threat: most commonly a bacterial or viral pathogen
  2. Antibodies target/neutralize these pathogens by recognizing the shape/size of unique protein molecules on the pathogen’s surface. These pathogen proteins are called “antigens.”
  3. When a human antibody correctly recognizes a pathogen antigen, the two molecules bind together. This “tags” the antigen and associated pathogen for further attack by other cells of the immune system (like white blood cells).
  4. The immune system begins to rapidly produce or “clone” more versions of the antibody that correctly “tagged” the pathogen. As more and more copies of this antibody pour into human blood/tissue, the immune system increasingly recognizes and kills the pathogen.

Today, almost every infectious disease is tied to antibody production. For example, when you get the flu, your immune system releases a range of antibodies in response to the flu virus. If all goes well, the antibody that best “tags” the virus’ specific antigens is “cloned.” More copies of this targeted antibody enter your bloodstream and the recovery process begins.

These same antibodies form the basis of the flu vaccine and related vaccines. In simple terms, a vaccine is made by taking an antibody already known to target a specific pathogen antigen – and injecting a small amount of this antibody into a patient. This “prepares” the patient’s immune system for the pathogen. Then, if the patient becomes infected at a later date, the immune system already knows exactly what antibody it should “clone” and rapidly release. The pathogen can then be neutralized and killed before it has a chance to spread.

OK. But what does this have to do with the theory of autoimmunity? In the 1930s-40s, researchers/doctors began to detect antibodies in patients with a range of chronic inflammatory conditions like lupus, rheumatoid arthritis, and multiple sclerosis. The natural next step would have been to search for chronic human pathogens whose presence could be tied to these antibodies. However, at the time (as described in my previous podcast), the human body was incorrectly believed to be largely sterile.

This confused many research teams working on the topic. How could they explain the presence of these antibodies when there were apparently no persistent microbes in the human body? They were forced to come up with their best guess as to what could be going on. They settled on a new concept that forms of the backbone of the theory of autoimmunity: the “autoantibody.” AKA, they postulated that in patients with these chronic inflammatory conditions, the immune system had somehow “broken down” or “gone crazy.” This confused immune system could then generate antibodies against antigens associated with the body’s own tissues. They named these hypothetical “self-targeting” antibodies “autoantibodies.”

Paul Ehrlich at his desk

At first the theory of autoimmunity and the idea that “autoantibodies” could exist met with resistance. Paul Ehrlich, the famous immunologist who had helped characterize antibodies in the first place, was particularly vocal. He wrote papers condemning the theory and coined the term “horror autotoxicus” to best counter it. Horror autotoxicus contends that the immune system has innate protection mechanisms that simply do not allow it to turn “against itself.”

But the scientific community wanted a consensus on the topic and needed simple guidelines on chronic inflammatory disease that could be given to doctors. Large conferences began to promote the theory of autoimmunity and detractors were increasingly pushed to the fringe. By the 1950s the theory started to be included in medical textbooks. Soon a growing number of “autoantibodies” were tied to different chronic inflammatory conditions. For example, patients with lupus test positive for an “autoantibody” that was named ANA. Rheumatoid arthritis was officially diagnosed if the “autoantibody” rheumatoid factor (RF) showed up on blood tests.

But a major question remained: if the theory of autoimmunity is correct, then what causes the immune system to fail so dramatically that it turns against the body’s own tissues? A number of theories attempting to explain this dilemma have been proposed over the years, none of which has been proven. One involves infection and is still frequently referenced today: the “pathogen/trigger” theory. It contends that certain well-characterized pathogens may infect a patient. Something about this temporary infection goes wrong and “triggers” the immune system to misfire and induce “autoimmunity.” The pathogen itself is somehow killed, but the immune system never recovers and the patient develops a full-fledged “autoimmune” disease.

The “pathogen/trigger” theory might have held more weight if the human microbiome had not been discovered around 2005. As described here, the discovery occurred when research teams started using new molecular tools to search for human microbes. The results of these new analyses were astounding: entire ecosystems of microbes were identified in the human body that had been missed by previous laboratory testing methods. Today we understand that trillions of microbes live in and on us – from the moment we are conceived in the womb until the day we die. These vast microbiome communities persist in every human body site – from the brain, to the liver, to the lungs and beyond.

This means that there are actually thousands, if not more, microbes/pathogens in the human body that could be tied to “autoantibody” release in patients with chronic disease. In other words, “autoantibodies” may just be regular antibodies created in response to pathogens that our testing methods were previously unable to detect. So, for example, when the “autoantibody” ANA is identified in patients with lupus, it could simply indicate that an unidentified pathogen plays a role in driving the lupus disease process. Also, it’s more likely that “autoantibodies” are created in response to chronic, persistent human pathogens than a temporary pathogen that the immune system somehow kills (as postulated by the “pathogen/trigger” theory.)

The possibility that “autoantibodies” are created in response to microbiome pathogens rather than “self” is strongly supported by the fact that “new” bacteria, viruses and other microbes continue to be identified in the human body. For example, Stanford researcher Stephen Quake recently detected thousands of never-before identified bacteria and viruses in human tissue/blood. In fact, 99% of the microbes he identified were previously unknown to science.

Also, for decades, “autoantibodies” have been regularly detected in patients with no signs of autoimmune disease that are instead suffering from an infection. For example, high levels of “autoantibodies” like rheumatoid factor, ANA (and others like ASCA, annexin-V and Anti-PL) have been identified in patients with bacterial, viral, and parasitic infections ranging from hepatitis A/B to Q fever to Rickettsia.

E. gallinarum under a microscope

A more recent study by researchers at Yale came to a similar conclusion. The team studied E. gallinarum, a bacteria they identified in the human gut, liver, spleen and lymph nodes. In models of genetically susceptible mice, the researchers found that E. gallinarum initiated the production of “autoantibodies,” activated T cells, and inflammation. Moreover, this “autoantibody” production stopped when they suppressed E. gallinarum’s growth with the antibiotic vancomycin and/or with a vaccine against the microbe. The team also identified E. gallanarum in the livers of patients with “autoimmune disease”, but not in healthy controls. One media headline on the study read: “The enemy within: Gut bacteria drive autoimmune disease.”

Research by Stanford’s Mark Davis supports the findings. Davis and team used a new testing method to obtain T cell sequences from the tissues/blood of patients with colon cancer, MS, Lyme disease, and ME/CFS. In all four diseases, they found that the T cells were activated and “cloned’ in a manner not observed in healthy subjects. According to the theory of autoimmunity, these cloned T cells would indicate “autoantibody” production. But Davis suggested that associated antibodies are likely formed “originally against some pathogen peptide.” This definitely makes sense since since, especially in Lyme disease, we know pathogens drive the disease process.

Interesting right!? But at this point I should bring up an important concern. Some researchers are still convinced that “autoantibodies” can target human tissue. If this is the case, the situation can easily be explained by a concept called “molecular mimicry.” Molecular mimicry refers to the fact that pathogen proteins and human proteins are often very similar in size and shape. This means that an antibody created in response to a pathogen protein/antigen might accidentally target a similarly structured human protein/antigen. This “collateral damage” could result in an inflammatory response towards that human tissue.

Think of molecular mimicry this way: Let’s say you’re a solider in an army. You and your fellow soldiers all have red uniforms, blonde hair, and stand about 6 feet tall. You confront the enemy on a battlefield only to realize that their soldiers also have red uniforms, blonde hair and are about 6 feet tall! Even though their red uniforms are shaped somewhat differently than your own, at a certain distance the shape becomes blurred. This means that when the battle begins, you have trouble telling who’s on your side and who’s the enemy. Occasionally you end up accidentally shooting a member of your own army. These two armies can be compared to similarly shaped pathogen antigens and human antigens that might generate “collateral damage” in “autoimmune disease.”

Ample research supports this “molecular mimicry” model. For example, one research team found that B cells infected with Epstein Barr Virus secrete antibodies that also react with human antigens like albumin, renin, and thyroglobulin. Another Canadian research team found that almost 20,000 proteins created by the hepatitis C virus have a high level of structural similarity to human proteins. Researchers in India identified tens of thousands of possible interactions between proteins created by Salmonella, E.coli, Yersinia, and similarly shaped human proteins.

All this research strongly suggests that in 2018 we no longer need the theory of autoimmunity. It seems Paul Ehrlich was right a century ago when he argued for “horror autotaxicus.” In patients with a range of chronic conditions, activated B cells, T cells and the antibodies they produce are likely targeting newly discovered human pathogens rather than “self.” This means the concept of the “autoantibody” is incorrect. In lieu of “autoimmunity,” we should focus on better characterizing pathogens in the microbiome and better studying their activity and survival mechanisms. For example, Mark Davis is planning to further study the activated T cells in his samples. He hopes to correlate the T cell activity with the presence of specific pathogens (and the antibodies created in response to their presence). Excellent, excellent idea!

Most of the top-selling drugs in the world are immunosuppressive

At this point you might say: “Aw, well it looks like theory of autoimmunity will die over time, no huge rush.” But unfortunately the situation is very urgent. That’s because a large chunk of the pharmaceutical industry is focused on creating drugs based on the theory of autoimmunity. These drugs shut down extremely important parts of the human immune system in an effort to stop “autoantibody” production and related inflammation. In fact, these immunosuppressive drugs are the top-selling medicines in the world, generating billions of dollars of revenue each year.

If “autoantibodies” are created in response to pathogens rather than “self”, these drugs are actually hurting the long-term health and microbiome balance/health of patients with “autoimmune disease.” This is almost certainly why patients taking immunosuppressive drugs tend to get sicker over time, to the point where they often fall ill with a second or third inflammatory condition while taking the medications. In fact, rampant use of immunosuppressant drugs is likely a primary factor driving the current epidemic of chronic disease (the incidence of nearly every “autoimmune condition” is on the rise).

That means we stand at a crossroads. This paper published just last year describes an entire new generation of immunosuppressive drugs currently being developed by pharmaceutical companies. Some of these new drugs appear to shut down the human immune system even more profoundly than the ones prescribed today.

OR, we can move in a new direction of drug/treatment discovery. We can ditch the theory of autoimmunity and instead develop treatments that SUPPORT the human immune system. And/or treatments that better target key pathogens and, in turn, promote balance, health and diversity of the human microbiome. Then, maybe…just maybe we can create a future where new treatments target the ROOT CAUSE of human inflammatory disease instead of just palliating symptoms. And then maybe…just maybe a growing number of patients with chronic inflammatory disease could reach a state of actual recovery and remission.

 

 

Antibiotics, immunosuppressive drugs and the downfall of the human immune system

March 31st, 2018 by Amy Proal

Hey! Today I’ll discuss two more factors that can debilitate the human immune system (allowing the microbiome to better cause disease). They are antibiotics (when used too frequently) and immunosuppressive drugs: two of the most common medical interventions of our time. Most of us are familiar with antibiotics. By immunosuppressive drugs I mean medications like Humira, Prednisone, Ritauximab, and Enbrel. These and similar drugs block or disable parts of the human immune response in an effort to temporarily palliate inflammatory symptoms.

It’s important to note that most antibiotics and immunosuppressive drugs were developed before mainstream science “discovered” the human microbiome around the year 2005. In other words, they were created/formulated during a period when the human body was assumed to be largely sterile. The antibiotic Penicillin was discovered by Scottish scientist Alexander Fleming in 1928. By the mid 1940s it was regarded as a wonder drug, especially during World War II when it saved countless soldiers from dying of infected wounds.

After World War II, antibiotics continued to be perceived as miracles, and it’s easy to understand why: diseases like pneumonia, scarlet fever, bacterial meningitis, gonorrhea and a range of ear, skin, urinary and throat infections could finally be treated! Life expectancy increased as antibiotics allowed patients to survive an ever-growing range of these specific bacterial infections. And since the human body was considered sterile, it was assumed that antibiotics only killed the single bacterial pathogen they were prescribed to treat. They were subsequently prescribed with increasing frequency, and for minor infections like colds or sore throats (especially in children).

Alexander Fleming discovered Penicillin in 1928

Then, in the early 2000s, new molecular tools were developed that identify microbes by their DNA/RNA rather than their ability to grow in a laboratory Petri dish. When researchers turned these new tools on the human body, they discovered thousands of microbes in human tissue/blood that had been missed by previous techniques. With each new molecular analysis, mainstream science realized that the human body is the opposite of sterile! Instead, humans harbor vast ecosystems of bacteria, viruses, bacteriophages and other microorganisms. The human gut alone harbors trillions of microbes. These complex microbial communities were named the human microbiome – the “discovery” of which marks one of the most important turning points in the history of science.

Almost every facet of human health and biology must be re-evaluated to account for the human microbiome. When it comes to antibiotics we face a stark reality: Current antibiotics like penicillin have such a broad range of activity that they kill most bacteria in the human microbiome in addition to any single pathogen they are prescribed to treat. The situation could be compared to dropping an atomic bomb on an entire continent in an effort to target a group of rebels in one small city.

What are the implications of this? Research on the topic is still in early stages. But we know two things:

  1. Health, diversity and balance of the human microbiome are key to prevention of human inflammatory disease
  2. Microbiome populations decimated by antibiotics can recover, but sometimes not to their full, previous capacity

For example, in this Ted talk, researcher Rob Knight charts how an antibiotic prescribed to treat an infant’s ear infection impacted several of the child’s early microbiome communities. The antibiotic caused “a huge change in community structure” that “set back many months of (microbiome) development.” Interestingly, the infant’s microbiome eventually recovered to a place similar to that of untreated subjects. But as Knight suggests, it’s hard to imagine that there are no longer-term implications associated with the event (especially if the infant is administered even more antibiotics over time). And, as a society, are we taking more antibiotics? We sure are! From 2000-2015 approximately 65% more antibiotics have been sold globally (accounting for 34.8 billion doses in 2015). In the USA, 47 million of these antibiotics are prescribed annually for viral infections that don’t even respond to antibiotics.

With those numbers in mind, it’s no surprise that a second major antibiotic issue has also surfaced. Antibiotics have been prescribed with such frequency that many major pathogens have developed resistance against them. As discussed in previous podcasts, pathogens can easily change their gene expression to survive under difficult circumstances. Many human pathogens have done exactly that – they have acquired genes or mutated in ways that allow them to remain alive in the face of our most commonly used antibiotics. This has created a range of antibiotic-resistant pathogens often referred to as “superbugs.” Many of these “superbugs” are deadly (Acinetobacter baumannii, Pseudomonas aeruginosa, and MRSA for example). In fact, the CDC estimates that these and related “superbugs” kill at least 23,000 Americans a year (for comparison, about 38,000 Americans die every year in car crashes).

“Superbugs” have developed resistance to most common antibiotics

The situation is regarded as a global catastrophe. According to the CDC, the rate at which new strains of antibiotic-resistant bacteria have emerged in recent years “terrifies public health experts.” Many consider new “superbug” strains to be just as dangerous as emerging viruses like Zika or Ebola. Britain’s chief medical officer, Sally C. Davies, has described drug-resistant pathogens as a national security threat equivalent to terrorism. The problem is compounded by the fact that a large number of bacteria in the human body have yet to even be characterized and named. Who knows what our overly zealous use of antibiotics is doing to these understudied bacteria! It’s very possible that heavy antibiotic use may be pushing MOST bacteria in the human body into a more virulent, resistant state.

Add to this a third major antibiotic problem. Over the past decades, farmers started feeding antibiotics to many of the animals we consume as food (cows, chickens, pigs etc). Why? Antibiotics cause the animals to gain weight, increasing the amount of meat that can be sold to consumers. Again, few people paused to consider the long-term or chronic consequences of saturating our food supply with antibiotics. Instead, approximately 80% of antibiotics sold in the United States are used in meat and poultry production. This has created an amazing atmosphere for the development of virulent pathogens that may better resist antibiotics. In addition, genes conferring antibiotic resistance are regularly transferred from farm animals into the human food supply. If ingested, these antibiotic resistance genes can theoretically be transferred and “taken up” by almost any bacterial species in the human body.

To make matters worse, antibiotics were added to the food supply during the first time in history where the average 1st world inhabitant started eating meat imported from around the world. For example, the USA imported 3.01 billion pounds of beef in 2016, from 22 countries across the globe (from Europe, to South America, to China and Thailand). Consumers are subsequently exposed to meat-associated pathogens from around the world – basically a potential cocktail of global pathogens. For example, a study by researchers in Germany found high levels of S.aureus and E.coli in pork/poultry imported into the EU, with other pathogens like Salmonella and Enterobacteriaceae present in lower amounts.

The above questions the interpretation of studies that tie meat consumption to chronic inflammatory conditions like heart disease. If a negative association between the two is identified, is the meat itself to blame? Or are pathogens in the meat to blame? (or a combination of both and/or other variables). The relatively unexplored possibility that pathogens in meat may promote chronic disease is supported by the fact that heart disease is increasingly tied to infection and microbiome dysbiosis. A recent study even found that lipids or fat molecules in arterial plaque can be created by persistent bacteria. Also, to be fair, the possible impact of food-borne pathogens on chronic disease applies to most foods – for example, what do the range of pesticides and chemicals sprayed on plants across the globe do to the microbes that live in/on the produce we consume?

Now I’ll take the topic one step further, by posing a rarely asked but extremely important question: “Where has the human immune system been during the modern era of antibiotics (1940-today)? Because, as I’ve argued in previous blogs/papers, the healthy, activated human immune system has incredible defense strategies for keeping antibiotic resistant bacteria or “superbugs” in check.

After the turn of the century, a range of chronic conditions were linked to persistent inflammation. This chronic inflammation generally results when the immune system attempts to target pathogens. However at that time (as mentioned) the human body was incorrectly assumed to be sterile. Researchers subsequently developed the “theory of autoimmunity” – the belief that in diseases like Crohn’s, Multiple Sclerosis and arthritis, the immune system “loses tolerance” and attacks its own tissues.

Most of the top-selling drugs in the world are immunosuppressive

By the 1960s-80s the theory of autoimmunity had gained incredible traction. Over 80 different conditions were deemed “autoimmune” in origin. The result? Pharmaceutical companies created drugs specifically designed to shut down key parts of the human immune system. These drugs, which temporarily palliate symptoms, have been prescribed so aggressively that today they are the top-selling medications in the world. For example, the potent immunosuppressive drug Humira is literally the top-selling drug in the world: generating more than 18.4 billion dollars in revenue in 2017.

This means we have been using antibiotics at the same time that we have deliberately compromised the immune systems of a growing chunk of the human population. In fact, it’s no coincidence that most “superbugs”  form or are identified in elderly, sick and/or hospitalized patients; patients much more likely to be on cocktails of immunosuppressive drugs/steroids. This trend escalates when severely immunocompromised patients are gathered in one place: a recent analysis found that pipes under NIH’s ICU rooms tested positive for bacterial plasmids that confer resistance to carbapenem antibiotics. The pipes were described as a “seething hookup zone for antibiotic-resistant bacteria.” Another report found that many veterans of the Iraq War suffered from antibiotic-resistant A. Baumannii infections. But the “superbug” was almost always identified in wounded veterans with complex injuries. Indeed, the report clarifies that,  “A. Baumannii  is not particularly virulent. If you’re in good health and (it) were to get into a wound it might not be a problem. It’s a problem for people who are the weakest of the weak.”

The most common treatments for “non-autoimmune” conditions are also immunosuppressive to various degrees. Opioids, antidepressants and antipsychotics dampen the immune response (especially when administered over long periods of time). Certain forms of birth control have been shown to lower immunity. Cancer chemotherapies substantially weaken the immune system. Indeed, childhood cancer survivors are prone to a wide range of health problems later in life including organ damage, cognitive deficits and high blood pressure. These long-term health issues are similar to those observed in patients administered “classical” immunosuppressive drugs. Even in animals given antibiotics for weight gain – do the overcrowded/unsanitary conditions at many farms favor “superbugs” by lowering animal immunity?

All in all, we are living in an era of mass immunosuppression. This has created a field day for antibiotic resistant and virulent pathogens! Such pathogens can easily thwart the human immune system because our drugs are “knocking it down” for them. We are greatly assisting the enemy.

Most concerning is that the “theory of autoimmunity” no longer holds up in the era of the microbiome. A growing body of research suggests that “autoantibodies” are created in response to microbiome pathogens rather than “self.” In addition, “autoimmune” conditions are increasingly linked to microbiome dysbiosis and not a defective immune response. So we are using immunosuppressive drugs to treat patients based on a failing and outdated paradigm. For more on this topic please read this paper.

To combat antibiotic resistance we must subsequently re-evaluate the “theory of autoimmunity” and the immunosuppressive drugs that have stemmed from its promotion. And we must do so NOW. We cannot afford to wait the decades it usually takes for an outdated theory to be replaced by a new, improved paradigm (Thomas-Kuhn style). Too many lives and the health of the entire public are at stake.

How else can we fix the situation? At least in the short-term, antibiotics and immunosuppressive drugs should be considered last resort treatment options. Patients should exhaust all other treatment options BEFORE being prescribed these medications. We must prioritize diet/lifestyle changes that the average doctor does not yet include in treatment plans. Although this may sound simple, it’s easier said than done when drug companies spend millions of dollars a year marketing immunosuppressive medications directly to physicians.

In the longer-term we should replace immunosuppressive therapies with new treatments that support the immune system and encourage balance + diversity of the human microbiome. This would require another major paradigm shift that I describe more here.

This last paradigm shift is essential because we NEED some antibiotics. When administered in a careful, responsible fashion antibiotics still save countless lives. Antibiotics are also a valuable tool for treating chronic inflammatory conditions that are increasingly tied to infection. For example, this article describes how a young boy with crippling OCD used antibiotics to treat a chronic strep infection. His OCD symptoms diminished to the point where he is able to function in society again.

I have several suggestions for how to best move forward:

  1. Antibiotics should be re-branded as complex, immunomodulatory drugs: Few people realize that antibiotics have profound effects on the immune system in addition to their antimicrobial properties. For example, many commonly used antibiotics (rifampin, cephradine, tetracycline, clindamycin, and penicillin among others) can bind directly into the human PXR nuclear receptor: a receptor that controls how the body detoxifies foreign substances. The PXR in turn modulates CYP3A4 – an enzyme key to controlling the human immune response. These and related interactions may explain why antibiotics like minocycline are regularly used as “anti-inflammatory” or “immunomodulatory” drugs in patients with arthritis and other rheumatic conditions. 

So imagine if every time an antibiotic was prescribed doctors were required to say: “I’m giving you a powerful medication that will impact your entire microbiome and immune system in profound ways still not fully understood by science.” This would give people pause before prescribing and/or popping an antibiotic for the common cold.
  2. We should keep this complexity in mind when developing new antibiotics: The superbug crisis has set off a wave of research aimed at creating new antibiotics. Many research teams working in this vein are trying to develop antibiotics that would target specific bacterial pathogens in lieu of the entire bacterial microbiome. This makes sense, but part of the trend involves taking parts of the human immune system and trying to make them into new antimicrobial drugs.

For example, researchers at Vanderbilt University recently made a fascinating discovery: human milk oligosaccharides (HMOs) in breast milk possess strong antimicrobial and antibiofilm activity against several superbugs (Eg: A. baumannii and MRSA). Several research teams responded to the finding by suggesting that these HMOs should be made into new antibiotics. While this makes sense in theory it also runs a large risk – any pathogen that develops resistance against these HMOs will have developed resistance against a key component of the human immune system itself.

The pathogen A. baumannii

What makes the above scenario likely to occur is that the human body constantly adapts its antimicrobial defenses to keep up with the changing nature (evolution) of virulent pathogens. For example, in this interview, Harvard researcher Robert Moir describes how the human immune system regularly changes the structure of the human antimicrobial peptide cathelicidin in response to different pathogen threats (each different form of cathelicidin is called an oligomer). The flexible nature of these oligomers means that when a pathogen mutates to survive in the face of the peptide, the peptide can change its own structure/function to “fight back.”

Human drugs based on only a single form of a human antimicrobial peptide/molecule would lack this flexibility, making resistance against them much more likely.

Ok that’s it for today. I’ll leave with the following message. Let’s not get so worried about current “superbugs” that we rush into making new antibiotics without fully considering long-term consequences. We’ve already done that once right? As the saying goes, “Fool me once, shame on you; fool me twice, shame on me!” The same goes for phage therapy – another proposed alternative to antibiotics. In this interview, Texas A&M researcher Ry Young does a great job explaining how we can move forward with phage therapy in the most proactive and responsible fashion.

Also, not everyone is surprised by the recent surge in antibiotic resistance and superbugs. Certain communities of researchers have been warning about the possibility since the 1960s. I will also do another podcast that talks about these researchers and several of their key findings – findings that should still be taken into consideration today.

The power of intracellular pathogens: how they “take down” the immune system and dysregulate human pathways

March 12th, 2018 by Amy Proal

A human white blood cell

Hey there! Last podcast I talked about how many “harmless” microbes in the human body are able to evolve to a state where they cause inflammation and illness. I explained that this “shifting” towards virulence (or disease) is much more likely to occur in people whose immune systems are compromised or debilitated. There are many factors that can negatively impact the health of the human immune system. I plan to discuss many of them in greater depth. But today I will focus on one major factor – the ability of key pathogens to slow and dysregulate the human immune system as part of their most basic survival strategies.

The most fundamental way that pathogens “take down” the human immune system is by infecting and living inside human cells – especially human white blood cells. Under conditions of health, these white blood cells traffic the body, where they engulf or digest pathogens that originate from both the internal and external environment. BUT, many key pathogens have evolved to thwart this process. They have evolved to remain alive inside these white blood cells; inside the very cells that are supposed to kill them! They can survive this way for long periods of time in a chronic, persistent state. Doing so clearly gives them a HUGE advantage or “edge” over their human host. If the situation was compared to a human war, it would be as if a soldier were able to inhabit the body of its opponent, and then make the opponent act in its own interest.

There are two main implications of this “intracellular persistence”:

1. The infected cell is unable to perform its basic immune system duties.
2. Any pathogen inside the infected cell can interfere with the way the cell “encodes” its human DNA.

Let me explain this second point in more depth. The center of every human cell has a copy of a person’s DNA. This DNA “codes” for the ~ 21,000 human genes that determine who we are and how we deal with our environment. These genes are eventually “translated” into proteins – proteins that direct our human pathways to correctly regulate the immune system, the nervous system, human metabolism and other critical aspects of human function. It follows that any pathogen able to persist in the center of a human cell can directly “mess with” the cell’s DNA and the vital human processes it regulates. The situation could be compared to a computer hacker. Imagine a hacker that not only breaks into your computer, but gains access to the programming code that directs your operating system and websites. By either directly altering this code, or changing the way the code is translated into programs, the hacker can cause your computer to malfunction in an almost infinite number of ways.

Here’s an example of how a pathogen can dysregulate a human cell in the above fashion. A research team in Louisiana studied what happens when Cytomegalovirus (CMV) infects a human white blood cell. They found that CMV significantly changed how the cell expressed its human genes. In fact, the virus “turned up” the expression of 583 human genes, and “turned down” the expression of 621 human genes. These changes were so profound that the infected white blood cells actually changed into an entirely different type of cell – an “inflammatory” cell that promoted CMV’s ability to infect neighboring cells. No wonder then that, as the paper points out, chronic CMV infection is associated with retinitis, gastrointestinal disease, hepatitis and pneumonia – but also with chronic inflammatory conditions like multiple sclerosis and atherosclerosis (heart disease).

Cytomegalovirus (CMV)

A recent study by researchers at Stanford confirms how broadly CMV can “re-program” the human immune response. The team studied identical twins and isolated cases in which one twin was infected with chronic CMV and the other was not. They tested the activity and function of 204 different parts of the human immune system in each twin. These 204 immune system “parameters” included the amount/frequency of certain immune cells, the ability of such cells to signal, and the concentrations of immune system proteins called cytokines. They found that, in general, 119 of these immune system “parameters” were altered in the twins with CMV. In fact they calculated that, in people infected with CMV, “the lifelong need” to control the virus causes approximately 10% of all T cells in the body to become directed against it. This led the team to conclude that the immune response is “very much shaped by the environment and most likely by the many different microbes an individual encounters in their lifetime.”

OK – but how exactly does this major pathogen interference happen? All human pathways that control the immune response, metabolism, cognition etc are regulated by receptors – protein molecules that control pathway signaling. In very simple terms, receptors function in a “lock and key” fashion. Imagine that you want to get into a very secure house. You might have to first open locks on an outside door. Then you might open or adjust a series of other locks on inner doors and/or other checkpoints before finally entering. Now, picture these locks as receptors associated with a human pathway. Under conditions of health, very specific human proteins bind into each receptor. These proteins are analogous to the keys in my house example. If the correct human protein or “key” fits into the correct human receptor or “lock,” the receptor/protein “complex” changes shape in a way that allows that part of the pathway to correctly move forward. This process happens over and over at each receptor “checkpoint” until the pathway fulfills its final, correct role in the body.

Now consider this: many pathogens have evolved to create proteins that are very similar in size/shape to human proteins (this redundancy is sometimes called molecular mimicry). If a pathogen with this ability survives inside a human cell, its OWN proteins may begin to “bind” into human receptors. Gradually, the human proteins meant to control a particular receptor may be increasingly replaced by these similar “enemy” or pathogen proteins. What happens? The slightly different shape of the pathogen proteins cause the receptor to act/signal differently than it would under conditions of health.

An excellent example of the above (that I’ve discussed in several published papers) is the ability of pathogens to dysregulate activity of the human Vitamin D Receptor (VDR). The VDR is an extremely important receptor located at the heart of every human cell. It controls the expression of almost 1000 human genes. It also controls the activity of very important parts of the human immune response. These include TLR2 (a protein the body uses to identify and target pathogens), and several kinds of antimicrobial peptides (natural antibiotic molecules the body uses against infectious threats).

It comes as little surprise then that the fungus Aspergillus fumigates has evolved to create a protein or gliotoxin that can bind into the VDR and directly slow its activity. This results in tremendous survival gains for Aspergillus – the altered VDR can no longer correctly express many of the human genes it regulates. The expression of TLR2 and the antimicrobial peptides under VDR control also diminishes. Now, Aspergillus can flourish in the face of the host’s weakened immune defenses.

In fact slowing VDR activity (and subsequently parts of the human immune system) is such a logical survival mechanism that many other key pathogens have also evolved ways to dysregulate the receptor. When Epstein Barr virus infects human B cells it dramatically lowers VDR activity. Mycobacterium tuberculosis slows VDR activity by a factor of ~3.3. Borrelia burgdorferi, Cytomegalovirus, and Mycobacterium leprae also slow VDR activity to varying degrees. HIV almost completely “shuts down” the VDR. I should be clear that some of these pathogens don’t create proteins that directly bind into the VDR. They interfere with the VDR receptor in ways a little too hard to explain here:) But you get the overall trend right!?

Now factor this in: the human body’s pathways are tightly interconnected. So any change in human signaling has “flow-on” effects that impact yet other pathways and systems. In the case of the VDR, the receptor’s activity is tied to that of related receptors that control hormone signaling (especially the estrogen-beta receptor). It follows that the “original” intracellular infection may also prevent these related receptors/pathways from functioning correctly.

But let’s go back to the immune system, and continue to use the VDR pathway as an example. Let’s say CMV infects a person’s B cells and lowers VDR activity. The person’s immune response suffers as a result. Now, it becomes easier for that person to pick up OTHER pathogens from the external environment. Or, pathogens already in that person’s microbiome find it easier to act in a more virulent fashion. These “new” pathogens may also infect the person’s cells, where the interference they cause further slows the immune response. A “snowball effect” may ensue, where each “new” pathogen begins to facilitate the survival of the next.

A successive infection “snowball” could drive many forms of disease

In many of my papers I’ve referred to this snowball effect as “successive infection.” When I think of successive infection I literally picture a snowball rolling down a hill. The snowball begins with one intracellular infection, and then grows, as more and more pathogens take advantage of the dysregulated atmosphere in the body. As more pathogens “join” the snowball, the disease state that the snowball represents pushes the human body farther and farther away from a state of health.

It’s important to realize that once the “successive infection” process has started, environmental factors besides infection can “add” to the snowball. These include chemical exposures, stress, immunosuppressant drugs, over-used antibiotics and any other factors that cause either the immune response or microbiome balance to suffer. In fact, any of these other factors can “start” the snowball too.

I realize this is a major claim, but in my mind, a successive infection snowball could drive almost any form of inflammatory disease. That’s because such wide-range of different pathogens and negative environmental exposures can contribute to the process. Pathogens are also able to infect so many parts of the human body (nerves, brain tissue, lung tissue). If the snowball represents disease, the specific illness any one person develops would stem from their UNIQUE infectious history, their unique medication history, their unique chemical exposure history, and the many, many different ways these and other variables can interact with/feed into each other.

If you think that’s a stretch, consider this: almost every analysis of the human microbiome identifies new microbes never before detected by previous studies. For example, just a few months ago, Steven Quake at Stanford identified thousands of “new” microbes in human tissue and blood. Many of them are potential intracellular pathogens we didn’t even realize could survive in humans. This led Quake and team to state that these novel microbes “may prove to be the cause of acute or chronic diseases that, to date, have unknown etiology…”

Also, research teams continue to discover new examples of how pathogens might interfere with human pathways. For example, a team in Indiana just identified viruses that create proteins similar in size/shape to the human protein insulin. They found these viral proteins could bind into human insulin receptors that determine how the body correctly regulates blood sugar levels. The viruses that create these insulin-like proteins are regularly detected in human blood and fecal samples. The team concluded that “since only 2% of (human) viruses have been sequenced, this study raises the potential for discovery of other viral hormones which, along with known virally encoded growth factors, may modify human health and disease.”

So I’ll end on the following note: never underestimate the “power” of intracellular infection. Human microbiome studies that fail to account for possible intracellular pathogens may miss part of a larger, more important picture of disease. For example, the gut microbiome dysbiosis now tied to many inflammatory conditions may be a downstream result of intracellular infection in the blood or other tissues. AKA: the widespread drop in immune system activity driven by intracellular pathogens may “set the stage” for an atmosphere that favors gut microbiome imbalance. This is certainly the case in HIV/AIDS, for example (for more context read this blog post).

Ok phew! That’s it for now. Next podcast I’ll go more into immunosuppressive drugs, antibiotics, and other factors that can also compromise the human immune system and/or the human microbiome. Talk then!

Towards a more nuanced view of the human microbiome: pathogens and evolution

March 1st, 2018 by Amy Proal

Vast microbiome communities persist in human tissue and blood (Image: hyperbiotics.com)

Podcast transcript:

People often ask me for a quick description of the human microbiome. I appreciate their interest, although it’s actually a difficult request: the microbiome is extensive and capable of vast range of behavior. Nevertheless I’ll usually say something like this:

“When I refer to human microbiome I’m talking about the trillions of bacteria, viruses, fungi, bacteriophages and other microrganisms now understood to persist in complex communities in all human tissue and blood.”

But beyond that: how do I best summarize microbiome ACTIVITY? Aka: What are these microbes in and on us are doing?? I’ve learned that most friends expect a response along these lines:

“The microbes in and on us are our friends. They work night and day to defend us from disease, improve our metabolism, break down our food, control our digestion, educate our immune systems and beyond.” In such a description, all the microbes we harbor are portrayed as allies or even heroes: organisms that continually devise ways to benefit or support our health and well-being.

This kind of description is often accompanied by a specific mindset: the belief that microbiome pathogens (those that cause disease) are of secondary importance to those that “help” us. And that to strongly focus on the presence of these myriad pathogens is to give microbes in general an overly “bad reputation.”

In my opinion, this portrayal the microbiome is too one-sided: At the very least it should be accompanied by an asterisk stating something like: “this is an attempt capture microbiome behavior under conditions of extreme health.” And it’s true, of course, that people with extensive microbiomes (take Olympians!) can function with incredible vigor and ability.

But the vast majority of us sustain a range of minor to major inflammatory health issues. Some people deal with acne, others have trouble tolerating or digesting certain foods. Insomnia, anxiety, metabolic problems…are all extremely common in today’s human population. Beyond that, we are living in the midst of an epidemic of chronic disease, in which the incidence of nearly every well-characterized inflammatory condition is on the rise (diabetes, obesity, heart disease, the “autoimmune diseases” to name just a few). In fact, an estimated 60% of Americans suffer from at least one chronic condition (and that number only includes people who have been officially diagnosed).

A growing number of these inflammatory conditions/symptoms are tied to microbiome dysbiosis: cases in which a microbiome community shifts towards a state of imbalance. This dysbiosis can be severe: pathogens tend to outcompete their more symbiotic neighbors, leading to drops in community diversity that promote an inflammatory environment.

This means that in the vast majority of people, the microbiome is acting in a nuanced fashion. Some of the microbes we harbor may perform health-related functions, BUT a significant number of others may be acting as pathogens. In fact, certain pathogens are so effective at promoting chronic symptoms that they can “push” the microbes around them towards a dysfunctional state. These pathogens are sometimes called “keystone pathogens.” A good way to remember that is that the activity and gene expression of keystone pathogens are KEY to promoting disease.

In my opinion then, it is vital that we continually recognize and encourage the general public to understand that persistent, chronic, pathogens can also play a central role in any microbiome community. To do so is not giving microbes a “bad reputation.” It is simply accurate. In fact, the current, alarming epidemic of chronic disease begs that we study these persistent pathogens with more urgency than ever before.

It also helps to interpret microbiome activity via the lens of evolutionary biology. When microbes are described as “friendly” people often assume that there is actual intent on their part to help us. In reality, any symbiotic or “commensal” microbe/human relationship is still driven by the most basic tenants of evolution: “survival of the fittest” and “reproductive fitness.” The goal of any microbe in/on our bodies is to stay alive and pass its genes (encoding its particular traits and characteristics) to the next generation. So if a microbe is, for example, breaking down our food, it’s because doing so also benefits its own ability to survive and reproduce. The fact that such behavior also helps its human host is simply an evolutionary coincidence.

In most ecosystems on earth (like the rainforest), commensal relationships are actually rare. This is partly because one species in a commensal relationship has only to slightly change its gene expression and/or metabolism for the relationship to shift, and begin to favor one species over the other. This “shifting” happens very easily with microbes because they can quickly adjust their gene expression patterns: they turn certain genes “on” or “off” in order to functional optimally under different environmental conditions. Microbes can also – through a process known as “gene transfer” – acquire completely new genes: genes that allow them to function in totally new ways. And, as with most organisms, a chance mutation in a microbe’s genetic code (their DNA or RNA) can endow it with yet other novel traits and abilities.

Take E.coli. Strains of E.coli are detected in almost every analysis of the human microbiome, even in studies conducted on healthy people. This makes sense, since people digest seemingly innocuous E.coli strains on a regular basis. But most of us are also familiar with E.coli’s ability to cause severe food poisoning and other forms of illness. This range of E.coli behavior is due to the fact that it can evolve to persist in many, many different states – some WAY more pathogenic (or able to cause disease) than others. Take a study done by researchers in Portugal. The team found that when “commensal” E.coli were exposed to human white blood cells, they rapidly mutated and changed their gene expression: the commensal E. coli evolved, in less than 500 generations, into virulent, or disease-causing, clones. When these virulent clones were injected into mice, the mice suffered an increase in inflammatory symptoms.

This “virulence”, or ability of a microbe to cause disease, is dictated in large part by the activity of genes that express “virulence factors”: molecules that help bacteria, viruses and other microbes better persist inside the cells of the immune system, better penetrate host tissue, better obtain key nutrients from the host, better produce toxins…and better engage in many other behaviors that cause human health to suffer.

E.coli as seen under a microscope.

Every microbiome community (even in healthy individuals) harbors microbes capable of expressing these virulence factors. These include potential pathogens present in the earliest microbiome communities that an infant inherits from its mother (microbes generally acquired via the placenta, vaginal canal, and/or breast milk.) In other words, every human is “seeded’ with potential pathogens from the beginning of life.

It follows that pathogens capable of expressing virulence factors can evolve and “emerge” from microbes already present in a “healthy” microbiome community. Let me give you an example. Researchers in England recently published a paper called “Severe infections emerge from commensal bacteria by adaptive evolution.” The team studied the bacterial species S.aureus. Like E.coli, S.aureus is frequently detected in the nasal (nose) microbiome of healthy individuals. However, S.aureus can mutate and change its gene expression so profoundly that it can also cause life-threatening infections.The English research team found that these life-threatening strains of S.aureus could easily descend from commensal S.aureus bacteria already colonizing a person’s nose. In other words, depending on conditions in the body, S.aureus is able to mutate from a harmless microbe to a major pathogen within the same person. This progression towards virulence involved an increase in the activity of genes that promote toxin production, abscess formation, immune evasion and many other processes that harm the human host.

Also key to the study’s findings was that, in the majority of cases, this within-person evolution towards S.aureus virulence occurred in subjects that had “risk factors for infection”: these included a weakened immune response, co-morbid conditions (the subject was sick with one or more inflammatory diseases), and/or medical interventions (subjects had undergone surgery or a procedure).

That brings me to my main point. To best understand microbe behavior we must not ignore the elephant in the room: the human immune system. The human immune system, and its extensive army of immune cells and antimicrobial molecules, plays the greatest role in determining how the microbes in any human ecosystem act, survive, and behave. Lately I’ve been describing this relationship with the following analogy:

Imagine a school. The children in the classroom are different microbes in a human microbiome community. The teacher and her support staff (like the principal) are the human immune system. Some of the children in the classroom are trying hard to get work done. Others are not so studious and hope to cause trouble. But these potential “bad students” don’t act out when the teacher is in the room and they can be disciplined by the principal and other staff. HOWEVER – if the teacher leaves the room, and her support system dwindles, these “bad influence” students can easily change their behavior to cause trouble. Without supervision they stop doing work. Further, they begin to encourage the better students around them to join them in promoting disorder. Little by little, even the best students find it hard to behave in the unsupervised classroom environment.”

For example, take the bacterial species Lactobacillus (which is added to many common probiotics). It is often regarded as a purely “good” microbe. However, as this article on the vaginal microbiome points out, some strains of Lactobacillus help maintain a healthy, acidic vaginal environment. BUT other strains can put women at a higher risk of acquiring a sexually transmitted infection. Indeed, Willa Huston, a professor at the University of Technology Sydney states the following about Lactobacillus and vaginal health: “It depends on the type of Lactobacillus, it might depend on the function of that Lactobacillus…and perhaps how it’s interplaying with the genome type of the woman.”

If you’re not convinced, let me give you a great example of how a “good” microbiome community can go bad (evolve in a direction of virulence). We’re all familiar with the fact that our teeth can develop cavities. The process that drives this cavity formation is called periodontitis. Periodontitis is now understood to be an infectious condition in which different oral microbes work together to damage tooth tissue and bone.

But aside from teeth brushing, what factors determine if a tooth develops a cavity or remains healthy? A team in Boston addressed this question by studying how the entire oral microbiome changes its gene expression during cavity progression. They found that community-wide gene expression did not change in teeth that remained cavity-free. But, the oral community dramatically altered its gene expression in sites that progressed to periodontitis. These changes involved genes that control oxidative stress, iron transport, sulfur metabolism, intracellular survival, and bacterial movement among many others. Cavity formation was also correlated with increased expression of virulence factors and higher levels of viral activity.

The team concluded that the entire oral microbial community, and not just a few select pathogens, drives the increase in virulence that leads to periodontitis. In effect, under conditions of increasing immune suppression, imbalance and inflammation, the whole oral microbiome community appeared to act together as a pathogen. This was supported by the fact that, in cavity sites, groups of microbes not usually considered to be pathogens expressed a large number of the disease-promoting virulence factors. Two of these microbes were S.mitis and S.intermedius –  bacterial species usually associated with dental health that are commonly described as “good.”

Single microbes can form into communities called biofilms.

Many cavity forming microbes also team up to persist in communities called biofilms. These biofilm communities are surrounded by a matrix that protects the different microbes inside from the human immune system. Participating microbes use electrical and chemical signals to communicate. This allows them to split up jobs important for their own survival – for example, virulent bacteria may protect the biofilm from outside intrusion, while other bacteria on the inside focus on obtaining nutrients for the community.

Few people would argue that microbes in a biofilm are “helping” their human host. They have instead joined forces in a manner that promotes their own survival over ours. It’s not surprising then that biofilms are connected to an increasing number of inflammatory conditions. In fact, some researchers estimate that the majority of bacteria in the body persist in a biofilm state.

So the way I see it, the human immune system and the human microbiome are actually in a continual state of competition and evolution. In healthy people the immune system has gained “an edge” over the microbiome. It is successfully able to keep microbiome pathogens in check. In inflammatory disease however, the opposite happens. The microbiome – or pathogens (or groups of pathogens) – begin to gain “an edge” over the human immune system…and its various defense strategies. This last situation is especially true in our modern world where an increasing number of environmental exposures and drugs are wearing down the human immune system.

And that’s what I’ll be talking about in one of my next podcasts: What factors most knock down or debilitate the human immune system in our modern society? And how can we help bring our immune systems back to a state of better activity and health? – a state that will help keep our microbiomes in a state of balance.

I will also do another podcast that explains more about how pathogens, and the metabolites they produce, are able to interfere with the human immune system and human pathways that control metabolism.

Interview with Ry Young: Phages and Phage Therapy – Pros/Cons and History

February 17th, 2018 by Amy Proal

Ry Young is a Professor in the Department of Biochemistry and Biophysics at Texas A&M University. He is also director of the Center for Phage Technology. Since the early 1970s Ry has studied bacteriophages: viruses that infect bacteria. He is now one of the world’s leading bacteriophage experts. His lab investigates bacteriophages as models for a new class of antibacterials. He has also participated in several “phage therapy” cases – in which bacteriophages are used as therapeutics to treat pathogenic bacterial infections.

Background information:

Tom Patterson phage therapy case: A recent medical case in which phage therapy was successfully used to treat an antibiotic resistant infection. Please watch this TED Talk for more context.

The interview:

Amy: Hi Ry. Thanks for taking the time to speak with me. You’ve researched phages for decades. How did you get interested in the topic? 

Ry: That’s a tough question. I don’t remember a time when I wasn’t interested in phages. I was a graduate student at MIT in 1968. At the time, one of the founders of the Phage Church was named Salvador Luria. He won the Nobel Prize in 1969, along with Max Delbrück and Alfred Hershey, for work on phage genetics. Luria was my microbiology instructor at MIT and turned me on to phages. So I learned about phages from what they call the horse’s mouth – Sal was a very charismatic person.

Phages attach to the outer membrane of bacteria with a thin “tail,” and inject their DNA into the bacterial cell.

Soon after however, I got my Vietnam War draft notice. I left graduate school to spend three years in the Navy. When I got out I went back to graduate school and started working on molecular biology. But as a postdoc, I got an NIH fellowship and chose to work in the lab of a brand new Harvard faculty member, Mike Syvanen, who was a phage guy. In 1978 I started my own phage lab here at Texas A & M. What I like about phage biology is the rigor of the field. Phages are easy to work with and have powerful genetics. You can posit hypotheses, do experiments, and get quick results.

Amy: So when you started researching phages I assume there wasn’t much of a concept of the human microbiome? 

Ry: No no. We had no concept of that. In fact, when I started working here in 1978, I had little idea that I would be one of the last people to start a phage lab for about 15 years. The entire phage movement, or Phage Church, was disassembling itself during the late 1970s and 1980s. This was based on an attitude in which phages were valued strictly as model systems for genetic experiments; as organisms that could help us work out the fundamental rules of molecular biology. But even then, many phage researchers decided it was time to move away from phage genetics to study other model systems. So the people who went on to found important fields of genetics research – like C. elegans for neurobiology and zebrafish for developmental biology – were mostly all phage people who moved away from the field.

This shift was largely started by a researcher named Max Delbruck, who founded modern phage biology. He was a physicist and reductionist: and if you’re a physicist you study the hydrogen atom and then, when you’ve got a predictive understanding of that…you move up to helium and so on. In fact Delbruck himself moved away from phages in the 1950s. So when I started my lab, I was one of a new wave of researchers who were interested in phages per se, rather than using them as a tool to study something else.

Max Delbruck in the early 1940s.

Amy: I see. I didn’t know that.

Ry: If you’re gonna keep up with phage you should get a copy of a textbook called “The Bacteriophages.” It’s edited by Richard Calendar (he’s retired now in San Francisco). I wrote the book’s one-page foreword in 2006. As part of writing it, I did an analysis of  NIH grants for phage research over the past ~ 30 years. I compared 1972, the height of the phage era, with 2002 (30 years later). I discovered that the number of NIH grants on phages had dropped down enormously. The number of RO1s for real phage research had gone from over 200 in 1972 to less than 10 in 2002. Basically the field had died.

Amy: Yes. But now it seems that the field has recently been reborn. 

Ry: Yes. Which is a real problem because suddenly there’s all these people who want to research phages, but few labs set up for phage research.

Amy: Interesting. Does this situation have anything to do with phage therapy?

Ry: No. The first stirrings of renewed interest in phages (and phage therapy) occurred in the early 2000s. That’s because the terrible antibiotic resistance problem we face was becoming more well-known. Even then, we still didn’t know anything about the human microbiome. So there are two separate current justifications for the renewed interest in phage therapy – One: we’re not getting any good new antibiotics. Two: even if we did we shouldn’t be using them. It’s kind of a double whammy in that regard.

Also, since the time when phage biology mostly died out in the 1980s, all sorts of new technologies have emerged – like next generation sequencing, laser technologies and high resolution fluorescence microscopy. The development of cryo-electron microscopy was especially important, because resolution power became so great that we could finally see the details of the phages. Suddenly, phage-related questions that had previously been beyond anybody’s ability to think about could be studied. Not only that, there’s a huge tide of new genomic information that’s bringing home to everybody what an important role phages play in the biosphere.

Because, back in the 1930s, phage biology was classically so powerful that the phage research community would get ahead of itself. That’s what happened with the original phage therapy. In the 1930s phage therapy was pioneered by a guy named Felix d’Herelle. He was a force of nature and went all over the world promoting phage therapy. So thanks to him, phages became the first widely used biological therapeutic.

But in the 1930s science in general was premature –  we didn’t even know about DNA. And because there were no standards, and no FDA or anything like it, most of these early attempts at phage therapy were kind of hopeless. We’d have been a lot better off if d’Herelle had not discovered phages until the 1940s or 1950s, when the scientific community knew more about molecular biology.

But there are other arguments about why this early phage therapy eventually fell out of favor. Part of what caused the decline was personality driven. And a lot was economics driven, because cheap antibiotics like penicillin were also discovered in the 1930s. No biological treatment like phage therapy is cheap. You can’t mass produce phages like you can penicillin. Also, to treat a patient you need thousands of phages versus one penicillin.

Amy: But in parts of Europe and Russia phage therapy did continue, right?

Ry: The very best person in terms of the history of phage biology and phage therapy is Bill Summers at Yale. He’s a great guy to talk to. He’s gives the world’s greatest seminars. They’re funny but illuminating. He was a phage and virus person way back when he was a junior faculty member, but now he’s become a very prominent scientific historian. He’s written any number of articles and books about phages. He’s the only person who knows the whole story. And unlike some people who mercilessly promote phage biology, he can tell you the underside too. And there are some undersides.

Amy: I want to go into those undersides. But before that let me ask you: Now that the microbiome has been better characterized, how would you describe the role that phages play in the human body?

Ry: Oh I don’t think anybody knows right now. Most of the information we have about phages in the actual body is derived from huge metagenomic experiments. For example, they might sequence your gut microbiome and discover there’s an awful lot of phage sequences in there. But by far the most common phages found by such studies are lysogenic phages – the ones present as prophages inside bacterial DNA. That’s a little misleading because all you can tell from that data is that those prophage sequences are being carried by bacteria. You don’t necessarily know if  the phage in question is even functional, or if it’s actually floating around doing anything.

Bacteriophages seen under an electron microscope.

But I think it’s clear that any microbiome community can’t be understood without understanding the phages too. We know phages are going to play a prominent role in the microbiome – there’s no way it can be avoided. Phages clearly interact, both positively and negatively, with the other species. We also know that the human microbiome changes as people develop. But nobody knows how many of these changes are mediated by phages. One of the things about phages in the gut is that there’s a lot of bacteria in the gut microbiome. This crowded environment allows phages to have a massive and rapid effect. Because phages have to absorb bacteria to kill them, and this rate of absorption is proportional. It’s a two body collision. The more phages and the more bacteria you have, the faster those collisions happen. For example, 200 phages can get made out of one bacterial cell.

So it’s a population density issue: when you have lots of any particular bacteria they become suddenly vulnerable to a single phage particle in the population. Whereas if the bacteria are very dilute, you could have 10^5 bacteria X and 10^5 phage Y (that would be able to grow on X) in the same microbiome community, but they might never encounter each other.

Amy: Right. But phages have been detected outside the gut right?

Ry: Oh yeah. Anywhere where you find bacteria in the human body you’re going to find phages. And for every type of bacteria there’s a different phage profile. Phage biology was really started with the study of laboratory strains of E.coli and closely related bacteria like Salmonella. Probably 99% of phage biology research was done on those microbes in the pre-therapy days (including my own research). And with E. coli, we found that you can go into any sewer or pond and find all kinds of different E.coli phages, in their every possible lifestyle. You’ll find lysogenic phages, and then all kinds of what people call lytic phages. Still, much of the variety of phages for E. coli has still not been tapped – we keep finding new ones.

On the other hand take Staph aureus – a high profile bacterial target (MRSA is surging in hospitals etc). There’s actually very little phage biology for Staph aureus. There’s basically two lytic Staph aureus phages. One is called Phage K (a big myophage). The other is a little podophage (a phage with a short stumpy tail). That’s all there is. No matter where you go in the world, if you look for lytic phages for Staph aureus, you’ll only find Phage K, its cousins, or the little podos.

That makes one ask: Why does Staph have such little phage variety? It’s seems like there was a bottleneck way, way back in evolution and those two phages won. It’s unfortunate in some ways, because we would like to have a lot more different kinds of Staph phages. On the other hand, phage K may have “won” because it evolved in a unique fashion. For a phage to affect a bacterial cell, the bacterial cell wall has to have a receptor that the phage can bind to. The bacterial cell wall receptor phage K and its friends use is a molecule called teichoic acid. And this teichoic acid receptor is essential for Staph: it can’t survive without it. So Staph cannot mutate to lose the receptor.  That’s probably why phage K “won”: it learned to recognize that essential receptor and then all the other phages were out of luck.

Amy: So Phage K outcompeted all the other phages? 

Ry: Yes and because the little podo phage has a very narrow host range, phage K is the only phage that’s useful. So there are a bunch of companies that have made phage cocktails against Staph, and they all turn out to be Phage K (plus friends and family). If fact, phage K has probably been patented 30 times.

Ry:  Do you know about the Bayne-Jones-Eaton report of 1934 that supposedly was the death knell of phage therapy?

Amy: No I don’t.

Ry: In 1934, the American Medical Association commissioned two scientists (Stanhope Bayne-Jones and Monroe Eaton) to survey phage therapy, to see if there was any anything “real” to it. They report they produced was very long – it was published in three separate issues of JAMA. They did a thorough study of all the different reported uses of phage therapy. Their general conclusion was that there was no demonstrated therapeutic benefit. And that’s not saying that phage therapy doesn’t work, just that the science hadn’t been done to show it could. However, the report did have an asterisk which stated something like “maybe we’re being too harsh, it does look like phage therapy works for Staph aureus, but nothing else.”

They came to that conclusion because they didn’t know about molecular biology. And if you don’t know what you’re doing, phage K will still work, because no matter how badly you use it it’ll still work. So there’s plenty of good evidence from thousands of cases showing that phage therapy works for Staph infections – evidence that goes way back to the 1910s – 1920s. But the Bayne-Jones-Eaton report still states that phage therapy has no demonstrated benefit (especially if you just read the top line). Also, that was right about the time when antibiotics started becoming available. So that was it: game over for phage therapy.

Félix d’Hérelle

I recently read the Bayne-Jones-Eaton report in depth while preparing for a two-day FDA workshop on phage therapy in July. I did a historical overview to see what the regulatory landscape was back when it was originally written. My interpretation is that “the fix was in for phage therapy,” because Bayne-Jones had a conflict of interest.

In the report, Bayne-Jones actually refers to the “failure” of phage therapy as the “d’Herelle Phenomenon”. And when you start calling something by somebody’s name, you instantly recognize that Bayne-Jones had something in mind about d’Herelle. At the time of the report, d’Herelle had been in the United States as a professor at Yale. There, he did what he did everywhere: frustrate many of his colleagues – he had a real talent for making enemies. He was self-educated with no college degree, much less a Ph.D. I think he was always very defensive about that. So eventually d’Herelle left Yale and Bayne-Jones took over his position. In modern times that would have instantly disqualified Bayne-Jones as an author of the 1934 report: You take somebody’s job and then write a report on the previous guy’s work!? I don’t think that’s a recipe for fairness. Although the report mattered less because antibiotics were going to wipe out phage therapy anyway.

One consequence of this was that d’Herelle had very sad life. He got nominated for the Nobel Prize about 28 times, but always had enemies high up in the scientific hierarchy that would blackball him. Because by any standard he should have gotten the Nobel Prize. He completely invented the whole area of phage therapy.

Amy: It would be interesting if d’Herelle could see new molecular data on phages.

Ry: Oh gosh yeah. He did a lot of fundamental research with no molecular tools, the lessons of which have turned out to be true a hundred years later. But because he was on the outs with the hierarchy, he ended up gravitating to to the left. The book he wrote about phages was dedicated to Joseph Stalin! Not exactly the way to endure yourself to the academic community. He had a real talent for painting himself into a corner.

That’s how he ended up starting the Phage Institute in the Soviet Union. As a result of that he ended up under house arrest all during World War II, because the fascists took over France. It was almost like a bad airplane novel. So now, in 2018, there’s a new phage therapy era. But to me this era is a rebirth of d’Herelle’s original idea.

Amy: I see. So this current rebirth of phage therapy: What’s the climate here in the U.S.? I know the FDA doesn’t allow it. What are the reasons for that?

Ry: Actually the FDA has been nothing but positive about phage therapy. It’s just that no phage therapy cocktail has been put through FDA tests yet. But the FDA has been very proactive: they’re trying to figure out what to do next. In fact, starting with the Tom Patterson case, they’ve set up a system where if a physician reports an appropriate phage therapy case, the physician can get “expanded access” (what we used to called compassionate use) to use phage therapy. This expanded access can be authorized within a day.

So while the FDA is not going to open the floodgates and say, “OK you can start injecting phage into people, don’t even bother calling us,” they’ve agreed to this expanded access (especially in life and death situations). Most of the time expanded access is used for cancer patients – at the end of life, one might as well give experimental treatments a try. So many potential cancer treatments that aren’t ready yet for clinical trials are also used in the same way.

Amy: But in the USA do you have to exhaust all antibiotic options before being allowed to use phage therapy? 

Ry: I think in general that’s correct. Right now if you want to use something that is not approved by the FDA you have to at least say there’s no other treatment option. That’s not a phage-specific guideline but true for all experimental drugs.

Amy: Might that change in the future because of the current antibiotic resistance problem?

Ry: Yeah I don’t know. I doubt that phage therapy will take the place of antibiotics. In the foreseeable future phages are going to be last resort items, mainly because of FDA approval issues. But there are several other problems with phage therapy. One is that we’re not going to find a broad-acting phage like penicillin. And since you know about the microbiota you could say, “we shouldn’t try to find a phage like penicillin!”

Amy: Yes, that’s a benefit in my mind. Because penicillin acts like an “atomic bomb” to the microbiome. It depletes entire microbiome communities in an effort to target one bacterial species. 

Ry: Right exactly. It would be better if we had antibiotics that targeted the disease bug and not the rest of your microbiota. But one reason we don’t have any small molecule-specific antibiotics is that there was no demand for them in the past. Up until recently Medicine wanted broad spectrum antibiotics.

But now there’s going to be a market for specific antibiotic molecules. I’m confident that pharmaceutical science and the industry are going to try very hard to find mass producible molecules that can target Staph, Psuedomonas etc. Those antibiotic molecules would be “round pegs” that fit fairly well into our established FDA drug testing system. For example, if you can find molecule X that kills Staph aureus, and doesn’t kill your microbiome, that molecule will be of great value. Then, somebody will be able to afford to pay the $100 million to $300 million necessary to put that molecule through the FDA drug approval process. I also think that the lysin technology that’s derivative of phage biology will become relevant, because it’s also a cheaper treatment option than phage therapy itself.

Amy: What is lysin technology?

Ry: Lysin technology is part of the phage story. For 40 years the NIH has been funding me to work on lysis (although I have no stock or conflicts of interest). The last step of the phage infection cycle is called lysis. That’s when the bacterial cell blows up and progeny virons are dispersed. One of the proteins involved is an enzyme called endolysin, or lysin, which means “the enzyme from inside that causes the cell to lyse.” Bacteria are protected by cell walls. But if you degrade the cell wall, which is what the phage does, then the cell blows up and the virus particles come out. So these lysin enzymes have now been developed to work from the outside to target gram positive bacteria like Staph. You take the phage lysin, make a lot of it, and inject it.

Crystal structure of a bacteriophage endolysin.

Lysins are not small molecules, but they’re a lot smaller than a phage. So you can mass produce and patent lysins – two things you can’t do with phages. But I think lysin technology will only be useful for Gram positive bacteria. Gram negative bacteria and mycobacteria have tough outer surfaces that endolysin can’t get at. The mycobacteria have a wax-like coating outside the cell wall that’s very difficult to break open.

Amy: Interesting. Back to phage therapy. Imagine there was a perfect world where we had studied phages in much greater depth. There’s a person with a bacterial infection and you’re trying to find a phage to treat them. You’re allowed to proceed however you choose – how would that work? 

Ry: You have to use multiple phages, otherwise you will probably get resistance very quickly. The word resistance means one thing to you, and a different thing to phage biologists. When phage researchers say “resistance” we are referencing a very specific situation: a phage cannot attach to a bacterium because the receptor it targets on the bacterial cell wall is damaged or missing. To clarify, every phage has a bacterial receptor it targets, and almost all the time those receptors are non-essential for the infected bacterium. So the bacterium can simply delete the receptor by mutation, allowing it to become completely resistant to the phage.

Given that notion, if you want a Pseudomonas phage cocktail to work, you need to have at least three phages that would target every possible Pseudomonas strain they encounter. Each one of those phages would target a different bacterial host receptor. The typical frequency of a knockout mutation for bacteria is one in a million: not a very big number if you’re being treated with one phage (because there are millions of bacteria and phages in the system already, some of which are already resistant). So as soon as you kill off all the bacteria that aren’t, you’re right back where you started within a few hours. But, if you have two different phages attaching to two different receptors, then instead of 10 ^- 6 frequency of a bacterial knockout mutation, it’s 10^-12. If you have three, that’s 10^-18. And nobody has 10^18 bacteria in them, at least no one who’s alive:)

So I’m using your perfect world scenario. In my perfect world there’s a Pseudomonas cocktail that’s ready for strain X of Pseudomonas, and we know its receptor targets. We also know that the bacterial strain we’re trying to target has those three receptors on its surface. Now, we can use that phage cocktail with confidence: we’re probably not going to generate resistant bacteria. That’s my hardliner perspective.

So the use of single phages could create a problem similar to what’s happening with conventional antibiotics? (where treatment can select for mutant bacterial strains) 

Ry: Right. Except resistance to phage happens a lot more frequently and quickly than with conventional antibiotics. If a bacterial cell runs into penicillin, it’s going to die unless it evolves to mutate residues of a particular enzyme in the bacterial cell wall (very difficult). So the mutations that create resistance to antibiotics are actually very rare. The problem is that because we’ve overused antibiotics, and put so much selective pressure in humans, many bacterial disease strains have sucked up genes that were already in the dirt. These are genes that had already evolved to degrade antibiotics.

But bacterial resistance to phages is a major concern. In fact, when I was a graduate student working in the heyday of phage biology, the classical attitude was that the resistance issue rendered phage therapy a joke. My mentor (and other members of the Phage Church) called phage therapy “bizarre” because resistance can occur so quickly. Of course, at the time we were completely unaware of how many more phages existed that we could use for cocktails. This was way before the development of next-generation sequencing.

Amy: To summarize. An upside of phage therapy is the specificity of the bacterial target. But that’s also a downside. And this issue could be addressed by using phage cocktails in lieu of single phages?

Ry: As director of the Center for Phage Technology I get to have official positions which absolutely no one has to listen to. But I always dreamed of being able to make pronouncements:) It’s our considered opinion that no phage should be used in a human unless we know what its receptor is. Period. Full stop. There are plenty of people who disagree with me because they don’t want to go through the trouble of figuring out what the target receptor is. But in the long run I can’t imagine the FDA is going to countenance the use of phages without clear receptor targets. And of course the original history of phage therapy has a bad reputation. There were negative outcomes and bad publicity. So now, when we restart phage therapy 2.0, we need to do it right.

Here’s another problem: right now phage therapy is always used via “expanded access.” When the phone rings in my office it’s always a crisis in which somebody is dying. And I’m not going to tell a dying patient and their family requesting a phage that we need six months to figure out what phage to use. But if we keep operating under these rushed emergency conditions, we’re going to start using phages that all use the same receptor. Then we’re going to get resistance and somebody’s going to die. That could lead to serious legal issues. If someone dies after being given an experimental phage, lawyers could later say, “Doctor yes or no…did you send viruses to this sick man? And were these viruses approved for treatment!?”

Amy: But to use an expanded access treatment the patient has to sign a medical release form right?

Ry: Yeah but that doesn’t help a bit because a patient’s heir’s lawyers are not bound by the patient’s request. So lawyers could still come after you. They wouldn’t come after me because I haven’t got a dime. But they might come after my University. Also, a flipside of the specificity issue is that in order to have any expectation of phage therapy success, we need to know whether the bacteria in a given patient are actually sensitive to the phages we have. That means we need at least one day to screen.

Recently there was a phage therapy case involving a young woman. She had gotten a lung transplant and appeared to be cured from a severe Burkholderia infection. But then the infection came back. So along with 3-4 other labs we tested Burkholderia phages. We tested over 400 phages, but only found three, and all of them were lysogenic. It took us 3 days working 24/7 to do that. Unfortunately she passed away a few days later (for reasons not related to phage therapy, but still).

But if we had already had a refrigerator full of phages, and we had known the receptor for those phages, we could have done some very simple, quick experiments. Or you could use a robot to get the bacterial strain from the patient, and know within 24 hours which of your phages might work against it. And if those phages are already pre-purified they could quickly be in the mail. But right now, no such well-characterized stock of phages for any bacterial pathogen is available. Again, the main reason for that is there’s no market for it. Because as far as I know, with “expanded access” you can’t sell the experimental drug (phage): you have to give it away.

Amy: So again, in a perfect world, it seems like the phage therapy community could use some time to calmly prepare for emergency phage therapy scenarios?

Ry: Yes. We could take highly prioritized pathogens like Baumannii and Pseudomonas and create a well-characterized collection of phages for which we knew the receptor. It wouldn’t even be a multimillion dollar project – more like hundreds of thousands of dollars. Which sounds like a lot of money, but I wonder what Tom Patterson’s bill for eight months in the UCSD ICU was? Maybe something like $10,000 a day? So the money needed to generate phage libraries wouldn’t be that large compared to many related costs. But nevertheless, who’s going to pay for it? And is every reference hospital going to have a walk-in cooler with thousands of phage lysates or purified phages? The other possibility, and the dream of the phage therapy world, is to have phage that works against all targets. For Staph aureus that already mostly exists.

Amy: A superphage?

Electron micrograph images of phage K (American Society for Microbiology)

Ry: Phage K is a “superphage.” You can get a phage K that will eliminate 85% of all the Staph on its own. It would target all Staph, except for the fact that bacteria find ways to fight back against phages that aren’t related to “resistance.” Bacteria also have restriction enzymes and their CRISP-R-associated systems, for example. But I think the dream of a super phage is very close to reality with phage K and its relatives. For other bacteria though, we haven’t found many examples of bacteria with essential cell wall components that phages can be targeted to.

Amy: But we’re discovering new phages every day, right?

Ry: Yes. We’re early in the game. And what’s astounding is that all the technology we’re using to characterize phages (with the exception of molecular sequencing) is really the same that d’Herelle used back in the 1930s. Again, that’s because there’s no current market for phage therapy. But, if people think there could be a market down the road, a lot of progress on how to purify phages could occur. The process could become cheaper than it is now. But currently we make phages for therapy in the same way we do for laboratory experiments. And that’s not something you can do on a large scale.

Amy: It seems to me the FDA will have to “paradigm shift” the way they’re going to evaluate phages and other new biological drugs.

Ry: Right, because it’s not just phages. There’s a lot of other biologicals that are like square pegs in the round holes of a regulatory process geared towards developing organic chemicals. I’m hoping the FDA’s “expanded access” phage decision will increase the climate for public and physician awareness of phage therapy. Also, there’s been some progress.

Amy:  What about pressure from Biotech companies? There is a focus in the Biotech community towards the development of personalized treatment approaches.

Ry: Yes. And phages are the ultimate personalized medicine.

Amy: That makes me think the FDA’s current drug approval process is going to become a problem for a lot researchers and drug companies.

Europe is ahead of us in addressing this issue. Partly because the countries involved are much smaller scale countries. If you’re living next door to the Minister of Health, it’s a lot easier for you to get your ideas across. So Belgium, for example, is pretty far ahead of us in terms of making “expanded access” more regularized. They’re going make it easier for physicians to say “I’m going to use phages.” And there will hopefully be suppliers of good phage. Then, the physician will be able to buy/use phages and have protection against being sued.

I don’t think phage therapy will replace antibiotics though. I want to make sure you understand that. I think at least for the foreseeable future phage therapy is going to be a last resort thing. I would like to see a lot more basic phage biology funded. I think in order for phage therapy to best move forward, we also need a rebirth of basic phage biology research. Because almost everything we currently know about phage biology is based on just phages of E.coli (which is of course what I worked on myself.)

Amy: Yes. A recent study estimated that 30 billion phages may traffic the human body on a daily basis. It does seem like we need more research on all these “new” phages!

Ry: Right. DNA sequencing is nice to have, but it doesn’t substitute for doing experiments that help better understand phage activity and function.

Amy: Did you read the recent Georgia Institute of Technology study which showed that the host innate immune response seems to play a role in helping phage therapy work? Because if that’s the case,  then our widespread use of immunosuppressive drugs and steroids seems like a problem. Especially because such drugs are administered frequently in hospital emergency settings.

Phages interact with the human immune system (Roach et al, Cell Press)

Ry: So I suspect that phage therapy will be mostly useful for reducing initial loads of bacteria, under conditions where a patient harbors enormous amounts of a bacterial pathogen. Because once the bacteria get down to a certain level they don’t collide with the phage anymore. Phages are not small molecules, so they can’t penetrate every body niche to find a bacterial target. So I think that phages will be most useful for somebody who’s got a raging infection. You give them a first round of phages and a knock the bacteria down by two or three logs. Now you’ve got a chance for either the body’s own immune system or antibiotics to take over.

Amy: At least in that scenario antibiotics are being used in a careful situation. As opposed to many other ways in which they are over-prescribed today.

Ry: Yes. There’s a lot of speculation out there that many of our current autoimmune diseases, like Crohn’s, may be due to the fact that our generation (my generation) was the first to be massively dosed with antibiotics all throughout childhood. My generation was convinced that if you didn’t get an antibiotic prescription you hadn’t been treated right. It was the golden age of antibiotics.

Amy: Right. When antibiotics were discovered they were perceived as miracles, which they kind of were, especially during World War II. But that has created a situation where the average person has not been well-educated about how antibiotics work. For example, about the fact that antibiotics can’t kill viruses. 

I think that the American Medical Association is at fault for not educating the public better about antibiotics. I mean they really are. For example, you see all these TV commercials for erectile dysfunction or cancer drugs. There ought to be a way that some of that airtime could be used to educate the public about medical topics like antibiotics. Especially because most drugs advertised in these commercials were developed with NIH funding. So I think big drug companies owe at least that to the public. Also, physicians need to have the courage to say “You have a viral infection. I’m sorry but it can’t be treated with antibiotics.”

Amy: Yes. I know you’re considered a “hardliner” when it comes to the future of phage therapy, but I think the caution you advise moving forward in spot on. We don’t want to create a future situation with phage therapy that could repeat problems we’ve encountered with antibiotics. Understanding and correctly managing phage resistance will be critical.

Thanks so much for taking the time to speak with me. I learned so much from this discussion.   

20 Questions for Gurol Suel: biofilm electrical communication

January 29th, 2018 by Amy Proal

Gürol Süel is a Professor of Molecular Biology and the Associate Director of the San Diego Center for Systems Biology. His research team integrates quantitative biology approaches with mathematical modeling to identify principles of bacterial organization and coordination. Most recently, the team discovered a new form of bacterial communication that arises in biofilms: Ion channel mediated electrical signaling. For more context on this discovery, please read this article in The Atlantic Magazine.

Amy: Thanks so much for speaking with me. Your last few papers on electrical communication in bacterial biofilms blew me away. I think many other people feel similarly! How did you get interested in the topic? 

Gurol: We were very interested in trying to understand higher-order organization and behavior in bacterial communities. That’s because even though bacteria are unicellular organisms they’re not solitary. They actually live in dense communities (biofilms) that have certain features: for example they’re very resilient to stress and antibiotics.

We became interested in biofilm electrical signaling partially because we saw that in biofilms there was some kind of long-range metabolic coordination. We were having a hard time understanding how the bacterial cells could communicate so effectively over such long distances. It seemed that a simple diffusion-based mechanism (like quorum sensing) might not be sufficient to support this communication. My Ph.D. is in molecular biophysics. I also have a background in electrophysiology. For example, I used to do electrophysiology in fruit flies with photoreceptors. So drawing from that experience, I started wondering if there could be a different type of signaling process involved.

We started looking at amino acids critical for slow-range biofilm communication. The most important amino acid appears to be glutamate. Glutamate is very interesting because it’s also an excitatory amino acid in the human brain. In fact, about half of all brain activity is regulated by glutamate. Glutamate is also one of few amino acids that’s charged. So it can’t just get in and out of cells on its own because that’s energetically unfavorable. So you’ve got to have some kind of a proton motor force to actually take up the charged amino acids. And if you’re dealing with a proton motor force then the cell membrane potential is important.

Little by little we pursued this line of thought and realized that we were looking at some kind of ion channel purpose or function in this signaling process. And when you think about ion channels and membrane potentials you think about sodium and potassium – those are the most commonly utilized ions in biological systems. So we looked at both and saw that sodium wasn’t really doing anything. That wasn’t surprising because Bacillus subtilis, the microorganism that we work with, is not known to have any identified sodium channels.

Bacteria form communities called biofilms (Image: The Laboratory of Gurol Suel)

But we saw a lot of beautiful signaling and dynamics going on when we looked at potassium. Then, we took the suggested potassium ion channel and started making mutants. One thing led to another and we realized that these bacteria were communicating using potassium ion channels, in a way reminiscent of what neurons do in the brain with action potentials. Once we uncovered that, we realized that if you have these long-range electrochemical waves propagating through the biofilm, then the waves wouldn’t just stop at the edge but might propagate a distance.

That led us to the Cell paper where we said: What if there’s some kind of motile cells nearby? Would they be sensing or responding? Would they be affected by these types of signals being emitted by the biofilm? Pursuing those questions turned into a fun and intriguing study. Then we said OK – if that’s possible, what if there’s another biofilm further away: could the two biofilms sense each other and interact? That led us to the Science paper about time-sharing of limited nutrients.

Amy: Right. I enjoyed reading those papers because your experiments are so well organized. I’m interested in the redundancy between the way neurons signal and the way you found that bacteria in these biofilms are signaling. Do you think it’s possible that there are any interactions between human nerve signaling and these microbes? That they could tap into each other’s signaling?

Gurol: I mean, we don’t know. That’s the short answer. Because no one’s ever looked at that. Would it be possible? I guess it would be possible. I do know that extracellular potassium signals (the potassium ion channel mediated signals) are not specific to just neurons. We showed it now even in bacteria. But it’s also very important in plants. It’s very important in all kinds of other living organisms – because these ions are very generic, meaning they can affect extracellular charge in any membrane.

So it’s not inconceivable to think that if a bacteria releases some charged particles and there happens to be an excitable cell nearby – maybe a mammalian cell – that the mammalian cell might actually respond to that. I don’t think that’s super crazy. Right now it’s unproven but it’s something we think about.

Amy: Very interesting. What about external electromagnetic fields – do you think that our human electronic devices or any radio frequency networks we’ve set up as humans could be impacting biofilm communities?

Gurol: That’s a very good question. Again I don’t know for sure. But it’s well known that lots of organisms have electro-reception, meaning they can follow electromagnetic fields on our planet. Animals with electro-reception range from whales to birds. So if organisms can sense these fields, what you’re suggesting is probably not super crazy.

There are actually bacteria known to have small particles inside that are sensitive to magnetic fields. We also know there are documented cells that will respond to an electrical field, including bacterial cells. There’s also good work out there clearly showing that just like temperature, pressure and so forth, biological systems are sensitive to magnetic forces and electrical forces. That’s more or less well-established. What we don’t know is whether or not your cell phone is going to have anything to do with your gut microbiome or things like that. Those are the things that, to my knowledge, have not been clearly studied.

Amy: Not really. But interesting future research?

Gurol: Yeah. And as I said, there’s enough strong evidence out there showing that living organisms are sensitive to these physical external properties: pressure, temperature, pH, electrical fields, magnetic fields. That’s not a new concept. So we’ll just have to see. And I don’t know, do birds sit on electric wires because it tingles? Or is it just a good sitting place?

Amy: Haha. There was a study which showed that Interference from human electronics and AM radio signals disrupted bird migration…

Gurol: Yes I wouldn’t be surprised. I think there is enough information out there to at least create a good incentive to look at the topic in greater depth.

Amy: Yes. So I research the human microbiome. If someone asked you to describe the human microbiome knowing what you do now, how would you explain it?

Gurol: Maybe I would say that the human body is an ecological network. We’re existing together with our environment, and our environment is not just about whether it’s cold or rainy outside. Within our bodies we have countless organisms (not countless but lots of organisms) and bacteria are just one of them, in fact we probably have even more viruses than bacteria – nobody’s been able to track down everything. So we harbor a lot of organisms, especially in our gut. And I think it’s a complex interaction that we don’t fully understand. There’s always, of course, a little bit of hype – when we discover something new everyone gets excited, maybe even a little over-excited. But even if 10% of all the stuff out there on the microbiome turns out to be relevant that’s already a lot.

So I would say the microbiome is a little universe of an ecological network within our body. There’s things going on between all the bacteria. But then there’s also things going on between the bacteria and our cells, the human cells. And we have yet to fully appreciate how many and which parts of these processes are super-critical and which are processes that are interesting but maybe not as critical.

The human microbiome is an ecological network

For example, there’s always interesting studies about species composition of the microbiome – studies stating that composition is critical to this process or that process. But we have to remind ourselves that the composition of the microbiome is not a static thing. When I wake up and go to bed my microbiome is probably totally different depending on where I went to dinner and other variables.

So to some degree we can’t be super sensitive to species-specific microbiome changes, because then we would have traumatic, physiological problems throughout the day. That model just wouldn’t work. We also know that the microbiome can recover: you can take antibiotics and really flush out a lot of stuff but then you can very quickly recover your microbiome. So it’s going to be important to discern which processes have an effect but are not supercritical and which processes are actually very important. That’s the kind of stuff that makes it interesting for the future – to see where it leads us.

Amy: Many people research microbes in isolation and outside their natural environment. I know great insight can be gained from that work. But do you think there needs to be a larger push to research how microbes really act in vivo (in their natural environment)?

Gurol: Yeah. So “in vivo” is an interesting term. It’s very important when you’re dealing with something like the study of cell lines in the laboratory. Problems have appeared when people have studied these cell lines and then tried to claim things about cancer: because the cell line was taken out of its natural context. But when you’re dealing with bacteria to say “what is in vivo?” is not even very clear to me. Take the organism we work with – Bacillus subtilis. You have it right now on your eyebrows and on your skin. It’s also in your gut. It’s in the soil. It’s on your tablecloths. If you have fruits and vegetables in your fridge it’s in there. So which of those locations is “in vivo”…right? They are all very different conditions, but are all places where bacteria naturally occur.

So the question of “in vivo” is a blurred, fuzzy, and potentially misleading question: in the sense that we don’t actually know what to call “in vivo.” If you isolate bacteria in the lab it’s more clear what you’re doing. But to say “this is the proper in vivo context” – I’m putting out a little word of caution: these bacteria are capable of surviving under so many different conditions that we have to be careful.

But overall any good scientist would know that if you’re studying a cell you have to understand the context of the cell, meaning the environment of the cell. And if the environment of the cell includes other cells, and those may be the same species, or different species, then obviously that is something that needs to be taken into consideration. But one can still execute very important studies without being shackled to this vague notion of “what is in vivo?”

It’s especially important to remind people of this now, because as a scientific community are very concerned in trying to make sure that our data is reproducible, and that we have reliable results: that if I do an experiment somebody else can reproduce it. But the moment we get into these types of in vivo conditions the problem is very poorly defined. Even the eyebrow skin environment differs between two people based on fat content, pH and other variables. So we’re dealing with a lot of unknowns, and although we can pat ourselves on the back and say “hey we’re studying something in a more natural context,” we have to realize that it comes at a cost – the cost of having less defined conditions and a lack of understanding of certain things. So it’s a balance – there’s no right or wrong – but we have to understand the pros and cons of each of these approaches very carefully.

Amy: OK I see. Still, in any environment, the microbiome community should focus on studying microbe activity in addition to just species, right?

Gurol: Sure. Sure.

Amy: Ok, what about other microbes like viruses, phages, fungi. Do you think that they’re involved in this electrical of signaling as well?

Gurol: We already showed in at least one paper that two very evolutionary distant species of bacteria can communicate electrically – Pseudomonas and Bacillus. One’s gram positive and one’s gram negative, so they’re very different types of bacteria. That is an indication that this signaling is not just limited to a single species of bacteria. Although that would have been a great result too. Because then you would have to ask “how can it be that only one species evolved this thing!?” That would be kind of  interesting on it’s own also. But maybe not surprisingly that does not appear to be the case.

Bacteria use action potentials to generate electrical signals

So we are still at the ground level if you will. These discoveries are still quite recent in terms of scientific years – they’re the opposite of dog years that go extra slow. So time will tell how many other species signal electrically – which ones do, which ones don’t and what that all means. But again it goes back to the earlier question that you asked: how widespread could this be? It has something to do with the fact that the membrane potential is a very fundamental property of any cell. Any living organism that’s a cell has a potential across its membrane. It’s a very ancient set of physiological processes, so it wouldn’t be shocking in hindsight if we found that different species, and even organisms across many different kingdoms, can engage in some form of this communication.

Amy: Agreed. Can you tell me a little more about how you integrate mathematical models along with the study of actual biofilms to get your results?

Gurol: Sure, the mathematical models are quite important. We use them for two main purposes. One is to come up with very specific hypotheses. The nice thing about mathematical modeling is that you can make predictions that are hypothesis driven…more than hypothesis driven: that are mathematically supported. That is very important because not only does it mean that you can define the question you’re asking really well, but once you have experimental results you can also interpret them using the framework of a mathematical model in a way that is much more powerful.

The other reason we’re doing it is that mathematical models allow us to identify concepts, and concepts are very important because a lot of people are interested in “what is this gene?, or “what is this protein?” Those are tangible objects, and so people really like working on those things. Concepts are by definition intellectual things. That means they are not tangible, in the sense that you can’t touch it, you can’t say “here’s the protein.” But concepts are very important because they allow us to bridge across very different types of systems. So if we can use the same mathematical description to describe processes happening in very different organisms that is meaningful. You can say, “There seems to be something that is shared between two systems, and then you can say “Are they trying to solve a similar problem?” Maybe there’s some kind of evolutionary information there.

Hodgkin and Huxley

So identifying concepts is very relevant in finding connections between things that appear on the surface to be different. Like bacteria and neurons: if I you told ten years ago that they might have similarities, then people would chase you out of the room and say you’re crazy. But now we can use the mathematical formulas that were originally developed in the 1950s by the brilliant Hodgkin-Huxley team. They got a Nobel Prize for the work. They identified a mathematical framework and figured out that basic action potentials in neurons are driven by potassium and sodium flux across the membrane. We showed that you can use that same mathematical framework to explain and make predictions regarding electrical signaling in bacterial communities.

Amy: Yes absolutely. How have your results been received? It seems like pretty enthusiastically, but have you had trouble communicating this to people who have only studied microbes in isolation?

Gurol: No, I mean communicating work is a very important part of being a scientist, because knowledge not shared is like knowledge that almost doesn’t exist. If only one person knows about it, that doesn’t help anybody. So communication is critical. And when you’re communicating you cannot blame the audience for not getting something – you have to figure out how to communicate your results so that anybody can appreciate them. So we take great pride in trying to write papers that are accessible, even though obviously we have to deal with the fact that we’re communicating technical scientific work. I think this pays off in that if you’re able to explain WHY something is important, people can make their own judgement on whether it’s critical or not. The dangerous thing is if people don’t understand it and they’re just confused. Confusion leads to frustration. At that point it’s very difficult to decide if something is noteworthy or not.

Amy: I think you did a great job with communication. I like how you put the more complex math towards the back of the paper.

Gurol: We try to make sure that you can read the paper, and then if you are interested there’s more meat you can sink your teeth into. I’m sure there’s still much room for improvement but we try.

Amy: No, you did a really good job. Can you tell me little more about ion potentials and about how biofilms are able to coordinate behavior to time-share nutrients?

Gurol: Sure. So you mentioned our three latest papers that you liked. The first paper describes how potassium ion channels are utilized by bacteria in biofilm communities to send long-range electrical signals. These signals allow them to coordinate their metabolism. That was an exciting finding that set many other things into motion. In the next paper we described how these electrical signals could extend beyond a single biofilm community. If there’s planktonic motile cells swimming around (because biofilms exist in aqueous environments) it’s not crazy to think that they could be recruited into the biofilm community (even different species). We knew that bacterial motility is driven by proton motor force – the cells utilize an electrochemical gradient across the membrane to generate the energy required to churn flagella: swirly appendages that allow bacteria to swim. We said, “Well if you have electrical signals maybe they would have an effect on this process of motility.” And indeed, we found that that electrical signals can direct and attract motile cells from a distance to the community. That’s interesting because now you can see how electrical signaling can turn a single species community into one that recruits other species too.

We followed that finding up by saying, “OK so if there’s two biofilm communities – two physically separated communities at some distance – not super far away but, with respect to the size of a bacteria some significant distance – what would happen?”

We saw that if these communities share the same environment they become coupled – they can sense each other so to speak. This happens in two main ways. One is this electrical signaling which syncs communities under metabolic stress. But there’s also competition between two communities, where resources are limited and there’s only enough to sustain one community. We found that under such conditions, communities started modulating the coupling between them and transitioned to different types of dynamics: they began time-sharing the limited resources. Time-sharing is a well-known concept. Take vacation homes or cloud computing (where it’s called resource sharing). Another example would be if you have one toy and two kids that want to play with it. You have the kids take turns playing with the toy. It turns out this same strategy is being employed by these bacteria. It’s an exciting discovery because time-sharing is a very dynamic process. With vacation homes or cloud computing time-sharing is not trivial – somebody has to set up a calendar saying, “You take August I’ll take June.” So it requires some kind of higher organization or somebody who coordinates things.

Bacteria use flagella to “swim”

But we found that bacteria naturally time-share through their dynamic system behavior. They can actually transition from being in phase where they’re competing a lot (and that’s OK if there’s enough nutrients) to then switching to time-sharing behavior when nutrients are low. In that case, there’s nobody from the outside telling which colony to do what. The process naturally emerges through the interaction between the communities. It’s very interesting then that bacteria are capable of executing what appear to be nontrivial strategies for coping with limited resources.

Amy: It’s extremely interesting. Do you think we’ve underestimated bacteria? With each new study it seems that bacteria are able to do much more than we previously believed.

Gurol: I’m always amazed by any living organism. If you realize how long these guys have been around (especially bacteria) they’ve survived mass extinction, one after the other. Not just the extinction that killed dinosaurs, but also the earlier extinction where even more species disappeared.

So they’re extremely resilient. They’ve been able to colonize from the Arctic to the depths of the ocean. It’s fascinating. You know, sometimes I go to the east coast and I have trouble adjusting to the new environment! So I’m impressed by these bacterias capabilities. Also, I’m sure there’s still a lot we don’t know. It’ll be a fun future to figure out what else they can do.

Amy: I agree. I know this is jumping ahead but do you see any possible therapeutic interventions based on these findings? Maybe you could interrupt an electrical signal in a biofilm-driven illness?

Gurol: Sure. We really like doing basic research and then letting the findings lead us in those directions. But we are certainly not blind to the possibility that our discoveries may also have biomedical implications. We’re trying to be careful – we don’t want to put blinders on and just focus on one thing. But we do obviously realize that biofilms are a major public health threat. Biofilms are responsible for the majority of infections in clinics. There are people dying and billions of dollars at stake. So yes. We’re aware of these problems and are slowly trying to explore those avenues as well.

Amy: What are you going to study next?

Gurol: We’re still trying to understand some of the basics. We’re still very intrigued by this new form of communication between bacteria – what else can it do? At the other side, at the smaller scale, we’re also trying to understand if in addition to ion channels, there are other genes and proteins playing regulatory roles. So we’re diving both in the direction of more detailed depth and more rigorous research. But also going in the other direction to see “How widespread is this and what other interesting behaviors can come out of this?” I suspect this research will lead to more unexpected findings.

Amy: Is there a growing community of researchers who are getting interested in this? Or are you guys working mostly alone?

Gurol: No, there’s a growing community. In fact we’re going to have a first session on biofilm electrophysiology at the upcoming American Biophysical Society Meeting this February. There will be speakers from all kinds of different institutions and from around the world. So there’s definitely a community that’s building and I think this field has a lot of potential. It’s obviously a very small field only in its infancy, but we’re trying to draw in more people and create a connected community.

Amy: Yes. Some of papers you cite are from the 1970s. How did that earlier research play into your findings?

Gurol: As they say, we’re standing on the shoulders of giants. Smart people existed before we came along. There’s been beautiful studies done – going all the way back to Hodgkin and Huxley. In fact, I would go back as far as Luigi Galvani in the 18th century who did this beautiful experiment where he cut a frog in half and used electrodes to make the legs twitch. It really freaked people out and led to books like Frankenstein where the monster is brought to life with electrical currents. So this field goes back hundreds of years. Obviously a lot more people work on electrophysiology in the human brain because that’s an important organ and it’s easy to see the relevance. But people have looked from plants to fungi to bacteria because the ability of cells to use electrochemical potential across the membrane is so ancient.

Amy: Yes. So let’s say you were talking to a student who wanted to study biofilms or microbiology. What kind of skillset would you recommend this student get to best succeed in the field? 

Computers play a central role in the study of microbes

Gurol: First you have to learn how to think like a scientist, because once you understand how that process works you can work on anything. In terms of a more concrete skillset, the more diverse the better. If you have a unique background and have done things that others have not done then, in the future, maybe you can see things from a different perspective. You might discover something new. I’m sure my background in electrophysiology allowed me to think about biofilm signaling a little bit differently.

I’ll also say this, the future is as clear: we’re heading in a direction where being able to communicate in terms of a mathematical language is key. Also understanding large data sets and analysis – that’s not going away. So having no fear of computers and software and mathematics and physics: that will be important, because again these boundaries are blurring. I think being interdisciplinary is very important.

Amy. Yes. I wish I had a physics background. I’m jealous. Before we go would you like to leave people with any other message?

Gurol: We covered a lot of stuff. Hopefully we’ll have more stuff to talk about in the future. But I’m happy that people like you and others are taking note and finding our work interesting. In this day and age it’s very important for people in science and people in science communication to make work accessible and reach out to the public. Because the more we educate the general public and try to help them understand what science is and how it works, the better it is for our society.

Amy: I do. Thank you again for this fascinating conversation.

Immunostimulation: embracing a new treatment paradigm for chronic disease

January 12th, 2018 by Amy Proal

Think back to the last time you got the flu (virus). The fever, the runny nose, the aches, the sore throat – what causes these and related symptoms? Most flu symptoms are not driven by the virus alone. Instead, they result from a “battle” between the virus and the human immune system. Symptoms begin when the immune system recognizes the flu virus and creates inflammatory proteins called cytokines in an effort to target infected cells. If infected cells are successfully killed, more inflammation is generated as toxins and cellular debris enter the bloodstream. In addition, antibodies may be created in response to these cell and viral byproducts, again leading to a rise in inflammation.

Most cold medicines lower symptoms by suppressing parts of the human immune system

We don’t have antivirals capable of killing the flu virus. So we “treat” the illness by letting this immune system “battle” run its course. In most cases, the human immune system “wins” over time, and the inflammation caused by cytokines, toxins and antibodies drops. We begin to feel better and life goes on.

In some cases, patients manage the flu with over-the-counter medicines. These include Motrin, NyQuil, and antihistamines. In other cases a doctor may prescribe steroids or immunosuppressive medicines. These medicines lower symptoms but do nothing to target the virus driving the illness. In fact, these medicines “work” by shutting down various parts of the human immune response towards the virus. They “tone down” the battle between the immune system and the virus so that less inflammation is generated.

The above medications make patients FEEL better. But they may actually impede recovery from the flu by allowing the virus to survive with greater ease. For example, Canadian researchers found that anti-fever medications suppressed fever in patients with the flu, but also allowed flu viral particles to spread more easily from person to person. Indeed, the team estimates that the use of anti-fever medicines by flu patients contributes to a 5% increase in general flu cases and deaths.

Despite these negative outcomes, our entire medical system centers on immunosuppressive treatments that “knock down” parts of the immune system to suppress symptoms. Patients with autoimmune disease are regularly prescribed immunosuppressive medicines like prednisone, rituximab or TNF-alpha inhibitors. Humira – a TNF-alpha inhibitor used to treat arthritis and other “autoimmune” conditions – is the “best selling prescription drug in the world,” with a $38,000/year price tag per patient.

Why this focus on immunosuppressive therapies? Most immunosuppressive treatments were developed before ~2004, during a time when the human body was believed to be largely sterile. Under these conditions the “theory of autoimmunity” gained hold. If inflammation was detected in patients with a range of conditions it was assumed to be a result of the immune system “going crazy” and attacking human tissue.

The discovery of the human microbiome greatly challenges this “autoimmune” model of disease. We now understand that vast microbiome populations persist in every human body site – from the gut, to the brain, to the placenta, to the liver and beyond. An increasing number of “autoimmune”/inflammatory conditions are now tied to dysbiosis or imbalance of these microbiome communities. This means that in “autoimmune disease,” the human immune system may be attempting to target pathogens in the microbiome in lieu of attacking human tissue. Indeed, an increasing number of studies demonstrate that the “autoantibodies” used to diagnose autoimmune disease are often created in response to a range of bacterial, viral and parasitic infections.

This growing association between infection, “autoimmunity”, and inflammation helps explain the poor long-term outcomes associated with immunosuppressive therapies. Patients administered prednisone or TNF-alpha inhibitors tend to feel better in the short-term, but relapse is common, and often expected. Each relapse can require higher doses of immunosuppressive medication to get symptoms “under control.” Meanwhile, patients are at greater risk for developing a second or third inflammatory disease, and are more likely to suffer from acute infectious conditions like tuberculosis. Long-term health outcomes associated with prednisone are so poor that the slogan “pred ’til dead” is commonly invoked (patients who start prednisone often require higher and higher doses of the medicine until they die from the underlying disease).

This begs the question: what if we treated autoimmune disease in the exact opposite fashion? If microbiome dysbiosis contributes to autoimmune disease then treatments that SUPPORT the immune system could target pathogens driving inflammation. By addressing this infectious root cause of inflammatory symptoms, such treatments might induce actual improvement or even recovery.

How might patients with “autoimmune disease” or related inflammatory conditions respond to “immunostimulative” or immune-supporting treatments? Case histories from the turn of the century offer clues. In the early 1900s, mercury was used to treat syphillis: a sexually transmitted infection caused by the bacteria Treponema pallium. Mercury “deliberately stimulated the immune response” in patients with the disease. This resulted in a phenomenon known as the Jarisch-Herxheimer reaction (named after the researchers who characterized it). As the activated immune system targeted Tremponema pallium a “battle” not that different from that associated with targeting the flu virus ensued. Patients suffered a temporary increase in symptoms including fever, chills, myalgia, and headache as cytokines were released and debris from dying bacterial cells entered the bloodstream. However, if patients endured these symptoms they generally “turned a corner,” where symptoms subsided as Tremponema pallium was gradually eradicated.

In the 100 years since the Jarisch-Herxheimer reaction was described in syphilis, it has been further documented in patients administered immunostimulative therapies for a broad range of infectious conditions. These include Lyme disease, leptospirosis, brucellosis and tuberculosis. More recently, the term “immunopathology” has been used in place of Jarisch-Herxhimer to refer to “a systemic inflammatory response consistent with elevated immune activation.”

Immune activation and immunopathology in the treatment of HIV/AIDS

Over the past decade, immunostimulative treatments have been developed to treat HIV/AIDS and cancer. These therapies are also characterized by temporary symptom increases as the activated immune system attempts to target root causes of inflammation. Current HIV/AIDS treatment centers on highly active antiretroviral therapy (HAART). Patients administered HAART receive a cocktail of anti-retroviral drugs, each of which impedes the ability of the HIV virus to replicate and spread. Prior to HAART, the HIV virus survives by dramatically slowing key parts of the human immune response, including the CD4 cells that normally target infectious agents. This means that when the virus is “contained” by HAART, the immune system “wakes up” and identifies pathogens acquired during previous periods of immunosuppression.

IRIS leads to increased symptoms as the immune system “re-activates” (Source: Khan Academy)

What happens next is a form of immunopathology. The activated immune system starts to target pathogens it could not recognize before HAART was initiated. The patient begins to experience temporary increases in symptoms ranging from fever, to malaise, to neurological dysfunction. Symptoms wax and wane with time, but generally decrease as the immune system better targets a range of previously unrecognized pathogens. The HIV/AIDS community has named this process “Immune Reconstitution Inflammatory Syndrome”, or IRIS.

A number of well-known pathogens are linked to IRIS symptoms: the herpes viruses, cytomegalovirus, hepatitis B and C, M. tuberculosis and Mycobacterium avium among others. Often however, symptoms increase despite the fact that no pathogen can be identified on routine blood tests. This suggests that newly identified microbes, like many of the thousands recently detected in tissue/blood by Stanford researcher Stephen Quake, are also being targeted by the activated immune system.

Several key patterns have been observed in patients experiencing IRIS. One is the “unmaking” of infections that can range back to childhood. Let’s say a patient suffered from bacterial meningitis at age eight. The meningitis microbe may re-appear on IRIS-related blood tests thanks to the renewed immune “attack” against its presence. This supports the fact that pathogens acquired throughout life can persist in our microbiome communities, where they may contribute to chronic symptoms.

Second, patients experiencing IRIS often ‘develop’ autoimmune conditions as the immune system reactivates. These include sarcoidosis, diabetes mellitus, rheumatoid arthritis, lupus and Graves disease. This strongly suggests that pathogens targeted by the IRIS immune response also drive symptoms associated with these related inflammatory disease states.

Cancer therapies target tumors by activating the immune system

The latest cancer therapies also seek to activate the immune system. According to the American Cancer Society, these novel immunotherapies “stimulate your own immune system to work harder or smarter to attack cancer cells.” Cancer immunotherapy treatments include CAR-T therapies: treatments that remove disease-fighting T cells from a patient, genetically modify them to better recognize and attack tumor cells, and then add the activated cells back into a patient’s blood.

Cancer immunotherapy activates human T cells

Response to CAR-T immunotherapy results in serious immunopathology. Nearly all patients administered CAR-T therapy experience a rise in symptoms due to what has been named Cytokine Storm Syndrome or CSS. As implied by the name, CSS results when an immune system “battle” between activated T cells and cancer cells causes massive amounts of inflammatory cytokines to be released into the bloodstream. Resulting symptoms are characterized by fever and in more severe cases, renal insufficiency, pulmonary insufficiency and altered mental status. Sometimes CSS is so strong that patients die from the reaction. Again however, if patients endure/survive the treatment they often enter a state of remission or recovery.

Factors driving CSS are debated by the cancer community. Researchers more familiar with the concept of immunopathology regard CSS as an “on-target” effect of CAR T-cell therapy—that is, its presence demonstrates that active T cells are at work in the body.” In other cases however, CSS is described as a poorly understood “side effect” of immunotherapy. For example, the Washington Post recently published an article on CSS titled: “New cancer therapies have perplexing side effects.”

This “side effect” viewpoint fails to consider a growing body of research linking cancer to infection. For example, “dramatic, continual alterations in the microbiome” were directly responsible for tumor development in a model of colon cancer. Another study found significantly altered microbiome populations in human breast tumor tissue. This imbalance was correlated with decreased expression of key antibacterial response genes. Even signaling peptides created by bacteria have been shown to directly induce tumor formation.

It follows that CSS may result, at least in part, from an immune response towards infected tumor cells. Immunotherapy may also target pathogens that control tumor development by altering the activity of human metabolic pathways. If this is the case, CSS may be the cancer equivalent of IRIS in HIV/AIDS. This is supported by case histories showing that some cancer patients undergoing immunotherapy also develop “new” autoimmune/inflammatory conditions.

Immunotherapy results in Cytokine Release Syndrome (Breslin, 2007)

For example, the Washington post describes a patient named Diane Legg’s response to cancer immunotherapy, stating: ”Her therapy knocked back her cancer, and she’s glad she got it. But the drug also gave her “almost every ‘itis’ you can get: arthritis-like joint pain, lung inflammation called pneumonitis and liver inflammation that bordered on hepatitis, in addition to the uveitis.”

Did immunotherapy really “cause” Legg to develop these new illnesses? Or, as with IRIS, did the conditions arise due to the “unmasking” of pathogens acquired during earlier periods of illness? It’s also worth noting that patients undergoing immunotherapy often present with “new” bacterial, fungal and viral infections. One study identified 43 infections in 30 immunotherapy patients in the first month of treatment, with infections causing the deaths of two patients.

More research is needed to clarify how these infections correlate with CSS. To move forward, researchers developing immunotherapy treatments must be trained to understand the complexity and extent of the human microbiome capable of driving inflammation. This poses a challenge, since at the moment the immunotherapy and microbiome research communities are not well connected.

Managing CSS in cancer and IRIS in HIV/AIDS is also a great challenge for doctors administering the immunostimulative therapies. Most physicians are not trained to consider the microbiome beyond the gut. They are also not taught to understand the general concept of immunopathology. Indeed, palliative medicine has gained such traction that early trials of cancer immunotherapy did not even anticipate a CSS response. This New York Times article titled “When drug trials go horribly wrong” describes testing of an early immunotherapy treatment (TGN1412). After infusion of TGN1412, all six human trial volunteers faced CSS leading to “life-threatening conditions involving multi-organ failure.” According to the Times, “the outpouring of toxic molecules when T-cells are activated…could not have been predicted from prior animal studies using the drug.”

Physicians also suffer from a lack of solid immunopathology treatment guidelines. According to the Washington Post “Many doctors are not up to speed on how to spot and handle an immune system revved up by immunotherapy.” In severe cases of both CSS and IRIS, physicians are forced to prescribe antibiotics and antivirals to manage symptoms associated with infection. However these drugs kill only a fraction of bacteria and viruses capable of driving symptoms. This forces many physicians to “dampen down” symptoms with corticosteroids or other immunosuppressants, the use of which counters the point of treatment in the first place.

Immunostimulation can target root causes of inflammatory disease (Source: Ty Bollinger)

For immunostimulative therapies to truly succeed then, medicine would need to embrace an entirely new paradigm. Drug companies would turn their energy towards developing new antivirals, new antibiotics or antibiotic alternatives. They would attempt development of palliative medicines that help symptoms without destroying the immune response. Tests that better detect and characterize pathogens in the microbiome would be prioritized. Physicians and drug developers would incorporate knowledge of the microbiome into all treatment practices.

The success of immunostimulative therapies also hinges on the willingness of institutional review boards (IRBs) to accept immunopathology. IRBs decide whether patients are allowed to enroll in a particular drug trial. At the moment, many IRBs are unwilling to allow patients with “autoimmune disease” or non-fatal inflammatory conditions to test immunostimulative therapies. This is based on a “do no harm” mentality that does not support increasing symptoms in an effort to improve long-term health.

We must prioritize human microbiome health (Image: BioCote)

What these IRB boards may not realize is that most patients with “autoimmune disease” or non-fatal inflammatory conditions are more than willing to feel temporarily worse (even for years) if offered hope of actual long-term improvement. Serious “autoimmune/inflammatory disease” can feel like a living death. For example, a patient named Anne Ortegren recently committed suicide after decades of suffering from the neuroimmune disease ME/CFS. In a letter written before her death she stated, “A very important factor [in choosing to die] is the lack of realistic hope for relief in the future. It is possible for a person to bear a lot of suffering, as long as it’s time-limited. But the combination of massive suffering and a lack of rational hope for remission or recovery is devastating.”

This leads to a final consideration: the greatest hope for immunostimulation in ANY disease hinges on the ability of treatment to be initiated early, or in a preventative fashion. Medicine must learn to support the immune system BEFORE pathogens push the microbiome far out of balance. “Bringing back” the immune system after years of neglect inevitably leads to severe symptoms and complications. For example, the severity of CSS in cancer is directly related to the tumor burden of the patients (patients with fewer, smaller tumors experience less CSS). In contrast, immunopathology in early-stage disease can be easy to treat and tolerate.

That is why I have a vision: In this vision, medicine respects and supports the immune system from the earliest days of life (even in the womb). Microbiome health forms the cornerstone of new therapies, with treatment administered at the first sign of symptoms. New drugs that target pathogenic bacteria, viruses and fungi are central to therapy. The need for immunosuppressive medicines in chronic inflammatory disease drops…to the point where maybe, one day, they are largely regarded as a failed relic of Medicine’s past.

 

 

 

 

 

Interview with Robert Moir: Infection in Alzheimer’s/brain microbiome

December 18th, 2017 by Amy Proal

Robert Moir is an assistant professor of neurology at Harvard Medical School and Massachusetts General Hospital (Boston). He studies Alzheimer’s disease and other inflammatory conditions characterized by neurodegeneration. His research team has shown that the amyloid beta protein associated with Alzheimer’s “plaque” is a potent antimicrobial peptide. Please read this blog post for more context on this important discovery. 

Background information:

Antimicrobial peptides: “Natural antibiotics” created by the human immune system. They are able to kill a range of bacteria, viruses, fungi, and other pathogens.

Innate immune system: The branch of the immune system that creates antimicrobial peptides. These peptides and other innate immune system cells form the body’s “first line of defense” against infectious agents.

THE INTERVIEW

Robert hey! Thanks for taking the time to speak with me. First question: your discovery that amyloid beta is an antimicrobial peptide is HUGE. What made you decide to investigate its possible antimicrobial properties in the first place?

The idea came from a somewhat chance discovery. On Fridays I go through my walkabout time on PubMed (a website that catalogs scientific journal articles). I rove wherever impulse takes me. I read a paper about LL37, a well-described human antimicrobial peptide. It was obvious from this paper that LL37 and amyloid beta share clear similarities: both structural similarities and the ability to form amyloid (protein-like deposits that can accumulate in tissues in certain disease states known as amyloidopathies).

At the same time, my mentor Rudolph Tanzi (he’s in the office next door) had just gotten back results from a screen of Alzheimer’s linked genes. He got the results back literally the same day I read the LL37 study. Well, most of the Alzheimer’s linked genes he characterized were also innate immune system genes. We both looked at one another and thought: “Antimicrobial peptides and the innate immune system are the way to go.” Then we did a bunch of experiments that went nowhere. It turns out that several classical methods used to study antimicrobial peptides don’t work for amyloid beta.

In some cases amyloid beta’s ability to target certain microbes is more than 100-fold stronger than penicillin.

Then I went on vacation with my six year-old son to the White Mountains. James Kirby of Beth Israel Deaconess Medical Center had invited us, and the trip gave me the opportunity to get his feedback. He helped us develop assays to correctly test amyloid-beta’s antimicrobial activity. Key to this correct assay preparation was the fact that amyloid beta had to be in its more oligomeric forms (the biologically active form of the peptide). Once we got these assays right, we immediately found that amyloid-beta had very potent antimicrobial activity. In some cases its ability to target certain microbes is more than 100-fold stronger than penicillin.

Me: Wow very interesting. Can you explain a bit more about what it means for amyloid beta to have “oligomeric forms”?

Amyloid-beta is the chief name for a peptide that self-associates to build multiple differently shaped molecules, each with its own molecular structure and activities. Small proteins that can self-assemble and generate diverse structures like this are sometimes called “lego peptides.” Amyloid-beta assemblies are called “oligomers.” Each particular oligomer structure has unique antimicrobial activity. In this way, amyloid-beta spontaneously generates a population of diverse oligomers able to target a broad spectrum of pathogens, and even microbial toxins released during infection. This turns out to be key for amyloid-beta’s effectiveness as an antimicrobial peptide in the brain. Oligomers can target many different microbes while non-oligomeric amyloid-beta is only effective against a limited range of pathogens. This strategy is so effective that we have not been able to find a pathogen that oligomeric amyloid-beta can’t inhibit to some degree.

Me: So these oligomers are a little like antibodies? In the sense that the immune system creates a range of antibodies in response to an infectious threat, each with the ability to target different species/strains of microbe(s)? 

Yes, they are like a cheap man’s antibodies. But antibodies need whole cells in order to be produced and are metabolically complex and expensive to make. Amyloid beta’s ability to form into a wide range of different oligomers is a simpler, metabolically cheaper, and far more ancient immune strategy. Even very ancient animals like jellyfish (which are 500 million year old primitive multicellular organisms) have this ability to create antimicrobial peptides that recombine to increase molecular diversity.

Moir (right) with colleague Rudy Tanzi (left) (Image: Jon Chase/Harvard Staff Photographer).

What’s hard to believe is that many researchers still regard amyloid-beta’s generation and activities as an ‘accident’ and continue to develop therapeutic strategies around this idea. This assumption was reasonable back when amyloid-beta was discovered in 1984. The peptide was thought unique to the human brain and believed to be only generated under the disease conditions found in Alzheimer’s. In addition, amyloid-beta is made by cutting the peptide out of a larger precursor protein that is embedded in cell membranes. No other peptide was known to be generated this way, so it was considered something that must only occur under disease conditions.

However, it’s been known for over two decades that amyloid-beta is continually made in the normal brain throughout our lives – and not just the human brain. All vertebrates make amyloid-beta in their brain, most of them the exact same peptide as we make. Furthermore, the mechanism that generates amyloid-beta is now known to be a common cellular pathway that is involved in making many different important normal peptides. But, the assumption that amyloid-beta is junk persists, even though the key assumptions underpinning this idea have been disproved. I still frequently hear the ‘amyloid-beta is junk’ idea justified by an old argument that is demonstrably wrong. It goes something like this: “Since Alzheimer’s occurs in individuals past reproductive age (that is post-menopause) there is no evolutionary pressure to remove it from the genome.”

Sounds plausible, until you start looking beyond the narrow precepts of the Alzheimer’s field and consider amyloid-beta in a broader biological context. Only three species of animals undergo menopause: us, dolphins, and pilot whales. Most other animals reproduce to within 1-2% of when they drop dead – and many social animals live well into old age. And yet, all vertebrates make amyloid-beta, with over 60% making the exact same form of the peptide we do. Moreover, they have been doing this for over hundreds of million of years! (The human amyloid-beta gene is expressed in coelacanths, a family of ‘living fossil’ fish that date back over 400 million years). In fact this data would suggest the exact opposite of the ‘amyloid-beta is junk’ argument – it supports the idea that amyloid-beta contributes to survival fitness throughout life in all vertebrates.

Also, anyone with grandparents knows that they typically play an important part in helping successfully raise children. In biology it’s known as the “grandmother hypothesis.”  Simply put, the grandmother hypothesis says that menopause allows aging mothers with an increasingly high risk of death from childbirth to stop direct reproduction and pass on their genetic material to future generations by helping raise their grandchildren. Grandparents are not reproductively irrelevant!! Any gene that intrinsically causes dementia and negatively impacts grandparent survival and their valuable store of accumulated experience, is going to be selected against. The gene is going to change or be eliminated. But amyloid-beta is 100% conserved from 400 million years ago.

The human amyloid-beta gene is expressed in coelacanths (Image: American Museum of Natural History).

What all this is telling us is that amyloid beta must actually be very important. It must be at the heart of important biological processes.  Indeed, human amyloid-beta is one of the top most conserved proteins in all of biology (as far as I have been able to ascertain, second only to the protein ubiquitin).

Me: Yes it seems clear that amyloid beta must have an important role in human health and disease. How has your research moved forward based on that possibility?

We set out to test whether amyloid beta has antimicrobial activity. We found that if you add amyloid beta to a microbial broth it will inhibit or straight out kill a range of microbes. We published a paper with those and related results in 2010. It was greeted with mixed reception. Some researchers (mostly young researchers) were enthusiastic about it. Older researchers not so much. From these detractors we got feedback like, “That’s fine, but battery acid can kill organisms in a test tube too.” In other words, they were critical that the experiments were done in a test tube and not in a living organism. For most antimicrobial peptides, demonstrating potent microbial killing activity in a test tube is enough to establish identity (it’s a very difficult activity for a protein to pull off), but it’s not an unreasonable critique. Showing protective activity in a living animal is the gold standard for confirming a protein is an antimicrobial peptide.

So we set out to show that amyloid-beta could have antimicrobial activity in living organisms. We tested its activity in genetically modified mice, fruit flies, nematode worms, and cultured cells. The work took us four years to complete. Our keystone finding was that the ‘plaque’ amyloid-beta generates is as important as the peptide itself. These amyloid-beta “plaques” directly entrap and neutralize microbes. Then, by chemically generating a burst of oxygen radicals – bleach in lay terms – they destroy the trapped pathogen. Just to be on the safe side, the plaque remains intact in the brain, entombing forever any microbe that may have managed to survive. This “entrapment” mechanism is not unique to amyloid beta. For example, alpha-defensin 5 and other human antimicrobial peptides create amyloid structures that function similarly.

So to summarize, expression of amyloid-beta protects cultured cells and nematode worms from lethal infections. In unpublished work we have also confirmed that amyloid-beta also protects against infection in fruit flies. In mice, the most important animal infection model, over-expression of amyloid-beta is protective against bacterial and viral encephalitis (brain inflammation driven by infection). Finally, if you “knock out” mouse (murine) amyloid-beta the mice develop increased susceptibility to encephalitis. So what we now have is convincing data from animal models that that amyloid-beta functions in humans as an antimicrobial peptide.

Me: Has it been hard to advance these findings?

One side of the story has gotten the most attention. If amyloid-beta is an antimicrobial peptide, one plausible inference is that Alzheimer’s is caused by an infection or infectious processes. I’m kind of agnostic about that implication, in that Alzheimer’s could also be a disease where immune pathways have gone wrong. There are a number of examples of ‘sterile inflammatory’ disease in which an immune pathway has become dysregulated and pathological. No pathogen involved.

Alois Alzheimer

But having said that, there’s mounting circumstantial evidence suggesting that infection plays a role in the disease’s etiology. The first guy to support that was Alois Alzheimer himself. In the 1970s-80s many Alzheimer’s researchers thought infection played a central role. But the discovery of amyloid-beta in 1984 (ironically) shifted the focus away from infection. Amyloid-beta was assumed to be all bad, bad, bad, and its accumulation blamed for the disease. It’s a simple explanation to a complex problem, which made it attractive. But it’s increasingly at odds with emerging data. The drug company Merck’s last Alzheimer’s drug lowers amyloid beta levels but did not slow the disease.

Amyloid-beta alone does not give you Alzheimer’s disease. You also need inflammation and tauopathy, another pathology in brain. It may still be effective to control amyloid-beta if you get it early enough in the disease process. Controlling amyloid-beta may help slow the cascade of events that promotes the neuroinflammation that ultimately kills neurons in Alzheimer’s. But, the question remains: what is driving the deposition of amyloid plaques in the first place? Old models hold that amyloid-beta does it because it’s catabolic junk with an unfortunate propensity to form functionless plaques that induce inflammation. But…could plaques and neuroinflammation in Alzheimer’s actually be an immune response to a genuine immune challenge from microbes in the brain? Perhaps. If it is, then targeting microbes in the brain may be a better way to go. What is clear is that more data on the role of microbes in Alzheimer’s etiology is needed. At the moment the “amyloid is bad” idea continues to dominate and most academic efforts are still focused on this model.

Interestingly, we may be seeing the beginnings of a shift in the way “Big Pharma” is looking at Alzheimer’s. Their costly drug failures seem to be making them open to exploring alternative models, including the “antimicrobial protection hypothesis of Alzheimer’s” – which is what we are calling this new emerging model of the disease. For example, “Big Pharma” is exploring if neuroinflammation in Alzheimer’s can be dampened down independent of amyloid-beta production or plaque deposition. My colleague Rudy Tanzi has discovered that the gene CD33 is a big on/off switch for immune cells in the brain. Maybe switching the gene off could help patients manage early Alzheimer’s symptoms. Most current first-line anti-inflammatories wouldn’t have the same effect since they tend to target the adaptive immune system. Antimicrobial peptides and amyloid-beta are part of our much more ancient and primitive innate immune system (the “front-line troops” of the immune response). Companies are now pursing CD33 as a possible drug target.

Dr Doo Kim’s lab in research Unit at Massachusetts General Hospital has created a special three-dimentional human neuronal cell culture system that’s dubbed “Alzheimer’s in a dish” by the media. The system helps us scan innate immune system drug candidates. The technology accelerates drug screening and reduces cost more than 10-fold compared to conventional approaches. It should allow new potentially useful drugs to be identified much faster.

Me: Interesting. But I’m confused. Do you think amyloid beta should be removed in patients with Alzheimer’s (despite its antimicrobial activity)?

Maybe not removed, but it’s certainly a good idea to control it. Amyloid beta may be a little like cholesterol – heart attacks are exacerbated if cholesterol is in the wrong place at the wrong time. But if you remove cholesterol completely, other serious health problems arise. So as was done with cholesterol targeting therapies, I think the first goal should be to better understand what amyloid-beta is actually doing in the brain and develop strategies accordingly. That would mean reducing bad effects while preserving amyloid-beta’s role in immunity.

What may be happening is that initially, amyloid-beta rises to do battle in cases of brain microbiome dysbiosis (imbalance). Part of this response is amyloid-beta induced inflammation. But, prolonged activation of innate immune inflammation by amyloid-beta leads to tissue damage and neurodegeneration.

One thing is that any treatment aimed at managing amyloid beta would work best if administered in a preventative fashion. Again it’s a little like cholesterol and statins. After a third heart attack it rarely helps to give a patient statins. You would want to begin treating 10-20 years ahead of time to prevent this. But with amyloid-beta in Alzheimer’s we currently have no effective assays to predict who will get the disease and when to intervene. You can’t give a drug to everyone over 65.

Me: Wouldn’t it make the most sense to just target whatever infection(s) are causing amyloid beta to be produced in the first place?

Well yes, a longterm solution may be vaccination against the microbe giving you trouble. But here’s the thing about that: there are research groups that have been pushing the role of infection in Alzheimer’s for a long time, but different pathogens are identified in their studies. Herpes simplex virus 1 is a common candidate, but also chlamydia pneumonia. This suggests there is no single pathogen driving the illness. Instead, many different bugs may be involved in Alzheimer’s. For example, our studies have found that amyloid-beta has strong antimicrobial activity against the herpes viruses and these viruses are linked to increased plaque deposition, However, herpes virus are only detected in about 60% of Alzheimer’s cases. What about the other 40%?

So it’s clear that we’re not dealing with a “classical” infection.” The findings don’t support Koch’s postulates: the idea that only ONE microbe can cause ONE disease. But we’re moving away from this “classical” infection model. We’ve been taking a close look at what microbes are found in the brain and started what we are calling the ‘Brain Microbiome Project’.

Me: Your lab is also studying the brain microbiome!?

Yes. We’ve been scanning brains as part of collaborative work with Mt. Sinai. And we have found that even “non-sick” humans harbor over 200 organisms in the brain. Those numbers don’t even include the virome (viruses). And we know that a bunch of herpes viruses can also survive in the brain. There’s even vertical transmission of certain viruses in the human genome.

So infection in Alzheimer’s may be similar to what we’re seeing in conditions like Crohn’s disease. In Crohn’s the bowel is disrupted but the entire gut microbiome is involved. It’s not a single pathogen but disruption of a whole microbial community. Within that framework certain key pathogens may “push” the community out of balance and contribute to disease more than others. In Crohn’s it’s where the microbiome meets the innate immune system that things go wrong and host pathologies arise. That’s where the problem in Alzheimer’s may also lie: at the interface between microbes and a foot soldier of innate immunity – amyloid-beta.

Going back to the brain microbiome: the microbes don’t just sit there. They cooperate, they’re competitive, they interact with the host and each other: it’s a true microbiome. What this means for Alzheimer’s is there could be general dysbiosis (imbalance) of the brain microbiome. There’s a normal brain microbiome, but in Alzheimer’s something may go “out of whack” and some of the bugs go bad (they become bad players). It could be compared to ulcers. Ulcer formation is linked to the bacterium H. pylori. But H. pylori is actually also a member of the normal gut microbiome. That means H. pylori contributes to ulcers under certain negative conditions. This is tentative, but in Alzheimer’s maybe the herpes viruses also go out of control. That is the model we are exploring at the moment. 

The brain microbiome and gut microbiome communicate via the Vagus Nerve

Linked in with that model is the gut microbiome because the gut microbiome and the brain microbiome communicate a lot via the vagus nerve. There’s lots of traffic, with bacteria in the brain/gut talking to one another via this highway all the time. Some products of gut fermentation like Short Chain Fatty Acids (SCFAs) literally travel the Vagus Nerve (physical translocation). Immune cells in the brain need these gut microbial SCFAs to mature correctly. Conversely, certain bacteria in the gut live exclusively off chemicals generated in the brain that are transported to the gut. Vagus Nerve traffic may include bacterial signaling molecules called quorum sensing molecules. In this sense microbes in the gut and microbes in the brain may be “talking,” and possibly reaching decisions about what to do next.

So, what’s going on in the brain can have dramatic effects on the gut. But, the gut microbiome can also affect neurological functioning. For example, the disruption of the gut microbiome is now linked to depression – it’s a two-way axis.

And remember, because amyloid-beta can form a vast number of oligomers, it’s able to react against an large range of pathogens. So right now amyloid beta’s activity leaves open a big question mark as to the exact nature of the infection it may be targeting in Alzheimer’s or related conditions. Which means that vaccination against one pathogen in Alzheimer’s (that I mentioned as a possibility before) might prove too simple an approach. Instead, we may want to ask “Can we modulate disease progression by manipulating the microbiome and/or the gut/brain axis.”

Me: You’ve been studying amyloid beta’s antimicrobial activity against HHV6. Where is that study?

Yes, we’ve tested amyloid beta’s activity against herpes simplex virus 6 (HHV6). There are no good animal models of chronic HHV6 infection, so we are using the “Alzheimer’s in a dish” system to look at this at the moment. Each chip is like a little human brain, that is basically thinking and shooting signals back and forth. This makes the experimental model more human-like and allows for better testing. We found that HHV6 induced a large amyloid burden in the “Alzheimer’s in a dish” system within a day. Key to our findings is that these herpes viruses are “low and slow” microbes whose pathogenic activity ramps up with opportunity. This makes HHV6 extremely effective at seeding amyloid beta deposition over time, even over the course of decades. We hope to get funding from the Cure Alzheimer’s Fund to develop a Alzheimer’s disease mouse that can be infected with human HHV6 (the mice need to be ‘humanized’ for a receptor critical for HHV6 infectivity).

But you can’t formally read about the results of that study at the moment. We submitted a paper with the findings to the journal “Cell.” The journal insisted on publishing an online pre-print before the paper was formally accepted. That pre-print was critiqued by researchers who reject a role for infection in Alzheimer’s etiology – not that our study actually claimed that. It showed strong data that amyloid-beta protects against herpes in the lab. Then, Cell rejected it after 6-weeks. The situation has hurt that study tremendously. While I understand that scientific journals are motivated by a genuine belief that expediting dissemination of new and exciting data is a good thing, I would caution other research teams not to allow a pre-print to go up before the study is accepted for publication. It can go disastrously wrong and it’s not worth it. Not worth it at all. We are submitting the findings to another journal.

Having vented a little on this… I have to say that healthy skepticism is key for advancing science. My problem is not criticism – throughout my career legitimate concerns and criticisms have been invaluable for refining our ideas and showing the way forward. My problem is when strong data with no obvious flaws are rejected out of hand because they do not fit current dogma and are dismissed for perfunctory reasons.

Phew! It’s sure hard to get new findings published. With that in mind, how do you get funding for your research?

This past October, Moir and Tanzi spoke about infection/Alzheimer’s at this “Cure Alzheimer’s Fund” Symposium.

NIH funding is hard for us to get – they are somewhat risk adverse and typically fund studies that explore prevailing ideas: “Evolution not revolution.” Fortunately, we are supported by some foundations that are willing to take risks. One is the Cure Alzheimers Fund in Boston, whose founders include successful Venture Capitalists. They are used to high risk ventures. For them, if only 50% of their funded projects have a successful outcome that’s a good result – it’s much better than the success rate among new businesses! I also get funds from Good Ventures, which is part of the Open Philanthropic Project out of California.

Me: Have you read some of the recent studies that have detected a range of fungi in Alzheimer’s brains?

Yes. And amyloid-beta is strongly anti-fungal. But of course there are fungal communities in healthy brains too. The organisms we’re finding in the human brain are incredible. For example there are amoeboid worms! Up to 20% of humans harbor toxoplasmosis (the Toxoplasma gondii parasite) in the brain. We’ve even detected another worm in some of our brain samples that was previously only thought to infect dogs. Remember that the nasal bulb is a primary source of entry for these and other microbes. Interestingly, efferent nerves from the nasal bulb trace straight back to brain areas where amyloid-beta formation starts.

Have you seen the Lund University study showing that PrP (prion) protein is also a potent antimicrobial peptide?

Yes. And we’ve tested PrP’s activity in our own lab. It’s a strong antimicrobial peptide. But it goes beyond that – amyloid creation happens in a range of human inflammatory conditions. Diabetes is an amyloid disease (in both type 1 and type 2 diabetes amylin is created in the pancreas). At high levels, this amylin is toxic to pancreas islet cells and highly pro-inflammatory. But it’s also one of the most potent antimicrobials we have ever tested – it’s able to target most pancreatic pathogens, including E, faecalis, a common cause of pancreatitis. There’s also an amyloid generated in the heart that is linked to heart disease. So the potential importance of recognizing amyloid can play a normal and protective role in immunity has legs well beyond just Alzheimer’s.

Me: Do you talk to other research teams studying these other forms of amyloid?

Yes. We have multiple collaborations going on around the world. In fact I collaborate so often with other labs that I spend half my time on the phone. There’s both a collaborative and competitive atmosphere that helps drive things along. I am delighted we are no longer alone in pursuing this line of investigation.

That being said, most research teams aren’t thinking this way. And for many of these teams, accepting amyloid-beta’s role as an antimicrobial peptide is like trying to turn around an 800 pound gorilla on a dime. It’s a paradigm shift, so it will likely take years for the focus to shift.

For example, when we tried to publish our 2010 paper on amyloid beta’s antimicrobial activity, the peer review process was extremely frustrating. We submitted the paper to the journal “Nature.” From the start the editors seemed hesitant about publishing the findings. Then they consulted with an Alzheimer’s “expert”, who it seems from comments didn’t even bother to read the paper, but rejected it anyway. The most recent paper in 2016 was rejected six times without review. The reviews we did get from one top-tier journal were some of the most appalling I’ve ever read in my life. One reviewer kept asking why we did not see amyloid in our control worms – the ones that DO NOT express amyloid-beta.

After decades of rejection, Barry Marshall and Robin Warren won the Nobel Prize (Picture: Reuters.com)

Me: That is extremely frustrating. I find it interesting that the scientific community frequently references the story of Barry Marshall and Robin Warren. The two researchers discovered that h.pylori bacteria plays a key role in driving ulcer formation (during a time when ulcers were believed to be caused only by stress and spicy foods). When they first presented these results, they were ignored and in some cases even mocked by other research teams. It took decades for their new finding to be taken seriously. Then, in 2005 they were awarded the Nobel Prize. The story is often referenced as though the scientific community has learned from this experience and is now more open minded. But when I look at how your work has been received thus far I’m not sure I see much progress.  

Yes. Also, Barry Marshall was my old microbiology instructor when I studied at the University of Western Australia. He did struggle with recognition for a long time. There are all sorts of urban legends about how he kept the research going in the face of the wall they kept butting up against.

Me: True. And final question: What studies are you planning to do next?

In a next study, we hope to look at the actual microbiome in the brain. Then remove amyloid plaque from this tissue and see if we can identify the exact microbes the plaques have trapped. We’ll do this by seeing if we can recover the genetic material of the seed microbe.

Me: That’s amazing. I can’t wait to see what you find. I’m going to let you go because I want you to get back to work immediately:)

 

Cholesterol, fat, and human metabolism: a microbiome-based paradigm shift

December 15th, 2017 by Amy Proal

At the age of 64, after a morning playing golf, president Dwight D Eisenhower had his first heart attack. As Pulitzer Prize winning author Gary Taubes describes in his book “Good Calories, Bad Calories” Eisenhower’s heart attack “constituted a learning experience on coronary artery disease (CAD).” After the event, his doctors, considered the top experts in the field, gave the public a “lucid and authoritative description of the disease itself”, followed by twice-daily press conferences held on the president’s condition. Soon, most of America, particularly middle-aged men, became intently aware of dogma promoted by these “experts”: the notion that CAD is caused by eating foods high in fat and cholesterol.

Like much of the rest of the nation, Eisenhower began to avidly lower his fat and cholesterol intake. Yet this plan of attack was counterintuitive for Eisenhower who, before his heart attack, had none of these supposed risk factors connected with CAD. His blood pressure was only seldom elevated, his weight throughout his life remained around 172 pounds (considered optimal for his height). His total cholesterol was below normal – his last measurement before the attack was 165 mg/dl, a level that heart disease specialists today consider safe. He had even quit smoking six years earlier in 1949.

President Eisenhower after his first heart attack.

After gaining four pounds, the ex-president reduced the amount of food he ate for breakfast, then eventually sacrificed lunch completely. His doctor was mystified at “how a man could eat so little, exercise regularly, and not lose weight.” Eisenhower then renounced butter, lard, and cream. But despite these additional dietary changes his cholesterol levels began to rise. “He’s fussing like the devil about cholesterol,” wrote his doctor. “He has eaten in the last week only one egg, one piece of cheese.” It got to the point where Eisenhower’s doctor started to lie about his cholesterol levels in order to keep the president calm. At one reading, Eisenhower was told his level was 217 when it was actually 223. On his final day in office, he was made to believe his cholesterol was 209 when in reality it had soared to 259. Finally, in 1969 at the age of 78, Eisenhower died of heart disease. By that time he’d had six other heart attacks.

We now know that the medical experts advising Eisenhower and the American public were wrong. In CAD, lipids, or fatty molecules, do accumulate in the arteries of patients with the disease. Indeed, CAD results from atherosclerosis: a gradual hardening and clogging of the arteries due to lipid accumulation. However, a growing body of research demonstrates that these lipids are not sourced from dietary fat and cholesterol. As Taubes describes it, the long held dogma about heart disease, in which cholesterol clogging the arteries and excess body fat are viewed as culprits, “as though the fat of a greasy hamburger were transported directly from the stomach to the artery lining” is no longer support by scientific evidence.

“Consensus thinking” led to incorrect CAD guidelines

It’s worth reading Taubes’ full book to better understand the scientific climate that, over the past decades, incorrectly linked to heart disease to dietary fat and cholesterol. For one thing, regulatory bodies were attracted to the simplicity of the “dietary fat/cholesterol clogs arteries” disease model. It was easy to communicate and implied that basic nutritional guidelines could prevent the illness. The model was also promoted at numerous “consensus” conferences: large meetings at which evidence to the contrary was often blatantly excluded.

For example, the largest diet-heart trial ever carried out in the United States was not included in medical or political debates about the best diet for the America public. Because the results opposed what was becoming the consensus view on diet and CAD, they went unpublished, (they were later published in a small cardiology journal that very few people read). The trial, which included 9,000 residents of various mental hospitals found that men on a low-fat diet had a slightly lower risk of heart attacks, although women did not. Overall, patients who had eaten a low-cholesterol diet were associated with a greater risk of heart disease.

Ancel Keys on the 1961 cover of Time Magazine. There, he advanced the idea that dietary fat “clogs the arteries”

A scientist named Ancel Keys played a central role in perpetuating the belief that dietary fat/cholesterol clog the arteries. He famously linked dietary fat to heart disease after studying seven distinct populations around the world who ate diets relatively low in fat and also seemed to have a lower incidence of CAD. However, researchers at the University of California, Berkeley later found that Keys had chosen only six countries for his comparison though data was available from 22 countries. When all twenty-two were included in the analysis, the link between fat and heart disease vanished.

In fact, Keys theory implicating diet as the cause of heart disease appeared on the cover of Time Magazine in 1961 despite the fact that at the time, only two studies had directly tested the connection. One of these studies actually proved Keys wrong. It was a British trial, in which the fat content of the meals of a group of men who had previously suffered from heart attacks was reduced to 1/3 of its previous level. A control group continued to eat a normal diet. After three years the average cholesterol levels dropped from 260 to 235, but the recurrence of heart disease in the control and experimental groups was essentially identical. “A low-fat diet has no place in the treatment of myocardial infarction,” the authors concluded in 1965 in the Lancet medical journal.

Recent studies continue to disprove the “dietary fat/cholesterol” model  

Lipids are “fatty molecules”

Despite studies like that described above, the “dietary fat/cholesterol clog arteries” model for CAD became so popular that debate on the topic continues in 2017. Many physicians and policy makers still issue “heart-healthy guidelines” urging Americans to lower dietary cholesterol/fat despite ever-increasing evidence to the contrary.

For example, a 2016 study by researchers in Finland found that a relatively high intake of dietary cholesterol was not associated with an elevated risk of coronary heart disease. Another meta-analysis found no association between consumption of saturated fats and either coronary heart disease, ischemic stroke, type 2 diabetes, death from heart disease or early death in healthy adults. A different study even demonstrated an inverse association between saturated fat and stroke (i.e. those who ate more saturated fat had a lower risk of stroke).

The authors of the first meta-analysis conclude their paper by stating: “Coronary artery disease pathogenesis and treatment urgently requires a paradigm shift. Despite popular belief among doctors and the public, the conceptual model of dietary saturated fat clogging a pipe is just plain wrong.”

In 2014 Time Magazine reversed its position on dietary fat and CAD

In 2014, even Time Magazine published a cover story reversing its position on Ancel Key’s earlier claims. The article was titled: “Eat butter. Scientists labelled fat the enemy. Why they were wrong.”

So where do the lipids (fatty molecules) driving CAD come from?

This begs the million dollar question: if lipid accumulation in CAD is not sourced from dietary fat and cholesterol, then where do the lipids in arterial plaque come from!? The results of a seminal study by researchers at the University of Connecticut provide a novel “answer” to at least part of this question. Indeed, the team’s findings are so important that they are positioned to change the future of heart disease research.

The team, led by Frank Nichols, found that two important forms of lipid detected in diseased artery walls are created by bacterial members of the human microbiome. More specifically, both lipids (Lipid 430 and Lipid 654) are made by bacteria from the phylum Bacteriodetes: a family of bacteria so common to the human body that they represent 1/3 of bacteria currently able to be cultured from the human intestine.

The team arrived at their results by analyzing lipids in atheroma collected from patients at Hartford Hospital in Connecticut. They identified bacterial Lipids 430 and 654 in the samples, and distinguished them from human lipids by analyzing their chemical structure. Both Lipid 430 and Lipid 654 contain fatty acids with branched chains and odd numbers of carbons (a structure not typically associated with human/mammalian lipids).

In a press release about the study, Nichols stated the following about Bacteriodes bacteria: ”I always call them greasy bugs because they make so much lipid. They are constantly shedding tiny blebs of lipids. Looks like bunches of grapes.”

Recovery of Lipid 430 and Lipid 654 in common Bacteroidetes bacteria.

Interestingly, both Lipids 430/654 were shown to activate TLR2 – a protein at the heart of the immune system’s response towards foreign/infectious threats. This suggests that the human immune system recognizes Lipids 430/654 as foreign, and mounts a sustained inflammatory response to their presence. These results “jive” with the fact that atherosclerosis is now understood to be a serious chronic inflammatory disease.

Nichols and team contend that Lipids 430/654 are created by Bacterioides bacteria in the gut/mouth. Under such conditions, lipids produced by these microbes would travel to the arteries via the bloodstream. However, additional research is needed to confirm that bacteria capable of living directly in the arteries don’t additionally contribute to lipid creation. For example, one study found that periodontal bacteria in the mouth can directly invade human arterial skin cells in culture.

In addition, Stanford researcher Stephen Quake recently reported the presence of thousands of previously undetected microbes in human tissue/blood. Many of these “new” microbes may also produce lipids capable of collecting in the arteries. It’s also worth noting that dead white blood cells (macrophages) are often found near lipids in arterial plaque. Since many intracellular pathogens directly infect macrophages, these dead cells may represent those parasitized by infectious agents.

Bacterial lipids may disrupt human metabolic feedback pathways

The accumulation of bacterial lipids in human arteries helps account for the “plaque” and chronic inflammation characteristic of atherosclerosis. However, bacterial lipids may also promote various forms of disease by interfering with human metabolism.

An extremely important paper by Tony Lam and team at the University of Toronto details feedback pathways that control hormonal signaling between the human gut and the human brain. These pathways help the body regulate appetite control, energy expenditure, and other important mediators of human metabolism. Lam’s paper shows that, in conditions like diabetes, glucose levels are regulated by a complex network of these pathways – many of which sense hormone/nutrient production in the gut and relay this information to the brain via the nervous system.

Gut peptide hormones and regulatory signals are released along the length of the gastrointestinal tract. (Lam et al)

Most of the pathways described in Lam’s paper are too complex to detail here, but the CCK pathway provides a good example of their general function. CCK is a gut hormone released following a meal. It acts on local gut receptors to help the body regulate glucose levels. CCK is secreted in the small intestine in response to fatty acids or lipids. It conveys information about these lipids (amount, composition, structure) to the brain. The brain integrates this information with signals arriving from related pathways and signals back to the gut to adjust glucose levels accordingly.

Under conditions of health, the CCK pathway is tightly controlled by the human body. It is governed by a series of “checks and balances” that help keep glucose and other hormones/nutrients in a normal range. This begs yet another important question: what might happen to the CCK pathway (and related pathways) if foreign, bacterial lipids accumulate in the body?

While there are differences between human and bacterial lipids, the fatty molecules created by both species also share many common chemical structures (molecular mimicry). For example, the Nichols study found that Lipids 430/654 are similar enough in structure to human lipids that they can be “broken down” by the same human enzyme (PLA2).

This means that human signaling molecules may be able to “sense” the presence of bacterial lipids. In the case of the CCK pathway (in which CCK is created in response to lipid concentration) this could significantly change the information the hormone conveys to the brain: in simple terms, the brain would begin to adjust glucose levels based off a sum total of human lipids AND bacterial lipids.

Many of the Bacteriodes bacteria that create lipids are major human pathogens – eg. members of the genera Prevotella, Tannerella, Capnocytophaga and Porphyromonas (a driver of to tooth decay). If the human brain integrates lipids created by these pathogens into the input it uses to adjust glucose levels and other aspects of human metabolism, serious illness may result.

In summary, there are three main ways bacterial lipids can drive human heart/metabolic disease:

  1. Bacterial lipids are created (or travel) to human arteries where they may contribute to formation of arterial “plaque.”
  2. The human immune system reacts to these foreign bacterial lipids, which results in sustained, chronic inflammation.
  3. Human feedback pathways may incorrectly “sense” foreign bacterial lipids, and factor them into signals controlling human glucose levels and other aspects of human metabolism. This would “throw off” or “mess with” these human pathways in a manner that promotes disease.

Imagine the satisfaction of going back in time to tell President Eisenhower about the Nichols and team findings. While preliminary, they suggest that he could probably relax, eat a second egg, and feel a little less crazy. The same goes for millions of patients around the globe, who must “re-learn” basic heart disease guidelines – but in the context of research that may better address the “root cause” of their symptoms.