Presentation - Metagenomic symbiosis between bacterial and viral pathogens in autoimmune disease

Type: Conference presentation
Presenter: Amy Proal
Conference: 7th International Congress on Autoimmunity
Location: Ljubljana, Slovenia
Date: May 2010

An increasing number of viruses have been detected in autoimmune conditions. However, the persistence mechanisms of the human virome are not yet fully understood. Recent studies demonstrated that persistent bacterial species such as Mycobacteria and Borrelia are able to suppress expression of TACO, TLR2A receptor which is expressed on the surface of certain cells and recognizes native or foreign substances and passes on appropriate signals to the cell and/or the nervous system., and key endogenous antimicrobials by dysregulating the VDRThe Vitamin D Receptor. A nuclear receptor located throughout the body that plays a key role in the innate immune response. nuclear receptorIntracellular receptor proteins that bind to hydrophobic signal molecules (such as steroid and thyroid hormones) or intracellular metabolites and are thus activated to bind to specific DNA sequences which affects transcription.. The suppression of these endogenous antimicrobials allows the persistence and proliferation of opportunistic viral pathogens. Thus, any one autoimmune disease state is likely due to the gradual accumulation of a unique mix of both persistent bacterial and viral genomes. The majority of these genomes have yet to be fully characterized, leaving large gaps in our understanding of the full nature of the microbes that may contribute to autoimmune disease. Some autoantibodies have already been associated with pathogenic genomes and as more species are characterized, we should expect this trend to accelerate. Over the past seven years, we have observed the effects of an experimental therapy for autoimmune disease that uses the VDR agonistA substance such as olmesartan (Benicar) or 1,25-D which activates the Vitamin D Receptor and transcribes the genes necessary for a proper innate immune response. olmesartanMedication taken regularly by patients on the Marshall Protocol for its ability to activate the Vitamin D Receptor. Also known by the trade name Benicar. to prime the innate immune system to kill chronic pathogenic bacteria. Interestingly, upon restoration of VDR activity, the viral titers of patients on this treatment often drop in conjunction with bacterial death. This supports the hypothesis that components of the human bacterial microbiotaThe bacterial community which causes chronic diseases - one which almost certainly includes multiple species and bacterial forms. prevent the innate immune system from effectively targeting persistent viral pathogens. It also suggests a new therapeutic avenue by which olmesartan apparently enables the immune system itself to mitigate viral virulence.

It's great to speak here, and be in Slovenia. I'm going to just jump right in… Today I'm going to discuss how metagenomic research can advance our understanding of infection and autoimmunity. The term metagenomics refers to [the study of] the collective genomes of different microorganisms. And that's what I'm going to be discussing today - how, in the human body, many different microbial genomes can combine to cause a disease state.

For example, this chart shows the composition of three important phyla that persist in the skin.¹⁾ But the blue bars correspond - show the composition of the phyla in healthy skin, whereas the red bars show the composition of the phyla as detected in 13 samples taken from individuals with psoriatic lesions. So, clearly the communities of bacteria in healthy and psoriatic skin are very different.

So, how are these bacteria detected? Well, instead of just using cultivation techniques, standard cultivation methods, to find the bacteria, the researchers used 16S RNA sequencing. They identified over 1,925 clones in the skin.

And similar techniques - these are techniques that also rely on identifying bacteria by characterizing their DNA - such as shotgun sequencing, pyrosequencing, and single cell sampling are revolutionizing the field of microbiology right now. And they're actually opening a door to a tremendous era of discovery.

For example, in the psoriasis study, 84 of the clones likely represented novel species never before known to persist in the skin. And these tools allow us to study microbes in the tissues in which they persist.

So, over the past few years, thanks to these molecular tools there have been so many novel microbes discovered in the human body. And in fact, there have been so many that we now realize that just a fraction of these microbes can be successfully cultured if we would only use standard laboratory methods.

To emphasize my point, I've put together this highly scientific illustration. And this is, for example, according to current NIH estimates, how many microbes you'd likely see here if we just used standard laboratory tests to look at this group of hypothetical rascals. So you can understand how, before the advent of molecular technology, when all these guys in the background were not detectable, the notion that the human body was relatively sterile became commonplace. But today we realize that there are thousands more genomes in Homo Sapiens, and actually most of them have yet to even be named and characterized.

Well, that's why the NIH is currently running the Human MicrobiomeThe bacterial community in the human body. Many species in the microbiota contribute to the development of chronic disease. Project, and it's a correlate to the Human Genome Project, where they are funding essentially the top genomic centers in the country to continue studying the differences in populations between bacteria and other microbes in health and disease.

Well, with this in mind, it's entirely possible that in autoimmune disease the antibodies are not being created in response to self but are instead being produced in response to these pathogens.

Well, even tissues with the highest reputation for sterility such as, let's say, the amniotic fluid, are actually filled with a diversity of bacteria. In 2008 DiGullio and Relman at Stanford published a paper showing the presence of 18 different bacterial taxa in the amniotic fluid.²⁾ You can see here, on the x-axis, that the number of bacteria was inversely correlated with length of pregnancy. The positive predictive value of PCR for preterm delivery was 100%, which is a number not even heard of in science. So clearly, we're not just dealing anymore with HHV-6 or chamydia pneumonia, but hundreds and hundreds of other microbes. And, these microbes, they can persist in the blood and inside the cells of the immune system. For example, in patients with gastic ulcer disease, h.pylori doesn't just persist in the cells of the gastric mucosa, it also persists in peripheral blood.

Well, in this context, how does chronic inflammatory disease appear to develop? Well, we can examine this question at the molecular level. And, key to the process is that chronic bacteria and viruses can alter expression of the body's metabolites.

Take, for example, the Vitamin D ReceptorA nuclear receptor located throughout the body that plays a key role in the innate immune response. or VDR - it's a type 1 nuclear receptor. Well, it turns out that viruses like Epstein-Barr Virus and bacteria like Mycobacterium tuberculosis have evolved to survive in the same fashion - and that's by slowing activity of the VDR.³⁾ For example, MTb downregulates VDR activity by about a factor of 3.3. And, this slide shows VDR activity being downregulated actually, by almost a factor of 30 in the longer lasting cell lines of B cells infected with EBV.

So, basically - you might ask, why the VDR? Well, colloquially speaking, the VDR serves as a “gatekeeper” to the innate immune responseThe body's first line of defense against intracellular and other pathogens. According to the Marshall Pathogenesis the innate immune system becomes disabled as patients develop chronic disease. - So, it expresses Cathelecidin, it expresses these key endogenous antimicrobials, that the body needs in order to target intracellular pathogens. Cathelecidin - the beta-Defensins - it also expresses Toll-like receptor 2. In addition to that, it expresses at least 913 genes, many connected to autoimmune diseases and cancers. So, by dysregulating and slowing the VDR, Epstein-Barr Virus and Mycobacterium tuberculosis have actually evolved to slow the body's very defense mechanisms that would otherwise be actively working to render them dead.

And note that HIV persists in the same fashion. It completely overtakes the VDR in order to transcribe its own genome.⁴⁾ And, Borrelia (Bb) does as well. Live Borrelia downregulates the receptor by about a factor of 50, lysed by about a factor of 8.⁵⁾

So - this is such a logical survival mechanism on the part of these microbes that it's almost certain that other less well-studied microbes have also evolved to slow VDR activity, or the activity of other receptors involved in controlling the immune response.

Well, these persistence mechanisms result in a snowball effect. When the immune system slows, and a person acquires one pathogen, then it becomes easier for them to acquire yet another pathogen. And the immune system then acquires yet another pathogen. And these pathogens each slow the immune system in turn. And the pathogens - they can be viral, fungal, bacterial… and so on and so on. And this process is referred to as successive infectionAn infectious cascade of pathogens in which initial infectious agents slow the immune response and make it easier for subsequent infections to proliferate..

In the context of successive infection, an inflammatory disease state appears to result from the combined pathogenicity of the sum of microbes that any one person accumulates over the course of a lifetime. As human genes are upregulated or downregulated by components of these microbes, the human body shifts further and further away from its natural state of homeostasis.

And, infected cells increasingly struggle to correctly produce human metabolites in the face of all the proteins and enzymes that are being created by the pathogenic microbes. So, eventually, people either start to develop an inflammatory diagnosis such as one of the “autoimmuneA condition or disease thought to arise from an overactive immune response of the body against substances and tissues normally present in the body” diseases, or they simply begin to present with aches and pains and other symptoms of what is often deemed “normal” aging.

So, for example, over the past years, Epstein-Barr Virus has been detected in so many different proportions in so many disease states. Here's just a sample of conditions associated with EBV. For almost the last century, these results have served as the source of such confusion. But, if we view them through the lens of metagenomics and in the context of successive infection they make sense! Epstein-Barr Virus is just one component of a mix. In some cases it could be a precipitating factor for an autoimmune disease. Or, in other cases, it's just a pathogen that is acquired later on in the disease process, when the body's already dealing with the widespread immunsuppression that can be caused by the hundreds of bacterial pathogens that accumulate in the body. And we now understand that by altering nuclear receptor activity, viruses can aid the survival of bacteria and vice versa.

With this in mind, a “one microbe/one disease” paradigm is no longer viable and treatments for autoimmune disease that aim to eradicate only a single microbe - such as say, our purple friend here (EBV) - will at best succeed in only reversing a very small part of the overall disease process.

Well, it may also not be by accident that the uniqueness with which a patients' autoimmune disease symptoms develop parallels the incredible diversity of the pathogens that can persist in the human body. The following wheel shows how truly related chronic diseases are. Each “spoke” on the wheel represents a published study that's shown a significant statistical relationship between patients suffering from one disease and the next. And that is a lot of comorbidity and a lot of symptom overlap.

So, where do we pick up these pathogens? They're everywhere! They're passed from mother to child during pregnancy, from father to child in sperm, through familial contact, they're in injectable medicines, they're in donated blood. There are many vectors. Consider for interest though, a study came out recently. This is a group that tested for total bacterial diversity of four brands of cigarettes.⁶⁾ And they found fifteen different classes of bacteria and a wide range of pathogenic microorganisms in every single cigarette tested – even the Kools!

Finally, and I want to stress this, the model of successive infection has great implications for the way autoimmune disease should be treated. If microbes - both bacterial and viral - are driving the autoimmune disease state, then treatments that suppress the immune response can best succeed at offering short-term palliation. They offer temporary relief by slowing the inflammationThe complex biological response of vascular tissues to harmful stimuli such as pathogens or damaged cells. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. that would otherwise be generated if the immune system were actually targeting the pathogens. But instead the pathogens remain alive and they're able to proliferate - and they cause the person to become sicker over the long term.

Well, this being said, I work with the non-profit Autoimmunity Research FoundationNon-profit foundation dedicated to exploring a pathogenesis and therapy for chronic disease. and we, over the last six years have been working with a treatment for autoimmune disease that stimulates rather than suppresses the immune response in autoimmune disease. And, key to the treatment is the use of a VDR agonist which turns on - returns on - those key components of the innate immune system that I described before that were so important. And, the results of the treatment thus far are, at least in my opinion, fascinating, because we have patients who are not only feeling better, but present with objective markers indicating significant improvement. My colleagues will be talking more about the treatment in the vitamin D session and translational medicine session in hall C, I think, at 2 - and I really, highly encourage you to attend!

So, even when you leave this conference I hope you keep one important consideration - and honestly, this is a potential paradigm shift that we're dealing with here - in mind. When it comes to the immune responses in autoimmune disease: Don't palliate, stimulate!!

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