All posts tagged autoimmunity

The Epstein-Barr Virus – Could it be Causing Neuroinflammation in ME/CFS?

EBV has been a virus of interest since almost day one in chronic fatigue syndrome (ME/CFS). In fact, at one point, EBV was such a hot topic that ME/CFS was called for a time “chronic Epstein-Barr virus” disease.

Virion EBV

Epstein-Barr virus virions (circular centers). Virions are the form of the virus which infects other cells. EBV dUTPase is released when the process of creating virions is aborted…

While studies have generally failed to find evidence of EBV reactivation, EBV has never fallen out of the picture with ME/CFS and for good reason. For one, it’s entirely possible that researchers were looking in the wrong place to determine if EBV is an issue in this disease.  For another, EBV infection in adolescence or later and the infectious mononucleosis (glandular fever) it produces, is a common trigger in ME/CFS, and is a proven risk factor for multiple sclerosis.

Besides ME/CFS, researchers are continuing to assess the role EBV may play in many serious illnesses including multiple sclerosis (MS), systemic lupus erythematosus (SLE), Guillain-Barre Syndrome, several cancers,  rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), celiac disease, schizophrenia, and others.

Neuroinflammation, of course, is a hot, hot (pun intended) topic in both ME/CFS and fibromyalgia. Recent studies suggest neuroinflammation is present in both diseases and major studies are underway to validate that finding.

Nobody until now, though has attempted to complete the circle, and bring that “original gangster” in ME/CFS – Epstein Barr Virus – and the new guy in town – neuroinflammation – together.  Could EBV be causing or contributing to the neuroinflammation present in the disease?

Some History

Over 10 years of work by an Ohio State University team lead by Maria Ariza and Marshall Williams has been turning the EBV question in ME/CFS on its head. High levels of EBV, they believe, are not the problem in ME/CFS at all. In fact, their studies suggest that EBV may be at its most dangerous in ME/CFS not when it reactivates – but when it fails to reactivate properly.

dTUPase model

The Ohio State University dUTPase continuing NIH grant is in its 9th year.

By the time the impaired immune systems of people with ME/CFS have started knocking down EBV’s attempt at reactivation, the bug has already produced a potentially pathogenic protein called dUTPase. The Ohio State University researchers believe this protein may be wreaking havoc in a large subset of people with ME/CFS.

With the NIH supporting them every step of the way – their continuing grant on dUTPase is now in its 9th year – the evidence that this protein is contributing to ME/CFS (and other diseases) has continued to build.

In 2012, the group found evidence that the immune systems of people in a large subset of ME/CFS patients were indeed battling this protein. Just a year later they showed that even when viral loads of EBV were low, dUTPase could still be triggering a significant pro-inflammatory response. That finding suggested that failed prior attempts to link EBV reactivation to ME/CFS were barking up the wrong tree.

Two years later, they demonstrated that dUTPase was able to make its way into exosomes (now a major topic of interest in ME/CFS), cross the blood-brain barrier, produce major immune effects, and perhaps even promote further EBV infections.

Then a 2017 study added another herpesvirus long suspected in ME/CFS – HHV-6 – to the mix. That study found antibodies to dUTPases produced by both EBV and HHV-6 in almost fifty percent of the ME/CFS patients.  That suggested that the two herpesviruses might even be reactivating each other – a feature found in some very immune suppressed states including organ transplant patients and drug induced hypersensitivity syndrome.

Then again, really significant immune suppression in ME/CFS may not be a surprise. Up to 75% of ME/CFS patients were found to have low numbers of the B-cells designed to keep EBV in check in a recent study.

If the immune system wasn’t having enough trouble, in 2017 the first evidence of an autoimmune process involving EBV dUTPase was found in ME/CFS. Autoantibodies to the human dUTPases (humans produce a dUTPase as well) were found in ME/CFS – at much higher levels than in healthy controls (39% vs. 5%). That suggested that the immune response to EBV and HHV-6 dUTPase may have gone awry in some people with ME/CFS. Their bodies were now attacking their own human dUTPase.

The 2019 Study

In the present study we provide further evidence…. (that) dUTPase protein…could contribute to the development of a neuroinflammatory microenvironment in the brain(s) (of a subset of ME/CFS patients.)  The authors

Epstein-Barr Virus dUTPase Induces Neuroinflammatory Mediators: Implications for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Marshall V. Williams PhD; Brandon Cox ; William P. Lafuse PhD; and Maria Eugenia Ariza, PhD. Clinical Therapeutics March 2019

In 2019, the team took another step. In an earlier study they’d demonstrated that the EBV dUTPase protein could be causing or contributing to the symptoms present in ME/CFS. Since many of these symptoms can be produced by the brain, they next asked if the enzyme could be affecting the integrity of the blood-brain barrier (BBB) and other aspects of neuroinflammation.

There’s a pretty good reason to believe this might be the case. EBV, after all, has been associated with some pretty nasty neurological diseases. The virus loves to hang out in nerve cells and astrocytes, is a risk factor for M.S. and has, in fact, been found scattered throughout the astrocytes and microglial cells in MS patients’ brains.

The Ohio State University researchers plopped the dUTPase protein into a variety of cells and then determined how it affected the expression of genes that play an important role in maintaining the blood brain barrier (BBB) and the functioning of various brain cells (cerebral microvascular endothelial cells, astrocytes, microglia cells).

The big bug’s dUTPase protein turned out to be quite adept at tweaking genes and proteins associated with the BBB and neuroinflammation. It turned on 12 of 15 genes and 32 of the 100 proteins examined in vitro (in the lab) and 34 of the 84 genes examined in mice.

The fact that these genes play a role in BBB integrity/function, fatigue, pain synapses and their functioning as well as tryptophan, dopamine, and serotonin metabolism suggested that this enzyme, in or out of the brain, could conceivably cause widespread problems.

How the Blood-Brain Barrier Works

 

 

All in all, the protein appeared to be doing its best to find a way to get EBV into the brain. That’s perhaps not a surprise given how much EBV loves to hang out in neurons. As EBV dUTPase was down regulating the expression of genes dedicated to producing a tight BBB it was “strongly” inducing the expression of two cytokines (IL-6 and IL-1β) known to disrupt The BBB.

If EBV dUTPase gets inside the brain, it seems almost guaranteed to cause neuroinflammation.  Studies indicate it can trigger microglial cells and astrocytes (star-shaped immune cells in the brain) to produce potent pro-inflammatory cytokines (IL-6, IL-1β and TNF-α). It also prompts astrocytes to produce a substance (PTGS2/COX-2) associated with neuroninflammatory toxicity. Plus it’s able to alter the expression of genes associated with pain (GPR8451 and GCH152) and fatigue (TBC1D153) to boot.

In mice, it altered the expression of genes associated with cognition (synaptic plasticity, learning and memory).  One of the more intriguing findings, given the possible disruption of the kynurenine pathway in ME/CFS, was the protein’s potential to increase synthesis of a potent neurotoxin called quinolinic acid. Genes associated with the metabolism of two of the major neurotransmitters in the brain, dopamine, and serotonin, were also affected.

EBV dUTPase neuroinflammation

If EBV dUTPase has indeed been able to get into ME/CFS patient’s brains it seems almost guaranteed to cause neuroinflammation

All in all, EBV dUTPase is not a protein anyone wants hanging out in their head. It is, however, a protein that could potentially produce a lot of the problems found in ME/CFS.  This study demonstrated that the protein appears to have the capability to make its way to ME/CFS patient’s brains. Determining if it has will take further investigations, however.

It should be noted that the protein and its antibodies (or the autoantibodies to the human dUTPase) are not found in everyone with ME/CFS but the potential subset – ranging from 30% to 60% of those tested so far, is pretty darn large.

Plus, the virus is heavily implicated in the stress response. If you feel like your nervous system is over-reacting to, well, anything (or everything), EBV and this protein could be a factor. Of all the viruses, EBV and the herpesviruses love most to come out and play when one’s system is stressed.

In fact, Ron Glaser, one of the initiators of the EBV dUTPase research effort, demonstrated back in 1991 that EBV thrives in situations of psychological stress. Given the enormous stress people with ME/CFS are under, and the affects the illness has on both axes of the stress response, it makes sense that the virus might be continually trying to reactivate – and spilling it’s toxic protein into the bloodstreams of some people with this disease.

A Good-bye to a Pioneer

Ron Glaser

Glaser was shocked he couldn’t get his ME/CFS grant applications funded at the NIH

Ron Glaser was something of a legend in his own time. With his doctorate in pathology, his EBV citations alone total over 100. All told he published over 300 papers. Glaser co-founded Institute for Behavioral Medicine Research, which under his leadership brought in over 140 million in grant money over 20 years. At one point he was one of the world’s most cited authors.

His memorials mention his impact on the psychoneuroimmunological (PNI) field, his enthusiasm, (and the red and white Corvette he loved). What they don’t mention is that this leader also devoted time to a much neglected field called chronic fatigue syndrome. Glaser, in fact, took the time out of his busy schedule to sit on the now disbanded federal advisory committee for ME/CFS (CFSAC).

I vividly remember talking to him. He was not a man to mince words. An accomplished researcher with a long history of grant success, Glaser was first shocked, and then very angry at the rejections piling up for his ME/CFS grant applications. He just couldn’t understand it. Never in his decades of work had he experienced such a thing.

Stating, ironically, he couldn’t stand the stress (he did look like he was about to burst a blood vessel), he eventually moved on, but not before making his experiences perfectly clear to the federal advisory committee and everyone around him.

Glaser was not happy at not being able to work more in ME/CFS, but the work he did did not go for naught. Glaser first published on EBV dUTPase in 1985 and on EBV and ME/CFS in 1988 and his work lives on in Ariza and William’s studies on ME/CFS today. Check out a memorium to Ron here. 

Marshall Williams – On the Continuing Hunt for EBV dUTPase in ME/CFS

What about the connection between this protein and the presence of infectious mononucleosis/glandular fever in ME/CFS? Do we have any idea if the enzyme is more likely to be found in people who’s disease was triggered by IM or who had an acute, flu-like onset?

That is an excellent question. We are in the process of trying to obtain longitudinal serum samples from an IM cohort who developed CFS as well as age matched patients who had IM but never developed CFS. Hopefully, that may address this question.

EBV dUTPase exosomes

When EBV (lytic) replication is aborted it tosses EBV dUTPase into exosomes (circles with red marks) which, after binding to TLR receptors on immune cells, tells those cells to turn on proinflammatory and other genes (from Ariza, Williams and Glazer -https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069827)

This study demonstrated that this enzyme has the potential to disrupt the BBB and enter the brain – and as added bonus – perhaps helps get EBV into the brain. Is there any way to tell if this has actually happened in ME/CFS?

Not really at this point but maybe in the future. Screening CSF from ME/CFS patients for antibodies to the EBV-dUTPase or HHV-6 dUTPase might suggest potentially the presence of these viruses in the brain.

Exosome research is heating up in ME/CFS. Some anecdotal reports show that exosomes in the blood may be affecting energy metabolism and other functions. Could herpesvirus dUTPases be involved? Is there any more information on exosomes and EBV dUTPases?

We have not looked at energy metabolism but there are some reports in the literature that some herpesviruses including EBV and HHV-6 alter mitochondrial function. There is information concerning EBV products in exosomes but most of these have focused on proteins/microRNAs involved with latency.

What is next for your team? 

We are in the process of submitting a manuscript detailing a mechanism(s) by which the EBV-dUTPase and to a lesser extent the HHV-6 dUTPase alter germinal center function, which could contribute to autoimmunity in CFS patients. We will be continuing these studies as well as those regarding neuroinflammation. (B-cells manufacture autoantibodies in the germinal centers found in the lymph nodes and spleen)

Large NK Cell Study Points to Autoimmunity and Inflammation in Chronic Fatigue Syndrome (ME/CFS)

The observed differences in some of the subpopulations of T and NK cells between patients and healthy controls could define a distinct immunological profile that can help in the diagnostic process of ME/CFS patients, contribute to the recognition of the disease and to the search of more specific treatments. Rivas et. Al. 2018

Problems with natural killer (NK) cell functioning have been like an anchor in the storm for immunologists interested in chronic fatigue syndrome (ME/CFS). While other immune results like cytokines have flipped and flopped all over the place, the NK cytotoxic results have been solid. Almost every study has found that when given the chance to kill infected cells, the NK cells in ME/CFS patients poop out.  (The studies which have not found differences in NK cell functioning have tended not to use whole blood or used older samples – suggesting that something in the blood could be impairing NK cell functioning in ME/CFS.)

Dr. Daniel Peterson, Sierra Internal Medicine and Simmaron Scientific Advisor

The most extensive study – a year-long 2012 study involving Dr. Peterson and Griffith University in Australia – found reduced natural killer cell functioning at all time points. (Peterson has a long history of interest in natural killer cells; he was a co-author of the first study, over thirty years ago, to find deficient NK cell functioning in chronic fatigue syndrome (ME/CFS).)

NK cells are important because they maintain the lines of our initial immune defense, holding the fort, so to speak, until the big guns – the T and B cells- wipe out the infection. – They also regulate the immune response.

Normally our cells signal that they are infected by displaying peptide fragments from the pathogen (using MHC Class 1 molecules) on their surface. NK cells then hunt out and destroy these infected cells. However, some pathogens have learned how to prevent the cells they’ve infected from displaying these peptide fragments.

If NK cells and other parts of the innate immune response can’t hold back the invaders, the pathogens may invade more deeply into the body, potentially causing more problems before the adaptive immune response (T and B-cells) can kick in.

innate immune response

Mast cells, complement, phagocytic cells and natural killer cells man the early or innate immune response

A deficient early response to pathogens would then very likely translate into more symptoms. We don’t know when the problems with NK cell killing got started in ME/CFS, but if they were in place prior to the illness or occurred early in the illness they could have played a role in the inception of ME/CFS as people who have more trouble fighting off a pathogen; i.e. people with more severe symptoms, are more likely to come down with ME/CFS.

Once ME/CFS has begun, the inhibited NIK killing response could mean more trouble removing tumor and infected cells – particularly herpes virus infected cells- as people deficient in NK cells  have trouble fighting off herpes viruses.

NK cells, then, are vitally important, but attempts to identify issues other than cytotoxic killing abilities have been less successful. NK cells come in different types (cytotoxic and regulatory) and the balance of these subpopulations is important. Some studies have found differences in these subpopulations in ME/CFS and some have not.

Many of those studies, however, have been small and used less than stringent criteria for defining ME/CFS. A Spanish group decided to rectify those problems with a more definitive study which examined NK cell populations in a larger study (n=149) with patients who met the Canadian Consensus Criteria for ME/CFS. In order to ensure they captured all factors in the blood that might be whacking NK cells, they used whole blood and analyzed it within 6 hours of collection.

Then they tried to reverse engineer their results to see if a diagnostic test could be developed which simply charted which kinds of NK cells a person had. That was pretty good, but then they went further and asked if people who were worse off had different subpopulations of NK cells or more evidence of herpes virus reactivations (EBV, HMCV).

Association of T and NK Cell Phenotype With the Diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) Jose Luis Rivas,1,* Teresa Palencia,1 Guerau Fernández,2 and Milagros García1,3 Front Immunol. 2018; 9: 1028.Published online 2018 May 9. doi:  10.3389/fimmu.2018.01028

This larger, fresher (quick analysis of blood), stricter (CCC patients only) and more comprehensive study found differences where others had not – and plenty of them.  This group validated – with a high degree of certainty (p = 0.0075) – previous findings of  an increased subpopulation of NK cells (NK CD56++(high)) which, get this, excrete more cytokines (particularly IFN-y), possibly causing more symptoms, but which have low cytotoxic activity.  Because these cells have unusually long life spans and pump out cytokines that cause more T-cell proliferation, higher numbers of them could contribute to autoimmunity and inflammation.

These cells were particularly high in the group of patients whose illness began without evidence of an infection. The Spanish group suggested that activation of the stress response via the HPA axis and raised levels of catecholamines such as norepinephrine (adrenaline) could have triggered the expansion of this potentially autoimmune affecting natural killer cell subset.

No differences were found, however, in the levels of several receptors (NKp46, NKp30, NKp44) that have been found elevated in some autoimmune/inflammatory conditions (Sjogren’s Syndrome, Crohn’s disease) or reduced in chronic infections (HIV, tuberculosis, influenza, etc.).

CD 69

Increased levels of the CD 69 marker suggested autoimmunity may be present in ME/CFS

Reduced levels of a receptor (NKG2C) were very common (p<0.0001) in ME/CFS. When this receptor, which is only found in NK cells, is activated by the presence of virally infected cells, it triggers an expansion of NK cells. Not surprisingly, NK cells become dotted with this receptor in people with chronic herpesvirus and other infections (HCMV, EBV) but ME/CFS patients’ NK cells had consistently lower levels of this receptor than did the healthy controls.  The authors didn’t speculate why this occurred, but it could involve lower levels of infection in ME/CFS – something Ron Davis is finding in his severely ill cohort – or a problem responding to infections that are present.

That second possibility was buttressed by an inverse correlation found between a marker of infection (CD 57+) and the lower expression of a marker (NKp46) which is often reduced in herpesvirus infections. The authors suggested that the scenario found in ME/CFS (increased cd57+, lower NKp46, high NKG2C) could reflect HCMV (cytomegalovirus) reactivation.

Watch Natural Killer T-cells (red) Swallow Up Antigen Presenting Dendritic Cells (green)

 

Increased levels of the CD69 marker (p= 0.011) provided another suggestion that ME/CFS may be an autoimmune/inflammatory disease. This important marker, which is found on many immune cells, stimulates NK cell cytotoxic activity. More importantly, CD69 has been described as a master regulator for autoimmunity in rheumatoid arthritis (RA) through its upregulation of TGF-B – one of the very few cytokines that has usually been found increased in ME/CFS.

A “descent” in T regulatory cells similar to that found in autoimmune conditions such as lupus and RA was also found. Finally, an inverted Th17/T regulatory cell ratio, which is also found in autoimmune conditions like lupus, wrapped up the autoimmune connections found in this study.

Using a mathematical classification model, the group was able to correctly diagnose 70% of ME/CFS patients and healthy controls simply by using the findings from this in depth study of natural killer cell populations.

Conclusions

This large Spanish study of NK cell subpopulations found numerous irregularities in NK cell types in ME/CFS, several of which pointed to issues with autoimmunity and/or inflammation. As in other studies, this study indicates that larger is indeed better when it comes to studying ME/CFS.

The study validated prior findings of an unusually large set of NK cells which produce more cytokines – conceivably causing more symptoms and immune activation – but which are less effective at killing infected cells.  That finding seemed to jive with a picture of highly symptomatic ME/CFS patients who may have trouble fighting off infections.

While no differences were found in the levels of receptors which can be elevated in autoimmune conditions, several other findings suggested that NK cells may be fighting off herpesvirus infections or may be involved in autoimmune/inflammatory processes in ME/CFS.

Finally, using just NK cell subpopulation data, the authors were able to correctly identify 70% of patients and healthy controls, indicating that significant NK cell differences exist. All told, the study identified several natural killer irregularities that could participate in autoimmunity and dysregulate other parts of the immune system.