Is Chronic Fatigue Syndrome An Inflammatory Disease? The 2016 IACFS/ME Conference Overviews Pt II

The immune system's complexity reared its head again at this conference as Dr. Montoya showcased some of the findings coming out of his large immune studies at Stanford.  Montoya's assertions that chronic fatigue syndrome (ME/CFS) is similar to systemic inflammatory response syndrome and should be called an inflammatory disorder were intriguing indeed. It's still, however, hard understand what is going on in the immune system in ME/CFS.This is a long blog; if you just want the main findings a quickie overview is given at the end of it.

IMMUNE SYSTEM

Montoya's huge (584 person!) and impressive immune studies -  the largest ever done in this disease - dominated several presentations.  The studies are bit unusual in that they contained about twice as many healthy controls (n=392) as patients (n=192).  Montoya posted an impressive list of 30 researchers he's collaborating with at Stanford and elsewhere.He spoke of a complex immune situation often characterized by both up and down immune activation, but which strongly suggested chronic fatigue syndrome is an inflammatory disorder.

Cytokine Study

Cytokines are molecules produced by immune cells that regulate immune functioning in many ways. Montoya tested many cytokines (51) but only two popped out in the first run of this study. That was surprising; large studies are particularly good at finding small but still significant differences, but this study found few differences between the ME/CFS patients and healthy controls than some smaller studies.Lipkin and Hornig enhanced their cytokine study results by controlling for duration. The key for Montoya was severity. When he added severity to the picture, the immune findings popped out. In the more severely ill patients a rather eye-popping third of the 51 cytokines tested (leptin, CXCL-1, CXLC10, GM-SF, IFN-Y, GM-CSF, IL-4, IL-5, Il-7, IL-12p70, IL-13,  IL-17F, NGF, TGF-b, CCLI, SCF and TGF-a) - most of them pro-inflammatory in nature  - significantly increased.Montoya proposed that TGF-b, traditionally thought of as anti-inflammatory, may have been acting as a pro-inflammatory cytokine. That cytokine has shown up in several ME/CFS studies before.

Interlude: Cytokine Results Still All Over the Map

The results were encouraging, but cytokine results in this disease are still all over the map. For years researchers have thought they MUST be involved in ME/CFS, but cytokine results have been stunningly inconsistent.For example, while a 145 person Australian study did, like Montoya's study, find increased levels of cytokines (IL-10, IFN-γ, TNF-α), none of those cytokines showed up in Montoya's results.A 99 person study from the Klimas group measuring 16 cytokines found significant alterations in 10 of them (increased - LT-a, IL-1a, IL-1b, IL-4, IL-5, IL-6, and IL-12; decreased - IL-8, IL-13 and IL-15.) IL-4/5-were increased in Montoya's severe ME/CFS group, but IL-13 was decreased in the Klimas study and increased in Montoya's.Wyller’s recent large study of ME/CFS adolescents found no cytokine differences between those diagnosed with the Fukuda criteria cytokine and healthy controls. A Japanese/U.S. study found no evidence that either sleep deprivation or exercise effected cytokine levels as well.The large Landi/Houghton 179 person study of longer duration patients found mostly cytokine reductions instead of increases (reduced levels of IL-7, IL-16, VEGF-a, CX3CLI, CXCL9; increased CCL24). If most of Montoya's group were early-stage ME/CFS patients, that might help explain the differences, but we don't know that they were. (Montoya did state that he is going to filter for illness duration.)The Lipkin/Hornig cytokine study found increased levels of 16 cytokines in early or late duration patients vs healthy controls (IL-1a, IL-1ra, IL-4, IL-12p70, Il-13, CXCL8, TNFα, SFASL, CCL2, CCL3, CD40L, MCP1, TNFSF10, SCF, CFS1, and resistin).  Only three of those (IL-12p70, Il-13, SCF) were found elevated in the Montoya study; thirteen were not.An Australian study that tracked for severity in a different way from Montoya suggested that more severe patients do have higher cytokine levels. It found reductions in IL-1b, and increases in IL-7, IL-8 and IFN-y. Of those, IFN-y was increased in the Montoya study.In a much (much) smaller cytokine study published earlier this year, Dr. Fletcher's study suggested that dramatic shifts in immune functioning may occur over time. IL-a plays an important role in early ME/CFS and then declines. IL-8 levels were abnormally high early on but declined to lower than normal levels after a few years. Il-6 levels were low early on and elevated later. Ironically, the Montoya study didn't find any of these cytokines elevated in his severely ill patients.

Conclusion (?)

Until cytokine results achieve more consistency they’re clearly not going to get traction in the medical world.  The inconsistency seems surprising as most of these studies are from good labs. It’s possible, though, that subsets are mucking up the issue. Filtering for duration is clearly needed, and Montoya’s study suggested that filtering for severity is as well. The Klimas group’s Gulf War Syndrome study suggested that  gender may need to be accounted for as well.Dr. Peterson's atypical patient subset may throw another loop into cytokine results. Peterson's atypical ME/CFS subset group so dramatically affected cerebral spinal fluid results that it had to be excluded from the study altogether.  Could  this group be effecting blood cytokine results as well?Researchers are not going to stop studying cytokines – they’re apparently too enticing – and it's possible that studies underway may help us understand what is going on.  If Lipkin/Hornig can, in their study underway, replicate their cytokine results in different duration patients – that will be something. Ditto with several good day bad day studies underway. If Montoya can duplicate the Lipkin/Hornig duration results that would really be something. Time will tell.It's also possible that cytokine levels per se aren't as important as we might think. Broderick's models suggest that context is key; in the right context a factor can be important even if it's levels are not raised.  His models suggest that treatments targeting just two cytokines might be able to enable ME/CFS patients to exercise again. (See upcoming IACFS/ME treatment blog).Montoya's network analysis indicated that Il-1B - an important regulatory cytokine associated with increased pain - was the most important factor 24 hours after exercise.  That certainly makes sense given what we know about exercise and pain.Another possibility is that cytokines in the nervous system are more important than those in the peripheral blood. It’s thought, for instance, that cytokines must contribute to central sensitivity syndromes (CSS’s) such as fibromyalgia as well, but a similar issue with consistency apparently applies there. Staud has suggested that cytokines probably play a major in CSS, but only within the central nervous system.

No Biomarker Yet - An immune signature that shows up only in the more severely ill gives us clues about the illness but obviously isn't going to work as a biomarker.  But what would happen if Montoya essentially shoved those people into a more severe state by having them exercise? Would adding exercise to the mix make the more moderately ill patients look like more severely ill patients?

Montoya's Exercise Study

Would exercise make moderately ill ME/CFS patients in the throes of post-exertional malaise look like severely ill patients? The answer to that question was no.Montoya’s maximal exercise test produced opposite results from the cytokine study done in patients at rest.  This time, exercise reduced the levels of four cytokines (TNF-a, IL-8, CCL4, ICAM-1) while increasing the levels of only 1 (CXCL-10).Both TNF-a and IL-8 increase during exercise in healthy people, however. The fact that both went down in ME/CFS patients may be notable.  If immune exhaustion is present then perhaps one might expect cytokine levels to drop when the body is faced with an exercise stressor.A 2014 review of exercise studies reported that while exercise does appear to effect the complement system and gene expression and increase oxidative stress in ME/CFS, it does not appear to effect cytokines. Montoya's results suggested the opposite.

Genomics Study Suggests Chronic Fatigue Syndrome is an Inflammatory Disorder

At the Stanford Symposium, Montoya announced that the gene expression results indicated that ME/CFS was similar to a disease called systemic inflammatory response syndrome or SIRS. He repeated that assertion again; this time stating that ME/CFS was a "100% match" to SIRS.  (The abstract was a bit more cautious, stating that the gene expression results were "very similar" to it and similar diseases).The concept of SIRS came out of ten years of work at a Toronto trauma lab by Dr. William Nelson. SIRS is  a kind of cytokine "storm" - a term sometimes used in ME/CFS - which refers to a positive feedback cycle that results in higher and higher levels of cytokines.  SIRS also effects both pro and anti-inflammatory cytokine levels as well.SIRS refers to a state of systemic inflammation after infection or some other insult and can result in organ dysfunction and failure. Intriguingly, given the Australian metabolomic group's suggestion that the metabolomic results in ME/CFS are similar to sepsis, it’s closely related to sepsis.SIRS has other manifestations that some may find familiar. Increased heart rates, lower or higher than normal body temperatures, rapid breathing rates, and low white blood cell counts found in SIRS have also been found in ME/CFS. The rapid breathing rates, by the way, are associated with either increased metabolic stress due to infection or inflammation or may signal inadequate perfusion because of the onset of anaerobic cellular metabolism.Other possible links include fibrin deposition, platelet aggregation, and coagulopathies aka Dr. Berg's findings in ME/CFS some years ago. Dr. Montoya's immense gene expression study almost couldn't have uncovered a more interesting disease to link to ME/CFS.  How serendipitous as well - if this all turns out - that Ron Davis and some members of his Open Medicine Foundation team have done an enormous amount of work on sepsis.How is SIRS treated? In some ways (blood volume enhancement, anti-anaphylaxis drugs, selenium, glutamine, eicosapentaenoic acid, and antioxidants) that can be helpful in ME/CFS.

Epigenetic Modifications Point at Immune System and HPA Axis

Montoya's epigenetic study suggested an infection (or some other insult) had indeed occurred in ME/CFS. Greatly increased rate of methylation in ME/CFS patients' immune regulatory genes suggested some infection or other environmental insult had occurred.Other epigenetic modifications were found to affect HPA axis genes.  Given the strong interaction between the HPA axis and the immune system, it wouldn’t be surprising at all to find that some event had tweaked both the HPA axis and immune genes in many ME/CFS patients. (The Montoya group is currently engaged in a promising HPA axis study.)Other gene groups affected by methylation (epigenetic modification) include genes that play a role in, yes, metabolism.  One gene highlighted in a whole genome polymorphism study has been implicated in lactic acidosis (NUFS7). A polymorphism in this gene, which transfers electrons from NADH to CoQ10, could result in increased oxidative stress and reduced mitochondrial output.

Is Chronic Fatigue Syndrome an Inflammatory Disease?

Finding increased immune activation in severe ME/CFS patients, and with gene expression results a close match to SIRS, Montoya asserted that ME/CFS is an overactive immune disease and proposed that its new name should include the word "inflammatory." Montoya results suggest this, but it's hard to see how any consensus can be reached until we get more consistent results from the cytokine studies (???).

Pathogens

When asked about retroviruses, Montoya suggested there was no cheese down that tunnel. In several of his newsletters Montoya promised "exciting" new findings regarding pathogens but none were presented at this conference.

Allergy Study Reveals Intriguing Subset

Dr. Levine's allergy study was, for me, one of the surprise highlights of the conference. This nice big study demonstrated how valuable a resource the multi-site ME/CFS experts centers are, and how valuable a tightly integrated network of research centers will be.In one of the bigger ME/CFS studies to date, Levine queried 200 patients in five sites regarding the incidence of allergic symptoms/conditions and found that the presence of sinusitis and hives distinguished ME/CFS patients from healthy controls.  (My guess is that the presence of sinusitis is overlooked and understudied in ME/CFS).The fact that having either of those conditions resulted in patients experiencing more pain suggested that an immune process was ramping up their pain levels.   That hypothesis was strengthened when Levine found that this group also had a much, much higher incidence of migraine, tension headaches, back pain, neck pain, and fibromyalgia.  Plus they had more gut and inflammatory symptoms. Something clearly appeared to be driving a pain sensitization process in these patients.What is the tie that binds these findings together? Levine suggested it might be mast cell activation. Plus, Dr. Levine noted that both mast cells and neurons secrete two factors: nerve growth factor and substance P, known to increase pain. Then there's tryptase to consider. A recent study suggested that modification of a tryptase gene could be behind some cases of EDS, POTS, IBS, ME/CFS and FM. Another suggested mast cell activation may be occurring in ME/CFSThis is the kind of study that makes you wonder why the heck it hasn't been done before. The study was surely not expensive, yet it might illuminate much about ME/CFS.  It was funded by the Hitchens Foundation.

POSTER: RNase L Returns? Novel Isoform of Ribonuclease L Shows up in Fibromyalgia

The idea that an important immune enzyme called RNase L had been broken into pieces and was not only no longer working properly but was actually causing channelopathies and other issues raised a great deal of interest in ME/CFS the 2000’s. At some point work on the enzyme stopped but RNase L was not forgotten.In a surprise a Spanish group looked for and found the broken-up bits of the enzyme in fibromyalgia. The results were too variable for the 37 dKA form of the enzyme to be considered a biomarker but they did suggest that a subset of FM patients carried it.Even more surprising was their finding of another broken up bit of RNase L (70 kDa) which was almost totally associated with the FM patients (p<.0001). They’ve create custom-made antibody to identify it and will apparently keep working on it.

PATHOGENS

POSTER: EBV Rides Again

We've heard so much about EBV over the years that we forget what a special virus it is. It's' true that almost everyone has been infected with EBV, and most have no problem with it, but EBV is no walkover.When one is exposed to EBV later in life, it causes infectious mononucleosis (glandular fever) and is associated with several forms of cancer (Hodgkin's lymphoma, Burkitt's lymphoma, gastric cancer, nasopharyngeal carcinoma, central nervous system lymphomas). Evidence suggests that EBV infections result in a higher risk of many autoimmune diseases including dermatomyositis, systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, and multiple sclerosis. Lastly, while hardly mentioned in the medical world (ME/CFS is not even mentioned in the Wikipedia article) but foremost in ME/CFS patients minds, EBV is a well-known trigger of ME/CFS.EBV must have a multitude of tricks up its sleeve to contribute to so many illnesses. The idea that it plays a major role in ME/CFS has risen and fallen over the years. Right now, that idea seems to be more in its descendant phase, but as Dr. Klimas's study shows, it ain't over until it's over; EBV may still very much figure in this disease.Micro RNA's - small bits of RNA - regulate which genes get expressed. It turns out that EBV, tricky virus that it is, encodes viral miRNA's of its own. (EBV was the first virus found able to do this. Given the immense amount of EBV research being done (over 25 studies published in November alone) that was perhaps no surprise.)Peripheral blood mononuclear cells (PBMCs) were collected from ME/CFS patients and healthy controls before, during and after exercise, and various tests were done to assess EBV miRNA's.  Preliminary results suggested that ME/CFS patients’ cells express higher levels of EBV proteins than normal and thus might be more likely to support EBV reactivation.Plus some strange features emerged. The immune cells in ME/CFS tended to be smaller and have less volume (Ron Davis has found something similar). Instead of forming a classic "pump" shape the ME/CFS nuclei take on a puckered and wrinkled look as if they were aged. Plus, when a key immune transcription factor called STAT I gets activated, presumably by the virus, it ends up in the wrong part of the cell - a pattern indicative of viral reactivation.All of this suggests that EBV may be tweaking ME/CFS cells in strange ways and that the virus may still play a part in ME/CFS.

POSTER: A Better HHV-6 Test

It's clear that herpesvirus tests leave something to be desired and Nancy Klimas’ group is attempting to find a way to improve the diagnostic effectiveness of the Elisa test. The current test are provide only  yes-infected or no-not infected answers and are particularly unreliable at the high and low ends of the spectrum.This study, involving Dr. Govindan from Tufts University and four Florida researchers, used various statistical tests to see if they could develop a truly "quantitative” Elisa for HHV-6.The intercept they developed allowed them to accurately stratify patients, and showed that the HHV-6 intercept they produced was negatively associated with physical functioning; i.e. the higher the intercept - the worse the ME/CFS patients physical functioning was. This suggested that a) HHV-6 does contribute to the symptom burden in ME/CFS, and b) that this new test could aid doctors in determining when to apply antiviral therapies.

POSTER: Enterovirus Brain Infection Found

Dr. Chia's work to get the medical world to take enterovirus infections in ME/CFS seriously continues. He gave a workshop on enteroviruses and seemed to be in demand; every time I saw him he was engaged in conversation with a group of people.His poster highlighted the possible effects of enteroviruses in the most dramatic way. It told the story of a young man who first developed gut problems and then severe ME/CFS. Tests for herpesviruses were normal, but his Echovirus antibody levels were sky-high.  Stomach and colon biopsies stained positive for enteroviruses but enterovirus RNA was not detected in his blood (it often isn't).Unfortunately, the young man failed to respond to either alpha or gamma interferon or to SSRI's, benzodiazepines or acid suppressants. Repeated MRI's of his brain and spinal chord were normal. Six years into his illness, at the age of 29, he committed suicide.His ending was tragic, but his story was not over. His harvested brain provided clues as to what may have happened. Neither a brain culture nor an RT-PCR picked up signs of enterovirus, but a western blot found protein bands which were similar to those found in the young man's stomach biopsies (but different from those found in tuberculosis and lymphoma).Dr. Chia concluded that this finding replicated a similar finding dating back to 1994.  He concluded that the

"finding of viral protein and RNA in the brain specimens ….is consistent with a chronic, persistent infection of the brain causing debilitating symptoms. EV is clearly one of the causes of ME/CFS, and antiviral therapy should be developed for chronic EV infection."

Like herpesviruses, most enteroviral infections are passed off quickly, but like herpesviruses, enteroviruses are also associated with serious disorders including polio, meningitis, myocarditis, hand, foot and mouth disease and others. According to Wikipedia, treatment for enterovirus infections is primitive, consisting mostly of relieving symptoms such as pain as they occur.One hopes at some point an independent lab will take up Dr. Chia's work and give it the replication it needs and he deserves.

Conclusions

The cytokine findings are disappointingly inconsistent, but the immune system is a vast place and gene expression, epigenetic modeling and other studies continue to point a finger at it.  The Montoya studies should tell us much, plus the entry of noted researchers such as Ian Lipkin and Mady Hornig,  Maureen Hanson, Derya Unutmaz, Michael Houghton and Patrick McGowan into the field ensure that we'll be learning much more about the immune system in the years ahead.Marshall-Gradisnik's NCNED team is churning out immune studies at a rapid rate, Broderick's early modeling  studies suggest an immune focused 1-2 punch may knock out post-exertional malaise, and Fluge and Mella are testing another autoimmune drug, cyclophosphamide, in clinical trials.Both Fluge/Ron Davis believe an immune process may be targeting energy production in our cells, the same may be true for ion channels, and it's now clear that an autoimmune process is producing POTS in some patients. Every microbiome study thus far suggests altered microbial diversity and/or gut leakage into the blood could be sparking an immune response.The Simmaron Foundation's expanded spinal fluid study should give us a better handle on what's happening in the brain just as new techniques to measure the amount of neuroinflammation present in the brain come online.Finally, it's encouraging that researchers are getting serious about subsets - and finding them when they look for them.

Major Findings

• Increased levels of pro-inflammatory cytokines are associated with increased severity in ME/CFS;
• Exercise, on the other hand, appears to down-regulate cytokine levels in ME/CFS including several cytokines that are typically increased during exercise in healthy people;
• Gene expression results suggest ME/CFS is very similar to a sepsis-like condition called systemic inflammatory response syndrome (SIRS) which shares some other characteristics with ME/CFS;
• Epigenetic modifications suggest that events may have altered the expression of genes involved in both the HPA axis and immune systems in ME/CFS;
• One subset of ME/CFS with sinusitis and/or hives also falls prey to other pain sensitization type disorders such as migraine, fibromyalgia, headache and back pain. Mast cells could be implicated;
• A broken up form of RNase L, an important enzyme involved in fighting pathogens, showed up in fibromyalgia;
• Higher levels of EBV proteins in ME/CFS patients' cells plus structural abnormalities in their cells suggest EBV reactivation may occur more frequently in ME/CFS;
• A quantitative Elisa test suggests that HHV-6 contributes to the symptoms of ME/CFS as well;
• Enteroviral proteins in the brain of a young man with ME/CFS who committed suicide suggested that enteroviruses have infected the brains of some people with ME/CFS.