Ground Zero for Immune Findings

natural killer cells stress

Would a stressful event whack natural killer cells in ME/CFS

Natural killer (NK) cells are kind of ground zero with regards to immune functioning in chronic fatigue syndrome (ME/CFS).  Problems with the ability of NK cells to kill pathogens were identified as early as 1987 and those findings have largely stood the test of time.

Later problems in both stress response systems, the HPA axis and the autonomic nervous system cropped  up – both of which regulate immune functioning. Reasoning that activating both stress respone systems would cause the immune system to take a hit in this study the CDC used a stress test called a Trier Social Stress Test (TSST) to push those systems hard and then checked on natural killer cells.

Acute psychosocial stress-mediated changes in the expression and methylation of perforin in chronic fatigue syndrome. Falkenberg VR, Whistler T, Murray JR, Unger ER, Rajeevan MS. Genet Epigenet. 2013 Jan 28;5:1-9. doi: 10.4137/GEG.S10944. eCollection 2013.

The CDC examined natural killer cell levels, the gene expression levels  of perforin – a key factor in natural killer cell “killing” – and the methylation sites that affect perforin functioning.  Being able to tie activation of a damaged stress response system with poor immune functioning; i.e. natural killer cell functioning – would be a major step forward – particularly given the effects of stress on many people with ME/CFS.

stress methylation chronic fatigue syndrome

Would stress take out the genes that enable NK cells to kill infected cells?

Being able then to tie methylation  – a process in the body that turns on and off the expression of genes – to the poor natural killer cell functioning found (via measuring perforin expression) – could be a big step in figuring out what the heck is going with natural killer cells in ME/CFS.

The Trier Social Stress Test consists of a 10-minute public speak­ing and 10-minute mental arithmetic task in front of three trained staff members (TSST panel. It was guaranteed to raise stress levels.


Another Bizarre Finding

Natural killer cells appear to be exquisitely sensitive to stress and as expected the stress test boosted natural killer levels dramatically in both sets of patients.  Perforin expression – a key part of the NK cell killing machinery – increased 60% in both CFS and healthy controls simply by telling them they were about to take a stress test.

stress natural killer cells chronic fatigue

Another strange result

During the stress test perforin expression continued to increase in the healthy controls but decreased slightly in the CFS patients. Then perforin expression decreased in the healthy controls after the test but increased later in the CFS patients as time went on. To wit:

  • Perforin went up during the test and then decreased in the healthy controls.
  • Perforin went down during the test and then increased afterwards in the CFS patients.

During the stress test perforin levels should have increased in the ME/CFS patients but they tanked.  That would presumably leave ME/CFS patients more vulnerable to viruses during that stressful period. They normalized immediately after the test but an hour and a half after the stress test was over, however, they shot up dramatically. It was almost as if a delayed reaction was taking place (or it was a spurious finding.)

The take away here is that NK cells appear to have trouble  – like so many other parts of the body in ME/CFS – regulating their functioning properly in response to stress. . .

This pattern seems somewhat similar to what happens to autonomic nervous system functioning before and after exercise. The heart rates of ME/CFS patients are increased at rest but then poop out once the system is put under the stress exercise (chronotropic incompetence). Perforin expression was normal during rest  in this study (although other studies have found that it was decreased during rest),  then declined during stress  and then later bizarrely shot up again.

The researchers suggested that reduced perforin expression found in the ME/CFS patients during the social stress test could be due to something as simple as reduced NK cell recruitment – the more NK cells present – the higher the levels of perforin in the blood.  (Other studies, however, have found normal natural killer cell levels in ME/CFS)

Methylation (???)

The big question in the study was methylation.  Were perforin levels decreasing because epigenetic processes in the body were turning them off?  The CDC tested seven sites involved in the methylation of the perforin gene.  The finding that increased methylation was reducing perforin expression (in both groups) was an important one for the field. The main finding, however, was that methylation levels were similar between the two groups.

Subset Found

Splitting the ME/CFS group up produced more significant results

Splitting the ME/CFS group up produced more significant results

When the CDC broke the “CFS” group up into patients with low and high methylation rates, however, they found that CFS patients with high methylation rates had significantly lower perforin gene expression just after exercise.  The authors noted that these patients had high baseline methylation rates – and therefore reduced perforin levels from the beginning.

The findings were intriguing enough that one wonders what a study with a more select group of ME/CFS patients would have found.


This potentially quite important study dug deep into NK cell functioning. Being able to link altered methylation rates to stress induced reductions in perforin expression could have identified why natural killer cells are doing so poorly in ME/CFS patients. The authors did find some abnormalities but not the broad abnormalities they clearly expected.  The results are a bit murky.

The Achilles Heel

The CDC had a great hypothesis and went to some length to test it – this was probably a quite expensive study – but it’s Achilles heel  may have been the participants. The chronic fatigue syndrome patients in this study  met the empirical definition of CFS and were gathered up in a population-based study.  That presumably means they were gathered up using random sampling and if that’s so that’s problematic.

achilles heel chronic fatigue syndrome

One of the Achilles heels of the ME/CFS field – no agreed upon definition

Using patients from doctor’s practices at least ensures that patients are sick enough to see a doctor.  Population-based studies, on the other hand, rely on the veracity of questionnaires to find their patients. (The participants are usually later assessed by a doctor).   Lenny Jason’s recent finding that no less than 30% of healthy controls meet the Fukuda criteria (and 15-20% met the CCC and ICC definitions) puts a big question mark on population-based studies like these.

From prior CDC studies we know that many of these patients tend to drift in and out of meeting the criteria for CFS: they’re definitely on the fringes of the CFS population.

It’s hard to know what to do with this study. Does it reflect what’s going in the ME/CFS population at large? It’s simply impossible to know.

Unfortunately, the definition issues in this or other studies are hidden. The limitations of a study are usually provided at the end of the study, and, in fact, the CDC cites several limitations in this study.

The CDC has never, however, acknowledged that I can remember – and does not acknowledge in this paper –  the dramatic effects their population sampling approach or the definition they use may be having on the type of participants they’re studying. A CDC study suggesting that using the empirical definition could increase the prevalence of this disorder fourfold means many people who did not formerly fit the criteria for ME/CFS now do. That’s a major possible confounding factor that is never mentioned.

Researchers and doctors who don’t know the history of the illness will assume this group of patients is no different from others.  They will probably assume that methylation does not play a major role in the natural killer cell problems in ME/CFS.

It’s otherwise good studies like this that cry out the need for a good research definition.   Particularly in population based studies that are already identifying less ill cohorts it’s hard to see the reason to continue using a  definition that plucks out less ill patients.

The P2P draft summary suggested the Oxford definition be retired and that a consensus effort identify a temporary definition as work proceeds on a new definition. It’s time for everyone – the CDC, the UK researchers, NIH funded researchers and privately funded researchers to get on the same page. Thirty years into ME/CFS researchers are using no less than five different definitions in their studies: the Fukuda, Oxford, Empirical, CCC and up in Norway – Wyller’s definition.

That’s a recipe for disjointed and slow progress in a field that needs to be as efficient as possible given its low funding.




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