Could ME/CFS be associated with NFAT5 dysfunction?

[Leadtheway]

Hello all,

During my research, I've become interested in a specific protein, which is called NFAT5 (or also TonEBP) and which I think might be linked to ME/CFS.

Here is why I think it could be the case:

Response to osmotic stress

Ronald Davis developed a test that works by submitting cells from a patient to osmotic stress and observing their caracteristics:

our experimental results on 40 ME/CFS individual patients and healthy controls revealed that ME/CFS hyperosmotic-stressed samples (PBMCs in plasma) display a unique characteristic in their impedance pattern, and one that is significantly different than what was observed in the controls.

A nanoelectronics-blood-based diagnostic biomarker for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)

The transcription factor NFAT5 is known, among other, for its role in the adaptation to osmotic stress, especially by increasing the influx of osmolytes or the production of proteins (Heat Shock Proteins) that can protect the structure of other proteins from the destructuring effect of osmotic stress.

Osmolytes are small molecules (for example betaine, aka trimethylglycine) that can help counter osmotic stress without disturbing the normal processes of the cell.

Could the abnormalities observed in cells of CFS patients, when an osmotic stress is set up, be the consequences of a dysfunction of NFAT5 and of the ability to accumulate osmolytes to prevent the damage done by osmotic stress?

Immune system

NFAT5 is also known to be tightly linked to the immune system:

NFAT5 is an important regulator of the immune response. NFAT5 haploinsufficiency has been described as an immunodeficiency syndrome affecting both innate and adaptive immunity. EBV susceptibility might be another manifestation in the spectrum of this disease.

Not enough by half: NFAT5 haploinsufficiency in two patients with Epstein-Barr virus susceptibility

Thus, a dysfunction of NFAT5 could be a cause of EBV reactivation. Some patients suffering from CFS are also suffering from EBV reactivation.

It was also found that Coxsackievirus B3 could act against the NFAT5 protein (cleaving it) as it's inhibiting its replication. So we can wonder whether other pathogens would seek to eliminate NFAT5 from the cells they infect.

Glymphatic system

NFAT5 could also be linked to CFS as it has been hypothesized that the glymphatic system, which could be related to NFAT5 via the aquaporin AQP4, could be involved in CFS:

In the present paper, we speculate that glymphatic dysfunction, causing toxic build up within the central nervous system, may be responsible for at least some cases of chronic fatigue syndrome.

The putative glymphatic signature of chronic fatigue syndrome: A new view on the disease pathogenesis and therapy

A dysfunction of NFAT5, having for consequences an impaired glymphatic flow, could cause some cases of the CFS.

Inflammatory Bowel Disease (IBD)

CFS seems to be associated with digestive troubles called Inflammatory Bowel Disease (because IBD could be associated with intestinal permeability and thus leak of pathogens and inflammatory substances like LPS?). A study had the following conclusion:

The findings from this population-based retrospective cohort study suggest that IBD, especially Crohn’s disease, is associated with an increased risk of subsequent CFS.

Increased risk of chronic fatigue syndrome in patients with inflammatory bowel disease: a population-based retrospective cohort study

It is interesting to note that NFAT5 could also be involved in IBD:

Crohn's disease (CD) and ulcerative colitis (UC), which together comprise IBD, are believed to result from an aberrant immune response to commensal gut microbes, leading to chronic intestinal inflammation. Compared to healthy controls, we observed that NFAT5 mRNA expression was significantly reduced in patients with active UC and CD (Fig. 7), raising the possibility that NFAT5 and other components of the osmoadaptation pathway may be dysregulated in IBD.

Immunodeficiency and Autoimmune Enterocolopathy Linked to NFAT5 Haploinsufficiency

Could NFAT5 dysfunction be a common root cause of CFS and IBD?

Water homeostasis

Some studies demonstrated that CFS patients could have issues with water homeostasis. In particular, they seem to suffer from drastically lowered vasopressin (an antidiuretic hormone) compared with controls:

Therefore, it is proposed that altered water metabolism secondary to changes in AVP regulation is a pathophysiological component of certain chronic fatigue disorders. In support of this hypothesis are data demonstrating that low baseline levels and erratic secretion of AVP are present in individuals with post-viral fatigue syndrome (9). Individuals with chronic fatigue disorders appear to be more sensitive to fluctuating levels of AVP than non-fatigued individuals. For example, patients with chronic fatigue excreted 62% of a water load, compared with 97% in healthy subjects (9). Baseline plasma AVP levels in patients with post-viral fatigue syndrome were reported to be significantly lower (0.1 + 0.03 pmol/1) than in healthy controls (0.9 + 0.2 pmol/1) (9). A close correlation between serum osmolarity and plasma AVP levels was observed in healthy subjects but not in the chronic fatigue patients. The patients with post-viral fatigue syndrome also showed evidence of increased total body water content (9). Thus, these data are consistent with the hypothesis that chronic fatigue disorders may result from an exaggerated, or hyper-sensitive, response to AVP. Alterations in water metabolism are also a biologically plausible explanation for the observed lethargy, mental confusion, weight fluctuations and diffuse muscle and joint pain that is characteristic of CFS and related disorders such as fibromyalgia.

Chronic fatigue disorders: an inappropriate response to arginine vasopressin?

Another publication found that NFAT5 (aka TonEBP in the literature), seemed to be necessary for vasopressin production.

Therefore, our result support the idea that TonEBP is directly necessary, at least in part, for the elevation of AVP transcription in dehydration conditions.

Transcription Factor TonEBP Stimulates Hyperosmolality-Dependent Arginine Vasopressin Gene Expression in the Mouse Hypothalamus

Thus, an NFAT5 dysfunction in the area of the brain in charge of vasopressin production could lead to lowered levels of vasopressin, which is associated with CFS.

Post-exertional malaise

After physical exertion, muscles themselves could be a part of the cause of PEM as inflammation can happen after physical activity and could, if not properly controlled, cause a sickness feeling. NFAT5 seems to play a role in muscles' recovery as it allows cells to take osmolytes so that they help fight the stress:

NFAT5+/- mice displayed a defect in muscle regeneration with fewer myofibers formed at early times after injury. NFAT5 has a muscle-intrinsic function because inhibition of NFAT5 transcriptional activity caused both a migratory and differentiation defect in cultured myoblasts.

A combinatorial role for NFAT5 in both myoblast migration and differentiation during skeletal muscle myogenesis

Pro-inflammatory cytokines activated mitogen-activated protein kinases, nuclear factor κB as well as nuclear factor of activated T-cells 5 mRNA expression. In muscle biopsies from patients with polymyositis or sporadic inclusion body myositis, osmolyte pathway activation was observed in regenerating muscle fibers. In addition, the osmolyte carriers SLC5A3 and SLC6A12 localized to subsets of immune cells, most notably to the endomysial macrophages and T-cells. Collectively, this study unveiled that muscle cells respond to osmotic and inflammatory stress by osmolyte pathway activation, likely orchestrating general protection of the tissue.

Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

Could an issue with NFAT5 activation after physical activity lead to an inflammatory reaction which would be a cause of PEM?

Do you think it could be a credible hypothesis?
Thanks.

 

[Hoopoe]

The apparent dysregulation of blood volume and water in ME/CFS is interesting. However NFAT5 is probably just one of hundreds of genes that could have something to do with that. As far as I know, altered muscle regeneration has not yet been reported in ME/CFS (but since I'm going from memory, this could easily be wrong). There doesn't seem to be any specific link between NFAT5 and ME/CFS.

The patient with immunodeficiency due to NFAT5 haploinsufficiency had a clinical presentation that seems different from ME/CFS.

I should add that I'm just an interested amateur. So far the only described clinical presentations of genetic defects that I've found that resembled ME/CFS where mitochondrial (and maybe hormonal).

 

[Wyva]

I can't really add much, as I'm not medically trained but I'm interested in anything that may explain my fluctuating water weight issues. I'm really really tiny in every way but managed to gain 3 kg in 4 days last week for example, all water. That's huge. And that's on top of my already bad water retention of at least 5 kg. It fluctuates so much that I have to keep clothes of different sizes. Pretty horrible and I've never had this before my EBV infection and it started very suddenly back then, literally one evening. I swelled up so quickly and to such an extent that I rushed to the hospital in panic.

Several doctors examined me since, all of them said it is not edema, despite my rapid weight fluctuations and the fact that its distribution is really weird. I met with a lot of disbelief despite the fact that it has totally changed my normal body shape and I do look really oddly shaped with it when it is very bad. In the evenings I have elephant legs and a pregnant-looking belly, in the mornings my face looks distorted due to the amount of water ending up on it at night after sleeping lying down. It is not just a little bit puffy, it is abnormal looking, even my nose is visibly bigger. I really only look myself when there is very little extra water in me during some exceptionally good period (so extremely rarely). It can affect my looks so much that I tend to wear sunglasses outside even in rain to hide my weird puffy eyes and when my body looks really oddly disproportionate then I just prefer not to be seen by people.

The swelling can feel really uncomfortably tight in my mid-section, at other times it just feels like my mid-section is falling apart to pieces, almost like my organs are going to just fall out (this feeling must be due to the expansion of the tissue?).

So anyway, it sucks and doesn't seem to be common, so I'm interested in anything that might explain it. This might actually just be something I was unknowingly predisposed to regardless of ME/CFS and maybe the disease has affected me in a way that it brought it out. I don't know but whatever it is it is strictly connected to the fluctuation and start of my other symptoms.

So anyway, I cannot really address the hypothesis but I'm interested in anything that might explain why this weird fluid imbalance is happening to me.

 

[Kitty]

It is really odd, @Wyva, I've heard of people getting severe oedema with lupus before, but not with ME.

It's frustrating that no-one will take it seriously. I know strange symptoms can occur without any explanation being found, but they can also be a sign of an underlying issue. You'd think they'd want to check that out.

I used to get what felt like severe bloating premenstrually, but my weight only increased by 1kg (and at 5' 8", I'm not exactly tiny either). I can't imagine gaining 3kg in a few days!

 

[Hutan]

@Leadtheway, thank you very much for putting your hypothesis up for discussion.

My son and I also experience problems with water retention/swelling that seem to be related to PEM. It would be interesting to put people on a standard diet and water intake, and then have them do the two day CPET, monitoring weight, calf and abdomen circumference and water excretion via urine and NFAT5 levels throughout. There have been a number of reports of hypovolemia being common in ME/CFS.

Interactions between the host and pathogens are a good place to look for a cause of ME/CFS. I note that your hypothesis doesn't require people to have a particular problem with the NFAT5 gene - NFAT5-related issues could be a consequence of an infection.

The Coxsackie paper you mention is interesting - here's the author summary:

Coxsackievirus B3 (CVB3) is one of the predominant pathogens of viral myocarditis, which is a major cause of sudden death in children and young adults. CVB3 alters the expression of many proteins in host cells to facilitate its multiplication. Nuclear factor of activated T cells 5 (NFAT5) is known to be involved in the response to high salt concentration in body fluids but its role in viral infection is not currently understood.

Here, we showed that NFAT5 was cleaved into several fragments during CVB3 infection, which blocked the activity of the protein. We demonstrated that intact NFAT5 inhibited CVB3 multiplication, but that such antiviral activity was impaired by NFAT5 cleavage products, indicating that CVB3 cleaves the NFAT5 protein as a survival strategy. When we used a high concentration of saline or mannitol solution to induce NFAT5 production in CVB3-infected cells and mice, we found that viral multiplication was significantly reduced, suggesting that there is therapeutic potential for use of NFAT5 to combat CVB3 infection. Taken together, our findings have uncovered a novel anti-CVB3 role of NFAT5 and provided a promising drug target for CVB3-induced myocarditis.

A bit on how NFAT5 levels varied during coxsackie virus infection below. The authors later went on to identify particular viral proteins that were cleaving the NFAT5 protein. They found that a cleaved bit of the protein was binding to activation sites as the intact protein normally would, but not producing the activation - a particularly effective and dastardly way for the virus to shut down the host response.

First, we detected whether CVB3 infection would change NFAT5 level. It has been reported that NFAT5 expression is stimulated by p38 MAPK activation in response to hypertonic stress [47]. Considering that CVB3 infection activates p38 MAPK via phosphorylation [48], we were expecting to see NFAT5 upregulation and activation during CVB3 infection. Indeed, we observed a significant increase in NFAT5 protein level at early time points (~2 h pi) in HeLa cells infected with CVB3 (S5 Fig), but to our surprise, it decreased rapidly and was undetectable 4 h after infection. However, no NFAT5 mRNA decrease was observed during CVB3 infection, indicating that the reduction in NFAT5 protein level was not due to transcriptional arrest or mRNA degradation. We therefore switched our focus to the degradation and cleavage of NFAT5 protein during CVB3 infection.

However, the N-terminal fragment competes with the intact NFAT5 for DNA binding, and can act as a dominant negative mutant of NFAT5

And, another bit on how damaging the NFAT5 protein might work to make life easier for the virus:

The protective role of NFAT5 may be associated with the transcriptional activity of nuclear factor κB (NFκB). NFAT5 enhances NFκB activity by forming an NFκB-NFAT5 complex and enhancing NFκB’s binding to the κB elements of NFκB–responsive genes [19]. NFκB has been shown to influence numerous cardiovascular diseases and can protect cardiovascular tissues from injury [20]. More interestingly, NFκB has also been reported to promote host cell viability in CVB3 infection [21] and inducible nitric oxide synthase (iNOS), a downstream signal of NFκB, exerts inhibitory effects on CVB3 replication and decreases mouse mortality rate after viral infection [22]. Combined, these previous findings inspired us to link NFAT5 to anti-CVB3 activity, a virgin field for studies of both NFAT5 and viral myocarditis.

So, that paper is suggesting that the NFAT5 protein increases the activity of NFkB which has been shown to increase host cell viability in coxsackie infection, increase inducible nitric oxide synthase (iNOS), to reduce coxsackie virus replication and reduce host mortality. So, it's not be surprising that the coxsackie virus and other viruses have evolved to reduce the efficacy of the NFAT5 protein.

 

[Hutan]

If a virus was inactivating the NFAT5 protein and so causing a decrease in inducible nitric oxide synthase (iNOS), then that would be likely to reduce nitric oxide production. We have seen papers reporting just that in endothelial cells in people with ME/CFS.
e.g.
Endothelial dysfunction in ME/CFS patients, 2023, Sandvik, Mella, Fluge et al

I guess a major question is, how does an impact of a viral infection become chronic? Could a mostly latent viral infection be enough to cause such problems to the extent that the result is symptoms of the severity seen in ME/CFS?

 

[Hoopoe]

Wasn't there talk about aquaporins during the latest Invest in ME conference? They function to allow water to move in and out of cells.

 

[Leadtheway]

Interactions between the host and pathogens are a good place to look for a cause of ME/CFS. I note that your hypothesis doesn't require people to have a particular problem with the NFAT5 gene - NFAT5-related issues could be a consequence of an infection

Indeed, it doesn't necessitate a gene defect. I also think a NFAT5 dysfunction wouldn't be (if it's really associated in the first place) the root cause of the disease as you noted. I wonder if stress (in a broad sense) and infections that can be stemming from it, nutritional deficiencies, metabolic issues and toxicity could be it.

The apparent dysregulation of blood volume and water in ME/CFS is interesting. However NFAT5 is probably just one of hundreds of genes that could have something to do with that.

Indeed, it's probably not the only gene involved. I also believe it's a quite important gene, be it regarding water homeostasis or the immune system.

@Wyva It doesn't really explain variations of edema but according to the study on water homeostasis, ME/CFS seems to be associated with water retentions.

Thanks all for your replies and consideration for this hypothesis

 

[boolybooly]

Any reasonable idea is worth discussing but the above seems like a series of circumstantial associations.

It seems fair to say something is dysregulating homeostasis in ME, it doesn't mean that NFAT5 is causal and its association with these processes may be symptomatic not causal. The causal mechanism remains obscure.

My immediate thought is that specific gene associated causation of ME is the kind of question Decode ME will help answer even if a SNP is not required to get ME, as the presence of an SNP may change the predisposition to developing ME relative to the general population. That is why n=25k.

 

[Leadtheway]

it doesn't mean that NFAT5 is causal and its association with these processes may be symptomatic not causal

Indeed and that's why I'm rather cautious (I hope I am) when I'm talking about it, mentioning associations. See also my last message where I say I don't think it could be causal.

But even if it's not the root cause (in the case there is a confirmed association in the first place, but it's absolutely not proven), I believe it would be something valuable to know, because it would bring the focus on water homeostasis, which could be an important factor in the disease.