Preprint [Preprint] Exosome-associated Mitochondrial DNA Elevated in... ME/CFS and Stimulates... Microglia to Secrete IL-1β, 2023, Theoharides, Natelson et al

belbyr

belbyr

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Exosome-associated Mitochondrial DNA is Elevated in Patients with ME/CFS and Stimulates Human Cultured Microglia to Secrete IL-1β

https://www.researchsquare.com/article/rs-154011/v1

Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease that presents with fatigue, sleep disturbances, malaise and cognitive problems. The pathogenesis of ME/CFS is presently unknown and serum levels of potential biomarkers have been inconsistent.

Methods: Exosomes were purified from serum obtained from patients with ME/CFS before and after exercise and their content of mitochondrial DNA (mtDNA) was determined by quantitative PCR. Exosomes from both patients and controls were incubated with cultured human microglia and release of interleukin-1beta (IL-1β) was measured by ELISA.

Results: Here we show that serum mtDNA, associated with exosomes, is increased in ME/CFS after exercise. Moreover, exosomes isolated from patients with ME/CFS stimulate significant secretion of IL-1β from cultured human microglia.

Conclusion: These results provide evidence for a potential novel pathogenetic factor and target for treatment of ME/CFS.
 
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Theoharis C Theoharides; Irene Tsilioni - Tufts University school of Medicine
Benjamin Natelson - Icahn School of /medicine at Mt Sinai

Funded in part by the Ramsay award from the Solve Me/CFS Foundation

The writing is accessible.
These results were obtained using mtDNA pooled from 5 subjects from either control or ME/CFS subjects and not with mtDNA from each individual subject.
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The small size of the study is noted as a limitation. The pooling (for the stimulation of the cells to produce IL-1b) isn't great either, as we can't know if it was just one person in the ME/CFS group producing problematic exosomes.
 
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So, serum for an unknown number of people with ME/CFS before and after exercise and serum for an unknown number of healthy controls, presumably just before exercise.

The order in these charts is Healthy Controls: ME/CFS before exercise: ME/CFS after exercise

Figure 1b shows that total exosome associated protein in the serum was lower in the ME/CFS samples than the healthy control samples, both before, and even more markedly, after exercise. Unfortunately, Figure 1b doesn't have levels for healthy controls after exercise. I'm assuming that the individual dots are for samples from individuals people. This finding, by itself, does look interesting.

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But, the authors note that findings related to total exosome protein have been all over the place, with other studies finding more extracellular vesicular protein in the serum of people with ME/CFS. I note that exosomes are not exactly the same as EVs.
We isolated total exosomes (30–100 nm fin diameter) from serum samples obtained from ME/CFS patients and controls as we reported previously. 28 Interestingly, we reported less total EV-associated protein obtained from serum of ME/CFS patients than controls unlike two other studies that reported no significant difference in EV yields obtained from plasma of ME/CFS patients and controls. 29, 30 In contrast, two other studies reported a significant increase in EV content in the serum of ME/CFS patients and controls. 31, 32These apparent differences may depend on the activity status of the controls, use of serum or plasma, as well as method of EV isolation and purification, thus highlighting the need for standardization for future studies.
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While calling for standardisation of future studies, the paper tells us nothing about the activity levels of their participants, including, oddly, about the exercise that the ME/CFS participants were given between blood draws.



Figure 2 shows the level of serum mt-DNA (I believe MT-7S is a marker for mtDNA, it's a subset that must be easy to count). Before exercise there's almost no mt-DNA in the serum of 8/14 the ME/CFS people (again, I'm assuming each dot is a sample from a different person), which is quite different to that of the healthy controls (maybe 1/24). After exercise, the level of serum mt-DNA increases a lot for the ME/CFS people. But, we don't know what would happen for the healthy controls, as no data is given for healthy controls post-exercise.

It might be that serum mt-DNA is increases when cells have to work hard, and maybe the healthy controls expended more energy getting to the sampling clinic? Without the post-exercise data for healthy controls, I don't think this is telling us much.

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I don't see how this is publishable unless I have missed something. I do not see mention of a DNA loading control/housekeeping gene or any quantification of loaded DNA. For all we know there are different amounts of total DNA in each sample. Unless this is clarified to be an oversight in the methods section the qPCR results mean nothing. If they have normalised the qPCR results to total protein (I can't tell whether they have or not) this is an assumption that the amount of protein in an exosome, between individuals (including in a disease group), is consistently proportional to the amount of DNA present. I would say that this is a very unsafe assumption.
 
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[Crossposted]
I've got to stop, I'll have to come back to this. The last figure is Figure 3 which gives the amount of IL-1b produced by microglia cells exposed to the exosomes from healthy and ME/CFS people. These are the pooled samples. This study isn't feeling very solid. There might be something here but the study is too small and there are too many missing bits. Perhaps the final version of the paper will be better - at present there are no Figure captions for example.

Screen Shot 2023-04-28 at 12.27.36 pm.png
 
DMissa said:
I don't see how this is publishable whatsoever unless I have missed something. I do not see mention of a DNA loading control/housekeeping gene or any quantification of loaded DNA. For all we know there are different amounts of total DNA in each sample. Unless this is clarified to be an oversight in the methods section the results mean absolutely nothing.
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I'm not surprised that you say this, as, the study looked pretty patchy to even me, who doesn't know the technicalities. They may have standardised the amount of DNA applied to each sample of microglia. But yes, there's quite a lot that they have not told us.
 
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Hutan said:
They did know the concentration of the 7S DNA
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Did they? Without a standard curve in the qPCR or without a total DNA quantification assay, I can't see how they'd know this. The qPCR, as shown, can only indicate the 7S content in the pooled ME/CFS samples relative to the pooled control samples, irrespective of total DNA abundance. So it's not an absolute quantitative value.
 
It looks like they've applied whole exosomes, not just DNA to the cells, so this part should be unaffected by that issue. (The raw result anyway), any further interpretations that the response is due to the mtDNA specifically would to me seem unfounded due to the DNA quantification issue and due to the exosomes containing other stuff like protein. They've quantified the exosomes themselves (at least, by protein content), just not the DNA itself.

I'll alter some of my wording in the first comment with an edit because, as is, it could be read that the exosome treatment part of the results were also meaningless (it was not was I was referring to). I was focused on the problems with the qPCR in that comment.
 
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This is pretty technical. We're on the same page. :)

This is a very good example of why you can't have too much detail in a methods section. For all we know they've done this but not explained it. But it's absolutely necessary to apply any meaning to the outcome!!
 
Far too little data to make any useful conclusions from, and definitely too little to make any claims about potential treatment targets. Even if a much larger and better-done study verified a difference in mtDNA content, it could be very far downstream of ME's mechanism. It might just mean that PWME spend more time reading or watching TV.
 
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