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Maureen Hanson’s group at Cornel continues to pound out fascinating results. The latest one – a preprint, “Single-cell transcriptomics of the immune system in ME/CFS at baseline and following symptom provocation” – again demonstrates why the NIH-funded ME/CFS centers are so important – and why we want and need more of them. The study was large, complex, and innovative, and came up with some surprising results.  Plus, as the Hanson group is wont to, it used an exercise stressor. Let’s hope the RECOVER Initiative is watching.

Preliminary results from this study showed up in an IACFS/ME conference presentation that Health Rising covered, but we’re covering the full paper as it’s chock full of potentially new and important insights into ME/CFS.

Myoglobin, Platelets and Monocytes, Oh My: The IACFS/ME Conference Review #III

The Gist

  • This NIH-funded ME/CFS research center study was complex and innovative, and it showed in its possibly ground-breaking immune results.
  • Instead of assessing the gene expression of immune cells “in bulk” (that is – assessing the gene expression of all the immune cells at once), the Hanson research group – lead by Andrew Grimson and Jan Grenier – assessed them cell by cell – giving them much more fine-tuned results. 
  • The results were surprising. While the ME/CFS patients’ T-cells and NK cells were acting strangely, it was monocytes – immune cells ME/CFS researchers have paid little attention to over the years – that were the most dysregulated. 
  • Monocytes troll the bloodstream looking for signs that something is wrong. When they find it, they turn into macrophages which dive into the tissues and engulf pathogens and/or clean up the cellular debris left behind by an injury. 
  • The monocytes they found in ME/CFS (classical monocytes) were in an aggressive state, appeared to have been activated, and were ready to turn into macrophages.  Macrophages tend to be active in environments where chronic inflammation is present. 
  • Some of the monocytes were “diseased”, or dysregulated, while others were normal. Those diseased monocytes seemed to be making an impact: whether it was functionality or post-exertional malaise or another symptom – the more diseased monocytes a person had, the worse off they were. 
  • The authors stated, “ME/CFS patients experience continual improper recruitment of monocytes to one or more tissues”. Given the monocytes’ tendency to turn into a prime mover of chronic inflammation (i.e., macrophages), the finding suggests inflammation could play a major role in this disease.
  • Given the strengths of this study – a large, complex study employing ME/CFS patients from ME/CFS experts that used a more effective technique – it has the potential to alter how this disease is viewed immunologically.
  • Further studies are needed – and the Hanson group suggested that the same type of study is now being done in long COVID – but one wonders if they’ve uncovered a prime immune driver of this disease.
  • Another surprise came with platelets. Platelet activation has been found in long COVID, but this gene expression study suggested that platelets in ME/CFS were inactivated at baseline but – strangely enough – looked normal after strenuous exercise. No evidence of platelet activation was found. 
  • The authors suggested that exercise might have activated the inactivated platelets, which then got swept up in the microclots that exercise provoked, thus removing them from the circulation.
  • The study also found a downregulation of genes associated with ribosomes – the seat of protein synthesis in the cell – in a wide variety of immune cells.  That suggested that many of the immune cells in ME/CFS were in a quiescent state and underperforming.  
The authors noted that “Thus, at present, it is far from clear which components of the immune system are most relevant to ME/CFS.” That’s a remarkable statement given how long the immune system has been a focus. The authors pointed to a familiar culprit – small studies – and then added a new one that we haven’t seen before – the use of “bulk samples” in gene expression studies.

Most gene expression studies use bulk samples: i.e., they assess gene expression patterns across thousands, and up to millions, of cells at a time. The problem with this approach is that it may obscure biological abnormalities in specific cell types – and, in fact, the gene expression results in ME/CFS have been underwhelming to say the least.

In this study, Andrew Grimson, the leader of the study, introduced a different method. He and Jan Grenier – two experts in single-cell sequencing – assessed the gene expression or transcriptomics cell by cell (scRNA-seq) – thus getting a much more fine-tuned picture – and that made all the difference.

The study used Workwell’s exercise protocol to compare the gene expression of immune cells before and after exercise. Jared Stevens (Workwell), Betsy Keller (Ithaca), and Susan Levine MD were amongst the co-authors, and John Chia MD provided patients as well.

The Study

The authors reported the study introduced a “new and important resource to investigate immune dysregulation” in ME/CFS. Only small differences were present in the proportions of immune cells: i.e., ME/CFS is not caused by some out-of-control immune cell that’s wreaking havoc.

Next, they analyzed the number of “significantly dysregulated genes per cell type”. Since which genes are turned on or off tells what the cells are or aren’t doing, they were looking for cells that were acting strangely. While most immune cells were acting perfectly normally, a few cell types exhibited “strong signals of dysregulation”: i.e., they were acting strangely, indeed. They included some big movers in the immune system: CD4+ T cells (naïve and effector/memory subsets), monocytes, and (as expected) cytotoxic NK cells.

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That was all to the good: significant dysregulation across a few immune cell types provides a much easier target than widespread dysregulation.

Surprise Immune Cell Comes to the Fore

The strongest center of dysregulation occurred in monocytes – particularly classical monocytes. Monocytes are large, white blood cells that play a major role in inflammation. The classical monocytes that showed up in the ME/CFS patients aggressively target damaged areas. Elevated levels of genes associated with chemokine signaling, migration, and activation suggested the monocytes in ME/CFS are highly activated and on the move.

The high CCL4 expression suggested that the monocytes in ME/CFS were primed and ready to turn into macrophages. When monocytes find a damaged site, they leave the bloodstream and burrow into inflamed sites, where they turn into macrophages that then remove dead or dying cells, or cellular debris, and/or engulf (swallow) pathogens. That’s all good, but it also signifies inflammation. Macrophages, it should be noted, play a particularly prominent role in chronic inflammation.

Nath, it should be noted, found small blood vessel leaks in the brains of people who’d died from COVID-19. Macrophages had invaded the tissues found outside the blood vessels in an attempt to clean up the mess. One wonders, given the problems with blood flows, if something similar is happening in other tissues in ME/CFS.

(Bruce Patterson MD believes that the monocytes are attacking the endothelial cells in long COVID – and a major part of his protocol involves stopping them. While Patterson found low levels of CCL4 in his long-COVID patients, though, this study found high levels of it in ME/CFS patients.)

Has Bruce Patterson Cracked Long COVID?

Next came an exercise where they labeled each monocyte as diseased or normal. They found that people with ME/CFS patients contained both normal and diseased monocytes, and in a finding they called “notable”, people with more diseased monocytes tended to be worse off – across a wide range of symptom scores (functionality, general health, SF-36 physical component, PEM severity). The more diseased monocytes a person has, the worse off they were in many ways.

signal strength monocytes ME/CFS

An analysis of signal strength (blue lines) suggested that monocytes were having an outsized effect on other immune cells.

Next, an “interactome” analysis found the monocytes were interacting a lot with the two other immune cells the study pulled up – T and NK cells. That was a nice package, as many ME/CFS studies have focused on NK cells and T-cells.

This study suggested, though, that when it comes to the immune system, monocytes may be “it” in ME/CFS.  Nobody in the decades studying this disease has ever done a deep dive into monocytes/macrophages in ME/CFS. It looks like that is about to change.

In a Cornell ME/CFS Center Facebook post, Grimson – who is the co-PI on the Cornell Research Center grant – wrote that the aberrant monocyte migration

“could contribute to many of the symptoms of ME/CFS. This work sets up lots of questions that motivate our work now – where are the monocytes going in ME/CFS individuals, what is causing them to be dysregulated, and ultimately, can we reverse this dysregulation?”

and that

Beyond changes in gene expression in monocytes in ME/CFS, we also find changes in expression in other immune cells, which will be an invaluable resource for both us and other researchers as we build towards a comprehensive understanding of immune alterations in ME/CFS.

All in all, their findings suggest that “ME/CFS patients experience continual improper recruitment of monocytes to one or more tissues”.

This study checks all the boxes: it’s a large study of ME/CFS patients (derived, it should be noted, from ME/CFS experts), which used a more effective technique (single-cell gene expression), that ended up highlighting a factor (diseased monocytes) that impacted symptoms across the board. This finding clearly has the potential to redefine how we think about ME/CFS immunologically – and that’s good news.

The Platelet Paradox

Finally (and weirdly), the Hanson team found a factor that was abnormal at baseline but normal after exercise. Platelet gene expression was reduced at baseline but returned to normal 24 hours after exercise (!). This is the first factor I can remember that was abnormal at baseline but returned to normal after exercise.

Platelet activation may be a big deal in long COVID, and a recent study suggested it was present in ME/CFS as well. This study suggested the same but flipped it on its head.

The genes expressed by the platelets at baseline didn’t suggest platelet activation at all. In fact, it made them look like “older” platelets that were hardly eager to become activated. (Were they exhausted?). All in all, platelet activation was reduced at baseline, returned to normal 24 hours after strenuous exercise, and never showed evidence of increased activation.

The authors had two possible explanations for this bizarre finding. Exercise induced both platelet activation and microclot formation in people with ME/CFS. The activated platelets then adhered to the clots – thus removing them from the circulation – leaving only normal platelets behind. Or, it’s possible that exercise, in combination with clot formation or removal of the older-looking platelets, might trigger an influx of normal platelets.

Immune Downregulation

A gene set enrichment analysis (GSEA) found a downregulation of ribosomal protein genes and core translational machinery across multiple cell types. Since ribosomes are the center of protein synthesis, this seems to imply that a breakdown in protein synthesis had occurred across the immune system and that the cells were likely underperforming. Interestingly, a recent study found a similar pattern – broad immune cell quiescence in ME/CFS – with the exception of the monocytes.

Conclusion

Monocyte

They haven’t gotten much press, but this study suggests that monocytes might be the real immune problem in ME/CFS. (Image from Blausen – Wikimedia Commons)

A more effective technique of measuring gene expression that hasn’t been used before in ME/CFS produced some surprising results. Instead of the usual suspects – NK, T, and B-cells – it was the monocytes – which have never received much attention in ME/CFS – that showed up in spades. Was a prime mover of immune dysregulation in ME/CFS missed all this time? Time will tell.

Another surprising result was platelet activation, or rather, the lack of it. Instead, the gene expression study suggested that the platelets in ME/CFS at baseline were snoozers – hardly activated at all. After exercise, things, strangely enough, returned to normal for reasons that are unclear.

The study also uncovered a reduction of ribosomal gene expression (e.g., protein production) across many immune cell types in ME/CFS, suggesting that many immune cells are in a kind of quiescent, sleepy state – and not likely to rise to the occasion when stressors occur.

All in all, this study showed why NIH-funded ME/CFS research centers are potentially so important and why we need more of them: they have the ability to redefine how we see this illness.

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