Itaconate modulates immune responses via inhibition of peroxiredoxin 5, 2025, Tomas Paulenda et al

Well then I hope my results get published someday because they might surprise you!

 

Well, I think it goes without saying that we all appreciate your efforts! I remember how hard doing academic humanities work was with mild ME, I can't even imagine throwing lab experiements into the mix!

Are the lab experiements ongoing or are they still being planned/set up? Do you have any sort of idea of a timescale until you might have your answer?

It does sound like a plausible theory that would fit with my experience of deterioration, and definitely sounds worth pursuing.

Totally understand about the drugs. I think that's probably sensible given the direness and desperation of our situations.

 

I have a potential collaboration worked out where a generous research team might be able to carry out one preliminary experiment for me. Timeline is a few months at least.

In the mean time, I’ve been meeting with researchers at my university to try to get trained on some protocols. Likely I’d need to get a grant award before I can move forward with doing anything myself. I’ll probably have a better chance with a grant if that preliminary data comes back supporting my idea

 

This feels important. What stimulant were you taking that caused this change in the delay?

 

Vyvanse and adderall, vyvanse gets converted into adderall (dextroamphetamine) in the body. Both are monoamine reuptake inhibitors acting on dopamine, norepinephrine, and serotonin.

I suspect it is due to effects of norepinephrine on glucose uptake in tissues.

I do not know if other pwME who can tolerate ADHD stimulants have observed the same effect, it might just be me.

 

Max Artyomov was asked about the other PRDXs during the Q&A of his recent Harvard talk. He said that he believes other PRDXs respond the same as PRDX5.

 

Does this change in delay between activity and PEM affect all symptoms or just the symptoms associated with the activity? In other words, if the PEM episode was induced by cognitive work, is the PEM delay change to 3-4 hours only related to brain fog PEM? Or does the change in delay pertain to GI symptoms or POTS as well? How many times have you experienced this dramatic delay reduction while on monoamine reuptake inhibition?

 

Hard to give an exact answer since my entire experience of ME changed quite dramatically with the stimulants. Pre-stimulants I was mostly homebound, though if absolutely necessary I could be out of the house for a few hours.

It felt like I was being powered by some "backup" system--no flushing, heart racing, or other effects that I'd expect from classic sympathetic nervous system activation, but I did feel some kind of rush. I'd usually have insomnia that night. I'd get pretty classic PEM symptoms ~24 hours later: primarily general malaise and exhaustion, flu-like symptoms (fever, chills), brain fog.

After stimulants, I was doing more in general. It took less effort to get myself going and I went back to school part-time. Instead of delayed PEM, I got more immediate PEM after 3-4 hours. The primary symptoms were muscle pain, stiffness, weakness, full body "heat" (particularly at the base of my skull), and brain fog. This would continue for at least 24 hours, and would get noticeably worse once I finally got home and collapsed. I would only get that pre-stimulant delayed PEM if I tried to keep pushing for several days while I was already in that state.

Taking NSAIDs before the activity and then continuing for the next few days through the duration of that PEM would alleviate, but not eliminate, those immediate PEM symptoms. NSAIDs never did anything for pre-stimulant PEM as far as I remember, but I also never tried taking them before activity. This tells me that prostaglandin release is a differential factor in my experience of immediate vs. delayed PEM.

I never had GI symptoms or POTS. The only time I had delayed PEM induced by cognitive work pre-stimulant was when I was trying to keep up with a full university courseload (right before I dropped out). Post-stimulant, I would get that immediate pain/weakness/stiffness PEM if I had too many zoom meetings in a day, but it would usually resolve overnight. In those cases, I would have less specific pain in muscles than if I had been on my feet for too long, but I would still get full-body symptoms.

It's been consistent since I started the stimulants 5 years ago. I take them every day--the few times I've run out of them, I went back to my pre-stimulant illness dynamics.

 

Hi Jonathan, There is no publication. I'm just obsessed with finding a cure for this damn disease. The hypothesis has two parts: 1) the shunt itself (cis-aconitate-->itaconate-->itaconyl-CoA-->citramalyl-CoA and then a lyase (CLYBL) that brings the C5 pathway to its short circuit end: pyruvate and acetyl-CoA. This first part would explain energy inefficiency in ME because the "itaconate cycle" produces at most one NADH. 2) a chronicity mechanism - some pathway that makes the shunt a long term feature of central carbon metabolism in affected cells. My first guess about this was based on recognizing that Type I interferons are, themselves, ISGs. We measured plasma IFNa in HCs and MEs using the Quanterix Digital ELISA and, disappointingly, found no difference [again, not published, but reported at the Hinxton Invest in ME meeting 2 years ago]. Since you've invested the time to watch those videos on the "Janet Show", you've heard my concern that plasma measurements may be fruitless if the ME/CFS phenotype is restricted to only 10-15% of body cells. That ME/CFS is a cell autonomous disease is a strong inference from Cara Tomas' and Julia Newton's results in fresh or cryopreserved PBMCs showing resting ME/CFS VO2 (Seahorse) decreased by nearly half. If every cell in the body was this sick, then whole body VO2 would also be half normal. Various investigators have measured VO2 for whole human HCs and MEs. The Vermeulens' and David Systrom's are my favorite papers on this. Apparently depending on disease severity, they measure VO2 between 86% and 95% of normal resting VO2. This, of course, is not treated as a major result of those papers, but it should be because I do not see any way around the conclusion that only 10-15% of patient cells have the Tomas/Newton phenotype [again no paper, but reported in my talk for the NIH ME/CFS Roadmap Physiology Webinar]. If one accepts this conclusion, it's but a small extrapolation to the classic ME patient's "normal bloodwork." Blood concentrations of anything are weighted averages of contributions from all body cells. The endocrinologists have it easy because they can measure signals uniquely produced by a small fraction of body cells, but if we think that ME is a disease of central carbon metabolism, plasma measurements will carry very little information if only 10-15% of cells are sick.

For this same reason, I'm constantly worried that I'm not studying sick cells even though I am studying cells obtained from ME/CFS patients. Even at the level of scRNAseq, ME PBMCs are not all different from HC PBMCs. At another meeting, Andrew Grimson agreed with this point: "At the level of mRNA, most PBMCs are normal, not sick." This is why Andrew's PD subset of classical monocytes is so interesting.

Getting back to the positive feedback loop, the reason Tom's and Max's Nature Metabolism paper is so compelling is that it provides yet another positive feedback loop that could explain chronicity of the itaconate shunt. It even explains why we find increased mitochondrial superoxide (MitoSox) in ME mitochondria, something I could not explain as a consequence of the itaconate shunt alone. Professional immunologists like Tom and Max tell me its impossible for any feature of innate immunity to be sustained chronically. If that's true, the itaconate shunt hypothesis is toast. I would counter that in computer simulation, a broken off-switch or a positive feedback loop can make the impossible possible.

On the other hand, experiments are the classic way to turn a hypothesis to toast - who was it who said, "...a beautiful hypothesis destroyed by an ugly fact"? We have an ME PBMC phenotype in which permeabilized ME cells produce significantly fewer reducing equivalents (as measured by a tetrazolium redox dye) from a fixed amount of a TCA cycle fuel. We did this for multiple fuels, and, intriguingly, reducing equivalent production was most impaired when the fuel was cis-aconitate. I was, admittedly, pretty hopeful that this result implied the itaconate shunt was active, but we measured ACOD1 mRNA and CAD protein and neither was increased compared to HC. We're still in the process of measuring itaconate, itaconyl-CoA, and citramalyl-CoA. If we don't find them increased, then the itaconate shunt hypothesis is experimentally toast assuming that a PBMC phenotype is a cellular model of the ME disease mechanism. Vide supra.

 

Any chance those results are published or soon-to-be published? I'd be extremely interested to look at them. I'm sure other members on this forum are sick of me talking about it, but you may not have seen my posts: I've had a bit of a miraculous boost from a malic acid supplement (suddenly able to walk 2 miles with only mild pain and soreness you'd expect from being out of shape, for the first time in 6 years). A few others have reported similar effects, not to the same extent as me, though it's possible that the immediate benefits are dependent on already being on a stimulant.

I've been trying to follow up on what might lead to a malate-aspartate shuttle impairment in ME but since my background is entirely computational bio and I'm not part of a lab that studies ME, I'm a bit limited in what I can accomplish on my own.

 

True. But the evolution of biological feedback control comes at the cost of nonlinearity and nonlinear systems have features that linear systems do not. Prominent among those features is bistability. A simple definition of bistability is that the system can operate in two different steady states for the same set of inputs and parameters. There are solid examples of this in human metabolism. Those of you with long memories will remember that bistability was a key feature of my first hypothesis for the pathogenesis of ME, the IDO metabolic trap. If one of those stable steady states is pathological, evolution will not be aware of it because the same metabolic control system is also capable of normal health. The gene circuit producing the sick phenotype can also produce the healthy phenotype. Indeed, both phenotypes are inherent in exactly the same biochemistry.

This raises the thorny problem of predisposition. What is it that explains why some individuals suffer an infection or trauma that devolves to ME, and others, suffering the same insult, do not? In nonlinear control theory, the bistable nonlinear system needs an exogenous stimulus to transition form one stable state to another. Of course, this could be a case of I've got a hammer (nonlinear control theory) so everything is a nail (bistability). Still, the possibility that ME/CFS is a bistability disease seems worth exploring.

One possibility I find attractive is that predisposition to ME/CFS is acquired during a previous infection (one preceding the "trigger" infection). Many viruses have evolved non-structural proteins that in various ways sabotage the host immune system. By this mechanism, the ME trigger infection finds a host immune system not entirely defined by host evolution. Indeed, the saboteur virus may have delivered a Trojan Horse that disables the homeostatic, spring loaded return mechanism. The viruses have been evolving a lot longer than we have.

 

I have not seen every post, but I've seen enough to know that your experience aligns with where I'm planning to move if we succeed in rejecting the itaconate shunt hypothesis. When I say "aligns" I mean only that we have a remarkable result concerning the other shuttle that moves reducing equivalents from the cytosol to the mitochondria, namely the glycerol-3-phosphate shuttle.

 

That’s fascinating, considering some other parts of my experience. Malic acid alone gave me great physical energy at the expense of worse brain fog. Malic acid + coq10 (which didn’t do much for me on its own) alleviates the extra brain fog. I added coq10 precisely because I thought certain neural pathways might be preferentially reliant on G3P over MAS, though of course there might be other explanations given coq10’s role elsewhere.

I’d be very interested in those findings you mention!

 

By providing malate, you're not really activating the MAS because you're not transferring reducing equivalents produced in the cytosol to the mitochondria. Instead, you're providing an exogenous source of reducing equivalents in a form that can be directly imported by mitochondria. Same with G3P.

Was it Q or QH2 you took to alleviate extra brain fog? Are you on a statin?

 

I’m aware! I don’t think malic acid would “activate” MAS, just potentially compensate for a lack of reducing agents getting into the mitochondrion in the first place.

[edit: my thought is that if MAS activity is normally tightly regulated in brain cells, the problem supplementation would cause in a dysregulated system is increased use of ubiquinone at complex II, rather than at mGPD. That’s just a stab in the dark, though]

I also think it is plausible that a problem in one of several TCA or MAS enzymes could result in a lower rate of malate coming from either fumarate or oxoaloacetate via malate dehydrogenase leading to that lack of mitochondrial reducing agents.

From what I’ve read, cells that have sufficient malate levels don’t tend to take up that much exogenous malate. Of course, there could be other factors that might drive increased uptake.

My experiences indicate that a large portion of my ME/CFS symptomology is directly a consequence of that lack of mitochondrial reducing agents. Whether it’s global or only in certain cells, I don’t know, but I have my suspicions.

Do you think there’s an issue of FADH2 availability in the cytosol or specifically in cGPD or mGPD? Or reducing agents more generally? I was looking into G3P shuttle briefly but found that most pathologies involving it are usually compensated for by the MAS. I did not dive in that deep though so I’m certainly open to other information.

Ubiquinone, 3-4x normal dosages. No statin.

 

Hi Robert @RDP , thanks for re-joining the discussion.
I think this sort of dialogue could be very productive. I don't think anyone has quite got the answer here but people coming from different angles picking things apart might get us there. This is exactly how we picked apart rheumatoid system dynamics and just one new observation (which it turned out we had made ourselves five years earlier and missed) allowed us to fit a whole lot of known steps into an autonomous story. I need to read through your comments carefully and respond. My collaborator, Jo Cambridge, who also worked with Chris Armstrong on this, is closer to your position than I am, but I think we are pretty much agreed on the systems dynamics arguments. It is just working out how the pieces fit together.

 

I eventually became a professional immunologist having been a rheumatologist, macrophage and stromal cell biologist, histopathologist, embryologist and fluid physiologist along the way! That might make me a Jack of all trades but I think it taught me to have a group of people with lots of different knowledge bases - redox, antibody, macrophages, tissue mechanics, whatever. (Macrophage FcgammaRIIIa is induced by mechanical forces, which is why RA is an arthritis.) I suspect @jnmaciuch may be picking up the same vibes from a similarly diverse track.

I can see what Tom and Max may be saying, and it is related to my own concerns, but I don't think it is as simple as that. There is no independent innate immune response. Even newborn infants use antibody to forearm their macrophages and NK cells. As Donne said 'No cell is an island, unto itself'.

Coming from a histological and tissue structure training I tend to see everything situated in this context and I don't think we have thought enough about where we think the bad cell events really are in ME/CFS. I am writing something with Jo Cambridge and Jackie Cliff and both of them came back with the first comment - 'where is it?' I think it very plausible that metabolic shifts inside macrophages are crucial to the bad signalling that makes people feel ill but this bad signalling might be hidden away in places we have never looked. I don't think we even need muscle cells to be involved at all, although they may be.

Where I suspect I agree with Tom and Max is in that it is hard to see innate immune cells getting stuck on the wrong fork of a bistable pathway without some outside help. And especially if this problem is supposed to spread to cells all over. I think the evolutionary argument must be that a pathway that is bistable and can flip into a hypo metabolic state entirely without external help will not survive. Non-linear bistable pathways are everywhere in haematology and immunology but they all have switch points that have external control I think.

The counter to this argument would be the pyrin gene allele for familial Mediterranean fever perhaps. It may have advantages as a heterozygous allele but produces bouts of fever when homozygous. It might be that people with early onset ME/CFS have something analogous - a gene variant that allows a purely innate immune cell bistable pathway to stuck flipped. It is just that the dynamics of ME/CFS seem very odd for that. It is not a paroxysmal disorder, nor a purely progressive one. It seems that other forces must be having a variable permissive effect on a flip over very long periods. That looks to me like an 'adaptive' immune response, with the caveat that you can get learnt clonal expansion of T cells that work more on innate signals than antigen recondition.

So I like the basic idea of invoking a positive loop and I like the idea of interferons because they modulate ell behaviour without necessarily being directly pre-inflammatory. I am drawn intuitively to gamma interferon but I have an open mind.

 

One specific thing that I think may be putting people of the scent is that peripheral blood mononuclear cell metabolism probably tells us thing much. A monocyte or a B cell only does something of interest when it meets other cells in tissues - when it may expand its cytoplasm and metabolic machinery tenfold. The Catch22 is that what it does wrong in ME/CFS may depend on soluble factors that may be present in the original plasma but will be lost in standard tissue culture. They may also only be present at short range between interacting cells. So failing to find things maybe should not put us off completely.

One possibility I guess is that, as for lupus, there are people with such a high genetic predisposition to ME/CFS that they are bound to get it and quite likely by age 12. Maybe once we have some genetic clues very early onset cases should be looked at as a specific subgroup. It might be that these people have such a bistable pathway in their macrophages that you can actually study it in vitro in isolation regardless of soluble mediators in the medium.

 

This is fascinating:

I'd never considered that we should focus on early-onset cases. Ron Davis focused on the severely ill to get the biggest ME/CFS 'signal'. I wonder if early onset and severity are correlated and if not, why not.

 

Surely there must be some surveys that have data on age at onset and severity? Could we do some as hoc analyses on those?