Prolonged indoleamine 2,3-dioxygenase-2 activity and associated cellular stress in post-acute sequelae of SARS-CoV-2 infection, 2023, Guo et al

Prolonged indoleamine 2,3-dioxygenase-2 activity and associated cellular stress in post-acute sequelae of SARS-CoV-2 infection

Background
Post-acute sequela of SARS-CoV-2 infection (PASC) encompass fatigue, post-exertional malaise and cognitive problems. The abundant expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase-2 (IDO2) in fatal/severe COVID-19, led us to determine, in an exploratory observational study, whether IDO2 is expressed and active in PASC, and may correlate with pathophysiology.

Methods
Plasma or serum, and peripheral blood mononuclear cells (PBMC) were obtained from well-characterized PASC patients and SARS-CoV-2-infected individuals without PASC. We assessed tryptophan and its degradation products by UPLC-MS/MS. IDO2 activity, its potential consequences, and the involvement of the aryl hydrocarbon receptor (AHR) in IDO2 expression were determined in PBMC from another PASC cohort by immunohistochemistry(IHC) for IDO2, IDO1, AHR, kynurenine metabolites, autophagy, and apoptosis. These PBMC were also analyzed by metabolomics and for mitochondrial functioning by respirometry. IHC was also performed on autopsy brainmaterial from two PASC patients.

Findings
IDO2 is expressed and active in PBMC from PASC patients, as well as in brain tissue, long after SARS-CoV-2 infection. This is paralleled by autophagy, and in blood cells by reduced mitochondrial functioning, reduced intracellular levels of amino acids and Krebs cycle-related compounds. IDO2 expression and activity is triggered by SARS-CoV-2-infection, but the severity of SARS-CoV-2-induced pathology appears related to the generated specific kynurenine metabolites. Ex vivo, IDO2 expression and autophagy can be halted by an AHR antagonist.

Interpretation
SARS-CoV-2 infection triggers long-lasting IDO2 expression, which can be halted by an AHR antagonist. The specific kynurenine catabolites may relate to SARS-CoV-2-induced symptoms and pathology.


https://www.sciencedirect.com/science/article/pii/S2352396423002943

 

From what I’ve understood the IDO2, Kynurenine and Tryptophan measurements didn’t corroborate Davis & Phair’s theories. Was anything published or is the data available somewhere? The researchers of this paper are also ME/CFS researchers.

 

Interesting. These some of the researchers will research PEM in ME as well. So is it correct that this doesn't fit with Davis and Phair's work?

 

That I don't know, but I certainly would like to know how this data matches with that of Phair/Davis/Armstrong. My phrasing wasn't good, what I want to say is that Phair's and Davis' own measurements didn't corroborate their own theories (I think the Kynurenine trial was particularly dissapointing).

 

https://twitter.com/user/status/1684534163599241218

 

Overall there are some possibly interesting bits in the paper but they don't all hang together as a cogent story in my opinion. There is no single experiment that directly backs up results seen in another experiment and all look at slightly different things - small n~15 sample sizes throughout. The authors do 4 main experiments: 1) metabolomics of PASC kynurenine/tryptophan related metabolites 2) immunohistochemistry of PBMCs staining for IDO1 and IDO2 proteins, and markers for cell death (Cas3) and autophagy (LC3b) 3) metabolomics of respiration related metabolites and other pathways 4) Seahorse respirometry of PBMCs, measuring how well the mitochondria can produce energy aerobically.

metabolomics of tryptophan metabolites

They use a few different cohorts for different experiments. I like that in general they are comparing PASC patients with healthy controls who have at one time tested positive for COVID-19 rather than healthy controls generally. In this first metabolomics experiment (1) they compare recovered but hospitalised COVID-19 patients (blue) with PASC patients who had a mild infection (red). This could be important if it turns out the effect is due to differences between severe and mild infection rather than long covid. These groups are also not sex matched very well (n=15 per group 20% male in patients, 60% male in hospitalised-recovered). (apologies for doubling up plots from @EndME's post):

They use a different set of patients and controls for all their remaining experiments and the problems with sex matching and hospitalisation no longer apply - they use a cohort of recovered COVID-19 patients who had mild illness as controls which is good.

 

Immunohistochemistry

In their histology experiments (2) they show some stains where it looks like there is greater IDO-2 expression in PBMCs generally in at least one PASC patient compared to a healthy control. (CD14 is a marker for monocytes):


They don't quantify or test for differences between a patient and control cohort as a whole so it's not convincing to me, especially since the authors will probably have picked the most dramatic looking images. The authors say this about not quantifying:

They also stain for one of the metabolites screened above - 3OH-anthranilic acid. They possibly see an increase of this metabolite in at least one PASC patient compared to a control. In the metabolomics above you can see there is no significant difference between hospitalised-recovered and PASC and the groups overlap substantially, so all together I don't think this amounts to much.

 

Metabolomics of respiratory and other processes

The authors go on to do metabolomics of respiratory and other processes related metabolites (3). Unfortunately they don't multiple test correct but at least they are transparent about that and state it's for exploratory purposes. Kynurenine pops up just barely passing the 0.05 significance threshold without correcting for multiple testing. They test 219 metabolites and the most significant one they see is citric acid, which looks to sit at just over ~3.8 (-log10(pvalue)) => p=0.000158. If we do a quick bonferroni correction (which is an extremely stringent correction far beyond what's probably necessary) we get 219*0.000158 = adjusted_p = 0.035, so it would probably survive multiple testing correction.


Citric acid is central to the krebs cycle and therefore aerobic respiration generally so it would be consistent with seeing lower rates of respiration, which is what they claim in the next experiment

 

Seahorse Respirometry - Assaying mitochondrial aerobic respiration

Lastly they do Seahorse respirometry to assay aerobic respiration on n=9 PASC and n=14 mild-recovered controls and see a significantly reduced aerobic capacity both under normal conditions and also when the oxidative phosphorylation machinery are allowed to run unrestrained.

I'm fairly sure this phenotype has been seen before in at least one other paper. It's the opposite of what fluge and tronstadt saw in their 2016 paper in a different experimental system where they applied serum to cultured muscle cells. The systems and cell types are different of course, but I wonder if there's is some kind of adaptive mechanism where serum factors are trying to upregulate oxidative phosphorylation activity but ME cells themselves are downregulating - all open speculation.

I remember a post by @DMissa discussing issues with using seahorse on PBMCs. The assay is designed to work with adherent cells I understand so in this case PBMCs are attached to the surface of wells with poly-D-Lysine. Anyway I can't find their post but I wonder if they have any opinions on this.

 

Answer from the author on LinkedIn:

His longer answer wasn't captured well:

"Thanks Anil, I read this paper yesterday evening. Yes, not only IDO1, but maybe in synergy with IDO2, we have considered trying to test and compare this hypothesis among fatal COVID, PASC, and ME/CSF patients; unfortunately, I did not get the material from ME/CSF patients finally. It is really easy to verify if you have sections of muscle or brain. Anyway, as far as I know, Our collaborators, Brent's team, are continuing to work on similar clinical projects, including ME/CSF."

https://www.linkedin.com/feed/update/urn:li:activity:7090089540291317760?commentUrn=urn:li:commentactivity:7090089540291317760,7090296665969975296)

 

An article in dutch news which describes some of their research and how they're very keen on testing therapeutics but are encountering problems on that front https://www.volkskrant.nl/wetenscha...-wetenschappers-zitten-op-een-spoor~ba477769/.

 

Google translated versions without paywall.

https://docs.google.com/document/d/1RExZS00rQhPcmqgs-EcN67_UCDwN3u3guxueaL8PM3E/edit?usp=drivesdk

 

News article put through Google Translate with some manual adaptions:

https://nos.nl/artikel/2486149-expe...jn-in-beeld-als-antwoord-op-longcovidklachten




Experimental cancer medicine in the picture as an answer to long covid complaints

A new important step has been taken in research into long covid. Immunologist René Lutter of Amsterdam UMC discovered that an enzyme is the cause of fatigue in patients with long covid. An experimental cancer drug could halt the production of this enzyme.

Lutter and his team have been researching the blood of long covid patients and the organs of deceased patients with long covid for two years now. It is estimated that there are more than 100,000 people in the Netherlands who still have serious complaints years after their covid infection.


"We still have to test it all, let's be careful and don't give people false hope," Lutter immediately said in the NOS Radio 1 Journaal. Still, his findings shed a new look at long covid.

IDO-2 enzyme

The immunologist accidentally came across the enzyme IDO-2. He specializes in asthma. In patients with this lung disease, the IDO-1 enzyme can often be seen in the blood. He expected this to also be the case with long covid patients, but came across a brother. "To my surprise, I actually didn't see much of IDO-1. I only saw IDO-2."

The research team found out that the body produces the IDO-2 enzyme to fight a covid infection. This enzyme disappears in most people some time after a covid infection. In patients with long covid, the body continues to produce the IDO-2 enzyme, which keeps them overtired. The brake is off and that causes body cells to be damaged or die.



Lutter therefore found "an insane amount" of the enzyme in the organs of deceased covid patients. IDO-2 has also been found in the blood of all fifteen long covid patients currently being examined by the team.

Lutter discovered that a substance being tested on cancer patients may have an inhibitory effect on the production of the IDO-2 enzyme. He tested a small amount of the substance on blood samples from six patients. With each sample, the enzyme in the cells decreases very strongly, resulting in less damage to the cells.

Not yet tested on humans

The drug has not yet been actually tested on patients. It is therefore not yet clear whether it has the same effect in practice as in the laboratory. The study design is ready for this and the subsidy application has been submitted. Lutter wants to try four different doses of the drug in 36 patients.

Two major manufacturers produce the drug: Brystol Myers Squibb (BMS) and Bayer. "The study of the drug is in phase 1. That is a kind of safety study," Lutter explains.

That immediately creates a major hitch in the cable. Because the effect of the drug is still being researched, the manufacturers are reluctant to provide the drug. "Bayer is very interested. But because the study has not yet been completed, they are unsure whether they want to release the drug. Unfortunately, BMS does not want to respond," says Lutter.

It is therefore not yet clear whether the research team can obtain the drug. Lutter hopes to use the drug as soon as possible to see if it works.

'Not acceptable'

Michael Rutgers, chairman of patient organization PostCovid NL and director of the Lung Fund, thinks it is "not acceptable" that the drug is not yet available.

"These results show how important it is to continue research into long covid," Rutgers said. "It gives hope. This group of patients has had no hope for a long time. They will become greenhouse plants. And there are tens of thousands of them."


Rutgers wants to assist the researchers as much as possible. "We're going to see if we can help find money for the research." He also wants to mediate in the consultations with the pharmaceutical companies.

The ultimate hope is that the drug can reduce the complaints of patients with long covid. "We don't know if it works. But I do think it's a way to keep things from getting more serious," says Lutter. He hopes for full recovery in patients: "But we still have to be careful to say that."

 

BMS drug is IK 175

https://ir.ikenaoncology.com/news-r...nounces-initial-clinical-data-ik-175-program/


Bayer drug is BAY 2416964

https://www.healio.com/news/hematol...olerability-activity-in-advanced-solid-tumors

 

Rene Lutter gave an interesting talk about the findings during a symposium organized by C-support. I added auto-generated subtitles. Original video in the description on YouTube. They seem to have progressed a bit further than the paper I think.

https://www.youtube.com/watch?v=h_tD_B801ho