A multi-omics based anti-inflammatory immune signature characterizes long COVID-19 syndrome, 2022, Kovarik et al

[marcjr]

This thread starts with discussion of the preprint.
Now that the research has been published, the thread title has been changed and the abstract posted in post #13

Preprint
Multi-omics provide evidence for an anti-inflammatory immune signature and metabolic alterations in patients with Long COVID Syndrome – an exploratory study
Johannes J. Kovarik et al

ABSTRACT

Despite the increasing prevalence of patients with Long Covid Syndrome (LCS), to date the pathophysiology of the disease is still unclear, and therefore diagnosis and therapy are a complex effort without any standardization. To address these issues, we performed a broad exploratory screening study applying state-of-the-art post-genomic profiling methods to blood plasma derived from three groups: 1) healthy individuals vaccinated against SARS-CoV-2 without exposure to the full virus, 2) asymptomatic fully recovered patients at least three months after SARS-CoV-2 infection, 3) symptomatic patients at least 3 months after a SARS-CoV-2 infection, here designated as Long Covid Syndrome (LCS) patients.

Multiplex cytokine profiling indicated slightly elevated cytokine levels in recovered individuals in contrast to LCS patients, who displayed lowest levels of cytokines. Label-free proteome profiling corroborated an anti-inflammatory status in LCS characterized by low acute phase protein levels and a uniform down-regulation of macrophage-derived secreted proteins, a pattern also characteristic for chronic fatigue syndrome (CFS). Along those lines, eicosanoid and docosanoid analysis revealed high levels of omega-3 fatty acids and a prevalence of anti-inflammatory oxylipins in LCS patients compared to the other study groups. Targeted metabolic profiling indicated low amino acid and triglyceride levels and deregulated acylcarnithines, characteristic for CFS and indicating mitochondrial stress in LCS patients. The anti-inflammatory osmolytes taurine and hypaphorine were significantly up-regulated in LCS patients. In summary, here we present evidence for a specific anti-inflammatory and highly characteristic metabolic signature in LCS which could serve for future diagnostic purposes and help to establish rational therapeutic interventions in these patients.

https://www.medrxiv.org/content/10.1101/2022.07.11.22277499v1

Not sure if we are allowed to post images from the paper, but the last one summarizes well their findings.

 

[SNT Gatchaman]

Will read shortly, but: multi-omics in healthy/vaccinated vs Covid-recovered vs long Covid - Hallelujah!

 

[LarsSG]

"Targeted metabolic profiling indicated low amino acid and triglyceride levels and deregulated acylcarnithines, characteristic for CFS and indicating mitochondrial stress in LCS patients."

Oddly, these are only low in the LC group relative to the Covid recovered group, not low relative to the healthy controls (with the exception of one amino acid, valine). Other aminos were higher in LC than controls. Some of it could just be random since the groups only have 13 people in each (e.g. triglyceride could just happen to have been higher in a few members of the recovered group, which the intervals given suggest). It's certainly interesting, but lower levels relative to recovered patients seems kind of different from lower levels relative to healthy controls.

 

[dreampop]

Iirc Ron Davis said he found higher omega3s in me/cfs patients but postulated it was due to supplementation. Think it was in a video.

 

[SNT Gatchaman]

Iirc Ron Davis said he found higher omega3s in me/cfs patients but postulated it was due to supplementation.

To confirm our hypothesis regarding the role of fatty acids in LCS (see below) and to exclude an effect from oral supplementation, we analyzed samples from a fourth cohort including 10 healthy subjects which had not been exposed to SARS-CoV-2. For this purpose, these subjects received tablets containing 870mg Omega-3 (420 mg EPA and 330 mg DHA) twice daily in a prospective study design for one week

Increased levels of trans-DHA in long-covid patients are shown in an extracted ion chromatogram [...]. No increase in trans-DHA levels after nutritional supplementation of omega-3 capsules was observed in healthy controls.

(EPA is eicosapentaenoic acid and DHA is docosahexaenoic acid).

 

[Midnattsol]

(EPA is eicosapentaenoic acid and DHA is docosahexaenoic acid).

One week is not nearly enough to compare omega-3 levels to someone who has been supplementing regularly, blood levels can increase for weeks after starting supplementation. I would have given them supplementation at least a month, and might even have increased the dose to 3 g/day not a bit under 2 g/day.

Edit: There are also obviously individual differences and 10 people isn't a lot.

 

[SNT Gatchaman]

only alpha-1-anti trypsin (SERPINA1) and vitamin D binding protein were found significantly up-regulated in the LCS group. A larger number of proteins was found down-regulated, including the protease inhibitors SEPINA5 and SERPINF2, Biotinidase and the macrophage-associated proteins soluble CD14, ANG and Proteoglycan 4 (PRG4).

Finally, Vitamin D binding protein, actually up-regulated in plasma of LCS patients, may also be involved in macrophage functions, as it is a precursor for the so-called macrophage-activating signal factor.

A few points from a recent review article: Vitamin D Binding Protein: A Historic Overview (2020)

It developed early in evolution and is conserved.

DBP is found in nearly all vertebrate species with well-conserved structure over the 500 million span of vertebrate evolution. This is especially the case for the ligand (25OHD) binding cleft.

Two major functions —

The transport of all vitamin D metabolites (at a single binding cleft of the A domain of the protein). Due to the combination of high affinity for vitamin D metabolites and a high protein concentration, free concentrations of all vitamin D metabolites are extremely low.

Tight binding of actin, creating a DPB-Actin complex that avoids [harmful] actin polymerization in serum after tissue damage.

Its plasma concentration is stable and independent of many factors, including vitamin D levels.

DBP circulates in serum in much higher concentrations than the combined concentration of all vitamin D metabolites

Vitamin D deficiency or excess, or vitamin D resistance, idiopathic hypercalcemia of infancy, or osteoporosis and many other diseases (such as hyperparathyroidism or hyperthyroidism, sarcoidosis, cancer, Addison’s disease, or growth hormone deficiency) have no effect on serum DBP concentration. DBP is slightly increased in acromegaly and in some inflammatory diseases (such as in rheumatoid arthritis).

Concentration is increased in females.

In humans, exposure to estrogens increases serum DBP but androgens have no effects.

Concentration is increased with infection.

DBP is a positive acute phase reactant after infections or minor trauma. Severe trauma (e.g., hip fracture) or severe illness (such as patients requiring intensive care treatment) decreases serum DBP by more than 10%.

It binds extracellular actin and is involved in clearance of fibrillar actin.

Many actin-binding proteins regulate or control intracellular actin polymerization or disassembly, including profilin. The extracellular binding proteins are, however, mainly DBP and gelsolin (also known as brevin).

Actin can easily switch from monomeric into polymeric actin in the cytosolic milieu, thereby helping in building the intracellular organizations of cells. In the plasma milieu, actin monomers are rapidly transformed into polymeric structures, which could result in clogging the microcirculation much like fibrinogen/fibrin. This process could be accelerated by the aggregation of platelets as actin-ADP binds to platelet surfaces and acts as agonist for platelet aggregation

May effect macrophage function.

deglycosylation of DBP can activate DBP to become a macrophage activating factor (MAF). A membrane bound β-galactosidase present on the surface of immune B cells and a sialidase present on T cells would remove two of the three sugar residues of DBP/GC1 protein. This DBP would then be able to facilitate the differentiation of monocytes to become osteoclasts and even correct the osteopetrosis phenotype of mice

See also Vitamin D-binding protein as a biomarker to confirm specific clinical diagnoses (2019)

 

[Hutan]

A team entirely from Vienna, Austria:

Johannes J. Kovarik, View ORCID ProfileAndrea Bileck, View ORCID ProfileGerhard Hagn, View ORCID ProfileSamuel M. Meier-Menches, Tobias Frey, Anna Kaempf, Marlene Hollenstein, View ORCID ProfileTarik Shoumariyeh, View ORCID ProfileLukas Skos, View ORCID ProfileBirgit Reiter, View ORCID ProfileMarlene C. Gerner, View ORCID ProfileAndreas Spannbauer, View ORCID ProfileEna Hasimbegovic, View ORCID ProfileDoreen Schmidl, View ORCID ProfileGerhard Garhöfer, View ORCID ProfileMariann Gyöngyösi, View ORCID ProfileKlaus G. Schmetterer, View ORCID ProfileChristopher Gerner

There's acknowledgement that LC isn't just anxiety and stress. There's a good list of symptoms, although 'lack of physical fitness' would have been better replaced with 'post exertion malaise and easy fatiguability'. The severity of the disease is noted. Although LC is described as 'novel', they go on to recognise the similarity with CFS in general and MERS and SARS in particular. This is not the first paper to suggest that long term symptoms following SARS1 has been observed 'for up to two years'. I'm sure that the persistence of symptoms from MERS and SARS have been much longer than that - there must surely be a reference that records that. But, overall, I thought it was a really good start.

At first, the observed symptoms were mainly attributed to psychological conditions such as anxiety and stress in the affected individuals (4). However, it is now recognized that chronic persistence of COVID-19 symptoms after acute infection constitutes a novel somatic disease entity termed post-acute COVID syndrome (PACS) or Long-COVID Syndrome (LCS)

Typically, LCS patients suffer from general fatigue, lack of concentration (self-described as “brain fog”) and physical fitness, dyspnea, postural tachycardia as well as a broad range of other clinical symptoms throughout the whole organism, which severely impedes the quality of life (6).

These clinical presentations mirror the situations found in chronic fatigue syndrome (CFS) which can be secondary to infection with different viruses and were also found in other past coronavirus epidemics including Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). Especially for the latter symptom persistence for up to two years has been observed (7). Strikingly, development of LCS is not associated with disease severity and so far, only a few studies about potential risk factors and associated comorbidities have been published (8, 9).

 

[Hutan]

There are four groups:
H - healthy, vaccinated at least 3 months ago, no covid 19 infection - 13 people

HL - healthy, no covid 19 infection, given tablets containing 870mg Omega-3 (420 mg EPA and 330 mg DHA) twice a day for one week - 10 people

R - recovered from a Covid-19 infection at least 3 months ago - 13 people

LCS - had a Covid-19 infection at least 3 months ago and have lingering symptoms (notably fatigue (9/13) and/or cognitive difficulties (10/13), dyspnoea (9/13)) - 13 people

They describe this study as an exploratory study - the sample sizes are small at 13 people in each group. From the listed symptoms of the LCS group, I don't think they would all qualify for an ME/CFS diagnosis, although perhaps if they were assessed by a clinician who routinely diagnoses ME/CFS, the description of symptoms would be different.

The samples look fairly well matched in terms of gender and age, although the inclusion of people with significant health conditions is a bit odd (e.g. the LCS group includes a person with multiple sclerosis and a person with autoimmune thyroiditis).

 

[Hutan]

a broad panel of 65 cytokines, chemokines and soluble receptors associated with immune activation was assayed.

Remarkably, neither pro-nor anti-inflammatory cytokines were found significantly up-regulated in LCS patients compared to the other two groups

They make quite a lot of IL-18 levels being lower in the LCS group, but there's a lot of overlap with the other two groups- I'm not convinced there is a real difference in IL-18.



But, some of the the LCS group do look different from the healthy groups - look at the PCA and volcano plot. They just aren't presenting as a particularly well-characterised group - individuals in the LCS group seems to be fairly different from each other as well as different from the healthy groups, which isn't very helpful.



Serpina5 looks really interesting - the LCS are nicely clustered with lower levels. The clustering of BTD (biotinidase) in the LCS is nice, but there's a lot of overlap with the healthy groups, so, not so interesting. GC (inexplicably to me at the moment, 'GC' is actually Vitamin D binding protein) levels are statistically different between the recovered and Long Covid groups, but there is plenty of overlap between the Long Covid group and the healthy groups, so it's not looking very important to me.

 

[SNT Gatchaman]

GC (inexplicably to me at the moment, 'GC' is actually Vitamin D binding protein)

GC was its old name ("group-specific component") which reflected —

In 1961, when serum proteins were still mainly characterized by their electrophoretic mobility as α, β, or γ globulins, further identification of the major proteins led to the discovery a highly polymorphic protein with genetically defined small differences in electrophoretic mobility, and therefore named “Group-specific component” or GC.

The first major link between GC and DBP was made by Daiger et al. Indeed, from the perfect parallelism between the electrophoretic mobility of GC (measured by immunologic techniques) and the mobility of [14C]vitamin D, they concluded that GC is the major transport protein for vitamin D.

(from the review article linked above)

 

[Hutan]

Here's something about Serpina 5 - I'm posting it at least partly because I like the title:
Cell penetrating SERPINA5 (Protein C inhibitor, PCI): More questions than answers
A 2016 paper, perhaps coincidentally by two people also based in Vienna.

SERPINA5 binds glycosaminoglycans, phospholipids, and retinoic acid. Glycosaminoglycans and certain phospholipids can modulate its inhibitory activity and specificity. Studies suggest that SERPINA5 may play a role in hemostasis*, in male reproduction, in host defense, and as a tumor suppressor. However, its biological role has not yet been defined. So far SERPINA5 deficiency has not been described in man.

SERPINA5 can be internalized by cells and translocated to the nucleus. The internalization is dependent on the phospholipidphosphatidylethanolamine and on the intact N-terminus of SERPINA5, which functions as a cell penetrating peptide. Further functional analysis of intracellular SERPINA5 will contribute to our understanding of the biological role of this molecule.

*hemostasis:
Hemostasis is a complex network of cellular and humoral systems, involving the platelet system, the coagulation process, the anticoagulant, and the fibrinolytic pathways)

Edited to add: the authors of the paper that is the subject of this thread had this to say about SERPINA5:

SERPINA5 serves as antagonist of the protease furin, which is essential for viral entry into human cells (24). Thus, it is intriguing to speculate that alteration in the furin/SERPINA5 interplay – either due to genetic background of the individual or due to specific immune responses during SARS-CoV-2 infection – might provide a basis for the development of LCS.

 

[Milo]

Same author publishes this paper, different title:
A multi-omics based anti-inflammatory immune signature characterizes Long COVID Syndrome

It may be same paper, but edited after peer review? The abstract is not the same.

 

[Trish]

Same author publishes this paper, different title:
A multi-omics based anti-inflammatory immune signature characterizes Long COVID Syndrome

It looks like the same research, so the thread heading and opening post have been amended. The first section is called 'summary' rather than abstract.

Summary
To investigate long COVID-19 syndrome (LCS) pathophysiology, we performed an exploratory study with blood plasma derived from three groups: 1) healthy vaccinated individuals without SARS-CoV-2 exposure; 2) asymptomatic recovered patients at least three months after SARS-CoV-2 infection and; 3) symptomatic patients at least 3 months after SARS-CoV-2 infection with chronic fatigue syndrome or similar symptoms, here designated as patients with long COVID-19 syndrome (LCS).

Multiplex cytokine profiling indicated slightly elevated pro-inflammatory cytokine levels in recovered individuals in contrast to patients with LCS. Plasma proteomics demonstrated low levels of acute phase proteins and macrophage-derived secreted proteins in LCS. High levels of anti-inflammatory oxylipins including omega-3 fatty acids in LCS were detected by eicosadomics, whereas targeted metabolic profiling indicated high levels of anti-inflammatory osmolytes taurine and hypaphorine, but low amino acid and triglyceride levels and deregulated acylcarnitines.

A model considering alternatively polarized macrophages as a major contributor to these molecular alterations is presented.
________________

 

[Hutan]

Skim-reading this paper, the latest version of it now, there's a lot in it.

There is the issue with the selection of the Long Covid cohort - the definition seems to be chronic fatigue or cognitive dysfunction following Covid-19, plus one further chronic symptom including breathlessness, coughing or loss of the ability to smell. That has likely resulted in a bit of a heterogeneous LC cohort. Also the sample sizes are small.

Nevertheless, I still think this is an interesting study. I hope that replication of the key differential findings is underway in a more tightly characterised and larger sample.

it can be envisioned that a combination of presently described proteins (e.g. low SERPINA5), docosanoids (e.g. high DHA) and small metabolites (e.g. high hypaphorine) in patients with characteristic anamnesis and symptoms could help to identify and better define LCS. In this regard, large scale studies to assess the potential sensitivity and specificity of such scores, including the consideration of different SARS- CoV-2 strains, are clearly warranted.

In summary, here we present a distinct multi-omics signature demonstrating a prevalence of anti-inflammatory effector molecules combined with molecular patterns of characteristically altered metabolism detectable in plasma of LCS patients, offering a unique chance for diagnosis with selected molecular biomarkers and providing novel hypotheses about the pathophysiology of the disease, thus potentially aiding the development of urgently required treatment options.

I think it's an intriguing idea that levels of some molecules in LC people look like levels in healthy people who haven't had an infection, but quite different to levels of people who are functioning normally but are recovering from a Covid-19 infection. What if we aren't properly mounting healthy inflammatory responses, not even to minor challenges like exercise?

When we workout, we damage our muscles. Then, our bodies repair them and grow back even stronger. Inflammation comes into play during the repair process when the body increases blood flow to the affected area to replenish oxygen, fuel muscles, and clear out waste.

Thank you to the researchers and the funders.

This study was supported by the Faculty of Chemistry, University of Vienna, by grants from the Austrian Science Funds (FWF project #P-34728B) and the Medical Scientific Funds of the Mayor of the City of Vienna (project #COVID010).