Soluble IL-2R impairs muscle cell mitochondrial respiration in fatigued individuals with post-acute sequelae of COVID-19, 2024, Peterson Brown

[Mij]

Abstract

Post-acute sequelae of COVID (PASC) persist in many patients for weeks and months after recovery from initial SARS-CoV-2 infection. Recent evidence suggests that pathological changes in skeletal muscle may contribute significantly to ongoing pain and fatigue, particularly post-exertional malaise.

This study aimed to investigate the underlying mechanisms of PASC-related fatigue by examining skeletal muscle function and circulating factors in affected individuals.

We conducted a cross-sectional case-control study of patients with fatigue-associated PASC who had experienced mild to moderate COVID-19 without hospitalization. Skeletal muscle biopsies revealed reduced mitochondrial respiration and content in PASC participants compared to healthy controls. This lower respiratory capacity was accompanied by markedly elevated circulating levels of soluble IL-2 receptor alpha subunit (sIL2R), a T cell-specific receptor.

In vitro experiments demonstrated that sIL2R directly impairs mitochondrial oxygen consumption and reduces mitochondrial complex III subunit protein levels in cultured muscle cells. These findings suggest a mechanism linking systemic immune dysregulation to muscle-specific mitochondrial dysfunction in PASC.

This work provides new insights into the pathophysiology of PASC identifying sIL2R as a promising therapeutic target for addressing mitochondrial deficits in PASC-related fatigue and opening avenues for developing targeted interventions.

LINK

 

[Mij]

Why measure soluble interleukin-2 receptor levels?

The measurement of soluble interleukin-2 receptor (sIL-2R) levels in serum or plasma in adults has become a valuable tool for clinicians to assess immune function in vivo as part of the investigation, management, or prognosis of a wide range of diseases.

What makes sIL-2R an appealing biomarker?

Earlier, it was observed that elevated blood levels of sIL-2R are indicative of an ongoing immune response, which could potentially serve as a monitoring tool for a wide range of immune-mediated diseases. This initial concern about the generic nature of the response should not overshadow its significance.

In general, conditions characterized by excessive production of lymphocytes, known as lymphoproliferative disorders, exhibit higher sIL-2R levels compared to healthy individuals. Similarly, granulomatous diseases like sarcoidosis, where T-cell activation is a typical characteristic, also demonstrate elevated sIL-2R levels.

The consistent stability of sIL-2R levels throughout adulthood, along with minimal gender-related variations, further enhances its appeal as a biomarker.

However, it is important to consider individual variations in factors such as age, gender, lifestyle, and overall health when determining reference values and ranges.

Since there is no universally recognized standard set by the World Health Organization (WHO), the measurements presented in this table are dependent on the specific assay used. Nevertheless, it is recommended to calibrate assay kits against the international reference standard NIBSC 97/600.

The widespread adoption of sIL-2R measurement as a standard practice in the clinical setting will, of course, rely on the development of assays compliant with IVDR.

Nevertheless, a significant portion of the groundwork necessary for their introduction into the clinic has been accomplished, and sIL-2R ELISA kits are already accessible for research purposes.

As more efforts are focused on uncovering the potential applications of this ubiquitous and intricate biomarker, it can aid in diagnosing and monitoring autoimmune diseases.

LINK

 

[SNT Gatchaman]

findings could explain post-exertional malaise in PASC patients despite seemingly normal baseline muscle function. It suggests that mitochondria may function adequately under resting conditions but fail to meet increased energy demands during exertion. The link between post-viral fatigue and mitochondrial abnormalities in muscle has been recognized since the 1990’s. While our findings stem from COVID participants, they may have broader implications for other viral infections and post-viral care. Furthermore, although we focused on skeletal muscle, sIL2R might potentially impair mitochondrial respiration in other tissues, such as the central nervous system. Future research could explore whether sIL2R contributes to cognitive symptoms like "brain fog" in PASC patients.

This looks like it might be the major paper for the week to discuss.

We found that basal respiration was unchanged, but maximal respiration was significantly reduced after treatment with sIL2R when compared to vehicle (PBS). Administration of IL-2 had no significant effect on OCR when compared to vehicle, suggesting that the mechanism of action for sIL2R is distinct from known IL-2 pathways described for T cells. We assessed CCO activity and found that myotubes treated with sIL2R have significantly lower complex-IV activity. We performed a western blot analysis and found that complex III subunit protein (UQCRC2) was significantly lower with sIL2R administration. Mitochondrial proteins ATP5A (CV), SDHB (CII), and NDUFB8 (CI) were trending to be lower but did not reach statistical significance.

It would be valuable to address the mechanistic aspects of soluble interleukin-2 receptor (sIL2R) action on mitochondria in muscle tissue. While the exact pathway remains speculative, exploring potential mechanisms could provide a framework for future investigations. One possibility is that sIL2R interacts with membrane-bound receptors on muscle cells, initiating a signaling cascade that ultimately affects mitochondrial function. One such possibility is the β1-integrin, which may interact with extracellular IL2R to mediate intracellular signaling that regulates myoblast proliferation and differentiation. Another possibility is a mechanism of trans-signaling reminiscent of soluble IL-6R (sIL-6R), where the sIL6R-IL-6 complex binds to gp130 to mediate intracellular potentiation of proinflammatory pathways. Alternatively, sIL2R might cross the muscle cell membrane through yet-unidentified transport mechanisms, directly influencing intracellular processes.

Signalling? Potential link with FOXP4 / FOXP3?

Despite the methodological issues commented on in thread Tolerability and Efficacy of s.c. IgG Self-Treatment in ME/CFS Patients with IgG/IgG Subclass Deficiency: A Proof-of-Concept Study (2021, Journal of Clinical Medicine) their finding was that "responders" had lower sIL-2r.

 

[SNT Gatchaman]

sIL-2r is elevated in a number of conditions, including sarcoidosis as noted by Mij. Recent review article —

The soluble IL-2 receptor α/CD25 as a modulator of IL-2 function (2024)
Juliane Lokau; Lynn M. Petasch; Christoph Garbers

The pleiotropic cytokine interleukin-2 (IL-2) is an integral regulator of healthy and pathological immune responses, with the most important role in regulating the homeostasis of regulatory T cells. IL-2 signalling involves three distinct receptors: The IL-2 receptor α (IL-2Rα/CD25), IL-2R β , and IL-2R γ / γ c . While IL-2R β and γ c are essential for signal transduction, IL-2Rα regulates the affinity of the receptor complex towards IL-2.

A soluble form of the IL-2Rα (sIL-2Rα) is present in the blood of healthy individuals and increased under various pathological conditions. Although it is known that the sIL-2Rα retains its ability to bind IL-2, it is not fully understood how this molecule affects IL-2 function and thus immune responses.

Here, we summarize the current knowledge on the generation and function of the sIL-2Rα. We describe the molecular mechanisms leading to sIL2Rα generation and discuss the different IL-2 modulating functions that have been attributed to the sIL-2Rα. Finally, we describe attempts to utilize the sIL2Rα as a therapeutic tool.

Link | PDF (Immunology) [Open Access]

 

[SNT Gatchaman]

It has also been highlighted in pain "catastrophising" studies —

Elevated circulating soluble interleukin-2 receptor sCD25 level is associated with prefrontal excitatory-inhibitory imbalance in individuals with chronic pain: A proton MRS study (2024)
Ma; Subramaniam; Yancey; Farrington; McGlade; Renshaw; Yurgelun-Todd

Aberrant neuronal excitability in the anterior cingulate cortex (ACC) is implicated in cognitive and affective pain processing. Such excitability may be amplified by activated circulating immune cells, including T lymphocytes, that interact with the central nervous system. Here, we conducted a study of individuals with chronic pain using magnetic resonance spectroscopy (MRS) to investigate the clinical evidence for the interaction between peripheral immune activation and prefrontal excitatory-inhibitory imbalance.

In thirty individuals with chronic musculoskeletal pain, we assessed markers of peripheral immune activation, including soluble interleukin-2 receptor alpha chain (sCD25) levels, as well as brain metabolites, including Glx (glutamate + glutamine) to GABA + (γ-aminobutyric acid + macromolecules/homocarnosine) ratio in the ACC.

We found that the circulating level of sCD25 was associated with prefrontal Glx/GABA + . Greater prefrontal Glx/GABA + was associated with higher pain catastrophizing, evaluative pain ratings, and anxiodepressive symptoms. Further, the interaction effect of sCD25 and prefrontal Glx/GABA + on pain catastrophizing was significant, indicating the joint association of these two markers with pain catastrophizing.

Our results provide the first evidence suggesting that peripheral T cellular activation, as reflected by elevated circulating sCD25 levels, may be linked to prefrontal excitatory-inhibitory imbalance in individuals with chronic pain. The interaction between these two systems may play a role as a potential mechanism underlying pain catastrophizing. Further prospective and treatment studies are needed to elucidate the specific role of the immune and brain interaction in pain catastrophizing.

Link | PDF (Brain, Behavior, and Immunity)

 

[SNT Gatchaman]

And paediatric functional somatic disorders —

Inflammatory Markers in Children and Adolescents with Functional Somatic Disorders: A Systematic Review (2024)
Hansen, Anne Sofie; Rask, Charlotte Ulrikka; Kallesøe, Karen Hansen

Functional somatic disorders (FSDs) are common in children and adolescents. Findings suggest that low-grade inflammation has a role in the development and maintenance of pediatric FSDs. This systematic review included studies with original data on systemic inflammatory markers in children and adolescents with an FSD compared to individuals without an FSD.

The literature search identified 1374 articles. After assessment, a total of 15 studies met the inclusion criteria. In total, 41 serum or plasma cytokines were assayed in a population of 696 children and adolescents. Altered cytokine levels in patients with FSDs were reported in 12 studies, whereas three studies found no significant differences when comparing patients with FSDs and controls.

The cytokine levels were significantly elevated in nine studies (i.e., IL-2, IL-6, IL-8, IL-12 (p70), CRP, hsCRP, IP-10, MCP-1, sTIM-3, sCD25 and TNF-α). The findings indicate that inflammatory response may have a role in the pathophysiology of pediatric FSDs. However, the included studies showed limited quality with potential risk of bias, small study populations and a narrow spectrum of included FSDs, which limits the generalizability of the results.

To further explore the potential link between inflammatory markers and pediatric FSDs, future research using a longitudinal study design is recommended.

Link | PDF (Children) [Open Access]

 

[Hutan]

Thanks MIJ and SNT
This is a paper from mostly a University of Kentucky team, with varied backgrounds but a substantial focus on muscle biology.
NIH funding

They appear to have started with the idea that there is something in the blood of people with PASC that causes mitochondrial dysfunction:

Specifically, we suggest that certain circulating factors, most likely T-cell derived, are elevated in PASC and induce mitochondrial abnormalities [16-22].

They then tested substances in the blood of people with PASC, to find out which ones affected mitochondria,

To test this hypothesis, we conducted an analysis of plasma immune markers of PASC participants to identify candidates that may directly or indirectly impair muscle cell mitochondrial respiratory capacity.

They say that they found reduced mitochondrial content, impaired muscle function and elevated sIL-2R-a

Our findings revealed significant reductions in measures of mitochondrial content and impaired muscle energetics in individuals suffering from fatigue specific PASC. We identified the T cell-derived soluble IL-2 receptor alpha subunit (sIL2R) as the most significantly elevated systemic factor following recovery from SARS-CoV-2 infection.

 

[Hutan]

On the methods:
People were eligible to participate as soon as 4 weeks after the Covid-19 infection, and they just had to report ongoing fatigue. The average time after the positive test for the acute illness was only 59 days.

And the healthy controls were mostly historic pre-pandemic samples in a biobank.

Age for healthy participants (n=25, 40% female) was 28.0±14.0 years and for PASC subjects (n=11, 45.5% female) was 32±21 years.

Those are quite small sample sizes - only 11 for PASC. There was an unusually high percentage of male subjects.

So, whatever they find, unless there is a very clear distinction, it's not going to be super conclusive due to possible issues related to sample size and storage (does fresher material have higher levels of IL-2R-a than material stored for a while in a biobank?).

 

[Hutan]

On the results:

Activity
At this early stage of the illness, muscle size, fibre composition, and muscle strength were still similar to the healthy controls.
Three participants (27%) reported no change in activity after infection, of which two had been highly active, and one was intermediately active prior to infection.

Mitochondrial analyses - Reduced mitochondrial content claim

Permeabilized muscle fiber bundles were prepared from freshly isolated biopsies of the vastus lateralis muscle in both PASC and healthy participants (n = 10 healthy, 7 PASC).

Note that the sample size was even smaller for these investigations - only 7 people with PASC.

We sought to gain insight into muscle mitochondrial activity using common histological methods: succinate dehydrogenase (SDH) histochemistry, which is a surrogate indicator of SDH enzyme activity.
The intensity of SDH histochemistry was quantified as a percent of all fibers showing dark, intermediate, or light coloration. In the PASC group (n = 9), there was a significantly (p < 0.05) higher percentage of intermediate intensity fibers (Supplemental Fig 1C) than in the healthy group (n = 7), apparently suggesting lower mitochondrial activity.

There's the Supplemental Fig 1C above. Yeah, there does look to be a higher percentage of intermediate intensity samples, and they go on to say that it was mostly in Type 2 muscle fibres. But here is Supplemental Fig 1E, with the Type 2 muscle fibres:

That's pretty underwhelming. There are really only 3 PASC people with a higher percentage of intermediate intensity fibres, and the sample size is so small.

They go on to investigate mitochondrial function further:

The observation that SDH activity differed in type II fibers prompted us to explore potential differences in mitochondrial respiration between healthy individuals and those with PASC. To investigate this, we used high resolution respirometry (HRR) to measure mitochondrial oxygen flux (JO2).

They found:

State 3 respiration for complex I and combined complex I+II were significantly (p < 0.05) lower in PASC muscle compared to control (Fig 2A-B). Complex II respiration and maximal electron transfer (ET) capacity were not significantly different between groups (Fig 2C-D).

I think there might be something there in Figures 2A and 2B. There is overlap between PASC and the controls though, perhaps not everyone in the small PASC group has the same thing causing their fatigue.

Citrate synthase activity and cytochrome C oxidase activity were further measured in tissue homogenates (Fig 2E-F). We found that citrate synthase (CS, a proxy for mitochondrial content) was significantly lower, but cytochrome C oxidase (CCO, a proxy for complex IV activity) did not reach statistical significance (p = 0.0848, n = 7 healthy, 11 PASC). The ratio of CCO to CS was not significantly different between groups (Fig 2G), suggesting that mainly mitochondrial content and not activity was lower in PASC compared to healthy muscle.

Here are Figures 2E and 2F:

Again, maybe there's something there with lower CS (which they say is a proxy for mitochondria content) and CCO (which they say is a proxy for complex IV activity), but there is overlap. Note that the proxy for complex IV activity is not statistically significant here, but they note later (see SNT's post above (#3) where the authors say

myotubes treated with sIL2R have significantly lower complex-IV activity

So, they are potentially hanging their hat on a molecule that reduces complex-IV activity, but a reduction in complex-IV activity wasn't significant in the muscle biopsies. Maybe it would be significant with a bigger and better characterised sample?

Electron microscopy demonstrated several alterations in muscle mitochondrial morphology with occurrences of rupture as well as autophagosome localization near mitochondria (Supplemental Fig 2).

That's interesting. The two pictures of PASC mitochondria do indeed seem to have a lot more of what are identified as autophagosomes near them than the one picture from a healthy control. But, the difference is not quantified.

To determine if mitochondrial complex protein levels were stoichiometric, we performed western blot analysis (Fig 2H) of 4 key subunits of complex I (NDUFB8, Fig 2I), II (SDHB, Fig 2J), III (UQCRC2, Fig 2K), and V (ATP5A, Fig 2L). Quantification of those blots indicated complex II protein was significantly lower, and complex III approached significance (p = 0.0544, n = 7 healthy, 11 PASC). To determine if changes in mitochondrial content and function from SARs-CoV-2 infection were due to altered mitochondrial biogenesis, we measured synthesis rates of mitochondrial protein [42]. However, there were no statistical differences in the fractional synthesis rate (FSR) for muscle mitochondrial protein (Supplemental Fig 3), indicating that mitochondrial biogenesis was not impaired.

So, they quantified the amount of subunits of the complexes in the electron chain. Here are the charts:


And yep, with a bigger and better characterised sample, maybe there would be reductions in the complexes. But, this is not strong evidence.

So, it's frustrating. This is potentially important study, looking at mitochondria in muscles. But, it's so underpowered, and the requirement to have had fatigue for 4 weeks after Covid-19 is a pretty low bar when it comes to cohort selection.

The paper then goes on to look at sIL-2R levels.

 

[Hutan]

On the sIL-2R finding:
They tested a lot of things (65), IL-2R on the left does look interesting.

Circulating sIL2R is significantly elevated in PASC participants and can directly impair mitochondrial function in muscle cells in vitro.

To assess if sIL2R has an inhibitory effect on mitochondrial respiration, we treated C2C12 myotubes with human recombinant sIL2R or IL-2 for 24 hours and used a Seahorse Extracellular Flux Analyzer to assess oxygen consumption rate (OCR) (Fig 4A, n = 4).

They investigated the impact of SIL2R and IL-2 (separately) on the oxygen consumption rate of a type of muscle cell in the Seahorse machine.

We found that basal respiration (Fig 4B) was unchanged, but maximal respiration (Fig 4C) was significantly reduced after treatment with sIL2R when compared to vehicle (PBS). Administration of IL-2 had no significant effect on OCR when compared to vehicle (Supplemental Fig 4), suggesting that the mechanism of action for sIL2R is distinct from known IL-2 pathways described for T cells.

Figure 4 certainly is interesting. That's a big difference in oxygen consumption rate. They went on to find lower complex IV activity and lower complex III protein levels.

 

[V.R.T.]

Well that sample size is disappointing. I hope they try this with a much bigger cohort.