Long COVID involves activation of proinflammatory and immune exhaustion pathways, 2025, Aid et al.

SNT Gatchaman

SNT Gatchaman

Senior Member (Voting Rights)
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Now published - see post #6


Preprint
Persistent Activation of Chronic Inflammatory Pathways in Long Covid
Malika Aid; Katherine McMahan; Nicole Hachmann; Jessica Miller; Erica Borducchi; David Hope; Marjorie Rowe; Eleanor Schonberg; Siline Thai; Ai-ris Collier; Janet Mullington; Dan Barouch

Long Covid, or Post-Acute Sequelae of COVID-19 (PASC), involves a spectrum of chronic symptoms following resolution of acute SARS-CoV-2 infection. Current hypotheses for the pathogenesis of Long Covid include persistent SARS-CoV-2, activation of other viruses, tissue damage, autoimmunity, endocrine insufficiency, immune dysfunction, and complement activation.

We evaluated 142 participants, including uninfected controls (N=35), acutely infected individuals (N=54), convalescent controls (N=25), and Long Covid patients (N=28), by comprehensive immunologic, virologic, transcriptomic, and proteomic analyses.

Long Covid was characterized by persistent inflammatory pathways compared with convalescent controls and uninfected controls, including upregulation of IL-6 and JAK-STAT pathways as well as activation of coagulation, complement, metabolism, and T cell exhaustion pathways. Moreover, robust activation of these pathways during acute COVID-19 infection correlated with the subsequent development of Long Covid. In an independent validation cohort (N=47), Long Covid patients had higher levels of plasma IL-6R compared with convalescent controls and uninfected controls.

These data demonstrate that Long Covid is characterized by persistent activation of chronic inflammatory pathways, suggesting novel therapeutic targets and biomarkers of disease.


Link | PDF (Preprint: BioRxiv) [Open Access]
 
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This preprint was from May so I'm expecting it to be published soon. Summary quotes —

Methodology
  • In this study, we evaluate the immunologic and inflammatory responses in people with Long Covid compared with convalescent controls at 3-6 months and >6 months after initial COVID-19 infection, using immunologic assays, virologic assays, transcriptomics, and proteomics.

  • samples included uninfected controls (Uninfected; N=35), acutely infected individuals <1 month following COVID-19 infection (Acute; N=54), convalescent controls (CC; N=24), and Long Covid patients (LC; N=28)

  • Clinical symptoms in the Long Covid (LC) cohort included persistent fatigue, shortness of breath, exercise intolerance, brain fog, and abnormal smell and taste

  • Peripheral blood mononuclear cells (PBMC) were collected at 3-6 months (LC: N=25; CC: N=25) and >6 months (LC: N=19) following COVID-19 infection. Plasma samples were similarly collected at 3-6 months (LC: N=23; CC: N=8) and >6 months (LC: N=16; CC: N=6) following COVID-19 infection.
Results: Virology
  • first assessed SARS-CoV-2 neutralizing antibody (NAb) responses using pseudovirus neutralization assays and T cell responses by pooled peptide interferon-g ELISPOT assays against SARS-CoV-2 WA1/2020, Delta, and Omicron BA.1 in the CC and LC groups. We did not detect differences in NAb titers, but we observed higher Spike-specific ELISPOT responses to all three variants in the LC individuals compared with the CC individuals (P=0.015, P=0.006, P=0.002, respectively)

  • may reflect either more severe acute infection or could represent potentially persistent virus […] did not detect plasma SARS-CoV-2 in any CC or LC individuals by RT-PCR genomic or subgenomic viral load assays
Results: Transcriptomics
Viral reactivation
  • did not detect differences in viral reads for multiple viruses in the LC compared with CC groups, including human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV)
Whole transcriptome
  • Unsupervised cluster analysis using the whole transcriptome revealed a separation between the LC and other groups, while the CC group clustered with the uninfected controls
Inflammation, complement, coagulation
  • upregulation of multiple inflammatory markers in the LC compared with the CC groups and uninfected controls, including the signaling molecules LIFR and JAK2; chemokines CXCL2, CXCL3, and CCL3; cytokines IL10, NLRP3, IFNG, IL6, TNF, IL1B, IL1A; and complement and coagulation proteins C5, F3, and THBS1

  • compare the expression profiles in PBMC of the LC and CC groups at 3-6 months following acute COVID-19 infection. The LC group was characterized by higher levels of innate immune cell signatures for monocytes, macrophages, neutrophils and dendritic cells; complement and coagulation cascade signatures; and cytokine and chemokine signaling pathways, including IL6, IL8, IL10, IL12, IL17, JAK_STAT, and type I interferon pathways
NK cells
  • Genes associated with natural killer function such as KLRC4, KLRC1, KLRC2 and transcription factors such LEF1, GATA6, BACH2 were decreased in the LC group compared with the CC and uninfected control groups
T cells
  • We observed downregulation of certain T cell pathways in the LC group compared with the CC group (Fig. 4c), including T cell differentiation and activation pathways and T regulatory cell signatures (Fig. 5a-b). However, signatures of T cell exhaustion and PD-1 signaling pathways were significantly increased in the LC group compared with the CC group (Fig. 5b), suggesting that Long Covid is associated with T cell dysfunction. T cell signaling correlated inversely with IL6, JAK-STAT, and interferon signaling (Fig. 5c).
Metabolism
  • also observed increased metabolic activity in the LC group compared with the CC group involving amino acid and lipid metabolism
Most significant pathways
  • pathways that showed the most significant enhancement in the LC group compared with the CC group were the IL6, JAK_STAT, IL1R, mast cell, coagulation, complement, bile acid metabolism, ascorbate/aldarate, and leptin signaling. No differences in these pathways were observed between the CC and uninfected controls.
Results: Proteomics
  • similar differences between the LC and CC groups, consistent with the transcriptomic data

  • increased levels of immune cell signatures, such as neutrophils, monocytes and epithelial cells; cytokine signaling, including IL6, IL12, IL8, NFKPB, JAK-STAT, and corticotropin releasing hormone; complement and coagulation cascades; and certain metabolic pathways

  • pathways of cytotoxic T cell and NK_DCs cross talk signaling were downregulated

  • IL6 signaling in plasma inversely correlated with cytotoxic T cell, amino acid metabolism, and DNA damage pathways and directly correlated with metabolic pathways

  • During acute COVID-19 infection, increased IL6, complement, and cortisol signaling correlated with the subsequent development of Long Covid at 3-6 and >6 months

  • Feature importance analyses revealed that plasma level of IL6R, IL6ST, STAT1, STAT3, PTPN11 and MAPK1,3,4 during acute COVID-19 infection (< 1month) was associated with the subsequent development of LC at 3-6 and >6 months

  • To validate our findings in an independent cohort, we evaluated plasma levels of proinflammatory cytokines and chemokines and found increased levels of IL6R in individuals with LC (n=19) compared with CC (n=13) and uninfected controls (n=13), using both and ELISA and a meso-scale discovery (MSD) assay at 3-6 months and >6 months after COVID-19 infection
 
The pathways that showed the most significant enhancement in the LC group compared with the CC group were the IL6, JAK_STAT, IL1R, mast cell, coagulation, complement, bile acid metabolism, ascorbate/aldarate, and leptin signaling. No differences in these pathways were observed between the CC and uninfected controls.
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Just picking out those two items Replicated blood-based biomarkers for Myalgic Encephalomyelitis not explicable by inactivity (2024, Preprint: MedRxiv) said —

Considering all cases combined, 54 proteins are significant (FDR < 0.05). Among these are 7 complement proteins (C1RL, C2, CFB, CFH, CFI, CFP and CR2) of the innate immune system, whose levels are all elevated in ME/CFS cases, including CR2 (complement C3d receptor 2)
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and

ME/CFS cases also show increase in levels of leptin (LEP), which has a role in energy homeostasis
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A few previous references to leptin in ME/CFS (there are older ones) —

Distinct plasma immune signatures in ME/CFS are present early in the course of illness (2015, Science Advances)

Cytokine signature associated with disease severity in chronic fatigue syndrome patients (2017, PNAS)

with S4ME threads already for —

Association of circulating biomarkers with illness severity measures differentiates myalgic encephalomyelitis/chronic fatigue syndrome and post-COVID-19 condition: a prospective pilot cohort study (2024, Journal of Translational Medicine)

Proteomics and cytokine analyses distinguish myalgic encephalomyelitis/chronic fatigue syndrome cases from controls (2023, Journal of Translational Medicine)
 
Long COVID involves activation of proinflammatory and immune exhaustion pathways

Aid, Malika; Boero-Teyssier, Valentin; McMahan, Katherine; Dong, Rammy; Doyle, Michael; Belabbaci, Nazim; Borducchi, Erica; Collier, Ai-ris Y.; Mullington, Janet; Barouch, Dan H.

Abstract​

Long COVID (LC) involves a spectrum of chronic symptoms after acute severe acute respiratory syndrome coronavirus 2 infection.
Current hypotheses for the pathogenesis of LC include persistent virus, tissue damage, autoimmunity, endocrine insufficiency, immune dysfunction and complement activation.

We performed immunological, virological, transcriptomic and proteomic analyses from a cohort of 142 individuals between 2020 and 2021, including uninfected controls (n = 35), acutely infected individuals (n = 54), convalescent controls (n = 24) and patients with LC (n = 28).
The LC group was characterized by persistent immune activation and proinflammatory responses for more than 180 days after initial infection compared with convalescent controls, including upregulation of JAK-STAT, interleukin-6, complement, metabolism and T cell exhaustion pathways.
Similar findings were observed in a second cohort enrolled between 2023 and 2024, including convalescent controls (n = 20) and patients with LC (n = 18).

These data suggest that LC is characterized by persistent activation of chronic inflammatory pathways, suggesting new therapeutic targets and potential biomarkers of disease.

Web | DOI | PDF | Nature Immunology
 
Jonathan Edwards said:
No data given, just interpretation.
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They have quite a lot of graphs and charts in the article - what they claim to have found would fit with a lot of what we discuss here.

They claim to have found an increase in IFN-Y and IFN a/b, along with JAK 3 and complement c2 and c4, and they say:
IFNγ, IL-6, JAK-STAT and T cell exhaustion pathways correlated with clinical symptoms in the group with LC, including fatigue, shortness of breath and cognitive complaints (Fig. 2e).
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IL-6 has been coming up a lot lately...
 
V.R.T. said:
What do people actually mean when they say T Cell exhaustion?
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I have several emails in my inbox from immunologists complaining about inappropriate use of the term, funnily enough.

In my opinion the only remotely coherent definition for "T cell exhaustion" is in specific contexts like cancers and chronic infections in tissue. What it means is that when you sort T cells that specifically recognize an antigen and stimulate them with that same antigen, they produce a much lower level of cytokines/effector molecules compared to appropriate controls. This phenomenon has been decently associated with poorer outcomes in those specific contexts--likely due to impaired ability of T cells to kill infected or cancerous cells and control spread over time.

That impaired effector function was found to correlate with certain metabolic changes and expression of certain markers like PD-1 in the T cells, so that became known as an "exhaustion signature." It's only ever a small subset of T cells that show those changes and often its only observable in T cells collected from the specific microenvironment harboring the infection or tumor. We don't know exactly why it happens, but the best evidence we have indicates that it's probably a combination of local signaling from other cells at the site of infection/cancer and repeated TCR stimulation leading to transcriptional changes in the T cells.

But the term "exhaustion" constantly gets brought up outside of that very specific context, so people will claim "T cell exhaustion" based on total quantification of some of those cytokines or expression of markers like PD-1 without sorting for antigen-specific T cells. Those differences in cytokines may be real, but you really can't infer anything about T cells or pathogens from them. It's the same logic as surveying how many times any fire alarm goes off in two large apartment complexes, finding that the frequency is lower in one of them, and then blaming the difference on every (or some unspecified subset) of fire alarms in the building being faulty. 99% of mentions of "exhaution" in LC or ME/CFS research are in this category.
 
jnmaciuch said:
I have several emails in my inbox from immunologists complaining about inappropriate use of the term, funnily enough.

In my opinion the only remotely coherent definition for "T cell exhaustion" is in specific contexts like cancers and chronic infections in tissue. What it means is that when you sort T cells that specifically recognize an antigen and stimulate them with that same antigen, they produce a much lower level of cytokines/effector molecules compared to appropriate controls. This phenomenon has been decently associated with poorer outcomes in those specific contexts--likely due to impaired ability of T cells to kill infected or cancerous cells and control spread over time.

That impaired effector function was found to correlate with certain metabolic changes and expression of certain markers like PD-1 in the T cells, so that became known as an "exhaustion signature." It's only ever a small subset of T cells that show those changes and often its only observable in T cells collected from the specific microenvironment harboring the infection or tumor. We don't know exactly why it happens, but the best evidence we have indicates that it's probably a combination of local signaling from other cells at the site of infection/cancer and repeated TCR stimulation leading to transcriptional changes in the T cells.

But the term "exhaustion" constantly gets brought up outside of that very specific context, so people will claim "T cell exhaustion" based on total quantification of some of those cytokines or expression of markers like PD-1 without sorting for antigen-specific T cells. Those differences in cytokines may be real, but you really can't infer anything about T cells or pathogens from them. It's the same logic as surveying how many times any fire alarm goes off in two large apartment complexes, finding that the frequency is lower in one of them, and then blaming the difference on every (or some unspecified subset) of fire alarms in the building being faulty. 99% of mentions of "exhaution" in LC or ME/CFS research are in this category.
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Thank you, a very helpful response!
 
jnmaciuch said:
I have several emails in my inbox from immunologists complaining about inappropriate use of the term, funnily enough.

In my opinion the only remotely coherent definition for "T cell exhaustion" is in specific contexts like cancers and chronic infections in tissue. What it means is that when you sort T cells that specifically recognize an antigen and stimulate them with that same antigen, they produce a much lower level of cytokines/effector molecules compared to appropriate controls. This phenomenon has been decently associated with poorer outcomes in those specific contexts--likely due to impaired ability of T cells to kill infected or cancerous cells and control spread over time.

That impaired effector function was found to correlate with certain metabolic changes and expression of certain markers like PD-1 in the T cells, so that became known as an "exhaustion signature." It's only ever a small subset of T cells that show those changes and often its only observable in T cells collected from the specific microenvironment harboring the infection or tumor. We don't know exactly why it happens, but the best evidence we have indicates that it's probably a combination of local signaling from other cells at the site of infection/cancer and repeated TCR stimulation leading to transcriptional changes in the T cells.

But the term "exhaustion" constantly gets brought up outside of that very specific context, so people will claim "T cell exhaustion" based on total quantification of some of those cytokines or expression of markers like PD-1 without sorting for antigen-specific T cells. Those differences in cytokines may be real, but you really can't infer anything about T cells or pathogens from them. It's the same logic as surveying how many times any fire alarm goes off in two large apartment complexes, finding that the frequency is lower in one of them, and then blaming the difference on every (or some unspecified subset) of fire alarms in the building being faulty. 99% of mentions of "exhaution" in LC or ME/CFS research are in this category.
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I second @V.R.T. — that response is a home-run explanation. Thank you, @jnmaciuch

I've also had multiple back-and-forths with one particularly prickly immunologist who was obstinate in his refusal to expand on why any mention of immune exhaustion was (in his words) "alarmist misinformation". His go-to was always arguing from authority: "Leave it to the experts" (as if we'd solved Post Acute Infection Syndrome, which he had no interest in discussing — thanks for nothing, Marc Veldhoen).

I actually went so far as to ask r/immunology this week for leads on respected immunologists in a bid to break from the whole "COVID is nothing" vs "COVID is AIDS" false dichotomy. (In both cases, an oversimplication of a complex and still misunderstood disease).

Given that this study is under the aegis of Dan Barouch, an Oxford graduate in immunology with an M.D. from Harvard Medical School (as well as being the director of the Center for Virology and Vaccine Research at Beth Israel Deaconess Medical Center), one would think their use of "immune exhaustion" might actually be valid. (One of Veldhoen's go-tos is always "none of the researchers are even immunologists" — well, they are this time, buddy boy). I haven't looked at the nitty gritty and don't have the requisite training to correctly assess it.

What I do know, however, is that this field of medicine remains very much in flux and that dogma is no friend to scientific investigation. My hope is that these findings might eventually expand our understanding as to WTF is wrong with me—I mean, us.
 
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Covidivici said:
Given that this study is under the aegis of Dan Barouch, an Oxford graduate in immunology with an M.D. from Harvard Medical School (as well as being the director of the Center for Virology and Vaccine Research at Beth Israel Deaconess Medical Center), one would think their use of "immune exhaustion" might actually be valid.
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Unfortunately not, they only assessed genes/proteins that commonly change in those specific “exhaustion” contexts I mentioned. The signature was significantly differential but we can’t infer what it means.

I know plenty of well-trained immunologists who use the term willy nilly, and plenty of equally impressive immunologists who chastise them for it. Sometimes endlessly on email threads I’m CC’d on.
 
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jnmaciuch said:
Unfortunately not, they only assessed genes/proteins that commonly change in those specific “exhaustion” contexts I mentioned. The signature was significantly differential but we can’t infer what it means.

I know plenty of well-trained immunologists who use the term willy nilly, and plenty of equally impressive immunologists who chastise them for it. Sometimes endlessly on email threads I’m CC’d on.
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Beyond the immune exhaustion terminology misuse debacle, what is your read on this paper? They claim to have found some interesting stuff but I'm not qualified to assess whether they really have.
 
jnmaciuch said:
Unfortunately not, they only assessed genes/proteins that commonly change in those specific “exhaustion” contexts I mentioned. The signature was significantly differential but we can’t infer what it means.

I know plenty of well-trained immunologists who use the term willy nilly, and plenty of equally impressive immunologists who chastise them for it. Sometimes endlessly on email threads I’m CC’d on.
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Dammit.
But as the husband of an up-do-date ER physician, that very much checks out. I'm constantly taken aback at little intellectual rigour (formerly) overachieving people apply on a daily basis. The stories I hear.......
 
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I will also note—they did check [edit: IFN-g response of T cells to spike protein stimulation] (Fig 1). And, exactly the opposite from what you’d expect in an “exhausted” T cell, IFN-g production was higher in LC.

[Edit: so the specific T cell response to COVID does not appear to be blunted in LC, even if some of the genes associated with “exhaustion” are higher when you measure all PBMCs together. This means that whatever is happening in the T cells of LC, it’s not antigen-specific “exhaustion”]
 
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V.R.T. said:
Beyond the immune exhaustion terminology misuse debacle, what is your read on this paper? They claim to have found some interesting stuff but I'm not qualified to assess whether they really have.
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it’s pretty much the exact same type of study as a paper I co-authored https://www.s4me.info/threads/ident...-2025-gabernet-et-al.42638/page-2#post-640199

Just way smaller cohort and much more limited in what they assessed. Some of the signatures here did come up in our analysis (like IL-6), but the association was weaker and did not pass our feature selection thresholds so we could not highlight them. This study was very much designed with the intention of finding immune signatures—our study was capable of finding the same pathways, but other pathways ended up providing a much stronger signature and overshadowed the few theyre highlighting here.

I’d need more time to dig into the methods to judge how rigorous this was. I’m obviously going to be biased trusting my team’s methods and findings over these where they diverge
 
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Now that I’ve had a chance to look through the methods, it looks fine to me. The analysis uses appropriate tools and statistical tests as far as I can tell.

I would have liked to see a multi-omic integration method of their cytokine data, transcriptomics, and proteomics to facilitate feature selection. The way they use it here is basically validating signatures from one assay in another, which is a fine alternative, it just means the analysis is less unbiased discovery and more seeking specific evidence to confirm a general hypothesis of “immune dysregulation.”

The main takeaway is just that these associations are on the weaker side. The way they present the data as logFC/z scores and pathway NES is somewhat standard in the field but can serve to make weak findings look more impressive than they are. All in all it’s one more study showing slight differences in immune signaling pathways in LC, some of which have come up repeatedly between studies and many of which do not replicate. The usefulness mostly comes down to how stringently they defined LC, which I don’t see much information on in the methods and don’t have time to chase down.
 
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