Persistent serum protein signatures define an inflammatory subcategory of long COVID, 2023, Talla et al.

[SNT Gatchaman]

Persistent serum protein signatures define an inflammatory subcategory of long COVID
Talla, Aarthi; Vasaikar, Suhas V.; Szeto, Gregory Lee; Lemos, Maria P.; Czartoski, Julie L.; MacMillan, Hugh; Moodie, Zoe; Cohen, Kristen W.; Fleming, Lamar B.; Thomson, Zachary; Okada, Lauren; Becker, Lynne A.; Coffey, Ernest M.; De Rosa, Stephen C.; Newell, Evan W.; Skene, Peter J.; Li, Xiaojun; Bumol, Thomas F.; Juliana McElrath, M.; Torgerson, Troy R.

Long COVID or post-acute sequelae of SARS-CoV-2 (PASC) is a clinical syndrome featuring diverse symptoms that can persist for months following acute SARS-CoV-2 infection. The aetiologies may include persistent inflammation, unresolved tissue damage or delayed clearance of viral protein or RNA, but the biological differences they represent are not fully understood.

Here we evaluate the serum proteome in samples, longitudinally collected from 55 PASC individuals with symptoms lasting ≥60 days after onset of acute infection, in comparison to samples from symptomatically recovered SARS-CoV-2 infected and uninfected individuals.

Our analysis indicates heterogeneity in PASC and identified subsets with distinct signatures of persistent inflammation. Type II interferon signaling and canonical NF-κB signaling (particularly associated with TNF), appear to be the most differentially enriched signaling pathways, distinguishing a group of patients characterized also by a persistent neutrophil activation signature. These findings help to clarify biological diversity within PASC, identify participants with molecular evidence of persistent inflammation, and highlight dominant pathways that may have diagnostic or therapeutic relevance, including a protein panel that we propose as having diagnostic utility for differentiating inflammatory and non-inflammatory PASC.

Link | PDF (Nature Communications)

 

[SNT Gatchaman]

I don't think we had posted this as a preprint, which was from May 2022.

 

[Hutan]

We analyzed the serum proteome using the Olink Explore 1536 panel (see Methods, supplemental Data S2) to first interrogate specific pro- teins that distinguish PASC (at their first time point available ≥60-days post symptom onset), recovered (at their last time point available ≥60- days post symptom onset), and uninfected participants in our cohort. We identified 275, 25, and 14 proteins that were significantly differentially expressed (p < 0.05) between PASC, recovered, and uninfected groups respectively when each group was compared to the other groups (Supplemental Data S3). We noted that within the PASC group, there was variation in expression of the serum proteomic signature, suggesting that some have an inflammatory signature and others do not (Supplemental Fig. S2E), and highlighting the heterogeneous nat- ure of the disorder.

That's a big difference in the number of proteins that were differentially expressed between the PASC group (55 adults:21 men; 34 women); recovered group (24 adults: 9 men; 15 women); uninfected group (22 adults: 12 men; 10 women). The recovered group and the uninfected group are both relatively small, and the uninfected group differs in the sex ratio. The recovered group had a confirmed infection but no lingering symptoms.

We hence took an alternative approach, using unbiased clustering of the serum proteome across the entire cohort (PASC+recovered +uninfected) to find clusters of individuals that had similar serum proteome signatures regardless of their COVID-19 status or reported symptomatology. For this purpose, we used the first ≥60-day sample available for each PASC participant, the last available post ≥60-day sample for each recovered participant (to maximize the chance that proteome alterations had returned to baseline), and the solitary sam- ple from uninfected individuals

They next did an unbiased clustering analysis across all of participants. I really like that sort of analysis, as it removes some of the bias. If a difference pops out and it correlates with the group differentiation, then there is a chance that the difference is characteristic. I'm not quite so keen on them using the first sample from the PASC participants and the last samples from the recovered participants as that sort of negates the point of having infected controls, but I understand why they would do that. The analysis doesn't sound to have been simply doing a PCA with all the proteins though.

We used curated canonical pathways from the Molecular Signatures Database (MSigDB) and applied a rule-in statistical approach (see Methods) to identify pathways that distinguished PASC from both recovered and uninfected individuals15. This resulted in identification of 85 pathways that have a significant rule-in performance (p < 0.01).

They looked for pathways with affected proteins: 85 significantly affected pathways. They then accounted for overlapping genes, reducing the pathways down to 54 proteomic modules. The results are shown in Fig 1a, which is worth a look. The image is too big to copy, but here is the top part of it.




Have a look at Cluster 1 on the left. There's a high percentage of males and uninfected participants, no symptoms and low levels of enrichment of the pathway modules listed at the bottom right (denoted by the purple squares). In contrast, look at Cluster 4. There's a higher proportion of females and almost all of them have persisting symptoms. There's a high incidence of enrichment of the pathway modules.

It's worth noting though that not all of the participants with persisting symptoms reported fatigue (as shown in the section with white, grey and black squares. In fact, quite a few did not.

 

[Hutan]

Two of the clusters (4 & 5) showed marked enrichment for inflammatory modules including type I and type II interferon signaling, TNF signal- ing, NFκB signaling, and several others, while clusters 1, 2, and 3 lacked a distinct inflammatory protein signature. Inflammatory clusters 4 and 5 included predominantly PASC (91% and 80% respectively).

To assess if the differential serum proteomic signatures captured at the initial post-60 day time point persisted over time, we extended our analysis to include all longitudinal samples available for each participant. We found that PASC participants exhibiting an inflamma- tory protein signature continue to have that signature over time and that most participants remained in the same cluster throughout the longitudinal study period

This was interesting. Participants in the PASC group mostly stayed in the same cluster over time.

 

[Hutan]

It is possible that a poor immune response to SARS-CoV-2 may allow delayed clearance of viral particles that could increase the risk for developing PASC with persistent inflammation. To address this question, we evaluated SARS-CoV-2 receptor binding domain (RBD)- specific IgG titers in serum samples obtained 60 days PSO from PASC and recovered participants in each of the 5 clusters identified above.

There was no significant difference in RBD-specific IgG responses between previously infected COVID participants in any of the clusters (Fig. 1B). We also compared SARS-CoV-2-specific CD4+ and CD8+T cell frequencies17,18 between PASC and recovered participants from all clusters and did not identify any significant differences (Supplemental Fig. S3C & S3D).

They didn't find any difference between the identified clusters in SARS-CoV-2 specific IgG levels, suggesting that an inadequate immune response wasn't the problem.

We assessed if other covariates or clinical parameters such as age, gender, BMI, or underlying comorbidities were associated with having an inflammatory serum proteomic signature. While there were no overall significant differences in age or BMI at enrollment between PASC, recovered and uninfected participants (Supplemental Figs. S4A & S4B), BMI was significantly higher in the inflammatory PASC groups (clusters 4 and 5) compared to participants in the other non- inflammatory clusters (Fig. 1D). We noted that well-described BMI associated proteins like (Leptin (LEP) and Fatty Acid Binding Protein (FABP4)) and the Leptin signaling module were significantly increased in participants with high BMI (clusters 4 and 5) in our cohort (Fig. 1E, F)22–24. We also noted that interleukin-6 (IL-6), identified as part of the inflammatory signature associated with PASC, was positively corre- lated with BMI (Fig. 1E). These data suggest that BMI may be a risk factor for exhibiting an inflammatory PASC phenotype but does not account fully for the increased IL-6 observed in inflammatory PASC because a small subset of participants whose BMI was within the “healthy” range also had increased IL-6 as part of their inflammatory proteomic signature.

Clusters 4 and 5 (which they characterised as the two inflammatory clusters and the ones with most PASC participants) had more older people and more people with a high BMI, but they also included people with healthy BMIs and ages at the younger end of the range. Cluster 1, the healthiest cluster, didn't have anyone with a BMI over 30 and had relatively young participants.

 

[Hutan]

Here's Figure 2a which summarises the pathways with enriched expression in clusters 4 and 5. Surely there must be some clues in there somewhere, even though the PASC cohort is very noisy with respect to ME/CFS-like illness. There are lots of interesting-sounding pathways e.g. viral acute myocarditis, biomarkers for urea cycle disorders

The Leishmania pathway is enriched in both clusters. I'm assuming that pathway is the hsa05140 pathway: Leishmania is an intracellular protozoan parasite that changes host cell immune responses

Successful infection of Leishmania is achieved by alteration of signaling events in the host cell, leading to enhanced production of the autoinhibitory molecules like TGF-beta and decreased induction of cytokines such as IL12 for protective immunity. Nitric oxide production is also inhibited. In addition, defective expression of major histocompatibility complex (MHC) genes silences subsequent T cell activation mediated by macrophages, resulting in abnormal immune responses.

The Anthrax pathway is enriched in both clusters too. Information on how the host protein response might be affected by anthrax here:

A common strategy used by pathogenic bacteria to establish infection is to secrete protein factors that block intracellular signalling pathways essential for host defence. Some of these proteins also act as toxins, directly causing pathology associated with disease. Bacillus anthracis, the bacterium that causes anthrax, secretes two plasmid-encoded enzymes, LF (lethal factor) and EF (oedema factor), that are delivered into host cells by a third bacterial protein, PA (protective antigen). The two toxins act on a variety of cell types, disabling the immune system and inevitably killing the host. LF is an extraordinarily selective metalloproteinase that site-specifically cleaves MKKs (mitogen-activated protein kinase kinases). Cleavage of MKKs by LF prevents them from activating their downstream MAPK (mitogen-activated protein kinase) substrates by disrupting a critical docking interaction. Blockade of MAPK signalling functionally impairs cells of both the innate and adaptive immune systems and induces cell death in macrophages. EF is an adenylate cyclase that is activated by calmodulin through a non-canonical mechanism. EF causes sustained and potent activation of host cAMP-dependent signalling pathways, which disables phagocytes.

There do look to be substantial differences between the groups and clusters with respect to the pathways - for example:

 

[Hutan]

Here is part of 3b which shows the levels of selected proteins for each participant - this chart is for IFN-y (interferon gamma). The pink dots are for the PASC participants, black dots are the infected recovered, and the grey dots are for uninfected participants. IFN-y does look different, but there are a lot of people labelled as having PASC with normal levels.

It seems that a lot of the differentially expressed proteins are downstream of interferon.

In addition to IFN-γ, increased expression of chemokines and cytokines known to be regulated by IFN-γ including CXCL9, CXCL10, CXCL11 and IL-27 in cluster 4 suggests that it is functionally active. We also observed increased expression of IL-12 p40 (IL12B) and the IL-12 p40/p70 heterodimer (IL12A_IL12B) in cluster 4, which may drive expression of IFN-γ and an overall Th1 signature (Fig. 3A, B). While not as strongly differentially expressed in cluster 5, IFN-γ and IFN-γ induced chemokines are enriched compared to those that recovered from COVID or were uninfected (Fig. 3B).

Proteins were evaluated longitudinally in all available samples begin- ning from early acute infection to 275 days post-symptom onset (PSO). IFN-γ, IL-12 p40, and IFN-γ-driven chemokines were consistently ele- vated in inflammatory PASC relative to the other 2 groups

In addition to the IFN-γ related signature noted above, we also observed that TNF, TNF-driven cytokines and chemokines (including IL-6 and CCL7 (MCP3)), and several TNF receptor superfamily mem- bers were also increased in both inflammatory PASC clusters but most extensively in cluster 4 (Fig. 3A, E). TNF, IL-6, and CCL7 remained persistently elevated in all inflammatory PASC (clusters 4 + 5) over time when compared to non-inflammatory PASC or individuals who were infected but recovered (Fig. 3F). In addition to elevated cytokine levels, there is evidence for persistent inflammatory cytokine signaling based on the enrichment of proteins involved in TNF signaling, the IL-18 pathway, and the NF-κB canonical signaling pathway (Fig. 3G and Supplemental Fig. S10B).

It's suggested that interferon is driving the protein signature of cluster 5 too. The authors say that it is very hard to quantify systemic type 1 IFN, but suggest that it is producing the elevation of the downstream proteins they did find.

In inflammatory PASC cluster 5, we also noted a protein signature suggestive of persistent type I interferon signaling including elevation of proteins that are induced by type I IFNs during acute SARS-CoV-2 infection (SAMD9L, DDX58, MNDA, and LAMP3)28,29. Interestingly, these type I IFN associated proteins were increased at the earliest sampling timepoint available for analysis in inflammatory PASC and remained elevated for approximately 180 days post infection (Fig. 3H and Supplemental Figs. S8 & S9). Similarly, ssGSEA analysis showed enrichment for the pathway associated with regulation of IFN-α sig- naling in inflammatory PASC that followed a similar kinetic (Fig. 3I and supplemental Fig. S10C). We do not have a specific readout for sys- temic type I IFN levels since these are notoriously difficult to accurately quantify in circulation and the Olink assay only measures IFN-γ and IFN-λ1, however the accumulated evidence points to the persistent activity of type-I interferons in PASC individuals that exhibit signs of persistent inflammation.

Just a bit on interferons from Wikipedia:
These are cytokines released by cells in the presence of viruses. They are called interferons because they interfere with viral replication.

IFNs also have various other functions: they activate immune cells, such as natural killer cells and macrophages, and they increase host defenses by up-regulating antigen presentation by virtue of increasing the expression of major histocompatibility complex (MHC) antigens. Certain symptoms of infections, such as fever, muscle pain and "flu-like symptoms", are also caused by the production of IFNs and other cytokines.

So, they cause lots of downstream impacts as part of a response to a pathogen, and they could be potentially causing flu-like ME/CFS symptoms. There are lots of varieties of interferons, each with their own particular effects.

I'm pretty sure that interferons have been looked at hard in proteomic studies of ME/CFS. And there was the work on the side-effects of interferon therapy (including fatigue) for viral hepatitis. If the release of interferons is very localised in tissue, perhaps they are hard to find in blood.

The authors note the evidence from other studies for persistent SARS-CoV-2 virus and its proteins, noting that this might be causing the interferon response. That's the big question - if interferons are the drivers of symptoms, what is causing them to be released?

 

[SNT Gatchaman]

See also Chronic inflammation, neutrophil activity, and autoreactivity splits long COVID (2023)