Researchers in the United States and Costa Rica have identified differences in the proportions of monocyte subsets that occur in severe cases of coronavirus disease 2019 (COVID-19) following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and post-acute sequelae of COVID-19 (PASC), often referred to as “Long COVID”.
“The recent COVID-19 pandemic is a treatment challenge in the acute infection stage, but the recognition of chronic COVID-19 symptoms termed PASC may affect up to 30% of all infected individuals,” says Bruce Patterson from IncellDx Inc. in California and colleagues.
However, the underlying mechanism of this distinct immunologic condition that persists for three months or more following initial infection remains elusive, adds the team.
Now, Patterson and colleagues have reported differences in the proportions of monocyte subsets between cases of severe COVID-19 and cases of PASC.
They also report that the SARS-CoV-2 protein subunit S1 was present in non-classical monocytes among PASC patients for up to 16 months following initial infection. The viral spike protein mediates the initial stage of SARS-CoV-2 infection by binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2).
Furthermore, the study findings point to a potential therapeutic target to reduce the survival of S1-containing non-classical monocytes and the associated vascular inflammation.
A pre-print version of the research paper is available on the bioRxiv* server, while the article undergoes peer review.
Underlying cause of PASC remains a mystery
PASC is a disabling and sometimes debilitating condition that affects between 10% and 30% of individuals following SARS-CoV-2 infection. The number and extent of symptoms are highly variable, with some studies reporting more than 200 different symptoms.
While the underlying cause of PASC remains a mystery, some data have pointed to persistent tissue reservoirs of SARS-CoV-2 as a potential mechanism.
Patterson and colleagues recently identified characteristic immune cell subset abnormalities in PASC, with the predominant abnormality being elevated levels of monocyte subsets.
Monocyte subsets comprise three phenotypic and functionally distinct types. Classical monocytes exhibit the CD14++, CD16- phenotype, while intermediate monocytes express CD14+ and CD16+, and non-classical monocytes express CD14lo and CD16+.
“During pathologic conditions mediated by infectious/inflammatory reactions, the proportions of monocyte subsets vary according to the functionality of each specific subpopulation,” writes Patterson and colleagues.
What did the current study involve?
The team assessed kinetic differences in the proportions of monocyte subsets in patients with severe COVID-19 and patients with PASC.
The levels of both intermediate (CD14+, CD16+) and non-classical monocyte (CD14Lo, CD16+) were significantly increased in PASC patients compared with healthy controls. In contrast, neither of these monocyte subsets were elevated in cases of severe COVID-19.
Since studies have previously found that monocyte subsets can be infected by HIV, HCV, Zika virus, and Dengue fever virus, the researchers screened the patients’ peripheral blood mononuclear cells (PBMCs) for SARS-CoV-2 RNA.
They found that PBMCS contained SARS-CoV-2 RNA in 4 of 11 (36%) individuals with severe COVID-19, compared with 1 of 26 (4%) PASC patients. The one PASC patient that was positive for SARS-CoV-2 RNA had been infected 15 months previously.
Establishing the SARS-CoV-2 RNA reservoir
To establish the precise reservoir contributing to the positivity for SARS-CoV-2 RNA, the researchers performed high parameter flow cytometry with antibodies that define monocytic subsets and simultaneously stained the cells with an antibody for the SARS-CoV-2 spike S1 protein.
This revealed distinct subpopulations of SARS-CoV-2 S1-containing cells in the non-classical monocytic subset for 19 of 26 (73%) PASC patients up to 15 months post-infection and for 10 of 11 (91%) severe COVID-19 patients. In addition, neither classical monocytes nor intermediate monocytes expressed the SARS-CoV-2 S1 protein.
“The clinical relevance of monocyte activation in COVID-19 patients and the significance of these cells as a viral protein reservoir in PASC is supported by our data reporting the presence of S1 protein within non-classical monocytes,” says Patterson and colleagues.
Viral particles and proteins can enter monocyte subpopulations in different ways, and this appears to be regulated differently in individuals that will develop severe disease or PASC, says the team.
Non-classical monocytes may ingest virally-infected apoptotic endothelial cells
The researchers say that since non-classical monocytes have been proposed to patrol endothelial cell integrity, pre-existing CD14lo CD16+ cells may ingest virally-infected apoptotic endothelial cells, thereby increasing the proportion of non-classical monocytes containing the S1 protein.
“This mechanism is more likely to take place in the PASC group, where the S1 protein was detected 12 to 15 months post-infection, than in the severe group,” write the researchers.
Patterson and colleagues say that the persistence of circulating S1-containing non-classical monocytes for up 15 months post-infection among PASC patients suggests that certain conditions are required to maintain this cell population.
It has previously been shown that non-classical monocytes require fractalkine (CX3CL1) and tumor necrosis factor (TNF) to inhibit apoptosis and promote cell survival.
Interestingly, Patterson and team previously demonstrated high levels of IFN-γ levels in PASC patients, which can induce TNF-α production.
“Further, TNF-α and IFN-γ induce fractalkine production by vascular endothelial cells, creating the conditions to promote survival of non-classical monocytes,” they write.
The fractalkine pathway as a potential therapeutic target
Although non-classical monocytes are typically considered to be anti-inflammatory cells, the researchers say it was recently shown that these cells could acquire a proinflammatory phenotype.
Non-classical monocytes acquire hallmarks of cellular senescence, which induces an inflammatory state in these cells and promotes their long-term survival in the circulation, they write.
“Our data suggest that interruption of the CX3CR1/fractalkine pathway would be a potential therapeutic target to reduce the survival of S1-containing non-classical monocytes and the associated vascular inflammation,” concludes the team.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.