Neutrophil Dysregulation Observed In Severe COVID. IgA-Dominant Responses Drive Neutrophil Effector Functions Causing Disease Severity And Mortality

COVID-19 Immunology: A new study by researchers from Harvard medical School and MIT has found that neutrophil dysregulation is observed in severe COVID and that IgA-dominant responses drive neutrophil effector functions causing disease severity and mortality.

 
The study team collected 781 longitudinal blood samples from 306 hospitalized COVID-19+ patients, 78 COVID-19− acute respiratory distress syndrome patients, and 8 healthy controls, and performed bulk RNA-sequencing of enriched neutrophils, plasma proteomics, cfDNA measurements and high throughput antibody profiling assays to investigate the relationship between neutrophil states and disease severity or death.
 
The COVID-19 Immunology team identified dynamic switches between six distinct neutrophil subtypes using non-negative matrix factorization (NMF) clustering. At days 3 and 7 post-hospitalization, patients with severe disease had an enrichment of a granulocytic myeloid derived suppressor cell-like state gene expression signature, while non-severe patients with resolved disease were enriched for a progenitor-like immature neutrophil state signature.
 
Severe disease was also associated with gene sets related to neutrophil degranulation, neutrophil extracellular trap (NET) signatures, distinct metabolic signatures, and enhanced neutrophil activation and generation of reactive oxygen species (ROS).
 
The study team found that the majority of patients had a transient interferon-stimulated gene signature upon presentation to the emergency department (ED) defined here as Day 0, regardless of disease severity, which persisted only in patients who subsequently died. Humoral responses were identified as potential drivers of neutrophil effector functions, as enhanced antibody-dependent neutrophil phagocytosis and reduced NETosis was associated with elevated SARS-CoV-2-specific IgG1-to-IgA1 ratios in plasma of severe patients who survived.
 
Further in vitro experiments confirmed that while patient-derived IgG antibodies mostly drove neutrophil phagocytosis and ROS production in healthy donor neutrophils, patient-derived IgA antibodies induced a predominant NETosis response.
 
The study findings demonstrate neutrophil dysregulation in severe COVID-19 and a potential role for IgA-dominant responses in driving neutrophil effector functions in severe disease and mortality.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2021.10.04.463121v1
 
The study deals with the characterization of neutrophil phenotypes in an attempt to provide biomarkers that can predict severe disease in patients hospitalized with coronavirus disease 2019 (COVID-19).
 
To date, neutrophils have been reported to be hyperactivated in severe COVID-19, and some scientists think this phenomenon indicates that these cells are closely implicated in the underlying disease mechanism.
 
It has been known that neutrophils recognize pathogens via opsonic receptors, including the Fc receptors, indicating that neutrophil responses are affected by the disease. Neutrophils recognize antigen-antibody complexes, which may be important in subsequent effector responses.
 
Importantly neutrophils can eliminate pathogens bound to antibodies directly in a process called antibody-dependent neutrophil phagocytosis (ADNP). Alternatively, they undergo NETosis, a dedicated program of cell death in which neutrophils release their chromatin extracellularly as a web, modified by anti-microbial and highly reactive proteins such as myeloperoxidase, to form neutrophil extracellular traps (NETs).
 
This phenomena of NETosis also occurs in cancers, viral infections, and heparin-induced thrombocytopenia is driven mostly by interactions between antibodies and Fc receptors, modulated by factors such as the isotype of the antibody and the type of sugars attached to the antigen.
 
In COVID-19, NETs may be involved in heart attacks and immunothrombotic events.
 
Past studies indicate that the protein translation patterns and gene expression profiles in hospitalized COVID-19 patients that reflect neutrophil activation and degranulation are mortality risk markers.
The present research describes a study of blood neutrophils from a large group of such patients using several methods to delineate the dynamic neutrophil response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
 
The study findings showed that the absolute neutrophil count (ANC) was related to creatinine, C-reactive protein (CRP), lactate dehydrogenase, and D-dimer, all acute-phase reactants. The ANC was also strongly correlated with intubation risk, increasing from day 0 to day seven.
 
The SARS-CoV-2 virus elicits a robust interferon response, both IFNγ and IFN-α, along with cytokine production. Distinct neutrophil states were found to be linked to severe and non-severe COVID-19.
 
The study findings also showed that activated and inflammatory neutrophils were elevated in samples from severe COVID-19, while in other samples, the activation was reduced, and the number of neutrophils in circulation was decreased by day seven.
 
Interestingly, it was found from the day of hospital admission onwards, very early neutrophil states allowed the eventual severity of the disease to be predicted with great accuracy.
 
Significantly, several metabolic circuits went from being enriched on day 0 in those who would not survive to be enriched on day seven in the eventual survivors. This included delayed or dysregulated interferon signaling.
 
Certain studies have shown that this could lead to increased interferon activity, resulting in the activation of immune cells when the lungs are already heavily inflamed. This could promote an increased risk of death.
 
It was also observed that interferon-stimulated genes were also enriched early in survivors but late in those who died. The metabolic pathways showed the opposite direction, with fatty acid metabolism, the tricarboxylic acid cycle, and NADP pathways being enriched in fatal cases on day 0, while day seven enrichment corresponded to surviving patients.
 
Typically, proteins that enhance histone modification can only be detected via proteomics, as they are post-transcriptional modifications.
 
A detailed search for such markers showed them to be associated with disease severity at all time points across neutrophil subtypes.
 
The study findings also revealed that Cell-free DNA levels were also associated with severity and with ANC, as well as with immature activated neutrophil samples. Moreover, severe disease was associated with neutrophil degranulation and T cell inhibition.
 
Interestingly neutrophils also prevent T cell activation and proliferation.
 
The study team also found that only IgG:S immune complexes had a strong ADNP effect and induced reactive oxygen species (ROS) to a higher level in severe COVID-19 than non-severe. NETosis from healthy neutrophils was much higher when incubated with IgA than IgG, independent of severity.
 
However, among non-severe patients, MPO was reduced with IgA compared to IgG, indicating that the latter stimulates higher production to accelerate ROS via MPO.
 
The study team found that they could differentiate six neutrophil phenotypes in COVID-19 and other cases. These could differ significantly from severe and fatal COVID-19 patients to non-severe cases. The dysregulation of neutrophils coupled with the changes in phenotypes indicates a common mechanism in COVID-19 and other diseases.
 
Also it was observed in all cases, an interferon-driven phenotype was seen but went down over time to be replaced by a suppressive signature or a neutrophil progenitor signature in severe and non-severe COVID-19, respectively.
 
Higher interferon levels were found on days three and seven in fatal cases, making this a potential biomarker predicting severe disease.
 
The study findings showed that IgA and IgG have different effects on the neutrophil effector functions, with a high IgA1/IgG1 ratio in fatal cases relative to survivors. This suggests that the antiviral antibodies and neutrophil effector functions mediate severe outcomes in COVID-19.
 
The study team suggest an initial IgA-driven mucosal response to the entry of the virus, followed by IgA-secreting B cells flooding the bloodstream.
 
The study team concluded, “In summary, our study elucidates how circulating neutrophils and their interactions with soluble factors drive COVID-19 disease severity, providing insight into this crucial and abundant cell type. We propose a model of SARS-CoV-2 infection in which antibody profiles drive neutrophils either to aid in disease resolution through phagocytosis or contribute to tissue damage via NETosis. Further, we hypothesize that therapies which simultaneously aim to ablate suppressive G-MDSC-like neutrophils and prevent excessive NETosis in circulation have the potential to aid with disease resolution in severe patients.”
 
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