The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is strange in the breadth of severity in its clinical manifestations, ranging from asymptomatic to fatal infection. The ability to predict severe disease and thus intervene early is important, and has spurred the search for markers of this shift.
An interesting study by an international team of researchers presents evidence that an elevated level of expression of oxidant molecules may be correlated with severe infection and lung injury.
The researchers recently published their findings in the Free Radical Biology and Medicine.
Increased expression of oxidant genes
The scientists, using already reported transcriptomics from COVID-19 patients, modeled the expression of 125 genes related to biological redox reactions.
They found that seven genes related to oxidation were upregulated in inflammatory cells, both in the lung tissue and in circulating blood. These genes, namely, MPO, S100A8, S100A9, SRXN1, GCLM, SESN2, and TXN, were being expressed at elevated levels in leukocytes in severe COVID-19, but not in non-severe cases.
In more detail, these genes are the Myeloperoxidase (MPO), Calprotectin (S100A8/S100A9), Sulfiredoxin-1 (SRXN1), Glutamate-cysteine ligase modifier (GCLM), and two antioxidant genes: Sestrin 2 (SESN2) and Thioredoxin (TXN).
Both macrophages and CD8+ T cells from lung washings, and neutrophils from the peripheral circulation, showed higher expression levels for these genes. MPO, S100A8, and S100A9 were the most prominently upregulated oxidation markers.
The latter two were found to be elevated in the lung tissue of individuals with SARS-CoV-2 infection.
The top three
Subsequently, the investigators found that both MPO and the calprotectins were indeed the most elevated when assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT PCR), in both blood and saliva, in severe COVID-19 cases vs. asymptomatic patients.
MPO is a neutrophil gene responsible for encoding the enzyme that produces reactive oxygen intermediates. When neutrophils are exposed to oxidative stress, they form neutrophil extracellular traps (NETs), as well as bursting open their granules containing both MPO and calprotectin.
Calprotectin is a heterodimer of Migration Inhibitory Factor-Related Protein 8 and 14 (MPR8 and MRP14), encoded by S100A8 and S100A9. Calprotectin has an alarmin function, binding to toll-like receptor 4 (TLR4) to trigger innate immunity pathways like MAP-kinase and NF-kappa-B signaling. Thus, they affect inflammation, redox capacity and cell death pathways.
Calprotectin genes are expressed at high levels only in cells of myeloid origin and activated macrophages during acute and chronic inflammation.
Salivary biomarker
The ability to detect this elevation in saliva indicates their potential use as non-invasive biomarkers of severe COVID-19. While MPO and calprotectin in blood were increased about one log-fold more in severe vs. asymptomatic disease, the log-fold increase in saliva under the same circumstances was 0.3 log higher for S100A9, but one log and 2.7 log more for MPO and S100A8.
This increase was marked in SARS-CoV-2 infection compared to other respiratory virus infections, such as influenza A and respiratory syncytial virus. In the latter, increases in oxidative stress gene expression did not cross one log fold change.
Conversely, in both SARS-CoV-1 and SARS-CoV-2, the increase was more than one log-fold for seven and 27 genes, respectively. TXN, QSOX1, MAPK14, MPO, S100A9, and S100A8 were elevated with both these viruses, at over 1.5 log-fold change.
Where do these come from?
Neutrophils are among the key sources of pro-oxidant molecules and play a central role in COVID-19 severity. A previous study showed an increase in the number of neutrophils along with an elevation in the proportion of low-density neutrophils. In these cells, the level of pro-oxidant genes increased from 0.3 log to 1.8 log upwards, while antioxidant genes like JUNB, FOS and SOD2 were expressed at -0.7 log to -1.5 log downwards.
Macrophages and CD8+ T cells in lung washings also showed this change in oxidative gene expression.
The mechanism of oxidant stress begins with macrophage and neutrophil activation on exposure to viral pathogen-associated molecular patterns (PAMPs), releasing reactive oxygen species such as superoxide anions. These trigger the pro-inflammatory cascade of cytokines with further recruitment of inflammatory cells.
This is a double-edged weapon, capable of clearing the virus by the destructive action of ROS on infected host cells and the viral particles themselves. However, if the virus continues to replicate and more cells are infected, as occurs in severe COVID-19, the cell runs out of antioxidant buffer molecules. The resulting accumulation of ROS and the vicious cycle of increasing cytokine release in response to damage-associated molecular patterns (DAMPs) causes long-term oxidative stress, damaging the cell membranes and nuclear content.
Oxidative stress could also be the result of, for instance, increased deep breathing to compensate for lung injury caused by the virus and maintain an adequate level of oxygen in the blood.
What are the implications?
The close reflection of severe COVID-19 with the upregulation of oxidative stress genes in immune cells as well as lung structural cells is a phenomenon that requires more work to understand the contribution of oxidative stress to severity of disease following infection with this virus.
The increase in gene expression was consistently significant even after accounting for the influence of age, sex, body mass index and comorbidities (as measured by the Charlson Comorbidity Index score).
The cause of this oxidant stress may be the dysregulated innate immune response in COVID-19, with a relative increase in neutrophils and a decline in lymphocytes, coupled with the cytokine storm. The result of high tissue levels of oxidant molecules may include damage to the lung cells, from cell death to tissue necrosis, depending on the level of oxidation.
The ability to observe significant increases in the MPO and calprotectin genes in saliva in severe COVID-19 indicates their potential as biomarkers of this condition.
Secondly, these genes offer therapeutic targets which could slow down disease progression and prevent multiple features of severe disease such as clotting tendencies, cytokine storm and lung damage. Examples of such agents include NRF2 antagonists and paquinimod, or more selective MPO-directed antioxidants.
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