DISCUSSION
Our study showed that NE, global DNase activity and components of NETs are involved in the early and later steps of COVID-19. NE and histone-DNA were associated with clinical manifestations and biomarkers related to pulmonary damage, cardiovascular manifestations, renal insufficiency, and inflammation. Elastase >593 ng/mL was an independent predictor of multi-organ injury in multivariate analysis.
The dramatic increase of blood levels of NE and NETs in ambulatory cases show their involvement in the early host response to SARS-CoV-2, as previously observed for other viral pneumonia 4, 6, 11, 18. Alveolar epithelial cells from lungs infected with influenza virus stimulate NETosis 19. However, there is a debate on the detrimental vs. protective effects of NETs in acute respiratory distress syndrome (ARDS) 20, 21. The citrullinated H3 and cell-free DNA reflect the production of extracellular traps by neutrophils, granulocytes and monocytes21, 22. The dramatic increase of blood concentration of NE reflects more specifically the activation of neutrophils22, 23. Of note, NE concentration correlates better with alveolar inflammation than neutrophil count, in acute respiratory distress syndrome 23, 24.
NE, but not histone-DNA and MPO-DNA, was an independent predictor of multi-organ damage in COVID-19 patients. This result reflects the prominent role of NE in mechanisms of neutrophil innate immunity22. NE is dispensed in tissues and blood by degranulation or release with NETs 22, 25-27. The induction of ROS by viral infection activates MPO, which then activates the release of NE 27, 28. The highly significant correlation between NE and histone-DNA and MPO-DNA illustrates the role of NE in NET formation. Upon neutrophil stimulation, NE translocates to the nucleus, where it participates in histone degradation before it releases with the DNA/chromatin material of NETs 6, 28. Importantly, NE associated with NETs remains active and escapes the endogenous anti-protease activity of AAT 29. NE can produce tissue damages in lung, heart, liver, vessels and kidney and exerts prothrombotic effects in viral infection 6, 24, 29-35. The degradation of extracellular matrix (ECM) components by NE produces the same lung and vascular injuries as those observed in autopsy specimens of COVID-19 patients 32-37. The associations of NE and histone-DNA with SaO2 at hospital admission and CT score of lung damage of COVID-18 are consistent with their effects in other lung viral infections 6, 12, 38. Their associations with troponin-T, D-dimer and fibrinogen suggest their role in the prothrombotic effects reported in COVID-1937-39. The myocardial injury during COVID-19 is not clearly understood 40-42. The mechanisms include direct viral infection, thrombosis, microvascular and myocardial injury related to reduced oxygen delivery and release of cytokines32, 41, 43. The increased cTnT associated with NE could be secondary to coronary thrombosis and myocardial infarction and/or myocarditis 32, 41, 42. NE and NETs could also contribute to the association of heart injury through systemic effects in kidney and other organs 2, 43, 44. The associations of NE and histone-DNA with urea and creatinine suggest their involvement in the acute kidney injury reported in about 30% of COVID-19 patients31, 45-47.
Our study contributes to a better understanding of pathological mechanisms of COVID-19 by pointing out the key role of the disruption of neutrophil innate immunity during and after viral replication, as summarized in graphical abstract . IL-6, IL-8 and TNFα account among the cytokines predominantly associated with COVID-19 severity48. IL-6 and IL-8 are produced upon NF-κB activation of infected alveolar macrophages through mechanisms that probably involve Bruton Tyrosine Kinase (BTK) 49. This is illustrated by the promising clinical effects produced by the BTK inhibitor acalabrutinib in patients with severe COVID-1949. IL-8 act as neutrophil-activating chemokine through its binding to CXCR2, which is a major chemokine receptor of neutrophils 50. Therefore, the dramatic increased of NE in severe COVID-19 may be related to neutrophil activation by the IL8/CXCR2 pathways 51. LDH, ferritin and D-dimer are highly correlated with NE and could reflect the macrophage activation of COVID-19 47, 52. Conversely, NE and NETs could also play a role in macrophage activation and the processing of proinflammatory cytokines 3, 6, 34-36. We reported a dramatic decrease of global DNase activity in serum which could participate in NETosis through decreased degradation of circulating chromatin-DNA fragments (graphical abstract)53, 54-56. It is noteworthy that aDnase1–/– Dnase1l3–/– mouse model exposed to pathogens produces lung lesions similar to those observed in patients with respiratory distress and/or sepsis and autopsies of COVID-19 patients38, 57. Consistently, a recent study reported a decreased activity of DNase I in bacterial and viral pneumonia in children 58.
The sera of COVID-19 patients decreased the cell retention of NETs and increased the release of dsDNA of neutrophils from healthy donors. Similar results have been previously obtained with COVID19 serum using the cell dye SYTOX Green to quantify NETs 9. These results show that the NETosis can be triggered by endogenous stimuli released in blood by injured tissues such as DAMPs, including free dsDNA59.
Our study opens up therapeutic perspectives. The excessive NE activity reported in our study suggests evaluating the use of new generation potent NE inhibitors, including lonodelestat (POL6014), alvelestat, CHF6333, and elafin in COVID-19 8,25. The dramatic decrease of DNase reported in our study also suggests evaluating the use of recombinant deoxyribonuclease I (dornase alfa) and/or DNase 1L3 60-62. One expected effect is the release of NE from degraded NETs, with increased free NE in blood and subsequently improved efficacy of NE inhibitors28. For this reason, we think that the association of DNase inhibitors with NE inhibitors should be considered in the treatment of COVID-19. Another therapeutic option to be considered could be the use of inhibitors of CXCR2 to block the neutrophil recruitment and activation.
Our study suffered limitations. We could not systematically report all biomarkers in all patients. The recruitment and cross-sectional evaluation did not allow us to study the kinetics of NE and NETs by the follow-up of patients. However, we were able to compare the concentration of NE and NETs in ambulatory symptomatic patients recruited during the first week of COVID-19 and patients hospitalized during the second to fourth week of the disease. The multicentric recruitment in local hospitals and in regional university hospitals was a strength of our study for evaluating cases with contrasted disease severity. The exhaustive recording of clinical data allowed us to perform multivariate analysis of the association of NE and NETs with severity and multi-organ damage of COVID-19, after forced adjustment for medical history.
In conclusion, our study points out the involvement of NE, DNase activity, and NETs as components of the innate immune defense of neutrophils against virus infection during the first phase of COVID-19 and their key role in the severity of the acute respiratory distress syndrome and cardiovascular, renal and inflammatory systemic manifestations in the later step of the disease. They suggest evaluating NE, DNases 1 and NETs as potential therapeutic targets.