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.