Elastase and exacerbation of neutrophil innate immunity are involved in multi-visceral manifestations of COVID-19

Abstract

Background: Many arguments suggest that neutrophils could play a prominent role in COVID-19. However, the role of key components of neutrophil innate immunity in severe forms of COVID-19 has deserved insufficient attention. We aimed to evaluate the involvement of neutrophil elastase, histone-DNA, and DNases in systemic and multi-organ manifestations of COVID-19.

Methods: We performed a multicenter study of markers of neutrophil innate immunity in 155 cases consecutively recruited in a screening center, local hospitals, and two regional university hospitals. The cases were evaluated according to clinical and biological markers of severity and multi-organ manifestations and compared to 35 healthy controls.

Results: Blood neutrophil elastase, histone-DNA, myeloperoxidase-DNA, and free dsDNA were dramatically increased, and DNase activity was decreased by 10-fold, compared with controls. Neutrophil elastase and histone-DNA were associated with intensive care admission, body temperature, lung damage, and markers of cardiovascular outcomes, renal failure, and increased interleukin-6 (IL-6), IL-8, and CXCR2. Neutrophil elastase was an independent predictor of the computed tomography score of COVID-19 lung damage and the number of affected organs, in multivariate analyses. The increased blood concentrations of NE and neutrophil extracellular traps were related to exacerbation of neutrophil stimulation through IL-8 and CXCR2 increased concentrations and increased serum DAMPs, and to impaired degradation of NETs as a consequence of the dramatic decrease in blood DNase activity.

Conclusion: Our results point out the key role of neutrophil innate immunity exacerbation in COVID-19. Neutrophil elastase and DNase could be potential biomarkers and therapeutic targets of severe systemic manifestations of COVID-19.

Keywords: COVID-19; DNase; innate immunity; myeloperoxidase; neutrophil extracellular traps (NETs).

FIGURE 1

Neutrophil elastase (NE), myeloperoxidase‐DNA (MPO‐DNA), histone‐DNA, and (B) cell‐free dsDNA and total DNase activity in serum of 155 COVID‐19‐positive cases. Symptomatic ambulatory cases (n = 34) were recruited in screening centers and hospitalized patients (n = 122) in local hospitals (n = 43) and in medical departments (n = 65) and intensive care units (n = 13) of two regional university hospitals. The p‐values of NE concentrations between groups were as follows: healthy versus ambulatory <0.0001; healthy versus proximity hospital <0.0001; healthy versus medical department <0.0001; healthy versus intensive care <0.0001; ambulatory versus proximity hospital 0.7870; ambulatory versus medical department 0.0028; ambulatory versus intensive care <0.0001; proximity hospital versus medical department 0.0003; proximity hospital versus intensive care <0.0001; and medical department versus intensive care 0.0239. The p‐values of MPO concentrations between groups were healthy versus ambulatory <0.0001; healthy versus proximity hospital <0.0001; healthy versus medical department <0.0001; healthy versus intensive care <0.0001; ambulatory versus proximity hospital 0.1237; ambulatory versus medical department 0.8589; ambulatory versus intensive care 0.1276; proximity hospital versus medical department 0.1463; proximity hospital versus intensive care 0.5811; and medical department versus intensive care 0.1482. The p‐values of histone_DNA values between groups were healthy versus ambulatory <0.0001; healthy versus proximity hospital <0.0001; healthy versus medical department <0.0001; healthy versus intensive care <0.0001; ambulatory versus proximity hospital 0.0013; ambulatory versus medical department 0.0525; ambulatory versus intensive care <0.0001; proximity hospital versus medical department 0.2740; proximity hospital versus intensive care 0.0.0033; and medical department versus intensive care 0.0003. The thresholds (dotted lines) were evaluated in healthy controls recruited several months before the epidemy. The threshold of NE was estimated at 73 ng/ml and those of MPO‐DNA at 0.562 AU (450 nm absorbance units), histone‐DNA at 0.591 AU, total activity of DNase at 9.94 U/ml, and serum dsDNA at 95.60 ng/ml (C) NE, MPO‐DNA, and histone‐DNA according to the number of organs affected by COVID‐19. Data from (A), (B) and (C) were compared by the Mann‐Whitney test. The dashed lines represent the cutoffs defined by the mean +2 standard deviations for NE, MPO‐DNA, histone‐DNA, cell‐free DNA, and the mean—2 standard deviations for DNase activity

FIGURE 2

Associations of neutrophil elastase (NE) with components of neutrophil extracellular traps (NETs), clinical features, and biomarkers of severity and multi‐visceral harm of COVID. Significant associations of NE were reported with blood concentration of myeloperoxidase (MPO)‐DNA and histone‐DNA, blood counts of neutrophils, neutrophil/lymphocyte ratio, and blood biomarkers of multi‐visceral harm, including urea, SaO2, troponin‐T, lactate dehydrogenase (LDH), D‐dimer, and fibrinogen. Correlations were assessed by Spearman's rank correlation

FIGURE 3

Associations of neutrophil elastase (NE) with cytokines involved in COVID‐19 pathological mechanisms. Significant associations of NE were reported with interleukin‐6 (IL‐6), IL‐8, and neutrophil chemokine receptor CXCR2, but not with TNF‐α. Dotted lines represent the cutoffs reported for multi‐organ damage in receiver operating characteristic (ROC) analyses. These cutoffs were used for the concordance analyses. Correlations were assessed by Spearman's rank correlation

FIGURE 4

Forest plot reporting the summary of the receiver operating characteristic (ROC) analyses to assess the diagnostic accuracy of neutrophil elastase (NE), myeloperoxidase (MPO)‐conjugated DNA, histone‐DNA, cell‐free dsDNA, and DNase activity for the prediction of disease‐related outcomes. For each biomarker, a ROC analysis was performed using the following classification variables: intensive care unit admission, heart decompensation, liver injury, kidney injury, respiratory failure, blood oxygen saturation <85%, number of affected organs ≥2, and NEWS 2 score ≥5. For each ROC analysis, the summary results were reported using the area under the ROC curve, the 95% confidence interval, and the associated p‐value (Table S3)

FIGURE 5

Neutrophil innate immunity is a target of the systemic effects of COVID‐19. (A, B) NET formation and release of neutrophil dsDNA by sera of COVID‐19 patients. (A) Representative data of flow cytometry experiments showing NET detection as citrullinated H3 (H3cit) and myeloperoxidase (MPO) double‐positive neutrophils (top) and NE‐positive neutrophils (bottom), after incubation of neutrophils with sera. (B) Quantitative analysis of NET retention on neutrophils from a healthy donor incubated with control or patient sera. (C) Quantification of DNA release from neutrophils of a healthy donor stimulated with PMA to induce NET formation and subsequently incubated with 10% serum from controls or patients in the absence or in the presence of aprotinin. The amount of released DNA was considered as 100% in unstimulated neutrophils from the donor