Neutrophil Extracellular Traps Contribute to COVID-19 Hyperinflammation and Humoral Autoimmunity

Abstract

The coronavirus disease 2019 (COVID-19) is related to enhanced production of NETs, and autoimmune/autoinflammatory phenomena. We evaluated the proportion of low-density granulocytes (LDG) by flow cytometry, and their capacity to produce NETs was compared with that of conventional neutrophils. NETs and their protein cargo were quantified by confocal microscopy and ELISA. Antinuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and the degradation capacity of NETs were addressed in serum. MILLIPLEX assay was used to assess the cytokine levels in macrophages' supernatant and serum. We found a higher proportion of LDG in severe and critical COVID-19 which correlated with severity and inflammatory markers. Severe/critical COVID-19 patients had higher plasmatic NE, LL-37 and HMGB1-DNA complexes, whilst ISG-15-DNA complexes were lower in severe patients. Sera from severe/critical COVID-19 patients had lower degradation capacity of NETs, which was reverted after adding hrDNase. Anti-NET antibodies were found in COVID-19, which correlated with ANA and ANCA positivity. NET stimuli enhanced the secretion of cytokines in macrophages. This study unveils the role of COVID-19 NETs as inducers of pro-inflammatory and autoimmune responses. The deficient degradation capacity of NETs may contribute to the accumulation of these structures and anti-NET antibodies are related to the presence of autoantibodies.

Keywords: COVID-19; DNA-complex; DNase and autoimmunity; HMGB1; ISG-15; LDG; LL-37; NETs; SARS-CoV-2.

Figure 1

LDG subsets correlate with different biological features of COVID-19. A correlation matrix is depicted with all statistically significant variables. Correlations were addressed using the Spearman Rho with Bonferroni correction. LDG: low density granulocytes. SpO₂: peripheral oxygen saturation. LDH: lactate-dehydrogenase. CPK: creatine phosphokinase. PaFi: the ratio of the arterial partial pressure of oxygen (“PaO₂”) from the Arterial Blood Gas divided by the fraction of inspired oxygen (FiO₂).

Figure 2

LDG and NDG from COVID-19 patients are prone to spontaneous NET release. (A). Representative confocal microscopy image of the spontaneous NETs from COVID-19 neutrophil subsets according to disease severity. (B). LDG (n = 20) and NDG (n = 18) from COVID-19 patients have a higher percentage of NETs in comparison to healthy donor LDG (n = 3) and NDG (n = 10). (C). NETs secreted by NDG from COVID-19 patients have a higher length in comparison to COVID-19 LDG and healthy donor both NDG and LDG. Medians were compared using the Kruskal–Wallis test with Dunn’s multiple comparison test. The length of NETs is expressed in a logarithmic scale.

Figure 3

LDG and NDG from COVID-19 patients carry a distinctive pro-inflammatory protein cargo. (A). Representative confocal microscopy image showing a higher expression of ISG-15, LL-37 and HMGB1 in NDG NETs from patients with COVID-19 in comparison to healthy donors. (B–D). Cumulative statistics show a higher MFI of ISG-15, LL-37 and HMGB1 in LDG (n = 20) and NDG (n = 18) NETs from COVID-19 patients in comparison to NETs from healthy donors LDG (n = 3) and NDG (n = 10). Differences among medians were addressed with the Kruskal–Wallis test and the multiple comparison Dunn’s test.

Figure 4

The sera from patients with severe and critical COVID-19 have a deficient NET degrading capacity. (A). Representative confocal microscopy images of the NET degradation capacity of COVID-19 sera according to disease severity. Neutrophils from healthy donors (HD) were stimulated with PMA to induced NET formation and then incubated with 10% sera from COVID19 patients or HD. The deficient degradation capacity is corrected after the addition of human recombinant DNAse. B-C. Severe/critical COVID-19 patients (n = 16) have a deficient degradation of NETs in comparison to mild/moderate COVID-19 (n = 8) and healthy donors (n = 10) (B). The serum deficient degradation capacity of NETs observed in severe/critical COVID-19 patients (n = 16) is reverted after the addition of hrDNase and micrococcal nuclease (C). Medians were compared using the Kruskal–Wallis test and Dunn’s multiple comparison test.

Figure 5

Patients with COVID-19 have antibodies that recognize neutrophils and NET’s antigens. (A) Healthy donor neutrophils were seeded on coverslips and stimulated with 2.5 mM PMA to induce the release of NETs. The samples were incubated with 10% sera from COVID-19 patients or healthy donors. Sera from COVID-19 patients show anti-neutrophil and anti-NET antibodies. The antigens recognized by anti-neutrophils and anti-NET antibodies include those located in the cytoplasm (continuous arrow), nucleus (arrowhead) and NETs (discontinuous arrow). The autoantibodies were not found in healthy donors. As the isotype control, the samples were incubated solely with the secondary antibody. (B) Relative amount of anti-NET antibodies expressed as the optic density index (ODI). (C) Titers of antinuclear antibodies expressed in a logarithmic scale. (D) Titers of anti-neutrophil cytoplasmic antibodies. Medians were compared using the Kruskal–Wallis and Dunn’s multiple comparison tests.

Figure 6

Heat map of the expression of cytokines and chemokines after macrophages were stimulated with COVID-19 and healthy donor NETs. The proinflammatory response is mainly elicited by COVID-19 LDG (red box) and NDG NETs (yellow box), whilst none of the healthy donor NETs promoted the secretion of these cytokines and chemokines (green box). Heatmap based on hierarchical clustering by Ward’s method was constructed.