SARS-CoV-2 Immune Complex-Mediated Neutrophil Activation

Abstract

Understanding disease pathogenesis is essential to developing therapies in patients with infections that cause critical illness. Herein, we show that SARS-CoV-2-specific antibody levels and markers of neutrophil activation are associated with disease severity in patients hospitalized with COVID-19. We also provide a link between the adaptive and innate immune response by demonstrating an association between antibody levels and multiple markers of neutrophil degranulation and NETosis. We further demonstrate through a series of in vitro assays that SARS-CoV-2 antigen-antibody immune complexes can stimulate NETosis. Last, we discuss how this NETosis is more strongly associated with IgA immune complexes than IgG and can be ameliorated with spleen tyrosine kinase inhibition.

Keywords: COVID-19; antibodies; immune complexes; neutrophil extracellular traps; neutrophils.

Figure 1.

Neutrophil marker and antibody titer correlations. A, Inverse probability–weighted multiple linear regression models comparing markers of neutrophil activation and antibody titers with disease severity. The exponentiated coefficient of disease severity represented the ratio of mean biomarker levels in patients with severe/critical disease over moderate disease (mean ratio) and was used to quantify the association of disease severity with the biomarker. B, Heat map shows Spearman correlations between anti-spike and nucleocapsid IgA and IgG levels and markers of neutrophil activation. P values are shown in parentheses. ANC, absolute neutrophil count; IL-8, interleukin 8; MPO, myeloperoxidase; NET, neutrophil extracellular trap; S100A8, S100 calcium-binding protein A8; S100A9, S100 calcium-binding protein A9.

Figure 2.

NETosis quantification and immune complex correlations. The induction of NETs in the presence of SARS-CoV-2 spike immune complexes: A, over 8 hours; B, at a 4.5-hour end point. n = 11 patients with COVID-19 and 2 healthy controls repeated across up to 8 individual experiments per patient. A, Circles represent the mean, and error bars indicate the standard error of the mean (SEM). B, Horizontal black bar indicates the mean. C, Induction of NETs comparing monoclonal IgG vs IgA. Error bars indicate the SEM. D and E, Correlations (solid lines) between NET intensity and IgA and IgG immune complexes. Dotted lines and shading indicate 95% CI. NET induction is measured by total NET integrated intensity (green calibrated unit [GCU] × µm²). *P < .05. **P < .01. ***P < .001. A, P values for comparing mean values are based on 2-way analysis of variance with Tukey multiple comparisons. B and C, P values for comparing mean values are based on Kruskal-Wallis test with Dunn multiple comparisons or a paired t test. D and E, Correlation significance established by Pearson correlation coefficients. NET, neutrophil extracellular trap.