Inflammasome activation in neutrophils of patients with severe COVID-19

Abstract

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engages the inflammasome in monocytes and macrophages and leads to the cytokine storm in COVID-19. Neutrophils, the most abundant leukocytes, release neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of COVID-19. Our recent study shows that activation of the NLRP3 inflammasome is important for NET release in sterile inflammation. However, the role of neutrophil inflammasome formation in human disease is unknown. We hypothesized that SARS-CoV-2 infection may induce inflammasome activation in neutrophils. We also aimed to assess the localization of inflammasome formation (ie, apoptosis-associated speck-like protein containing a CARD [ASC] speck assembly) and timing relative to NETosis in stimulated neutrophils by real-time video microscopy. Neutrophils isolated from severe COVID-19 patients demonstrated that ∼2% of neutrophils in both the peripheral blood and tracheal aspirates presented ASC speck. ASC speck was observed in neutrophils with an intact poly-lobulated nucleus, suggesting early formation during neutrophil activation. Additionally, 40% of nuclei were positive for citrullinated histone H3, and there was a significant correlation between speck formation and nuclear histone citrullination. Time-lapse microscopy in lipopolysaccharide -stimulated neutrophils from fluorescent ASC reporter mice showed that ASC speck formed transiently and at the microtubule organizing center long before NET release. Our study shows that ASC speck is present in neutrophils from COVID-19 patients with respiratory failure and that it forms early in NETosis. Our findings suggest that inhibition of neutrophil inflammasomes may be beneficial in COVID-19.

Graphical abstract

Figure 1.

Circulating neutrophils from COVID-19 pneumonia patients demonstrate inflammasome activation and high nuclear citrullination. Plasma samples from healthy controls (n = 8) and patients with severe COVID-19 (n = 6) were evaluated for: (A) IL-1β, (B) IL-18, (C) H3cit, (D) dsDNA, and (E) MPO-DNA complexes. Values are means plus or minus SEM. Data were analyzed using Mann-Whitney U test; *P < .05, ***P < .0001. Neutrophils freshly isolated from healthy controls and patients with severe COVID-19 were stained with DAPI (blue) and (F) an anti-ASC antibody (red) or (I) with an anti-H3cit antibody (red). White arrows indicate the presence of the ASC speck. Nonspecific staining corresponds to immunostaining performed without primary antibodies. (F) Representative images from widefield microscopy of neutrophils. Scale bar, 10 μm. (G) Representative confocal microscopy images of an immunostained neutrophil and a PBMC from COVID-19 patient. Blue, DNA (DAPI); red, ASC antibody staining. Scale bar, 5 µm. (H) Quantification of neutrophils presenting a speck. (I) Representative images from widefield microscopy of neutrophils. Scale bar, 10 μm. (J) Quantification of neutrophils with nucleus positive for H3cit. Each dot represents 1 individual. Values are means plus or minus SEM. Data were analyzed by Mann-Whitney U test; *P < .005, **P < .001. (K) Correlation between the percentage of neutrophils positive for H3cit and percentage of neutrophils forming speck among all individuals healthy (▪) and COVID-19 patients (•). A nonparametric Spearman correlation test was computed. dsDNA, double-stranded DNA; H3cit, citrullinated histone H3; MPO-DNA, myeloperoxidase‐DNA; SEM, standard error of the mean.

Figure 2.

PBMCs from severe COVID-19 patients form ASC speck at similar frequency to neutrophils. PBMCs were stained with DAPI (blue), an anti-CD66b antibody (neutrophil marker, green) and (A) an anti-ASC antibody (red), or (C) with an anti-H3cit antibody (red). (A) Representative images from widefield microscopy of PBMCs. Scale bar, 10 μm. (B) Quantification of CD66b⁺ cells (neutrophils) in PBMC fraction. (C) Representative images from widefield microscopy of PBMCs. Scale bar, 10 μm. (D) Quantification of CD66b⁻ cells presenting a speck. (E) Quantification of CD66b⁺ cells (neutrophils) with nucleus positive for H3cit. Each dot represents 1 individual. Values are means plus or minus SEM. Data were analyzed by Mann-Whitney U test; *P < .05. SEM, standard error of the mean.

Figure 3.

Neutrophils recruited to the lung in patients with COVID-19 pneumonia have inflammasome activation and are predisposed to NETosis. Smears of tracheal aspirates were costained with DAPI (blue), an anti-CD66b antibody (neutrophil marker, green) and an anti-ASC antibody (red), or with an anti-H3cit antibody (red). Nonspecific staining corresponds to immunostaining performed without primary antibodies. (A) Representative images from widefield microscopy of tracheal aspirates from severe COVID-19 patients. White arrows indicate the presence of the ASC speck. Scale bar, 10 µm. (B) Quantification of CD66b⁺ neutrophils in tracheal aspirates. (C) Quantification of ASC speck–positive neutrophils in tracheal aspirates. (D) Quantification of H3cit⁺ neutrophils in tracheal aspirates. (E) A nonparametric Spearman correlation test was computed. SEM, standard error of the mean.

Figure 4.

Human neutrophils activate inflammasome early after LPS activation. Neutrophils isolated from healthy individuals (n = 5) were activated for different lengths of time with 5 μg/mL of LPS and stained with DAPI (blue) and an anti-ASC antibody (red). (A) Representative images from widefield microscopy of neutrophils activated for 30 minutes with 5 μg/mL of LPS. White arrows indicate the presence of the ASC speck. Scale bar, 10 μm. (B) Quantification of neutrophils presenting a speck at 30 minutes, 1 hour, and 4 hours after LPS activation. (C) IL-1β concentration measured in the supernatant of unstimulated (US) neutrophils or activated with LPS. Each dot represents 1 individual. Values are means plus or minus SEM. Data were analyzed by Mann-Whitney U test; *P < .05. SEM, standard error of the mean

Figure 5.

Inflammasome activation kinetics and localization during NETosis in mouse neutrophils. ASC citrine (green) reporter neutrophils were stained with far-red SiR-DNA (blue) or with far-red SiR-tubulin (red), and time-lapse DIC and spinning-disk confocal images were taken at 2- to 5-minute intervals for 4 hours. (A) Time series of image overlays of DIC (grayscale) and fluorescence of cell and DNA dynamics in neutrophils during NETosis in the presence of LPS. (A and F) Boxes around images indicate different cellular events (orange, speck formation; purple, microvesicle shedding [MV]; green, nuclear rounding; yellow, DNA release to the cytosol; black, extracellular DNA release). (B) Timing of cellular events relative to MV shedding under LPS activation. Each dot represents an individual cell. (C) Arithmetic means of percentage of cells forming ASC speck as a function of time in the absence (black curve) or in presence (red curve) of LPS added at time = 0 minutes. (D) Fluorescence intensity of ASC-citrine speck in neutrophils according to the time relative to MV shedding. Values are means plus or minus SEM. (E) Percentage of NETosis in cells forming speck (•) or in cells not forming speck (▪) under LPS stimulation. Each dot represents 1 independent experiment. Bar size represents the mean of 12 independent experiments. A Mann-Whitney U test was used; ***P < .001. (F) Time series of image overlays of DIC (grayscale) and fluorescence of ASC (green) and MT (red) in mouse neutrophils. White arrows indicate the presence of the ASC speck. Scale bar, 10 μm. MT, microtubules; SEM, standard error of the mean.