Kinetics of peripheral blood neutrophils in severe coronavirus disease 2019

Abstract

Objectives: Emerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID-19). We isolated neutrophils from the blood of COVID-19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation.

Methods: Multicolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co-culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) allowed us to investigate viral replication in neutrophils.

Results: Upon ICU admission, patients displayed high plasma concentrations of granulocyte-colony-stimulating factor (G-CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10^-, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non-metalloproteinase-derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID-19 neutrophils were hyper-responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS-CoV-2 failed to replicate inside human neutrophils.

Conclusion: Our study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID-19 patients, and supports the concept of an increased neutrophil activation state in the circulation.

Keywords: COVID‐19; chemokine; cytokine; emergency myelopoiesis; neutrophil; protease.

Figure 1

Quantification of neutrophil‐mobilising and neutrophil‐activating factors in plasma from patients with COVID‐19. Multiplex technology was used to measure concentrations of (a) G‐CSF, (b) CXCL12α, (c) CXCL1, (d) CXCL5, (e) CXCL8, (f) CXCL10 and (g) CXCL11 in plasma samples from COVID‐19 patients who stayed at the ICU [samples were collected during the first 48 h after admission (ICU – day 1; n ≥ 10), after one week (ICU – day 7; n ≥ 10) and upon discharge from the ICU (ICU – discharge; n ≥ 10)], COVID‐19 patients who were hospitalised in general wards (ward; n ≥ 13) and healthy controls (HC; n ≥ 7). Moreover, (h) CD26/DPP4 enzyme activity was determined in a substrate conversion assay. Bars indicate the median plasma cytokine/chemokine concentration (a–g) or CD26 activity (h) for each study group. Results were statistically analysed by the Kruskal–Wallis with Dunn’s multiple comparisons tests. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 for statistical differences between patients and controls. $P ≤ 0.05; $$$P ≤ 0.001; $$$$P ≤ 0.0001 for statistical differences between ICU – day 1 and other patient groups.

Figure 2

Polarisation responses of peripheral blood neutrophils from patients with COVID‐19. Neutrophils and plasma were collected from the peripheral blood of COVID‐19 patients who stayed at the ICU [samples were collected during the first 48 h after admission (ICU – day 1; n ≥ 7), after one week (ICU – day 7; n ≥ 5) and upon discharge from the ICU (ICU – discharge; n ≥ 7)], COVID‐19 patients who were hospitalised in general wards (ward; n = 9) and healthy controls (HC; n = 6). Neutrophils were resuspended in HBSS buffer, incubated in the presence of a chemotactic stimulus and fixed, upon which the percentage of polarised cells (as determined by the cellular shape) was determined microscopically. (a) Baseline polarisation was determined by fixing the cells immediately after purification. Stimulus‐induced polarisation was determined by incubating the cells for 3 min in the presence of the following stimuli: (b) vehicle; (c) CXCL8 (5 ng mL⁻¹); (d) CXCL8 (12.5 ng mL⁻¹); (e) CXCL12 (300 ng mL⁻¹); (f) TNF‐α (50 ng mL⁻¹); (g) CXCL10 (100 ng mL⁻¹); and (h) CXCL10 (300 ng mL⁻¹). Moreover, (i) healthy donor neutrophils were treated with plasma from COVID‐19 patients or healthy donors. Results are represented as percentage of polarised cells and were statistically analysed by the Kruskal–Wallis with Dunn’s multiple comparisons tests. *P ≤ 0.05 for statistical differences between groups indicated by horizontal lines.

Figure 3

Quantification of proteases, protease inhibitors and enzymatic activity in plasma from patients with COVID‐19. Plasma samples were collected from patients who stayed at the ICU [samples were collected during the first 48 h after admission (ICU – day 1; n ≥ 9), after one week (ICU – day 7; n ≥ 18) and upon discharge from ICU (ICU – discharge; n ≥ 18)], COVID‐19 patients who were hospitalised in general wards (ward; n ≥ 16) and healthy controls (HC; n ≥ 6). ELISA technology was used to determine concentrations of (a) neutrophil elastase, (b) TIMP‐1, (c) TIMP‐1/MMP‐9 complexes and (d) α‐2‐macroglobulin (α2M). In addition, (e) total gelatinolytic activity present in patient plasma, as determined by the degradation of a fluorogenic gelatine substrate, (f) gelatinolytic activity in patient plasma in the presence of the metalloproteinase inhibitor EDTA, (g) total MMP activity in patient plasma, as determined by the degradation of a fluorogenic MMP substrate, and (h) MMP activity in patient plasma in the presence of the metalloproteinase inhibitor EDTA were measured. Bars indicate the median value for each study group. The dashed lines indicate the lower detection limits. Open symbols indicate values above the upper detection limit. Results were statistically analysed by the Kruskal–Wallis with Dunn’s multiple comparisons tests. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 for statistical differences between patients and controls.

Figure 4

Phenotypical characterisation of peripheral blood neutrophils from patients with COVID‐19. Flow cytometry was used to evaluate the surface expression of (a) CD10, (b) CD16, (c) CXCR2, (d) CXCR1, (e) C5aR, (f) CD66b, (g) CD35 and (h) CD63 on neutrophils (gated as CD16⁺CD66b⁺ cells) from COVID‐19 patients who stayed at the ICU [samples were collected during the first 48 h after admission (ICU – day 1; n = 9), after one week (ICU – day 7; n = 9) and upon discharge from the ICU (ICU – discharge; n = 10)], COVID‐19 patients who were hospitalised in general wards (ward; n = 9) and healthy controls (HC; n = 6). Results are represented as percentages of positive neutrophils or median fluorescence intensity (MFI). Bars indicate the median value for each study group. Results were statistically analysed by the Kruskal–Wallis with Dunn’s multiple comparisons tests. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Figure 5

SARS‐CoV‐2 fails to replicate in human neutrophils and does not induce MPO release or cell death in vitro. (c, d) Neutrophils were isolated from the peripheral blood of healthy donors and immediately challenged with SARS‐CoV‐2 in vitro at a multiplicity of infection of 0.1 or vehicle‐treated. (a, b) Vero cells were used as a positive control. At 6 and 12 h post‐infection (p.i.), (a, c) cells and (b, d, e) supernatants were collected. Quantification of SARS‐CoV‐2 replication in (a) Vero cells, (c) neutrophils or (e) neutrophil cell culture supernatant by RT‐qPCR and shown as equivalent of plaque‐forming unit (ePFU) mL⁻¹. Plaque assays assessed infectious viral progeny in (b) Vero cells and (d) neutrophil supernatant in PFU mL⁻¹. (f) Bright‐field microscopy images at 12 h p.i. of SARS‐CoV‐2‐infected and mock‐infected neutrophil cultures. (g) LDH activity and (h) MPO activity were assessed 6 and 12 h p.i. in neutrophil culture supernatants. Each dot represents an independent measurement. Bars indicate median values. Samples were statistically analysed using the Mann–Whitney U‐test or analysis of variance (ANOVA) with Sidak’s multiple comparison’s tests.