DEspRhigh neutrophils are associated with critical illness in COVID-19

Abstract

SARS-CoV-2 infection results in a spectrum of outcomes from no symptoms to widely varying degrees of illness to death. A better understanding of the immune response to SARS-CoV-2 infection and subsequent, often excessive, inflammation may inform treatment decisions and reveal opportunities for therapy. We studied immune cell subpopulations and their associations with clinical parameters in a cohort of 26 patients with COVID-19. Following informed consent, we collected blood samples from hospitalized patients with COVID-19 within 72 h of admission. Flow cytometry was used to analyze white blood cell subpopulations. Plasma levels of cytokines and chemokines were measured using ELISA. Neutrophils undergoing neutrophil extracellular traps (NET) formation were evaluated in blood smears. We examined the immunophenotype of patients with COVID-19 in comparison to that of SARS-CoV-2 negative controls. A novel subset of pro-inflammatory neutrophils expressing a high level of dual endothelin-1 and VEGF signal peptide-activated receptor (DEspR) at the cell surface was found to be associated with elevated circulating CCL23, increased NETosis, and critical-severity COVID-19 illness. The potential to target this subpopulation of neutrophils to reduce secondary tissue damage caused by SARS-CoV-2 infection warrants further investigation.

Figure 1

Flow cytometric strategy to determine DEspR^high cells in the peripheral circulation. Freshly obtained blood cells were analyzed after erythrocytes lysis. (A) Flow cytometric plots showing gating strategy for cells expressing a high level of cell surface DEspR in control (upper) and COVID-19 (lower) subjects. (B) Graphical representation of data from flow cytometric analysis on the number of DEspR^high/SSC^high neutrophils in control (n = 12) and COVID-19 (n = 26) patients; Mann–Whitney test. (C) The number of DEspR^high/SSC^high neutrophils in control subjects and patients with mild (n = 6), severe (n = 9), or critical (n = 11) COVID-19. Kruskal–Wallis test, Dunn’s multiple comparisons test; P value is shown.

Figure 2

Expression of CD10, CD14, and CD16 on DEspR^high neutrophils in control subjects and COVID-19 patients. The expression of cell surface markers was determined in the subpopulation of peripheral blood SSC^high neutrophils (total neutrophils) or a subset of neutrophils with high expression of DEspR (DEspR^high neutrophils) in control (n = 12) and COVID-19 (n = 26) subjects. (A) Representative flow cytometric plots showing expression of CD10 on total neutrophils in control (upper) and COVID-19 (lower) subjects. (B) Number of CD10 positive (left) and CD10 negative neutrophils. Mann–Whitney test. (C) Flow cytometric plots demonstrating expression of CD10 on DEspR^high neutrophils in control (upper) and COVID-19 (lower) subjects. (D) Percentage of CD10 positive cells in a subpopulation of total neutrophils (Total) or a subset of DEspR^high neutrophils in control (left) and COVID-19 (right) subjects. Mann–Whitney test. (E) Percentage of CD10 positive (left) and CD10 negative (right) neutrophils in control subjects and COVID-19 patients. Mann–Whitney test. (F,G) Expression of (F) CD14 and (G) CD16 on total and DEspR^high neutrophils in control and COVID-19 subjects. The expression is represented by the mean fluorescence intensity that corresponds to the level of cell surface CD14 and CD16. ΔMFI was calculated by subtracting the mean fluorescence intensity of isotype controls from the mean fluorescent intensity of specific antibodies. Two-way ANOVA with Tukey multiple comparisons test; P values are indicated.

Figure 3

High neutrophil to lymphocyte ratio is associated with an increased number of DEspR^high neutrophils. (A) Neutrophil–lymphocyte ratio (NLR) in control (n = 12) and COVID-19 (n = 26) subjects. Mann–Whitney test. (B) Relationship between NLR and number of DEspR^high neutrophils. Spearman correlation coefficient is indicated. (C–E) Number of (C) CD3 T lymphocytes, (D) CD3/CD4 and (E) CD3/CD8 T cells in control and COVID-19 subjects. Mann–Whitney test. (F–H) Association between the number of DEspR^high neutrophils and (F) total T lymphocytes, (G) CD3/CD4, and (H) CD3/CD8 T cells. Spearman correlation coefficients and P values are indicated.

Figure 4

Level of circulating pro-inflammatory factors in control and COVID-19 subjects. Levels of circulating cytokines and chemokines were determined in platelet-free plasma in groups of control subjects (n = 12) and COVID-19 patients (n = 25). One patient with extracorporeal membrane oxygenation support was excluded from the analysis due to the potential effect of cytokine adsorption during ECMO therapy. Mann–Whitney test.

Figure 5

Association between DEspR^high neutrophils and CCL23. (A) Heat map associated with hierarchical clustering analysis of tested parameters in groups of mild (n = 6), severe (n = 9), and critical (n = 10) COVID-19 patients obtained using ClustVis 2.0. Columns with similar annotations are collapsed by taking the median inside each group. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage. Color keys show the relative strength of the signal in each cluster group. Annotations on top of the heatmap show clustering of the samples. AST aspartate aminotransferase, BMI body mass index, CRP C-reactive protein, DAPI+ 4′,6-diamidino-2-phenylindole viability dye positive dead cells, Est FiO2 estimated fraction of inspired oxygen, HOSP LOS hospital length of stay, ICU LOS intensive care unit length of stay, sCD73 soluble CD73. (B) Association between the level of circulating CCL23 and COVID-19 severity. Spearman correlation coefficient and P value are indicated. (C) The level of CCL23 in COVID-19 survivors (n = 21) and non-survivors (n = 4). Mann–Whitney test. (D,E) Associations between the level of CCL23 and number of (D) DEspR^high neutrophils and (E) total neutrophils in COVID-19 patients. (F,G) Relationships between the number of NETosing neutrophils and number of (F) DEspR^high neutrophils and (G) total neutrophils in COVID-19 patients. (D–G) Spearman correlation coefficients and P values are indicated.