Blood neutrophils from children with COVID-19 exhibit both inflammatory and anti-inflammatory markers

Abstract

Background: Perhaps reflecting that children with COVID-19 rarely exhibit severe respiratory symptoms and often remain asymptomatic, little attention has been paid to explore the immune response in pediatric COVID-19. Here, we analyzed the phenotype and function of circulating neutrophils from children with COVID-19.

Methods: An observational study including 182 children with COVID-19, 21 children with multisystem inflammatory syndrome (MIS-C), and 40 healthy children was performed in Buenos Aires, Argentina. Neutrophil phenotype was analyzed by flow cytometry in blood samples. Cytokine production, plasma levels of IgG antibodies directed to the spike protein of SARS-CoV-2 and citrullinated histone H3 were measured by ELISA. Cell-free DNA was quantified by fluorometry.

Findings: Compared with healthy controls, neutrophils from children with COVID-19 showed a lower expression of CD11b, CD66b, and L-selectin but a higher expression of the activation markers HLA-DR, CD64 and PECAM-1 and the inhibitory receptors LAIR-1 and PD-L1. No differences in the production of cytokines and NETs were observed. Interestingly, the expression of CD64 in neutrophils and the serum concentration of IgG antibodies directed to the spike protein of SARS-CoV-2 distinguished asymptomatic from mild and moderate COVID-19.

Interpretation: Acute lung injury is a prominent feature of severe COVID-19 in adults. A low expression of adhesion molecules together with a high expression of inhibitory receptors in neutrophils from children with COVID-19 might prevent tissue infiltration by neutrophils preserving lung function.

Funding: This study was supported by the Ministry of Science and Technology (National Agency for Scientific and Technological Promotion, IP-COVID-19-0277 and PMO BID PICT 2018-2548), and University of Buenos Aires from Argentina (20020170100573BA).

Keywords: Cell-adhesion molecules; Fcγ receptor; IgG; Inhibitory Receptors; Neutrophils; Pediatric COVID-19.

Fig. 1

Low expression of CD11b, CD66b, and L-selectin and high expression of HLA-DR and PECAM-1 in neutrophils from children with COVID-19. (a) Frequency of CD11b, CD66b, CD62L, HLA-DR and PECAM-1 positive cells in neutrophils from healthy (n = 22) and COVID-19 children (n = 65 for CD11b, n = 22 for PECAM-1 and n = 35 for all other markers). (b) CD11b and CD66b MFI analyzed in the cohorts described in (a). (c) Representative histograms of CD11b and CD66b expression. Gray filled histograms show the isotype controls. (d) Correlation between CD66b and CD11b expression (MFI) in neutrophils from children with COVID-19 (n = 32). Median and min to max of n donors are shown in a and b. P values were determined by Kruskal-Wallis test with Dunn's posttest, Mann-Whitney U test and Spearman correlation test: ** p < 0.01, *** p < 0.001, ****, p < 0.0001. Controls (white circles), COVID-19 (blue circles) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 2

High expression of LAIR-1, PD-L1 and CD64 in neutrophils from children with COVID-19. (a) Frequency of LAIR-1 and PD-L1 positive cells in neutrophils from healthy (n = 20) and COVID-19 (n = 21). (b) Representative dot plots of LAIR-1 and PD-L1 expression in a donor from each cohort are shown. Numbers indicate the percentage of neutrophils positive for the expression of LAIR-1 or PD-L1. (c) Frequency of positive cells for CD16, CD32 and CD64 expression in neutrophils from healthy (n = 22) and COVID-19 (n = 56). (d) Representative dot plots of CD16 and CD64 expression are shown. Data show the percentages of positive cells in gated neutrophils. Median and min to max of n donors are shown in a and c. P values were determined by Kruskal-Wallis test with Dunn's posttest and Mann-Whitney U test: * p <0.05, ** p <0.01. Controls (white circle), COVID-19 (blue circle) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 3

Concentration of inflammatory cytokines and NETs in plasma and neutrophil supernatants from children with COVID-19. (a) Plasma levels of IL-1β, IL-6 and IL-8 in healthy (n = 13) and COVID-19 (n = 40) quantified by ELISA. (b) Plasma levels of cell-free DNA and citrullinated histones H3 in healthy (n = 20 for cell-free DNA and n = 5 for citrullinated histones H3) and COVID-19 (n = 49 for cell-free DNA and n = 19 for citrullinated histones H3) were quantified by fluorometry and ELISA, respectively. (c) Isolated neutrophils (5 × 10⁶/mL) from healthy (n = 10) and COVID-19 (n = 40) were cultured for 18 h, and the spontaneous production of IL-1β and IL-8 was analyzed by ELISA. (d) The production of IL-1β and IL-8 by neutrophils stimulated or not with R848 during 18 h was determined by ELISA in paired samples from healthy (n = 5) and COVID-19 (n = 15). (e) Levels of cell-free DNA in the supernatant of unstimulated neutrophils (1 × 10⁶/mL) from healthy (n = 10) and COVID-19 (n = 30) cultured for 4 h at 37 °C were quantified by fluorometry. Median and min to max of n donors are shown in a-e. P values were determined by Kruskal-Wallis test with Dunn's posttest and Wilcoxon matched-pairs signed rank test: * p < 0.05, **** p < 0.0001. Controls (white circle), COVID-19 (blue circle). R848, TLR 7/8 agonist resiquimod (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 4

Neutrophil phenotype, cytokines and NETs levels in children with COVID-19 according to disease severity. (a–g) Children with COVID-19 (asymptomatic, mild and moderate) and MIS-C were classified as described in Methods. (a–d) Expression of CD11b, CD66b, LAIR-1 and PD-L1 in gated neutrophils was analyzed by flow cytometry in whole blood samples. (a) CD11b (asymptomatic, n = 13; mild, n = 37; moderate, n = 15 and MIS-C, n = 15); (b) CD66b (asymptomatic, n = 10; mild, n = 17; moderate, n = 8 and MIS-C, n = 19); (c) LAIR-1 (asymptomatic, n = 7; mild, n = 9; moderate, n = 5 and MIS-C, n = 16); (d) PD-L1 (asymptomatic, n = 3; mild, n = 14; moderate, n = 4 and MIS-C, n = 8). (e) Spontaneous production of IL-1-β (left) and IL-8 (right) evaluated in supernatants from unstimulated neutrophils (asymptomatic, n = 10; mild, n = 20; moderate, n = 10 and MIS-C, n = 10). (f) Plasma levels of cell-free DNA (left: asymptomatic, n = 13; mild, n = 24; moderate, n = 12 and MIS-C, n = 17) and citrullinated histones H3 (right: asymptomatic, n = 8; mild, n = 7; moderate, n = 4 and MIS-C, n = 8). (g) Levels of cell-free DNA in the supernatant of unstimulated neutrophils from asymptomatic, n = 7; mild, n = 16; moderate, n = 7 and MIS-C, n = 11. Dotted line depicts the median values found in healthy controls. Median and min to max of n donors are shown in a-g. P values were determined by Kruskal-Wallis test with Dunn's posttest and Mann-Whitney U test: * p < 0.05, ** p < 0.01, *** p < 0.001. Asymptomatic (light green circle), mild (dark green circle), moderate (yellow circle), MIS-C (red circle)(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) .

Fig. 5

CD64 expression and plasma levels of IgG antibodies directed to the spike protein of SARS-CoV-2 in acute and recovered children with COVID-19. (a) Expression of CD64 in gated neutrophils analyzed by flow cytometry (asymptomatic, n = 6; mild, n = 34; moderate, n = 16, recovered, n = 8 and MIS-C, n = 17). Dotted line depicts the median expression of CD64 in neutrophils from healthy controls. (b,c). Plasma levels of IgG antibodies directed to SARS-CoV-2 spike protein measured by ELISA. (b) Healthy donors (n = 40), children with COVID-19 (n = 174) and MIS-C (n = 21). (c) Asymptomatic (n = 29), mild (n = 102), moderate (n = 43) acute COVID-19, recovered COVID-19 (n = 8) and MIS-C (n = 21). Dotted line indicates the cut-off value in b-c. Median and min to max of n donors are shown in a-c. P values were determined by Kruskal-Wallis test and Mann-Whitney U test: * p < 0.05, ** p < 0.01, **** p < 0.0001. Controls (white circle), COVID-19 (blue circle), asymptomatic (light green circle), mild (dark green circle), moderate (yellow circle), recovered (orange circle), MIS-C (red circle)(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).