Twenty-seven hospitalized patients with PCR-verified SARS-CoV-2 infection were enrolled in the study. The median age of covid patients was 66 (range 48-90). There were 17 men (63%) and 10 women (37%) participants. The control group consisted of 6 male (50%) and 6 female (50%) healthy participants verified with a negative PCR test for SARS-CoV-2. The median age of covid patients was 33 years (range 25-58 years). Demographic and clinical characteristics of enrolled patients and healthy controls are summarized in Table 1.
COVID-19 is characterized by neutrophil hyperresponsiveness and elevated production of pro-inflammatory cytokines
Neutrophils from COVID-19 patients, compared to healthy individuals, were characterized by significantly higher expression intensity of neutrophil activation marker CD11b (mean fluorescence intensity (MFI) ± SD of 5448 ±2071 and 3475 ±1035, respectively; p<0.01; Fig. 1A). Fluorescence intensity and percentage of neutrophils positive for granulocyte activation marker CD66b were also significantly increased in COVID-19 patients compared to controls (1257 ± 408 and 605 ± 115.7, respectively; p<0.0001; Fig. 1B and median and interquartile range of 6.220 (4.6–10.3) versus 2.380 (1.83–3.98), respectively; p<0.0001; Fig. 1VS). In addition, we found that the plasma level of the pro-inflammatory cytokines, TNF-α, IL-6 and IL-8, central cytokines in antiviral responses and cytokines causing a cytokine storm phenomenon, was significantly higher in patients with COVID-19. 19 compared to healthy donors (TNF, median and interquartile range of 1.545 (1.165–2.076) versus 0.6383 (0.513–0.730); p<0.0001; Fig. 2A; IL-6, median and interquartile range of 8.749 (3.79–21.79) versus 0.4955 (0.01–0.819); p<0.0001); Fig. 2B; IL-8, median and interquartile range of 20.19 (17.47–24.07) versus 15.07 (13.62–16.30); p<0.0001); Fig. 2VS).
Neutrophils from COVID-19 patients show increased survival compared to neutrophils from healthy donors
Neutrophil populations detected in the blood of COVID-19 patients showed, compared to healthy donors, significantly higher expression of CD47, which is a “don’t eat me” molecular signal that inhibits phagocytosis of the cell of expression and prolongs its survival (MFI ± SD of 2204 ± 631 and 1323 ± 231, respectively; p<0.0001; Fig. 3A). On average, 32% (± 13%) of neutrophils from COVID-19 patients expressed CD47 compared to 25% (± 15%) in healthy donors (p<0.05; Fig. 3B). The expression of CD36 which is an “eat me” signal was not significantly different between the groups (Fig. 3CD).
Low expression of CD49 on neutrophils in COVID-19 patients indicates increased migration into lung tissue
CD49+ neutrophils accumulate in the lungs during viral infections12. CD49 expression is also increased on the surface of aged neutrophils. COVID-19 patients had about 50% CD49+ blood neutrophils as opposed to healthy controls, where more than 80% of blood neutrophils expressed CD49 (49.3 ± 21.4 vs. 81.9 ± 14.6; p<0.0001; Fig. 4A). The MFI of CD49 on neutrophils was also lower than that of healthy controls. However, this result did not reach statistical significance (p= 0.051, Fig. 4B). The low expression of CD49 on neutrophils in the blood indicates that neutrophils upon SARS-CoV-2 infection largely migrate to peripheral tissues including the lungs and are young.
COVID-19 patients have high levels of NET markers in their plasma
Consistent with previous research, plasma levels of circulating cell-free DNA (cf-DNA) and cell-free nucleosomes were significantly higher in patients with COVID-19 compared to healthy individuals (median (IQR) of 98, 2 (88.7–120.3) vs. 78.9 (75.6–83.3); p<0.0001 and 0.47 (0.29–0.95) versus 0.16 (0.12–0.26); p<0.0001; Fig. 5A B). The expression of these markers indicates the formation of NET in the blood of affected patients.
High-dimensional cluster analysis reveals neutrophil clusters characterized by long-lasting hyperresponsiveness
To extend the results, we processed the data as described in the Methods section and mapped neutrophil populations on t‐SNE composite plots, which revealed clear locations of neutrophil populations in COVID-19 patients and controls. healthy. Phenographic analysis revealed 17 unique clusters in the t‐SNE space. Detailed cluster characterization is attached as a supplementary table 1. Figure 6 shows the distribution and location of neutrophils in all study participants (Fig. 6A), COVID-19 patients only (Fig. 6B) and healthy controls only (Fig. 6VS).
As can be seen in Fig. 6, neutrophil populations in COVID-19 patients are characterized by completely opposite phenotypes to those of populations seen in healthy controls. Neutrophils from healthy controls were grouped into groups 2, 4, 6, 8, 9, 13, 15 and 17 (Fig. 6B). Neutrophils associated with COVID-19 were mainly localized in groups 1, 3, 5, 7, 10-12, 14 and 16 (Fig. 6VS). Clusters of SARS-CoV-2 infected patients included neutrophils characterized by high expression of activation markers (CD66b, CD11b, CD62L) and “don’t eat me” markers (CD47).
Neutrophil populations of COVID-19 patients were plotted on the same t-SNE patterns with respect to gender (Fig. sevenA–C). The analysis revealed that male patients compared to female patients had significantly more neutrophils traced in group 3 (p< 0.01) which includes neutrophils with high “don't eat me” expression and activation markers (CD47, CD11b). On the other side, women had significantly more neutrophils traced in group 1 which includes unactivated mature neutrophils (CD16HighCD62LHigh).
Additionally, patients with peripheral blood markers of hyperinflammation (elevated CRP C-reactive protein levels and elevated ferritin levels) were characterized by an overrepresentation of neutrophils in groups 5 (representing premature unactivated neutrophils) and 1 (representing unactivated mature neutrophils). activated neutrophils). On the other side, patients with normal CRP level had significantly more neutrophils which can be classified as aged and pre-apoptotic (Fig. 8A).
Another analysis, comparing patients with severe disease defined as hospital stay greater than 7 days and milder disease with hospital stay less than 7 days, found that group 3 comprising hyperactivated neutrophils expressing “don’t eat me” markers is overrepresented in patients with severe disease (Fig. 8B) as well as in older patients (over 70 years old) (Fig. 8VS).
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