Phenotypic and functional features of innate immune system cells in COVID-19
Phenotypic features of monocytes, neutrophils, NK and NK-like innate lymphoid cells and, oxidative burst, phagocytosis and apoptosis of monocyte and neutrophils were assessed.
Pro-inflammatory monocytes with decreased HLA-DR expression in patients with COVID-19:  As antigen-presenting cells, monocytes constitutively express HLA-DR. In our study, the mean percentage of HLA-DR expression on monocytes was 86.2%±10.4 in the mild; 85.7%±10.7 in the moderate and 85.3%±8.8 in severe groups of patients. That was 94.5 %±3.6 in the healthy control group and HLA-DR expression on monocytes of all three groups was significantly lower in comparison to healthy controls (p<0.01; Fig 1a). Although there was no difference in the percentages of HLA-DR among the patients’ groups, Mean Fluorescence Intensity (MFI) of HLA-DR, which is a relative indicator of the number of molecules per unit cell, was 521 ± 293 in the mild; 299 ± 200 in the moderate and 294 ± 192 in the severe group of patients with COVID-19 (Fig 1b). Mild group MFI values were significantly higher in comparison to that of moderate and severe patients (p=0.02, p=0.05 respectively). HLA-DR MFI on monocytes of the healthy population was 752 ± 352 significantly different from all patients group (p=0.02 for mild, p<0.01 for both moderate and severe patients; Fig 1b). The gradual decrease of HLA-DR expression on monocytes of patients with COVID-19 can be considered as a gradual exhaustion of monocytes while disease progresses.
The expression of CD16 defines a subgroup of monocytes which is called intermediate, non-classical monocytes. It has previously been shown that they were pro-inflammatory monocytes (42). We demonstrated that monocytes of all groups of COVID-19 expressed CD16 whereas that expression was very low in the healthy control group (69%±23 in the mild; 57.7% ± 27 in the moderate; 52.6% ± 32 in the severe group of patients versus 8.7%±6 in controls, p<0.01 Fig 1c, d). That difference was also significant between the mild and severe cases (p<0.05), Monocytes from patients with COVID-19 tended to lose their CD16 during disease progression and that the mean percentage of CD16 on monocytes was negatively correlated with the clinical course (CC: -0.24; p = 0.03) has been shown. In the severe cases, that decrease might be a second proof of that monocytes have been exhausted, similar to losing their HLA-DR on their surfaces when the disease becomes serious.
Expressions of CD10 and CD16 on neutrophils from COVID-19 patients : As the markers of mature neutrophils, expression of CD10, and CD16 were measured on neutrophils. That CD10 expression 95.7%± 4 in the mild; 88%± 18 in the moderate; 74.9%± 29.5 in the severe group patients with COVID-19, and 92.8%± 13 in the controls have been found. CD10 expression was found to be significantly low in the severe group compared to the others (p<0.02; Fig 1e). When MFI of CD10 was measured; a consistent decrease has been found in all three groups (267±154 in mild patients, 174.5±110 in moderate, 114±70 in the severe group). But, the statistical difference has only been found in comparison to controls (773±356, p<0.01; Fig 1f). The mean percentage of CD16 on neutrophils from patients with COVID-19 was; 96.9%±2.8 in mild; 94%±11 in the moderate, and 96%±5 in the severe group of patients. No difference has been found between the groups even in comparison to controls (96%±5.5; p>0.05). MFI values for CD16 expression were 571±272 in the mild group, 385±210 in the moderate group and 536±248 in the severe group versus 1093±454. No difference was found among the groups. However, all was low in comparison to controls (p<0.01; Fig 1g, h). Therefore, we demonstrated that neutrophils of all patients with COVID-19 were phenotypically mature. The tendency to shed surface CD10 and CD16 could be a hallmark for their activation status.
Full competency in ROS production and phagocytosis of neutrophils and monocytes in patients with COVID-19:  The oxidative burst (OB) of neutrophils and monocytes is measured as a percentage value, as a result of the conversion of nonfluorescent dihydrorhodamine (DHR123) to fluorescent rhodamine 123 oxidized by the reactive oxygen species (ROS) produced in the stimulated cells. Percentage of OB in both monocytes and neutrophils of Covid-19 patients was greater than 95% similar to the control group indicating that no defect in ROS production of monocytes and neutrophils of COVID-19 patients (Fig 2). Similar results were obtained from the phagocytosis of fluorescent-labeled E.coli (>%90 in both monocytes and neutrophils). In addition to percentage results, the folds increase was calculated by dividing the MFI values from each stimulated sample to samples’ own initial MFIs. The folds increase in ROS production in the OB experiments was not different between the patients and controls (2.3±0.2 fold for neutrophil OB; 2.1±0.1 fold for monocyte OB in both groups). But, the folds increase in phagocytosis has found to be slighty low in patients with COVID-19 (2.07±0.1fold in the patient group vs 2.3 ±0.17 fold in the controls for neutrophils; 2.09±0.08 fold in the patient group vs 2.3±0.08 fold in the controls for monocytes; p=0.02). This decrease may be due to the fact that neutrophils are busy due to viral infections and its consequences such as clearance of necrotic and apoptotic cells. Thus, neutrophils and monocytes of patients with COVID-19 were shown to be fully competent in ROS production.
Apoptosis of monocytes and neutrophils: No statistical significance has been found in early and late apoptosis of monocytes and neutrophils between the patients with COVID-19 and controls (data not shown).
CD3-CD8+CD56+NK like innate lymphoid cells: While evaluating CD3 vs CD8 plots during lymphocyte subset analysis, it has been noticed that there was an increase in CD3- CD8+ quadrant. Then, it was shown that this population was CD56+(Figure 3a, b).  The percentages of CD3-CD56+cells gated on CD8+ cells were calculated in all patient groups and controls. This was 18.2% ±10 in the control group, 33.8%±14 in the mild; 33.8%±22 in the moderate and, 41%±19 in the severe group (Fig 3c). Thus, we demonstrated that the percentage of CD3-CD8+CD56+ NK like innate lymphoid cells were significantly high in patients with COVID-19 (p<0.001).
NK cells: As bridging cells between innate and adaptive immune systems, the percentages and absolute numbers of NK cells (CD3-CD16+CD56+) were analyzed. Results for all three groups of patients with COVID-19 and controls were given in Table 2. Although there was an elevation tendency in NK cells in the severe group and, remarkably high percentage of NK cells was observed in some of the severe cases, no statistical difference has been found among the groups (Figure 4c). The absolute number of NK cells was found to be low in all groups of patients in comparison to healthy controls (p<0.03, Table 4)  Thus, it was shown that the percentage of NK cells was in the normal range but, their absolute numbers in all groups of COVID-19 patients were as low as approximately 50% of controls.
In the evaluation of phenotypic and functional features of the innate immune system in COVID-19 demonstrated that the presence of fully functional and active monocytes and neutrophils, and the normal or an increased percentage of bridging cells such as NK and NK like innate immune cells, respectively. However, the decreased absolute number of NK cells was not accorded with their percentage values.