Flow cytometric evaluation of the neutrophil compartment in COVID-19 at hospital presentation: A normal response to an abnormal situation

Abstract Coronavirus disease 2019 (COVID-19) is a rapidly emerging pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Critical COVID-19 is thought to be associated with a hyper-inflammatory process that can develop into acute respiratory distress syndrome, a critical disease normally mediated by dysfunctional neutrophils. This study tested the hypothesis whether the neutrophil compartment displays characteristics of hyperinflammation in COVID-19 patients. Therefore, a prospective study was performed on all patients with suspected COVID-19 presenting at the emergency room of a large academic hospital. Blood drawn within 2 d after hospital presentation was analyzed by point-of-care automated flow cytometry and compared with blood samples collected at later time points. COVID-19 patients did not exhibit neutrophilia or eosinopenia. Unexpectedly neutrophil activation markers (CD11b, CD16, CD10, and CD62L) did not differ between COVID-19-positive patients and COVID-19-negative patients diagnosed with other bacterial/viral infections, or between COVID-19 severity groups. In all patients, a decrease was found in the neutrophil maturation markers indicating an inflammation-induced left shift of the neutrophil compartment. In COVID-19 this was associated with disease severity.


INTRODUCTION
In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan City, Hubei Province, in China. 1 Since then, the virus has spread globally, causing a pandemic of coronavirus disease 2019 . The disease severity greatly varies between patients, ranging from mild complaints to intensive care unit (ICU) admittance and death. Groups at risk for severe disease are the elderly, patients with chronic diseases, and individuals suffering from obesity. 2 Severe disease is associated with both pulmonary manifestations and, albeit less abundant, nonrespiratory symptoms. 3 The disease can become life threatening when tissue function becomes critically compromised, such as seen during respiratory failure and acute respiratory distress syndrome (ARDS). In addition, cardiovascular complications can also lead to pathology in the intestine, heart, brain, and renal tissue. 4 The underlying mechanism leading to tissue damage in COVID-19 is still uncertain, but a toxic combination of abnormal coagulation, systemic thrombosis, thrombo-embolisms, and hyperinflammation is thought to mediate critical disease in COVID-19 patients. 5,6 The ARDS found in COVID-19 patients is characterized by decreased oxygenation and rapid respiratory failure. 7 The pathophysiology of ARDS is normally mediated by malfunctioning of the neutrophil compartment leading to accumulation and specific activation of these cells in the pulmonary tissue, which in turn causes collateral damage characterized by destruction of epithelial and endothelial cells. [8][9][10][11][12] Tissue damage is caused by neutrophil-driven mechanisms that are normally employed to kill microorganisms, which include production of reactive oxygen species, degranulation of toxic proteins and enzymes, and netosis. 10,13,14 This leads to increased vascular permeability and protein-rich alveolar edema causing decreased gas exchange and hypoxemia. 15 Many COVID-19 patients meet the Berlin definition for ARDS, 16 but it remains to be elucidated whether ARDS seen in COVID-19 patients is similar to the archetypal ARDS seen in other patients. Although both are characterized by bilateral consolidations and severe hypoxia, COVID-19-mediated ARDS seems to have a later onset than "classical" ARDS and is more often associated with a relatively normal lung compliance. 8,17 Also, the role of neutrophils in COVID-19-mediated ARDS is still unclear.
A variety of increased proinflammatory cytokines (TNF-, IL-1 , IL-6, IL-8) is found in the blood of COVID-19 patients, suggesting a proinflammatory state 18 and a hyper-activation of the immune system. 19 Therefore, several studies have suggested that ARDS in COVID-19 is mainly caused by a cytokine storm and that anti-inflammatory drugs might be helpful. 20 However, direct evidence that COVID-19-related ARDS is characterized by inflammation is missing. 21 Alternatively, COVID-19-associated ARDS might be caused by pulmonary edema via a dysfunctional bradykinin metabolism. This latter explanation was put forward as the angiotensin-converting enzyme 2 (ACE2) receptor, important in bradykinin1-9 inactivation, is the main porte d'entrée of SARS-CoV-2 in airway epithelium and its expression is decreased upon virus entry into the epithelial cells. 22 Interestingly, bradykinin1-9 can also be inactivated by neprilysin (CD10), which is an important activation and differentiation marker expressed by neutrophils, possibly linking neutrophils with bradykinin-induced pulmonary edema. 23,24 The increase in expression of activation markers on neutrophils is generally used to study the role of neutrophils in vivo in health and disease. [25][26][27] However, these cells are notoriously sensitive to ex vivo manipulation and it is therefore essential to minimally manipulate the cells ex vivo. We have recently shown that neutrophils become activated in the blood collection tube relatively quickly (<1 h) after venipuncture even before processing, which masks essential information about the state of the cells in vivo. 28
Thereafter, the blood tube was placed in the automated AQUIOS CL "Load & Go" flow cytometer (Beckman Coulter, Miami, FL, USA) that was located at the point of care in the emergency department. This was done as fast as possible after blood drawing, because neutrophils become easily and quickly activated ex vivo. 28 Healthy control blood obtained from the Mini Donor Service (Mini Donor Dienst) at UMCU was analyzed in a similar manner. Healthy control subjects were chosen based on donor availability. COVID-19 measures implemented in the hospital caused a shortage of available donors and therefore these could not be matched to the study patient population for age or gender.
Thus, blood from healthy controls was drawn at the out-patient clinic of UMCU, which is in close proximity to the emergency department and sample handling times were similar to those of patients included in this study. The median age of the healthy control subjects was 24 yr (interquartile range [IQR]: 23-27) 33.3% female and 66.7% male.
Healthy control subjects were not on any medications for chronic diseases that could impact study results.

Flow cytometry analysis by automated AQUIOS CL "Load & Go" flow cytometer
The AQUIOS CL combines robotic automated sample preparation with automated analysis of single cells using flow cytometry. 28 A cassette filled with blood tubes is placed in the machine. Hereafter, the device reads the barcodes on the blood tubes, automatically mixes and pipets the blood, and proceeds with the antibody staining. After 15 min of incubation, the blood is lysed by the addition of 335 l of lysing reagent A followed by 100 l of lysing reagent B. Lysing reagent A is a cyanidefree lytic reagent that lyses red blood cells. Lysing reagent B slows the reaction caused by reagent A and preserves the white blood cells for measurement in the flow cell. Finally, the prepared sample is aspirated for analysis. Absolute white blood cell count is based on an electronicvolume measurement.  All patients were projected into the model and a mean DAMACY score was calculated.

Clinical characteristics of suspected COVID-19
Patients who tested SARS-CoV-2 positive by PCR at any point during Critical disease was defined as severe disease with ARDS at any point during admission. ARDS was defined as SpO 2 /FiO 2 ≤ 315, 35 where FiO 2 is fraction of inspired oxygen.
Baseline characteristics included age, sex, body mass index (BMI), onset of symptoms, ICU admission, death, and duration of admission.
Vital functions during admission were collected from the moment

Statistics
Variables are presented as frequencies and percentages for categor- Multidimensional analysis was performed by application of the DAMACY algorithm as described by us earlier. 33 The results of all statistical analyses are shown in Supporting Information Table S1.

Study approval
For this study, a waiver for formal ethical approval was provided by ). CD64 was also tested in this panel but is not shown here because it is not discussed further in this article; (B) gating strategy for analyzing the blood samples from healthy controls and patients. Flow cytometric analysis was done using FlowJo analysis software (Tree Star Inc.)

No signs of hyperinflammation in the neutrophil compartment during COVID-19 at hospital presentation
In order to investigate the involvement of a hyperactivated neu- groups at hospital presentation (see Fig. 5A, C, E, G). Remarkably, expression of CD11b and CD62L was also not higher in COVID-19 patients when compared to healthy controls (Fig. 5). Furthermore, CD11b was significantly lower in patients with bacterial infections, other viral infections and in the combined COVID-19 group compared to healthy controls (see Fig. 5 and Supporting Information The longitudinal data supported the finding that no clear activation of neutrophils was evident at presentation in the hospital as the expression of CD11b was relatively low at this early time point (Fig. 5F), whereas CD62L was high (Fig. 5B). Interestingly, during admission the neutrophils acquired a more activated phenotype char-

Association of COVID-19 with the presence of young mature neutrophils in the peripheral blood
Acute inflammatory diseases are normally associated with the presence of young neutrophils (banded cells) and neutrophil progenitors in the peripheral blood. 30,36,37 However in COVID-19 patients, the absolute number of promyelocytes in the blood of COVID-19 patients did not significantly differ from the number in healthy controls (Fig. 6A).
The absolute number of myelocytes was significantly lower in all COVID-19 groups compared to healthy controls as can be seen in Even after exposure to fNLF, the expression of CD10 by neutrophils from COVID-19 and other disease groups stays significantly lower compared to healthy controls (Fig. 7G). The longitudinal data of CD16 indicated that expression of this marker slightly increased over time in some patients, but generally stayed in the low range (see Fig. 7B).
Surprisingly, CD10 expression was low during the early days after onset of COVID-19 symptoms and continued to stay low after stimulation with fNLF (Fig. 7F, H)). CD10 expression generally seemed to normalize with time, although values remained relatively low compared to healthy controls.

Multidimensional analysis of the complete dataset
In data shown thus far, the analysis was one and 2D and was carried out by multiple gating. This approach might have missed correlations that were not expected. Therefore, we analyzed the complete dataset by the algorithm DAMACY 34 that allows comparison of flow cytometric profiles between individual patients. An algorithm was used in the DAMACY analysis to extract putative COVID-19 or other diseasespecific cellular differences when compared to healthy controls. Based on these differences a DAMACY score was calculated. A higher score indicated more deviation from healthy control samples. The DAMACY score clearly discriminated between healthy controls and infectious diseases including COVID-19 ( Fig. 8 and Supporting Information Table S1.5). However, no significant differences were found between severities of COVID-19 or between COVID-19 and other infectious diseases. The changes seen in neutrophil marker expression were therefore not COVID-19 specific, but indicative of the presence of disease in general.

Maturation dissociation in systemic infectious disease
Despite the fact that the overall expression of CD16 was decreased in COVID-19 patients, no clear CD16 dim /CD62L bright subset was found in these patients suggesting a low number of banded cells in the blood of these patients. This is in marked contrast to acute inflammatory conditions such as noted during experimental endotoxemia [38][39][40] and multitrauma. 41 tion during presence in the blood collection tube. 28 In our previous research we showed a specific activation of neutrophils during prolonged (>30 min) presence in a blood tube. 28 The sensitivity of this artificial activation might be more prominent for cells that are primed by disease in vivo. This situation is completely different during acute inflammation associated with the presence of banded neutrophils such as that found after LPS challenge 30 and during severe trauma. 10 During acute inflammation a clear induction of CD11b expression was found 10 (also see Supporting Information Fig. S1). It is tempting to speculate that the difference between acute and more chronic inflammation might be caused by homing of activated cells to the tissues. 48,49 Our longitudinal data are also suggestive of this. Upon presentation at the ER, neutrophils from COVID-19 patients were characterized by a young (CD10 dim /CD16 dim ) population with a low activation phenotype (CD11b dim /CD62L high ). It is tempting to speculate that activated mature cells may have migrated to the tissues leaving behind relatively young nonactivated neutrophils, a situation also found in trauma patients. 49 Interestingly, this situation seemed to normalize with time as the patients recovered, leading to a counterintuitive activation of the neutrophil compartment characterized by a rise in CD11b expression and a lower expression of CD62L (Fig. 5B, F). Also, an increase in cellular age was detected (higher CD10 expression). The apparent increase in myelocyte numbers over time in the peripheral blood of COVID-19 patients (Fig. 6D) is supportive of the activation of neutrophilopoiesis in the bone marrow. These data also imply that an acute inflammatory event may have occurred prior to admittance to the hospital. This hypothesis has yet to be tested in a new study on mild COVID-19 patients.
The approach employed in this study also led to the surprising find- to the patients included in this study, it is important to emphasize that the expression of nCD10/neprilysin on neutrophils measured by automated flow cytometry is not dependent on the age of the donor. 28,59 Expression of CD10/neprilysin is high as seen in neutrophils in healthy control blood ( Fig. 7E and Marini et al. 54 ). Interestingly, the low expression of neutrophil CD10 is already present at the start of the COVID-19 symptoms (Fig. 7F) 53 In addition, lack of ACE2 might be involved in the thrombotic complications observed in COVID-19 patients. 22 In parallel with the ACE2-mediated breakdown, bradykinin1-9 can also be metabolized by CD10 (neprilysin), 24 which is a well-known maturation and activation marker expressed by neutrophils. 56  patients at presentation. This study entails neutrophil activation measured only in the peripheral blood with limited number of markers. It is possible that neutrophils are activated in the tissues (e.g., lung tissue), which cannot be investigated without invasive lung/tissue biopsies.
It could also be that there was neutrophil activation in the peripheral blood, but that this did not result in neutrophil surface receptor (CD11b and CD62L) changes. This study also did not investigate the possible role of netosis in COVID-19 patients and the disease pathology, a concept that has been put forward in COVID-19 literature. 61,62 The putative absence of hyperinflammation in COVID-19 patients