key: cord-0741010-vqrkpqan
authors: Zhang, Dan; Guo, Rui; Lei, Lei; Liu, Hongjuan; Wang, Yawen; Wang, Yili; Qian, Hongbo; Dai, Tongxin; Zhang, Tianxiao; Lai, Yanjun; Wang, Jingya; Liu, Zhiqiang; Chen, Tianyan; He, Aili; O'Dwyer, Michael; Hu, Jinsong
title: COVID‐19 infection induces readily detectable morphologic and inflammation‐related phenotypic changes in peripheral blood monocytes
date: 2020-10-11
journal: J Leukoc Biol
DOI: 10.1002/jlb.4hi0720-470r
sha: 48b0269293984e8256373cc45afb80f77da9007a
doc_id: 741010
cord_uid: vqrkpqan

Excessive monocyte/macrophage activation with the development of a cytokine storm and subsequent acute lung injury, leading to acute respiratory distress syndrome (ARDS), is a feared consequence of infection with COVID‐19. The ability to recognize and potentially intervene early in those patients at greatest risk of developing this complication could be of great clinical utility. In this study, we performed flow cytometric analysis of peripheral blood samples from 34 COVID‐19 patients in early 2020 in an attempt to identify factors that could help predict the severity of disease and patient outcome. Although we did not detect significant differences in the number of monocytes between patients with COVID‐19 and normal healthy individuals, we did identify significant morphologic and functional differences, which are more pronounced in patients requiring prolonged hospitalization and intensive care unit (ICU) admission. Patients with COVID‐19 have larger than normal monocytes, easily identified on forward scatter (FSC), side scatter analysis by routine flow cytometry, with the presence of a distinct population of monocytes with high FSC (FSC‐high). On more detailed analysis, these CD14(+)CD16(+), FSC‐high monocytes show features of mixed M1/M2 macrophage polarization with higher expression of CD80(+) and CD206(+) compared with the residual FSC‐low monocytes and secretion of higher levels of IL‐6, IL‐10, and TNF‐α, when compared with the normal controls. In conclusion, the detection and serial monitoring of this subset of inflammatory monocytes using flow cytometry could be of great help in guiding the prognostication and treatment of patients with COVID‐19 and merits further evaluation.

and CD206 + compared with the residual FSC-low monocytes and secretion of higher levels of IL-6, IL-10, and TNF-, when compared with the normal controls. In conclusion, the detection and serial monitoring of this subset of inflammatory monocytes using flow cytometry could be of great help in guiding the prognostication and treatment of patients with COVID-19 and merits further evaluation. 

In this study, 34 cases of COVID-19 patients (see Supplemental Table 1 for supporting information) in the period from 18 February 2020 to 24

April 2020 were studied from Xi'an No. 

Peripheral blood samples from the first 2 days of COVID-19 patients on admission were collected in K 3 -EDTA-containing tubes for laboratory assessments according to the doctor's instruction. The blood samples were processed within 6 h after collection. antibody and the secondary antibody as described above. Samples stained with the secondary antibody alone were used as a negative control.

A full list of antibodies used in this study is included as Supplemental 

Pseudovirus 

The peripheral blood smears were made and stained with Wright's stain using a SP1000i (Sysmex, Kobe, Japan) automated smear-makerstainer system. Examination of stained blood smears was semiautomated with a digital cell morphology system CellaVision DM96

(CellaVision AB, Lund, Sweden), following the manufacturer's instructions.

To investigate the potential effects of FSC-low monocytes and FSC- Cox models were also fitted to adjust the potential effects of age and gender.

Statistics values are presented as means ± SD. Data were analyzed by using GraphPad Prism version 6.04 (GraphPad Software, San Diego, CA, USA). An unpaired t-test was used to compare the mean between 2 independent groups. A one-way ANOVA was used to compare the 

To better understand the impact on the immune response of SARS-CoV-2/COVID-19, we originally sought to investigate changes of immune cells in the peripheral blood in COVID-19 patients using flow cytometry. Unexpectedly, in the tested COVID-19 patients, we found the presence of a specific population (FSC-high) right next to the population of monocytes, when using the FSC and SSC parameters (Fig. 1A) .

In contrast, this population is virtually absent in healthy donors ( Fig. 1B and C). To validate the nature of the FSC-high population, we further performed Wright-Giemsa staining on blood smears. As shown in Fig. 1D , we confirmed the presence of an increased number of larger, atypical, vacuolated monocytes, not normally seen in the peripheral blood of healthy individuals. Table 1 shows that while the percentages of monocytes in COVID-19 patients are still in the normal range from 2% to 8% in total WBC count, there was an increased proportion of larger and atypical monocytes.

Next, we analyzed the expression of the phenotypic markers on both FSC-low and FSC-high populations using flow cytometry. As shown in Figs Fig. 2D and E, the expression levels of M1-specific cytokines IL-6 and TNF-in monocytes from COVID-19 patients were significantly higher than that of healthy donors. Similarly, M2-specific cytokine IL-10 was found to be highly expressed by the monocytes from COVID-19

patients, but no statistically significant difference was found. Collectively, these findings may suggest that the peripheral monocytes in COVID-19 patients are inflammatory, and start to differentiate into macrophages. (Fig. 4A) . Moreover, we also found that the monocytes in the peripheral blood of healthy donors and COVID-19 patients are ACE2 positive (Fig. 4B-D) . However, the levels of ACE2 on the peripheral monocytes from COVID-19 patients are significantly lower than healthy donors (Fig. 4E) . Importantly, by using a pseudovirus system expressing viral spike protein, we further demonstrated that SARS-CoV-2 could direct infect these ACE2-positive monocyte cell lines and primary peripheral monocytes (Fig. 5 ).

As shown in Fig. 6 , significant differences in time to discharge from hospital were identified between COVID-19 patients with low and high levels of FSC-low monocytes% ( 2 = 9.87, P = 0.0017; Fig. 6A ) and FSC-low/FSC-high monocytes% ( 2 = 12.08, P = 0.0005; Fig. 6B ).

The differences were still significant after being adjusted for age and 

Of the 34 COVID-19 patients included in this study, 5 were admitted to the intensive care unit (ICU). Focusing on the total number of monocytes and the percentage of FSC-low and FSC-high, we observed a noticeable difference from the non-ICU patients. As shown in Fig. 6C,   3 representative ICU patients have lower number of total monocytes; of these, the FSC-high subsets are the majority. Moreover, the average length of hospital stay of the 5 ICU patients is 40.60 ± 7.89, significantly longer than the average 15.68 ± 2.67 days of the non-ICU patients (P < 0.0001).

Here, we report our findings in 34 patients with confirmed COVID- Chinese group also succeeded in detecting the virus in the serum, 21 suggesting that with the use of more sensitive assays, viremia may be more common than previously thought. Notably, the detectable serum SARS-CoV-2 viral load was found to correlate with drastically increased IL-6 levels in COVID-19 patients tightly. 22 In peripheral blood, there is a unique monocyte subpopulation, which are highly inflammatory and enormous chemokine producers implicated in cytokine storm. 28 These data strongly suggest that targeting migration and differentiation of monocyte/macrophage, or selective cytokine blockade might improve the treatment outcomes of COVID-19. 29, 30 Indeed, Tocilizumab, an IL-6 receptor antibody approved for treatment of rheumatoid arthritis as well as CAR-T-associated cytokine release syndrome, has been conducted in the treatment of severe COVID-19 patients with promising results. [31] [32] [33] Other treatments for cytokine storm, such as the IL-1 receptor antagonist Anakinra and the JAK inhibitors, may also prove to be useful for treating COVID-19. 34, 35 Given the central role that monocytes appear to play in COVID-19

infection, it is important to recognize the limited information provided by most routine automated blood analyzers. Indeed, the contribution of monocytes to the patient's pathology may be overlooked as patients with severe disease can be monocytopenic. 6 This may well reflect the migration of the inflammatory monocytes/macrophages into the lungs and other affected organs. Although morphologic examination of peripheral blood films revealed somewhat larger, atypical, vacuolated monocytes, these findings are not very specific. We have shown that In conclusion, in patients with severe COVID-19 infection, monocyte activation and the associated inflammatory response is associated with characteristic changes that can be rapidly identified by a simple blood-based flow cytometry-based assay, and serially monitored.

Although we acknowledge the limitations of our study, given the small sample size, we feel nevertheless that our findings could be of great help in guiding prognostication and treatment of patients with COVID-19 and merit further evaluation and confirmation in future studies.

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The authors thank Dr. Peng Li for help with RT-PCR analysis of SARS-CoV-2 in the blood samples from COVID-19 patients. This study was 

The authors declare no conflicts of interest.

https://orcid.org/0000-0002-6173-7140 Jinsong Hu https://orcid.org/0000-0003-0565-4567