Frontline Science: COVID-19 infection induces readily detectable morphologic and inflammation-related phenotypic changes in peripheral blood monocytes

Abstract

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.

Keywords: COVID-19; flow cytometry; forward scatter; monocyte; morphology.

Graphical abstract

FIGURE 1

Flow cytometry analysis of the peripheral blood identified a FSC-high population in COVID-19 patients. (A and B) Representative pictures of flow cytometry FSC/SSC parameters showing a specific population in the peripheral blood of COVID-19 patients (A) and healthy donors (B). (C) Statistical analysis of the percentage of FSC-low and FSC-high population in healthy controls and COVID-19 patients (HD n = 16, patient n = 34, ****P < 0.0001). (D) Representative pictures of peripheral blood films showing the FSC-high monocytes in COVID-19 patients (×1,000 magnification)

FIGURE 2

Flow cytometry analysis of the expression of the macrophage markers on the monocytes in peripheral blood of COVID-19 patients. (A) Representative flow cytometry results show the expression of monocyte/macrophage-related markers in the FSC-low population in healthy donors. (B) Representative flow cytometry results show the expression of monocyte/macrophage related markers in FSC-low and FSC-high population in COVID-19 patients. (C) Statistical analysis of the expression of the macrophage markers on the monocytes in healthy donors and COVID-19 patients (HD n = 8, patient n = 14, *P < 0.05, **P < 0.01). (D) Representative intracellular staining flow cytometry results show the expression of M1/M2-associated cytokines. (E) Statistical analysis of the expression levels of M1/M2-associated cytokines in peripheral blood monocytes from healthy donors and COVID-19 patients (HD n = 6, patient n = 15, *P < 0.05, **P < 0.01)

FIGURE 3

The percentage of the 3 kinds of monocyte subsets (classical/intermediate/nonclassical) in FSC-low and FSC-high populations. (A–C) Representative flow cytometry results show the percentage of the classical/intermediate/nonclassical monocytes in FSC-low of healthy controls (A), FSC-low of COVID-19 patients (B), and FSC-high of COVID-19 patients (C). (D) Statistic analysis shows that COVID-19 patients had a reduction in classical monocytes with a higher proportion of intermediate and nonclassical monocytes, compared with the healthy controls (HD n = 9, patient n = 21, *P < 0.05)

FIGURE 4

SARS-CoV-2 receptor ACE2 is positively expressed on the surface of the monocytes. (A) Representative flow cytometry results show the expression of ACE2 on monocyte/macrophage cell lines (n = 3). (B and C) Representative flow cytometry results show the expression of ACE2 on monocytes in peripheral blood of the healthy controls (B) and COVID-19 patients (C). (D and E) Statistical analysis shows that patients had lower expression of ACE2 on their monocytes, compared with the healthy donors (HD n = 3, patient n = 13, *P < 0.05, **P < 0.01, ****P < 0.0001)

FIGURE 5

FNV-SARS-CoV-2-S pseudovirus infects monocytes. (A) Representative flow cytometry results show the infection percentage of the different monocyte and macrophage cell lines. The infected GFP-positive cells were detected at the timepoints of 8 (for U937 and THP-1 cells) or 16 (for RAW264.7) hours after the pseudovirus infection (n = 3). (B) Representative flow cytometry results show the infection of pseudovirus to cultured primary peripheral monocytes. Sixteen hours after the infection, the PBMCs were stained with anti-CD14 to gate on monocyte (n = 3). (C) Statistical analysis of the transduction efficiency of FNV-SARS-CoV-2-S pseudovirus in different monocytes

FIGURE 6

Kaplan–Meier (KM) survival curves obtained from time to discharge from the hospital of the COVID-19 patients stratified by the amounts of FSC-low monocytes and ratio of FSC-low/FSC-high monocytes. (A) KM survival curves stratified by the amounts of FSC-low monocytes. (B) KM survival curves stratified by the ratio of FSC-low/FSC-high monocytes. (C) The typical FSC/SSC images of the 3 ICU COVID-19 patients