COVID-19 infection induces readily detectable morphological and inflammation-related phenotypic changes in peripheral blood monocytes, the severity of which correlate with patient outcome Dan Zhang 1*, Rui Guo 2*, Lei Lei 1*, Hongjuan Liu 3, Yawen Wang 4, Yili Wang 5, Hongbo Qian 2, Tongxin Dai 2, Tianxiao Zhang 6, Yanjun Lai 7, Jingya Wang 8, Zhiqiang Liu 8, Tianyan Chen 9, Aili He 10#, Michael O’Dwyer 11,12#, Jinsong H
Abstract Background: 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. Methods:We performed detailed flow cytometric analysis of peripheral blood samples from 28 COVID-19 patients treated at Xian No.8 Hospital and the First Affiliated Hospital of Xian Jiaotong University in early 2020 in an attempt to identify factors that could help predict severity of disease and patient outcome. Findings: While we did not detect significant differences in the number of monocytes between patients with COVID-19 and normal healthy individuals,we did identify significant morphological and functional differences, which are more pronounced in patients requiring prolonged hospitalization and ICU admission. Patients with COVID-19 have larger than normal monocytes, easily identified on forward scatter, side scatter analysis by routine flow cytometry,with the presence of a distinct population of monocytes with high forward scatter (FSC-high). On more detailed analysis, these FSC-high monocytes are CD11b+, CD14+, CD16+, CD68+, CD80+,CD163+, CD206+ and secrete IL-6, IL-10 and TNF-alpha, consistent with an inflammatory phenotype. Conclusions: 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.
"The FSC-high population in COVID-19 expresses macrophage markers Next, we analyzed the expression of the phenotypic markers on the FSC-high population using flow cytometry. As shown in Figure 2A-B, these cells are strongly positive for CD14 and CD16, suggesting that they belong to the monocyte lineage. More importantly, macrophage markers CD11b, CD68, CD80, CD163, CD206 are all expressed in this population. Since CD80 is considered a marker typical of M1 polarization, and CD163 and CD206 are considered to be typical of M2 polarization, it suggests that the FSC-high population contains both M1 and M2 macrophages. To further confirm this suspicion, we performed intracellular staining to validate the expression of M1/M2-specific cytokines in the FSC-high cells. As shown in Figure 2C-D, M1-specific cytokines IL-6 and TNF-α were found to be secreted, while on the other hand, the M2-specific cytokine IL-10 was also expressed by cells in the FSC-high population. Collectively, these findings strongly support that the FSC-high population in COVID-19 are composed of macrophages that have a mixed M1 and M2 polarization..."....SARS-CoV and SARS-CoV-2 receptor ACE2 is strongly expressed in monocytes ACE2 has been shown to be the entry receptor for both the SARS-CoV and SARS-CoV-2.7-10Speculating that direct viral infection could trigger the observed changes in monocytes, we further investigated whether monocytes express ACE2 and therefore could be infected by the SARS-CoV-2. By performing flow cytometry staining ACE2 on human monocytic cell lines THP-1 and U939, as well as murine macrophage cell line RAW264.7, we demonstrated that all these monocyte/macrophage cells are ACE2 positive (Figure 4A). Moreover, we confirmed that the monocytes in the peripheral blood of the healthy donors and COVID-19 patients are positive (Figure 4B-D). More importantly, we found that the expression level of ACE2 on the monocytes/macrophages in COVID-19 patients is significantly lower than healthy donors (Figure 4E)..... Inflammatory cytokines such as IFN-α, GM-CSF, IL-6 or TNF-α can also stimulate the differentiation of monocytes to macrophages. We have found that treatment high doses of IL-6 or TNF-α are capable of inducing macrophage differentiation in normal peripheral blood samples within 2 days, whereas GM-CSF plus IFN-α requires 5-7 days (data not shown). ...This may well reflect migration of the inflammatory monocyte/macrophages All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.8 into the lungs and other affected organs. While morphological examination of peripheral blood films revealed somewhat larger, atypical, vacuolated monocytes, these findings are not very specific. We have shown that simple assessment of FSC by flow cytometry in the context of COVID-19 infection can rapidly identify those patients with an increasing proportion of large, activated, IL-6 and TNF secreting monocytes, who have severe disease and are at greatest risk of ICU admission. In contrast, patients with a high proportion of normal monocytes have better prognosis with earlier recovery and discharge from hospital. These findings appear to be relatively specific for COVID-19 as we have not seen a similar pattern in patients with other viral illnesses, such as H1N1 influenza, HIV, or Hantaanvirus. 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. While 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."
https://www.medrxiv.org/content/10.1101/2020.03.24.20042655v1.full.pdf