Effect of ORF7 of SARS-CoV-2 on the Chemotaxis of Monocytes and Neutrophils In Vitro

Abstract

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most significant public health threat worldwide. Patients with severe COVID-19 usually have pneumonia concomitant with local inflammation and sometimes a cytokine storm. Specific components of the SARS-CoV-2 virus trigger lung inflammation, and recruitment of immune cells to the lungs exacerbates this process, although much remains unknown about the pathogenesis of COVID-19. Our study of lung type II pneumocyte cells (A549) demonstrated that ORF7, an open reading frame (ORF) in the genome of SARS-CoV-2, induced the production of CCL2, a chemokine that promotes the chemotaxis of monocytes, and decreased the expression of IL-8, a chemokine that recruits neutrophils. A549 cells also had an increased level of IL-6. The results of our chemotaxis Transwell assay suggested that ORF7 augmented monocyte infiltration and reduced the number of neutrophils. We conclude that the ORF7 of SARS-CoV-2 may have specific effects on the immunological changes in tissues after infection. These results suggest that the functions of other ORFs of SARS-CoV-2 should also be comprehensively examined.

Figure 1

Expression of ACE2 and distribution of ORF7 in A549 cells. (a) Expression of ACE2 in A549 cells (qRT-PCR). (b) Expression of ACE2 in A549 cells (Western blotting). (c) FLAG-ORF7 expression in A549-ORF7 cells (Western blotting). (d) Cellular distribution of ORF7 in A549-ORF7 cells (immunofluorescence microscopy).

Figure 2

Effect of ORF7 on the expression of cytokines and chemokines in A549 cells. (a) Relative to control cells, A549-ORF7 cells had increased expression of IL-6, CCL2, and IFN-β (all P < 0.01), decreased expression of IL-1α, IL-8, and TNF-α (all P < 0.01), but similar expression of IL-1β and IFN-α (both P > 0.05). CCL7 and IL-10 were undetectable (qRT-PCR). (b) Activation of innate immunity by SeV/VSV infection increased the expression of CCL2 and decreased the expression of IL-8 in A549-ORF7 cells compared to control cells (both P < 0.05; qRT-PCR). (c) A549-ORF7 cells had increased levels of CCL2 and decreased levels of IL-8 relative to control cells (both P < 0.01; ELISA).

Figure 3

Effect of ORF7 on chemotaxis of neutrophils and monocytes. (a–d) Neutrophils were identified as CD11b+ cells and monocytes as CD14+ positive cells (flow cytometry). (g–n) Chemotaxis of monocytes and neutrophils (upper chamber) in response to A549-ORF cells (lower chamber) was examined by staining (Transwell assay). (e, f) Quantitation of chemotaxis results showing increased monocyte chemotaxis and decreased neutrophil chemotaxis (both P < 0.01).