Severe Acute Respiratory Syndrome Coronavirus 2-Induced Immune Activation and Death of Monocyte-Derived Human Macrophages and Dendritic Cells

Abstract

Studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients and experimentally infected animals indicate a critical role for augmented expression of proinflammatory chemokines and cytokines in severe disease. Here, we demonstrate that SARS-CoV-2 infection of human monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells was abortive, but induced the production of multiple antiviral and proinflammatory cytokines (interferon-α, interferon-β, tumor necrosis factor, and interleukins 1β, 6, and 10) and a chemokine (CXCL10). Despite the lack of efficient replication in MDMs, SARS-CoV-2 induced profound interferon-mediated cell death of host cells. Macrophage activation and death were not enhanced by exposure to low levels of convalescent plasma, suggesting that antibody-dependent enhancement of infection does not contribute to cell death. Together, these results indicate that infection of macrophages and dendritic cells potentially plays a major role in coronavirus disease 2019 pathogenesis, even in the absence of productive infection.

Keywords: COVID-19; IFN; SARS-CoV-2; cell death; macrophage.

Graphical abstract

Figure 1.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) abortive infection of human monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MDDCs). A and B, Human peripheral blood mononuclear cells (PBMCs), MDMs, and MDDCs were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 2. Infected immune cell subsets were identified by flow cytometry using an anti-SARS-CoV-2 N protein antibody. C, SARS-CoV-2 N protein expression in SARS-CoV-2–infected MDMs treated with an anti–human angiotensin-converting enzyme 2 antibody or its isotype control. D–G, MDMs (D, F, and G) or MDDCs (E) were infected with SARS-CoV-2 or severe acute respiratory syndrome coronavirus at MOI of 2, followed by washing with phosphate-buffered saline (G) or not (D–F), before addition of 10% fetal bovine serum–Dulbecco’s modified Eagle’s medium. At indicated time points, culture media were collected and cells were treated with Trizol for RNA extraction. SARS-CoV-2 N RNA expression was determined by quantitative polymerase chain reaction (D, E, and G). Infectious viral particles in culture medium were detected by plaque assays (F and G). B and D–G, Data are shown as mean ± standard error of the mean and are representative of 3 independent experiments (n = 4–8). *P < .05; **P < .01. See also Supplementary Figures 1 and 2. Abbreviations: ACE2, angiotensin-converting enzyme 2; APC, antigen-presenting cell; Ctrl, control; FBS-DMEM, fetal bovine serum–Dulbecco’s modified Eagle’s medium; FSC-A, forward scatter area; HPRT, hypoxanthine-guanine phosphoribosyltransferase; IC, isotype control antibody; IgG, immunoglobulin G; LOD, limit of detection; MDDC, monocyte-derived dendritic cell; MDM, monocyte-derived macrophage; PBS, phosphate-buffered saline; PFU, plaque-forming units; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 2.

Coronavirus disease 2019 convalescent plasma (CP) blocks the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human monocyte-derived macrophages (MDMs). SARS-CoV-2 was incubated with CP or control plasma as described in the Materials and Methods before infecting human MDMs. A and B, N protein expression in SARS-CoV-2–infected MDMs treated with CP or control plasma was determined by flow cytometry at indicated time points. C, SARS-CoV-2 N RNA expression in MDMs was detected by quantitative polymerase chain reaction. B and C, Data are shown as mean ± standard error of the mean and are representative of 3 independent experiments (n = 4–6). *P < .05; **P < .01; ***P < .001. Abbreviations: APC, antigen-presenting cell; CP, convalescent plasma; Ctrl, control; HPRT, hypoxanthine-guanine phosphoribosyltransferse; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 3.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–infected human monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MDDCs) produce multiple cytokines/chemokines. MDMs (A) or MDDCs (B) were exposed to severe acute respiratory syndrome coronavirus (SARS-CoV) or SARS-CoV-2 (with or without convalescent plasma treatment), or ultraviolet-inactivated SARS-CoV-2. RNA was extracted at the indicated time points postinfection. Cytokine/chemokine levels were determined by quantitative polymerase chain reaction as described in the Materials and Methods. Data are shown as mean ± standard error of the mean and are representative of 3 independent experiments (n = 4–6). *Difference between the SARS-CoV-2 and SARS-CoV groups (P < .05). Abbreviations: CP, convalescent plasma; HPRT, hypoxanthine-guanine phosphoribosyltransferase; IFN, interferon; IL, interleukin; MDDC, monocyte-derived dendritic cell; MDM, monocyte-derived macrophage; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF, tumor necrosis factor; UV, ultraviolet.

Figure 4.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of human monocyte-derived macrophages (MDMs) results in interferon (IFN)–dependent cell death. A, Representative transmission electron micrographs of SARS-CoV-2–infected human macrophages from mock and SARS-CoV-2 groups. N: nucleus, C: cytosol; bar = 1 µm. Images shown are representative of 3 independent donors. Insets show progressive loss of mitochondrial integrity as infection proceeds. B, SARS-CoV-2–infected MDMs were fixed at 96 hours postinfection (hpi) and prepared for confocal microscopy as described in the Materials and Methods. Some wells were treated with anti-alpha/beta interferon antibody (α-IFNAR) antibody at 12 hpi. The expression and co-localization of SARS-CoV-2 N protein and caspase-3 are shown. C and D, Virus was incubated with convalescent plasma (final concentration: 1%) or cultures were treated with α-IFNAR antibody at 12 hpi. Isotype matched antibodies were included as controls. Dead MDMs were identified at 96 hpi using a LIVE/DEAD cell viability assay kit with flow cytometry as shown in Supplementary Figure 3A (C). SARS-CoV-2 N protein RNA expression in MDMs was detected by quantitative polymerase chain reaction at the indicated time points postinfection (D). Data are shown as mean ± standard error of the mean and are representative of 3 independent experiments (n = 5). *P < .05; ***P < .001. See also Supplementary Figure 3. Abbreviations: α-IFNAR, anti alpha/beta interferon receptor antibody; CP, convalescent plasma; Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindole; hpi, hours postinfection; HPRT, hypoxanthine-guanine phosphotransferase; IgG, immunoglobulin G; MDM, monocyte-derived macrophage; n/a, not applicable; NP, nucleocapsid protein; n.s., not significant; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.