key: cord-0919455-9phiaa4l
authors: Zheng, Jian; Wang, Yuhang; Li, Kun; Meyerholz, David K; Allamargot, Chantal; Perlman, Stanley
title: SARS-CoV-2-induced immune activation and death of monocyte-derived human macrophages and dendritic cells
date: 2020-12-05
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa753
sha: 04dbf436933eb4de4c4d46c4c1f6b794d0fbafb7
doc_id: 919455
cord_uid: 9phiaa4l

Studies of SARS-CoV-2-infected patients and experimentally infected animals indicate a critical role for augmented expression of pro-inflammatory chemokines and cytokines in severe disease. Here, we demonstrate that SARS-CoV-2 infection of human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) was abortive, but induced the production of multiple antiviral and pro-inflammatory cytokines (IFN-α, IFN-β, TNF, IL-1β, IL-6 and IL-10) and a chemokine (CXCL10). Despite the lack of efficient replication in MDMs, SARS-CoV-2 induced profound IFN-mediated cell death of host cells. Macrophage activation and death was 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 COVID-19 pathogenesis, even in the absence of productive infection.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19, is characterized by severe pneumonia, and multiple systemic complications (multi-organ damage including endothelialitis, thrombosis, myocardial damage, etc.) [1] .

Although respiratory tract epithelial cells are the primary target cells of SARS-CoV-2 [2] , other respiratory tract-resident cells, such as macrophages in alveoli and pulmonary hilum lymphoid tissue, also contain SARS-CoV-2 antigen [3] . Also, circulating monocytes migrate to multiple tissues and in the context of other infections, have been implicated in the spread of virus to distant organs and tissues [4] .

In addition, myeloid cells also orchestrate host immune responses to virus infections.

Alveolar macrophages sense and respond to microbial threats by producing bioactive molecules to eliminate pathogens and promote tissue repair [5] , while the infiltration of circulating myeloid cells, especially pathogenic inflammatory monocyte-macrophages (IMM), may result in elevated lung cytokine/ chemokine levels, vascular leakage, and even impair virus-specific T cell responses [6] . A dysregulated macrophage response may promote excessive neutrophil infiltration and tissue damage, leading to macrophage activation syndrome (MAS) and increased morbidity and mortality [7, 8] . The beneficial or deleterious role of lung-resident or infiltrating macrophages is determined largely by their secretion of cytokines and chemokines [9, 10] .

Previous studies of SARS and Middle East respiratory syndrome [11] coronavirus showed that human macrophages and dendritic cells were potent sources of pro-inflammatory inflammatory mediators [4, 6, 12] . SARS-CoV abortively infects these myeloid cells [13] , but whether MERS-CoV infection is abortive or productive is controversial [12, 14] . SARS-CoV-2-infected macrophages or dendritic cells could potentially produce multiple pro-A c c e p t e d M a n u s c r i p t 5 inflammatory cytokines and chemokines, such as interleukin (IL)-6, IL-8, tumor necrosis factor (TNF) and C-X-C motif chemokine ligand-10 (CXCL-10), contributing to local tissue injury and pathogenic systemic inflammatory responses (cytokine storm) [15] [16] [17] . Proinflammatory mediator induction (IL-6, IL-8 and type I, III interferons (IFN) among others) enhances Nuclear Factor [18] -kB-mediated activation and possibly, immune dysfunction, and results in multiple organ failure in severe COVID-19 cases [19] [20] [21] .

In this study, using human circulating monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs), we showed that SARS-CoV-2 abortively infected both types of cells in an angiotensin-converting enzyme (ACE) 2-dependent manner. Abortive infection induced expression of higher levels of cytokines and chemokines (IFN-, TNF, IL-6, IL-8, IL-10 and CXCL-10), leading to type I IFN-mediated cell death. In comparison to SARS-CoV, SARS-CoV-2 induced high levels of most cytokines and chemokines and more cell death.

Written informed consent was obtained from subjects to obtain plasma for this study.

The study was approved by the Institutional Review Board of the University of Iowa (IRB (#202003554 and #201402735).

Human peripheral blood samples were obtained from anonymous donors at the DeGowin Blood Center at the University of Iowa. Consent forms were approved by the University of Iowa's Institutional Review Board. To obtain monocytes, PBMCs were isolated by density gradient centrifugation through Ficoll-Paque PLUS density gradient media A c c e p t e d M a n u s c r i p t 6 (Cytiva) and cultured at a seeding density of 1×10 6 cells/ml in RP-10 media (RPMI-1640 medium (Invitrogen) supplemented with 10% FBS (Atlanta Biologicals), 2mM l-glutamine) and 5ng/ml M-CSF (eBioscience) (macrophages) or 100ng/ml GM-CSF (PeproTech)+50ng/ml IL-4 (PeproTech) (MDDCs) at 37°C with 5% CO2. After 96 hours, the plates were washed with Hanks's balanced salt solution devoid of divalent cations (Invitrogen) to remove nonadherent cells. Adherent cells were then trypsinized, pelleted and cultured for 10 days. 

Human PBMC, MDMs or MDDCs were infected with SARS-CoV-2 or SARS-CoV at MOI=2 in Dulbecco's modified Eagle's medium (DMEM, GIBCO, Grand Island, NY)), 37℃.

1 hour later, the culture medium was replaced with 10% FBS-DMEM with or without washing with PBS. In some experiments, virus was inactivated by UV. Inactivation was confirmed by plaque assay as described below.

Virus titers in culture medium were determined using Vero E6 cells (grown in 10% 

After fixation with 2.5% glutaraldehyde in 0.1M Na cacodylate, the cells monolayers were treated with 1% OsO4 containing 1.5% potassium ferrocyanide followed by 2.5% 

Cell lysates were collected from cells lysed in Nonidet P-40 cell lysis buffer [22] supplemented with a protease inhibitors cocktail [22] and a phosphatase inhibitor cocktail [22] . Protein samples were run on 4%-20% SDS/PAGE gels and transferred to activated 

High titer COVID-19 convalescent plasma (IC 50 =1:1480, determined by a neutralizing titer assay using a luciferase-expressing SARS-CoV-2 S protein pseudovirus system as previously described [23] ) and control plasma were collected (IRB approved protocols #202003554 and #201402735). The plasma was diluted at 1:5 (final concentration:10%) or 1:50 (final concentration: 1%) in DMEM and mixed 1:1 with SARS-CoV-2 for 1 hour at 37 o C. The virus-antibody mixtures were then adsorbed to MDMs for 1 hour at 37C.

To block viral entry, MDMs were incubated with -ACE2 monoclonal antibody (AF933, R&D) or isotype control IgG at a final concentration of 10g/ml for 1 hour prior to SARS-CoV-2 infection.

To block type I IFN signaling, -IFNAR monoclonal antibody (clone TAB-722, Creative Biolabs) or isotype control IgG was added to cells at a final concentration of 10g/ml at 12 hours after SARS-CoV-2 infection.

Total RNA was extracted from MDMs using Trizol (Invitrogen) according to the manufacturer's protocol. Following DNase treatment, 200 ng of total RNA was used as a template for first strand cDNA. The resulting cDNA was subjected to amplification of 

A Student's t test was used to analyze differences in mean values between groups. All results are expressed as mean±SEM. P-values of <0.05 were considered statistically significant. *, P < 0.05; **, P < 0.01, ***, P < 0.001. M a n u s c r i p t

To determine SARS-CoV-2 infectivity of human immune cells, peripheral blood mononuclear cells (PBMCs), and in vitro-differentiated myeloid cells (MDMs or MDDCs) from healthy donors were infected with SARS-CoV-2 at MOI=2. 24 hours later, the expression of SARS-CoV-2 nucleoprotein (N protein) in immune cell subsets was determined by flow cytometry. As shown in Figure 1A (hpi) and decreased 10 (MDMs) and 100 (MDDCs) fold over the following 96 hours ( Figure   1D and E). Notably, viral genomic RNA (ORF1ab, gRNA) levels were much lower in MDMs and MDDCs compared to both Vero E6 and Calu-3 (See Supplementary Figure 2A and consistent with previous studies [13, 24] , SARS-CoV also resulted in abortive infection of MDMs and MDDCs.

To further distinguish ACE2-mediated entry from non-specific virus uptake by MDMs, virus was incubated with media containing 10% or 1% human COVID-19 convalescent plasma (CP, original titer: 1:1480) or control plasma from a healthy donor before infection.

CP treatment decreased the percentage of SARS-CoV-2 N protein + MDMs (Figure 2A and B), and viral RNA expression ( Figure 2C ). In addition, we found no evidence of enhanced macrophage infection when cells were treated with a low dose of CP (final concentration:

1%, neutralizing titer 1:14.8) ( Figure 2C ).

Since MDMs-derived cytokines and chemokines play critical roles in limiting viral infection and in pathogenic processes, we evaluated cytokine and chemokine mRNA production by virus-infected MDMs ( Figure 3A ) and MDDCs ( Figure 3B 

To determine whether abortive infection in combination with elevated expression of proinflammatory cytokines and chemokines resulted in additional phenotype changes such as cell death, MDMs were infected and examined for ultrastructural changes by transmission electron microscopy [18] . The nucleus and cytoplasm of infected macrophages showed Figure 3C ).

Here, we demonstrate that human MDMs are abortively infected by SARS-CoV-2, in agreement with a previous report [24] . We additionally show that MDDCs do not support productive SARS-CoV-2 infection. Levels of intracellular viral RNA and protein were very low in both cell types and we detected no virus or viral replicative structures by TEM. We also show that entry is ACE2 and S protein-dependent, since anti-ACE2 antibody ( Figure 1C [25] . Of note, in disagreement with a recent report [26] , we found no evidence that T cells, B cells or NK cells ( Figure 1A) were infected by the virus.

A c c e p t e d M a n u s c r i p t 15 Remarkably, given the low expression level of viral RNA and protein, SARS-CoV-2 infection invoked potent cytokine and chemokine production by human MDMs and less by MDDCs (Figure 3 ). Chemokine and cytokine expression were not induced solely by virus binding to the ACE2 receptor because they were not induced by UV-inactivated virus ( Figure   3A ). Whether viral RNA or protein expressed during abortive replication signal through tolllike receptors (TLRs) or retinoic acid-inducible gene (RIG)-I-like receptors to activate IFNand NF-kB-dependent pathways will require further investigation. Several SARS-CoV-2 proteins have been reported to activate or counter the innate immune [18, 27] but at this point, it is not known whether these proteins, double stranded viral RNA or the intrinsic process of virus replication (e.g., membrane distortion to form sites of RNA replication [28] ) are most important in this process. Further, we assessed cytokine/chemokine RNA expression longitudinally, and found that infection of MDMs induced higher expression of IFN-, TNF, IL-6, IL-8, IL-10, CXCL-10 and similar expression of IFN- and IL-1 when compared to SARS-CoV ( Figure 3A ). Of note, neither virus was as potent inducer of proinflammatory molecule expression as H5N1, H1N1pdm, or MERS-CoV [24] . Since mortality in COVID-19 patients is associated in some cases with dysregulated and elevated pro-inflammatory molecule expression [1, 3] , this observation suggests that other cells, such as lung epithelial cells, or uninfected bystander myeloid cells are major contributors to expression of these molecules. Nonetheless, since macrophage-derived cytokines and chemokines contribute to both host defense and tissue damage, these results suggest that even abortive infection of myeloid cells could contribute to a hyperinflammatory milieu. For example, CXCL10 plasma levels are highly associated with COVID-19 severity and may predict disease progression [29, 30] . TNF levels were found to correlate with the abundance of pro-inflammatory CXCL10 + CCL2 + macrophages in severe COVID-19 [31] , which might exacerbate inflammation in a positive feedback loop.

A c c e p t e d M a n u s c r i p t 16 IL-6 and IL-1 expressions were also shown to correlate with disease severity [16, 17, 30] . Although blocking IL-6 therapy was postulated to improve clinical outcome [32] [33] [34] , recent controlled trials did not support its efficacy in treatment of COVID-19 patients [35] .

IL-1 signaling blockade using Anakinra, was also found to provide benefit in some COVID-19 cases [36] [37] [38] [39] . However, confirmation of IL-1 blockade efficacy will require larger controlled trials.

We also demonstrate that human MDMs-derived IFN- participated in cell death in an autocrine or paracrine manner after SARS-CoV-2 infection ( Figure 4C ). Similarly, IFN- treatment of human alveolospheres [2] cells resulted in cell death [40] . Induction of caspase 3 suggests that MDMs underwent apoptosis. However, this conclusion needs further validation because caspase-3 is also associated with necrotic/pyroptotic cell death in a few settings [41] . These results are in contrast to studies of apoptosis in MDMs infected with a common cold human coronavirus (HCoV-229E), which is dependent on viral replication, and not TNF-related apoptosis-inducing ligand (TRAIL), FasL, TNF or caspase activity. HCoV-229E infection of PBMC-derived myeloid cells shows preferential killing of dendritic cells but not macrophages [42] . The lower expression of pro-inflammatory cytokines in SARS-CoV-2-infected MDDCs, compared to that of MDMs, might contribute to their lower rates of death ( Figure S4C In summary, these results illustrate some of the complexities of the pathogenesis of infections caused by SARS-CoV-2 and other human respiratory CoV. Even when the infection is abortive, the consequence may be expression of pro-inflammatory mediators that contribute to pathogenic outcomes. These results reinforce the need for anti-viral agents since infectious but not UV-treated virus causes macrophage activation and cell death and for agents that dampen the host immune response, diminishing the exuberant inflammatory response, to which dying macrophages contribute. M a n u s c r i p t 

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The authors have no competing interests, financial or non-financial.

We thank Dr. Thomas Gallagher for critical review and discussion of the manuscript. This