key: cord-0863930-v3higj6g
authors: Labzin, Larisa I; Chew, Keng Yih; Wang, Xiaohui; Esposito, Tyron; Stocks, Claudia J; Rae, James; Yordanov, Teodor; Holley, Caroline L; Emming, Stefan; Fritzlar, Svenja; Mordant, Francesca L.; Steinfort, Daniel P.; Subbarao, Kanta; Lagendijk, Anne K; Parton, Robert; Short, Kirsty R.; Londrigan, Sarah L.; Schroder, Kate
title: ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication
date: 2022-03-22
journal: bioRxiv
DOI: 10.1101/2022.03.22.485248
sha: 55bd92607f0fabc1a55af3b0fde894f54a5d5c89
doc_id: 863930
cord_uid: v3higj6g

Macrophages are a major source of pro-inflammatory cytokines in COVID-19. How macrophages sense the causative virus, SARS-CoV-2, to drive cytokine release is, however, unclear. Here, we show that human macrophages do not directly sense and respond to infectious SARS-CoV-2 virions because they lack sufficient ACE2 expression to support virus entry and replication. Over-expression of ACE2 in human macrophages permits SARS-CoV-2 entry and early-stage replication and facilitates macrophage pro-inflammatory and anti-viral responses. ACE2 over-expression does not, however, permit the release of newly synthesised virions from SARS-CoV-2-infected macrophages, consistent with abortive replication. Release of new, infectious SARS-CoV-2 virions from ACE2 over-expressing macrophages only occurred if anti-viral mediator induction was also blocked, indicating that macrophages restrict SARS-CoV-2 infection at two stages of the viral life cycle. These findings resolve the current controversy over macrophage-SARS-CoV-2 interactions and identify a signalling circuit that directly links macrophage recognition of SARS-CoV-2 to restriction of viral replication. One sentence summary ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication.

, despite reports indicating that these cytokines circulate at 125 high levels in vivo during SARS-CoV-2 infection (24, 25) . By contrast, HMDM showed a 126 robust secretory response to synthetic viral mimetics such as the toll-like receptor (TLR) 7/8 127 ligand, R848 (TNF, IL-6) and the melanoma differentiation-associated protein 5 (MDA5) 128 ligand, transfected poly I:C (pIC) (CXCL10) (Figure 1A) . mRNA analyses revealed that 129 SARS-CoV-2 did not upregulate HMDM expression of Ifnb1, Ifnl1, Cxcl10, Il6, Tnf or Il1b 130 at 2h or 24h post-infection (p.i.), at either low (0.5) or high (5) MOI, while these genes were 131 robustly induced by R848 and pIC ( Figure 1B) . Thus, HMDM do not respond to SARS-132

CoV-2 exposure in vitro, even at the high MOI of 5. including during entry. As ACE2 is the primary receptor facilitating SARS-CoV-2 176 attachment to host cells, we next investigated whether ACE2 expression could be a 177 determining factor in macrophage resistance to SARS-CoV-2 infection. We assessed ACE2 178 mRNA and protein levels by qPCR and immunoblot, and observed low Ace2 mRNA 179 expression, and no ACE2 protein expression in HMDM isolated from 4 individual donors 180 ( Figure 3A, B) , even when HMDM were treated with IFN ( Figure 3B) . Similarly, ACE2 181 protein was not detected in airway macrophages isolated from BAL macrophages of 3 182 individual donors ( Figure 3C) . We used lentiviral transduction to overexpress either ACE2 183 (untagged) or a control protein (mScarlet, mSc) in the THP-1 monocyte cell line and 184 differentiated these to macrophage-like cells with phorbol-myristate-acetate (PMA). We 185 confirmed that ACE2 overexpression in THP-1 cells resulted in readily detectable ACE2 186 mRNA and protein ( Figure 3A, 3B) . The surface protease TMPRSS2, which is required for S 187 protein cleavage, was also readily detected at the protein level in HMDM and THP-1-ACE2 188 cells ( Figure 3B) . 189 190 We next explored whether insufficient ACE2 expression is the primary block to SARS-CoV-191 2 replication in macrophages. We thus challenged THP-1-ACE2 with SARS-CoV-2 (MOI 192 0.5 and 5) and compared viral replication and release to THP-1-mSc and Calu3 control cells. 193

There was a significant increase in cell-associated viral RNA in THP-1-ACE2 cells at 24h 194 p.i. at both MOIs, before plateauing from 24 to 72h p.i., indicating cell susceptibility and 195 early-stage viral RNA replication (Figure 3D, 3E) . By contrast, viral RNA levels did not 196 increase in the THP-1-mSc control cells at any timepoint ( Figure 3D, 3E) , similar to 197 observations in HMDM and BAL macrophages ( Figure 2B, 2E, 2F) . As expected, in Calu3 198 cells which support viral replication and virion release (28), SARS-CoV-2 RNA levels 199 continued to increase over the time course (Figure 3D, 3E) . 200

To determine whether the increased cell-associated viral RNA in THP-1-ACE2 cells was 202 coupled to the release of infectious virions, we performed plaque assays on cell-free 203 supernatants to measure release of infectious viral particles, represented by plaque forming 204 units (PFU). Despite increased cell-associated viral RNA, we observed a decrease in 205 infectious viral particles in THP-1-ACE2 cells at 48 or 72h p.i. compared with input virus 206 levels at 0h (Figures 3F, 3G) . Similarly, we observed a decrease in infectious viral particles 207 in THP-1-mSC cell-free supernatants (Figure 3F, 3G) . In contrast, Calu3 cells were 208 productively infected at the low MOI (MOI 0.5) over the same time course, indicated by a 209 significant increase in infectious viral particles present in cell-free supernatants between 0 210 and 72h p.i. (Figure 3F ), though at high MOI (MOI 5) detection of new infectious virions 211 peaked at 24h ( Figure 3G ). This suggests that while some new virions are produced in THP-212 1-ACE2 cells, a secondary block may serve to limit virus production in these cells. Together, 213 these data suggest that ACE2 expression is necessary for SARS-CoV-2 entry and new viral 214 RNA synthesis in macrophages but is not sufficient to support productive viral replication. 215

Thus, macrophages have two blocks to productive SARS-CoV-2 replicationlack of ACE2 216 expression, plus an additional mechanism downstream of viral entry and replication that 217 limits release of infectious virions. Additional SARS-CoV-2 entry receptors beyond ACE2 are reported, such as the C-type 222 lectin receptors (CLRs) (23). As our assays to measure viral protein and RNA were unable to 223 distinguish whether virus is intracellular or extracellular ( Figure 2B ,2D), we next determined 224 whether SARS-CoV-2 can still bind and enter HMDM, despite lack of ACE2 expression. We 225 used transmission electron microscopy to determine the sub-cellular location of incoming 226 virions. To capture these events, we used a high MOI of 20. HMDM ( Figure 4A, A') and 227 THP-1-ACE2 ( Figure 4B) showed internalised, intact virions within the phagosomal system 228 after 1h infection. In THP-1-ACE2 cells but not HMDM, we also observed virions bound and 229 potentially starting to fuse at the plasma membrane ( Figure 4C A key question in SARS-CoV-2 pathogenesis is which host cells sense SARS-CoV-2 to 304 trigger inflammatory cytokines and anti-viral mediator release. Given that macrophages are 305 primary candidates for sensing and responding to SARS-CoV-2 (7), a second, unresolved 306 question is whether SARS-CoV-2 can infect and productively replicate in human 307 macrophages. This study elucidates the requirements for macrophages to sense and respond 308 to SARS-CoV-2. 309

We demonstrate that primary HMDM do not respond to SARS-CoV-2 challenge, despite 311 taking up virus through phagocytosis. We found that both BAL macrophages and HMDM do 312 not express ACE2 protein, rendering them resistant to early-stage SARS-CoV-2 replication. 313

In turn, ectopic expression of ACE2 rendered macrophages susceptible to SARS-CoV-2 entry 314 and replication and able to mount ensuing robust pro-inflammatory and anti-viral responses. 315

Intriguingly, ectopic ACE2 expression was not sufficient for macrophages to efficiently 316 release new virions, and thereby increase viral titres. In this context, productive virion release 317 can be rescued by disabling macrophage interferon production and signalling. Thus Nevertheless, our viral stocks did not trigger cytokine release from human primary 356 macrophages or THP-1 cells, unless ACE2 was ectopically expressed to allow SARS-CoV-2 357 entry and early-stage replication (Figure 1A, 1B, Figure 5A ). This suggests that human 358 macrophages do not sense infectious SARS-CoV-2 virions unless they are instructed to 359 upregulate ACE2 expression. We anticipate that this observation will hold true for ACE2-360 dependent SARS-CoV-2 variants, including Omicron (B.1.1.529 and BA lineages), and 361 indeed any beta-coronaviruses that utilize ACE2. Consistent with this, SARS-CoV, which 362 also requires ACE2 for entry, does not trigger macrophage cytokine responses (38). 363 364 We also observe that SARS-CoV-2 does not replicate in human macrophages and does not 365 trigger productive virion release. This is in line with multiple reports of abortive SARS-CoV-366 2 infection of macrophages in vitro (8, 9, 20, 21) . While these studies observe that 367 macrophage infection results in decreasing viral RNA levels (8, 9, 20) (Figure 3F, 3G, 5A, 6D) . This mechanism appeared to block virion 389 production at a stage beyond genome replication (as measured by viral RNA, Figures 3D-E) In the inflamed, SARS-CoV-2-infected lung, we expect that macrophages will sense cell 409 

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A: HMDM and THP-1 cells were analysed by qPCR for ACE2 mRNA expression, 905 with each data point showing an independent donor or experiment (n=3). B: HMDM 906 were stimulated with IFN (10 ng/ml) for 6 h and protein extracts were analysed by 907 immunoblot, alongside extracts from THP-1 cells (WT, THP-1-ACE2, THP-1-mSc). 908 C: BAL macrophages from 3 donors were adhered overnight and lysed. Expression of 909 ACE2 in BAL macrophages was analysed by immunoblot, relative to a loading 910 control (Calnexin). Lysate from A549-cells overexpressing ACE2 were used as a 911 positive control. D-E: Cells were infected with SARS-CoV-2 at MOI 0.5 or MOI 5.