Activated lung interstitial macrophages are a prominent site of SARS-CoV-2 infection and focus of inflammation

A group from Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA, etc. has identified activated interstitial macrophages as a prominent site of SARS-CoV-2 viral takeover and focus of inflammation.

Here, an experimental model of SARS-CoV-2 infection was proposed to allow systematic interrogation of the early molecular events and pathogenic mechanism of COVID-19 at cellular resolution in native human lung tissue.
To define the early events of SARS-CoV-2 infection in human lung, thick sections (~300-500 µm “slices”) of fresh lung tissue were prepared from therapeutic surgical resections or organ donors, and placed the slices in culture medium containing DMEM/F12 and 10% FBS. Then those tissue slices were infected with SARS-CoV-2 (USA-WA1/2020) at a multiplicity of infection (MOI) of 1 for two hours, and the cultures continued for 24 or 72 hours to allow infection to proceed.

To characterize viral and host gene expression during SARS-CoV-2 infection, slices were dissociated and analyzed by single-cell RNA sequencing. Multiplexed single molecule fluorescence in situ hybridization (smFISH) of the infected lung slices were also performed to simultaneously detect positive strand viral RNA (S gene probe), negative strand viral RNA (replication intermediate, Orf1ab gene probe), the canonical viral receptor ACE2, and markers of the infected cell types detected in scRNA-seq.

The results indicate that the most susceptible lung target of SARS-CoV-2 and focus of inflammation is activated interstitial macrophages. In this newly characterized lung macrophage subtype, viral RNA amplification results in host cell takeover with viral transcripts comprising up to 60% of the total cellular transcriptome. During takeover, there is cell-autonomous induction of an interferon-dominated inflammatory response, including induction of chemokines that can recruit local innate immune cells expressing the cognate receptors (CCL8, CCL2, CCL13, CXCL10). Takeover also induces expression of cytokine IL6, the potent inflammatory molecule central to cytokine storm. Thus, SARS-CoV-2 infection and takeover of interstitial macrophages and interferon-dominated induction of this suite of chemokines and cytokines forms a focus of lung inflammation and immune infiltration, which we propose initiates the transition from COVID-19 pneumonia to ARDS.

To explore the mechanism of SARS-CoV-2 entry into human lung macrophages, a modified recombinant Spike-pseudotyped lentivirus system was applied. Treatment of purified lung macrophages with hydroxychloroquine, a lysosomal protease inhibitor, or cytochalasin D did not block infection by the lentivirus across a wide range of concentrations. This indicates that Spike-mediated entry into lung macrophages does not require phagocytosis. Then, neutralization assays using three potent anti-Spike monoclonal antibodies (mAbs) were evaluated. Although each of these mAbs robustly inhibited lentivirus infection of HeLa-ACE2/TMPRSS2 at nanomolar concentrations, none reduced lentivirus infection of purified lung macrophages. Thus, this means that spike-mediated entry into lung macrophages occurs by a potentially novel mechanism that does not require phagocytosis or the ACE2-interacting receptor binding motif of the SARS-CoV-2 Spike protein.