Activated interstitial macrophages are a predominant target of viral takeover and focus of inflammation in COVID-19 initiation in human lung

Abstract

Early stages of deadly respiratory diseases such as COVID-19 have been challenging to elucidate due to lack of an experimental system that recapitulates the cellular and structural complexity of the human lung, while allowing precise control over disease initiation and systematic interrogation of molecular events at cellular resolution. Here we show healthy human lung slices cultured ex vivo can be productively infected with SARS-CoV-2, and the cellular tropism of the virus and its distinct and dynamic effects on host cell gene expression can be determined by single cell RNA sequencing and reconstruction of “infection pseudotime” for individual lung cell types. This revealed the prominent SARS-CoV-2 target is a population of activated interstitial macrophages, which as infection proceeds accumulate thousands of viral RNA molecules per cell, comprising up to 60% of the cellular transcriptome and including canonical and novel subgenomic RNAs. During viral takeover, there is cell-autonomous induction of a specific host interferon program and seven chemokines (CCL2, 7, 8, 13, CXCL10) and cytokines (IL6, IL10), distinct from the response of alveolar macrophages in which neither viral takeover nor induction of a substantial inflammatory response occurs. Using a recombinant SARS-CoV-2 Spike-pseudotyped lentivirus, we show that entry into purified human lung macrophages depends on Spike but is not blocked by cytochalasin D or by an ACE2-competing monoclonal antibody, indicating a phagocytosis- and ACE2-independent route of entry. These results provide a molecular characterization of the initiation of COVID-19 in human lung tissue, identify activated interstitial macrophages as a prominent site of viral takeover and focus of inflammation, and suggest targeting of these macrophages and their signals as a new therapeutic modality for COVID-19 pneumonia and progression to ARDS. Our approach can be generalized to define the initiation program and evaluate therapeutics for any human lung infection at cellular resolution.

Precision cut lung slices: an integrated ex vivo model for studying lung physiology, pharmacology, disease pathogenesis and drug discovery

“…An early study utilized various strains of infectious bronchitis virus, which are avian coronaviruses, to examine infection of the lung with these viruses [32]. An understanding of the mechanisms driving the inflammation associated with SARS-CoV2, as well as replication mechanisms and pathologic features of the disease have been achieved using PCLS [33][34][35]. Other laboratories identified effective therapeutics that could target SARS-CoV2 infection in PCLS [36][37][38][39][40].…”

The use of single-cell RNA-seq to study heterogeneity at varying levels of virus–host interactions

“…This can also be seen as an advantage, as both productively, and nonproductively, infected cells can contribute to overall pathology and disease progression. This method was used to support tropism of SARS-CoV2 in both human and animal tissues, including ex vivo human lung models, not only confirming angiotensin converting enzyme 2AU : Pleasenotethat}ACE2}hasbeenfullyspelledo (ACE2) as the predominant receptor, and identifying cell-specific expression of this receptor, but also demonstrating ACE2-independent entry into certain cell populations [83][84][85]. Not limited to SARS-CoV2, cells susceptible to infection have been studied using scRNA-seq for astrovirus, rubella, and influenza, among others [80,86,87].…”