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Deciphering the initial steps of SARS-CoV2 infection in whole human lungs identifies macrophages as primary targets and reveals subset-specific responses.

ABSTRACT: Several in vitro models have been established to mimic the initial interaction between lung cell types and SARS-CoV-2, mainly using explant-based techniques that generated contrasted results about the cell types that were infected. These contrasting results could be due to biases in the various tissue-culture conditions. Furthermore, all these systems do not respect the spatial tissue architecture and the anatomic constraints that shape cell type interactions with viruses. In this study, we used whole human lungs maintained alive ex vivo for 10 h, based on a technique used in lung transplantation (ex vivo lung perfusion or EVLP), to analyze the first steps of SARS-CoV-2 infection in the lung. Five human donor lungs declined for transplantation were perfused and ventilated at 37°C and three of them were infected with SARS-CoV-2 using nebulization of the Wuhan lineage and D614G variants. Single-cell RNA-sequencing (scRNA-seq) using 10x Genomics 3' (v3 Chemistry) has been performed on these samples.

ORGANISM(S): Homo sapiens

PROVIDER: GSE246128 | GEO | 2024/07/04

REPOSITORIES: GEO

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Project description:Ischemia reperfusion (IR) injury is a key complication following lung transplantation, and it remains to be fully resolved. Ex vivo lung perfusion (EVLP) has shown its significance as a platform to assess and repair marginal donor lungs prior to transplants with good clinical outcomes. Recently, it has been shown that Glutamax supplementation to Steen solution can improve basic cellular function as well as porcine EVLP duration with stable lung quality. To better understand the molecular mechanisms of IR injury, acellular EVLP, and Glutamax supplementation during EVLP, transcriptomic changes in cell culture models simulating IR and EVLP have been investigated. Briefly, human pulmonary microvascular endothelial cells (HPMEC) and human lung epithelial cells (BEAS-2B) were cultured in DMEM+10%FBS, then were perfused with Perfadex at 4°C for 18H to simulate cold ischemic time (CIT). Subsequently, the cells were perfused in either DMEM+10%FBS (IR model) or in Steen solution or Steen with 4mM Glutamax (EVLP models) for 4H. RNA samples were collected for CIT, IR, and two EVLP conditions, and sequencing data were analyzed for differentially expressed genes and pathways. HPMEC and BEAS-2B cells showed significant changes in gene expressions for IR and EVLP models. In pathway analyses, IR models of both cell types had up-regulated pro-inflammatory responses and down-regulated cell metabolism, and there was up-regulation of epithelial process and/or vascular activation. Steen alone EVLP models exhibited down-regulation of cell metabolism, with an absence of inflammatory and vascular or epithelial signals, while Steen with Glutamax exhibited similar down-regulation of cell metabolism as well as down-regulated inflammatory and vascular process. The commonly used EVLP perfusate, acellular Steen solution, can silence the activation of pro-inflammatory signaling in HPMEC and BEAS-2B cell culture models. This finding may yield insights into how acellular EVLP has been successful in safe preservation of the donor lungs during organ evaluation and repair prior to lung transplantation.

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Deciphering the initial steps of SARS-CoV2 infection in whole human lungs identifies macrophages as primary targets and reveals subset-specific responses.

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