Precision cut slice lung model to study early host viral pathogenesis of SARS-CoV-2 infection

In a recent study posted to the bioRxiv* preprint server, researchers applied their previously developed precision-cut lung slice (PCLS) model to study the initial events in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenicity in humans.

SARS-CoV-2 infects and replicates in the airways with subsequent pulmonary damage. Unfortunately, among hospitalized patients, host-SARS-CoV-2 interactions have been found to go on for several days or weeks. This makes it a challenge to decipher the initial responses of the host to SARS-CoV-2. Although animal models have benefitted in this context, they have not recapitulated human complexities completely.

About the study

In the present study, researchers applied their previously designed PCLS model in mice to investigate the initial host-SARS-CoV-2 interactions in humans.

Inflation of a human pulmonary lobe was performed with a 2% low melting point (LM) agarose, and PCLSs were obtained. The slices were directly infected with SARS-CoV-2 for three days and subsequently subjected to immunofluorescence and flow cytometry analyses. For further investigations, cellular staining was performed for double-stranded RNA (dsRNA) and SARS-CoV-2 spike (S) protein. In addition, the PCLSs were infected by either Influenza A virus (IAV) or SARS-CoV-2 to appreciate SARS-CoV-2-induced changes in pulmonary composition and gene expression. 

To investigate the effects of SARS-CoV-2 on myeloid cells, the alveolar macrophages (AMs), situated within the air cavities and hence directly exposed to SARS-CoV-2, were analyzed for angiotensin-converting enzyme 2 (ACE2) expression.

To study the capability of SARS-CoV-2-exposed AMs to produce and release novel viruses, bronchoalveolar lavage (BAL) of the human lungs was performed post which SARS-CoV-2 and IAV were incubated with BAL cells [multiplicity of infection (MOI) 0.1 or 1]. Plaque assays were performed to determine the production of viral loads by the BAL supernatant. A similar quantity of viruses was incubated only with media to serve as controls. After 48 hours of incubation, cell-free supernatant was obtained and used to infect Vero E6 cells with SARS-CoV-2 ancestral USA-WA1/2020 strain or Delta strains and MDCK (Madin-Darby Canine Kidney Epithelial Cells) cells with IAV. After 24 hours of infection, the cells were assessed by flow cytometry and S staining.

In addition, endotracheal aspirates (ETA) were collected from seven intubated coronavirus disease 2019 (COVID-19) patients and subjected to single-cell ribonucleic acid (RNA) sequencing (scRNA-seq) for comparing the PCLS model findings with the clinical COVID-19 scenario and characterize the influence of SARS-CoV-2 on particular cell populations. Lastly, the differential gene expression among infected and uninfected AMs from SARS-CoV-2-positive ETA samples was analyzed.

Results

ACE2-expressing AMs demonstrated productive SARS-CoV-2 infection, in contrast to IAV neutralization. In comparison to IAV, SARS-CoV-2 showed poor interferon responses in the infected myeloid cells. ETA samples obtained from COVID-19 patients confirmed the PCLSs observations. This indicated an immediate depot of myeloid cells for SARS-CoV-2 in the human lungs.

In the immunostaining analysis, the infected PCLSs showed staining for S in epithelial cells (EpCAM+) and ACE2. However, three days post-infection, <10% of epithelial cells were positive for S and dsRNA. Flow cytometry and imaging analyses showed that SARS-CoV-2 produced infection in pulmonary epithelial cells, albeit low in magnitude. S colocalized to CD45+ ACE2+ cells, and likewise, dsRNA was observed in the cells, supporting that the immune cells are either infected by SARS-CoV-2 or destroy SARS-CoV-2 by phagocytosis.

Clusters with eight populations of lymphocytes (B and T) and non-immune cells and four myeloid cell populations were observed. Within 24 hours, SARS-CoV-2 reads were enriched with myeloid cells. While IAV reads showed scattering, SARS-CoV-2 showed selective tropism for AMs and pulmonary myeloid cells [cluster of differentiation (CD) 3-, CD 19-, CD 14+, CD 45+, human leucocyte antigen-DR isotype positive (HLA-DR+), which included monocytes, DCs, and macrophages. The myeloid cells also showed significant dsRNA and S signals after 48 to 72 hours of SARS-CoV-2 infection.

The scRNA-seq analysis showed that the key IAV targets were fibroblasts and epithelial cells, in accordance with the PCLSs observations. Post IAV infection, the most prominent change was a decrease in pulmonary epithelial cells and fibroblasts. Contrastingly, SARS-CoV-2 did not produce any significant trend in pulmonary non-immune cells, compared to controls. While IAV destroyed immune cells with a resultant reduction in cells in myeloid cells, SARS-CoV-2 gradually increased the myeloid fraction, with an approximately 50% increase 72 hours post-infection compared to controls.

After 48 hours of SARS-CoV-2 infection at MOI 0.1, S was detected in <10% AMs, and increasing the MOI to 1 could not substantially increase S+ AMs. This indicated that the cells were protected at high titers. AMs showed high viability at both MOI values, indicating that SARS-CoV-2 did not induce cell death of AMs. This finding was in contrast to that noted in monocytes of COVID-19 patients, in whom approximately 25% of AMs were SARS-CoV-2-positive and demonstrated upregulation of interferon-stimulated genes (ISG).

Notably, BAL cells in the donor’s lungs showed AM enrichment, with CD169+, CD45+, and HLA-DR+ cells, and lacked epithelial cells (<1%). Of the S+ AMs, most were ACE2+ and showed a substantial reduction in S+ AMs on using an ACE2 blocking antibody. This indicates that ACE2 mediates the entry of SARS-CoV-2 into AMs. Incubating BAL cells and AMs with SARS-CoV-2 led to viral amplification and ISG induction for the ancestral and Delta strains at MOI 0.1 and 1, albeit 10-fold lower than IAV.

Overall, the study findings showed that SARS-CoV-2 has a selective tropism for myeloid cells in the human lungs. The study also provides evidence of productive SARS-CoV-2 infection by the ancestral and Delta strains in the AMs with a low concomitant immune response, indicative of a depot effect wherein the protective immune mechanisms are hijacked to facilitate SARS-CoV-2 replication.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.