Study shows SARS-CoV-2 exhibits differential tropism for nasal epithelial cells with age

In a recent study published on bioRxiv* preprint server, researchers explored the relationship between inflammatory goblet cell development and the age-related pathophysiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Background

The nasal epithelium is the primary barrier against inhalation infections, while nasal epithelial cells (NECs) are the primary target for coronavirus disease 2019 (COVID-19) infection. Infection of upper respiratory tract cells can spread distally, leading to extensive alveolar damage and long-term consequences such as the incidence of pulmonary fibrosis.

Current research indicates that, to some extent, a preactivated antiviral interferon (IFN) state in the upper airway epithelium protects children against infection. This may partly explain why children are better protected, but it does not fully explain the increased age-related risk of infection in adults. The investigation regarding epithelial infection and subsequent repair against COVID-19 may result in innovative treatment methods for COVID-19 and any future developing respiratory virus risk.

About the study

In the current study, researchers assessed changes in cell topography and function of pediatric, adult, and elderly nasal epithelial cells infected with SARS-CoV-2.

For this investigation, fresh nasal brushes were obtained from humans. Four paired nasal brush samples from children were used for single-cell ribonucleic acid sequencing (scRNA-seq) analysis. The team used the SARS-CoV-2 isolate (hCoV-19/England/2/2020) for the infection. The Vero E6 African green monkey kidney cell line was used for viral propagation.

Additionally, the team quantified viral load by plaque assay, and viral copy number was quantified by real-time quantitative polymerase chain reaction (PCR). Then, the phenotypic consequences of infection on epithelial cells were estimated in all age groups using a combination of live cell microscopy, proteomics, immunofluorescence labeling and gene expression analyses.

Results

In healthy and uninfected control cultures, the proportions of several epithelial cell types varied with age. The proportion of basal/progenitor subtypes (KRT5hi) in adult and aged culture datasets was 36.5% and 35.3%, respectively, while that in pediatric culture datasets was 13 .8%. Elderly and adult cultures had the highest number of basal 1 and 2 cells, which were virtually non-existent in pediatric cultures. All age groups demonstrated comparable apical differentiation, such as mucus production (MUC5AC) as well as similar amounts of cilia, with no noticeable change in ciliary beat frequency (CBF) and no altered cell motility with age.

The most obvious change in the pediatric cultures was the increasing number of goblet cells, particularly the goblet 2 subsets. This indicated a change in cellular state from secretory cells found in aged and adult cultures to goblet cells with higher levels of bactericidal/permeability-increasing fold-containing group A member 1 (BPIFA1).

Importantly, although the team observed no disparity in protein levels associated with viral entry factors in the samples with age, pediatric cultures had the highest transmembrane protease serine 2 (TMPRSS2) expression, which is required for priming of the SARS-CoV-2 spike (S)26 protein, and the highest levels of angiotensin-converting enzyme-2 (ACE-2), which consisted of the viral S protein binding site necessary for viral entry.

Secretory and basal cell types 2 also showed the highest expression of TMPRSS2 and ACE2 in cultures of adults and elderly patients. This indicated a change in age-related susceptibility to viral infection to goblet cell secretory cells. Other viral entry factors, such as basigin (BSG), neuropilin-1 (NRP1), NRP2, cathepsin L(CTSL) and FURIN showed similar patterns to ACE2 and TMPRSS2.

The most notable cellular alteration after infection was the appearance of inflammatory goblet 2 cells, missing in sham-infected pediatric samples, but not in aged or adult cultures. Additionally, inflammatory goblet cell type 2 had higher amounts of IFN-induced proteins with tetratricopeptide repeats-1 (IFIT1), IFIT3, and CXCL10 than other goblet cell subtypes, indicating a significant association with type I IFN signaling.

Furthermore, infected aged epithelial cell samples showed an elevation of SARS-CoV-2 infected basal cell quarters compared to sham cultures, indicating mobilization (proliferation) of aged-specific progenitor cells in response to the SARS-CoV-2 infection.

The team also noted that aged NECs infected with SARS-CoV-2 showed a significantly faster rate of wound healing than infected pediatric cultures, suggesting an increase in cell motility, which is another symptom of faulty repair mechanisms. The healing rate of samples from elderly people infected with SARS-CoV-2 was remarkably faster than that of their mock-infected equivalent cultures. Pediatric NECs infected with SARS-CoV-2 exhibited a significantly delayed healing rate compared to their corresponding sham infectious cultures.

Conclusion

Study results demonstrated that the tropism of SARS-CoV-2 for nasal epithelial cells varied with age, with infection preferred from pediatric goblet or adult secretory cells. Infected pediatric goblet cells showed a significant innate SARS-CoV-2 antiviral response driven by interferon and less infectious virions compared to cultures from older adults. These findings provided new insights into the pathophysiology of age-associated COVID-19 and demonstrated how defective repair increases SARS-CoV-2 infection in the elderly.

*Important Notice

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