Age-specific nasal epithelial responses to SARS-CoV-2 infection

Despite effective vaccines, age remains the single greatest risk factor for COVID-19 mortality. Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rarely develop severe disease, while the mortality in infected people over 85 years is currently as high as 1 in 10. Nasal epithelial cells (NECs) are the primary target of SARS-CoV-2, and understanding their viral response is crucial as infection of upper airway cells can progress distally, leading to diffuse alveolar injury with respiratory failure and long-term complications including lung fibrosis. Initially, it was thought that higher viral entry factor expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) in adults could explain increased severity, but such differences between children and adults remain uncertain. Children may alternatively be protected by a pre-activated antiviral state in the upper airways, but this does not fully explain the increased risk with increasing age. In addition, most in vivo studies so far were unable to identify early cellular responses, since in almost all cases the exact time of infection was unknown, symptom onset was variable, and research sampling usually occurred only a few days after testing positive for SARS-CoV-2. This study investigated the effects of early SARS-CoV-2 infection on human NECs from healthy children (0–11 years), adults (30–50 years) and older adults (>70 years). NECs were cultured at an air-liquid interface (ALI) and either subjected to mock infection or infected with SARS-CoV-2 for up to 3 days. This setup was used to examine epithelial-intrinsic differences in function, viral replication, gene and protein expression. The researchers aimed to reveal age-specific epithelial responses, independent of immune cells, focusing on interferon (IFN) response and the potential role of older adult basaloid-like cells in viral replication and fibrotic signaling pathways.

The researchers reviewed existing literature highlighting the disparity in COVID-19 severity between children and older adults. Previous studies suggested potential explanations such as differences in ACE2 and TMPRSS2 expression, or a pre-activated antiviral state in children. However, these explanations were incomplete and lacked the resolution of early cellular responses due to limitations in in vivo studies, including unknown infection times and variable symptom onset. The existing literature lacked a comprehensive understanding of the age-specific cellular mechanisms driving the differential responses to SARS-CoV-2 infection. This paper aimed to address this gap by focusing on early epithelial responses in a controlled in vitro setting.

The study involved the collection of nasal brushings from healthy paediatric (0-11 years), adult (30-50 years), and older adult (≥70 years) donors who tested negative for SARS-CoV-2 and reported no respiratory symptoms. These brushings were used to culture differentiated human nasal epithelial cells (NECs) at an air-liquid interface (ALI). The cultures were then either mock-infected or infected with an early-lineage SARS-CoV-2 isolate for up to 72 hours. A multifaceted approach was used to analyze the cellular responses: * **Single-cell RNA sequencing (scRNA-seq):** This technique was used to profile the cellular composition and gene expression of NECs from different age groups before and after infection. * **Immunofluorescence staining:** This technique was employed to visualize the localization of viral proteins and other markers of interest within the NEC cultures. * **Transmission electron microscopy (TEM):** This technique was used to observe the ultrastructure of infected cells and to visualize viral particles. * **Proteomics (mass spectrometry and Western blot):** These techniques were used to quantify the abundance of proteins in the apical fluid and cell lysates of infected cultures. * **Viral load quantification (plaque assay):** This technique was used to measure the number of infectious viral particles produced by infected cultures. * **Viral genomic sequencing:** This was performed to assess viral replication and detect mutations. * **Wound healing assay:** This was used to investigate the effects of epithelial repair processes on viral replication. Statistical analysis included multiple paired t-tests, one-way and two-way ANOVA with Tukey’s multiple comparisons test, zero-inflated Poisson models, and gene set enrichment analysis (GSEA). The study also integrated existing in vivo scRNA-seq datasets from COVID-19 patients to validate in vitro findings.

The study revealed significant age-specific differences in the cellular response to SARS-CoV-2 infection in nasal epithelial cells: **Age-related differences in cellular landscape:** The researchers found age-related differences in the proportions of various cell types in healthy NECs. Adult cultures had a higher abundance of basal/progenitor cell subtypes than paediatric cultures. Paediatric cultures exhibited a higher abundance of goblet cell types, particularly goblet 2 cells, while older adult cultures were thicker with a distinct spiral morphology. **Differences in viral replication:** Total viral RNA did not significantly differ across age groups; however, the number of infected cell types and the overall spread of the virus differed. Older adult cultures showed significantly greater viral spread and significantly higher infectious viral titres compared to paediatric cultures. Ciliated cells were primary viral replication centers across all ages. **Age-specific effects of infection:** SARS-CoV-2 infection led to decreased culture thickness and epithelial integrity in adult and older adult cultures, accompanied by increased basal cell mobilization and epithelial escape. In contrast, paediatric cultures exhibited a unique response with a significant increase in goblet 2 inflammatory cells characterized by high expression of interferon-stimulated genes (ISGs). These goblet cells showed a bias in viral reads toward the 5' end, suggesting incomplete viral replication. **In vivo validation:** Integration of in vivo COVID-19 datasets confirmed the in vitro findings. Goblet inflammatory cells were enriched in both paediatric and older adult COVID-19 patients, while basaloid-like 2 cells were most abundant in older adult COVID-19 patients. The in vivo findings validated the relevance of the in vitro model. **Interferon response:** Paediatric cultures exhibited a stronger interferon response, particularly in the goblet 2 inflammatory cells, which showed high expression of ISGs and seemed to restrict viral replication. **Pro-fibrotic response in older adults:** Older adult cultures showed an increase in basaloid-like 2 cells, which are associated with pro-fibrotic markers and epithelial-mesenchymal transition (EMT). These cells were also associated with upregulation of alternate viral entry receptors. Importantly, stimulating wound repair in older adult cultures increased the expression of these basaloid-like 2 cell markers, accelerated wound healing, and amplified SARS-CoV-2 infection, suggesting that these cells contribute to viral spread and disease progression. Specific quantitative data points are included within the detailed results section of the study.

This study provides valuable insights into the age-specific mechanisms underlying SARS-CoV-2 pathogenesis. The in vitro model successfully recapitulates key aspects of the in vivo response, demonstrating the importance of early epithelial responses in determining disease severity. The strong interferon response in paediatric cultures, leading to incomplete viral replication, contrasts sharply with the pro-fibrotic response and enhanced viral replication observed in older adult cultures. The shift in cell tropism from goblet cells in children to secretory cells in older adults highlights the age-dependent susceptibility of different nasal epithelial cell subtypes to SARS-CoV-2. The identification of basaloid-like 2 cells as key players in viral spread and disease progression in older adults has significant implications for understanding the increased mortality risk in this population. The findings underscore the need for age-specific therapeutic strategies targeting the different pathogenic mechanisms involved in COVID-19.

This study demonstrates age-specific tropism of SARS-CoV-2 in nasal epithelial cells, with distinct cellular responses driving the varying severity of COVID-19 across age groups. Children exhibit a strong interferon response leading to incomplete replication, while older adults display enhanced viral replication linked to basaloid-like 2 cells and impaired epithelial repair. These findings offer crucial insights into age-related COVID-19 pathogenesis and pave the way for developing age-tailored therapeutic interventions. Further research should focus on elucidating the specific molecular mechanisms involved in the interaction between SARS-CoV-2 and these age-specific cell types and explore targeted therapies based on these findings.

The study primarily utilized an in vitro model, which may not perfectly capture the complexity of the in vivo immune response. The sample size, while robust for the in vitro component, might be considered limited for definitive conclusions about the in vivo aspects. There's inherent variability between individual donors, which is addressed in part by pooling and the use of statistical methods; however, the study design cannot account for all individual variations. The in vivo analysis relies on integration of existing datasets with different protocols and might have slight discrepancies with the in vitro model. Future studies are needed to expand on these in vivo findings with a larger, more homogenous dataset.