The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in millions of infections and deaths. A significant concern is the development of Post-Acute Sequelae of SARS-CoV-2 (PASC), also known as "long COVID," affecting 3-11.7% of infected individuals. PASC is characterized by a range of symptoms, including fatigue, cognitive dysfunction, and respiratory issues, persisting for over 12 weeks post-infection. Respiratory symptoms, such as shortness of breath and dyspnea, are particularly prevalent in hospitalized patients, even months after the acute phase. The underlying mechanisms responsible for respiratory PASC are not fully understood, although lung fibrosis has been implicated. However, fibrosis alone cannot fully explain the observed respiratory symptoms. Incomplete or protracted alveolar regeneration is another potential contributing factor. SARS-CoV-2 infection causes diffuse alveolar damage (DAD), characterized by the loss of alveolar epithelial cells (AT1 and AT2) and subsequent inflammation. Alveolar regeneration requires progenitor cells to replace lost AT1 cells. While AT2 cells were previously thought to be solely responsible for AT1 regeneration, recent research using mouse models suggests the involvement of airway progenitor cells after severe lung injury. A key intermediate in AT2 to AT1 cell differentiation is the alveolar differentiation intermediate (ADI) cell, characterized by CK8 expression, a specific morphology, and upregulation of genes involved in EMT and cell cycle exit. ADI cell accumulation has been observed in various lung injury models and is linked to unresolved hypoxemia and fibrosis in COVID-19 patients. The persistence of these cells is a critical aspect of PASC that is poorly understood due to the limited availability of sequential human samples. Animal models are essential for studying the sequential phases of SARS-CoV-2 infection and evaluating potential therapeutic interventions. Syrian golden hamsters provide a suitable model, exhibiting transient and non-lethal COVID-19-like disease. This study aimed to comprehensively characterize the alveolar regeneration process in SARS-CoV-2-infected hamsters, focusing on the role of ADI cells and airway progenitors.

Existing literature highlights the significant impact of PASC on COVID-19 survivors, particularly respiratory complications. While lung fibrosis is recognized as a potential sequelae, protracted alveolar regeneration is also proposed as a major mechanism underlying persistent respiratory symptoms. Studies using mouse models have advanced our understanding of alveolar regeneration, identifying the role of both AT2 cells and airway progenitor cells in repairing alveolar structures following injury. The discovery of the ADI cell as a crucial intermediate in AT2-to-AT1 cell differentiation has added another layer to this understanding. Previous research has shown that ADI cell accumulation is observed in various lung injury contexts, and a pathological persistence of these cells is implicated in severe COVID-19 and potentially PASC. However, the lack of longitudinal human studies necessitates the use of animal models to investigate the complex interplay of cellular processes during alveolar regeneration after SARS-CoV-2 infection. Syrian golden hamsters, owing to their susceptibility to SARS-CoV-2 and their development of a non-lethal, transient disease, emerged as a valuable model for this research. The literature lacks detailed characterization of lung epithelial regeneration in this model following SARS-CoV-2 infection, making this study crucial in bridging the gap between human observation and preclinical investigation.

This study employed a Syrian golden hamster model of SARS-CoV-2 infection. Hamsters (n=10 per time point per group) were intranasally inoculated with either 104 plaque-forming units (pfu) of SARS-CoV-2 or PBS (control). Animals were euthanized at 1, 3, 6, and 14 days post-infection (dpi). Right lung lobes were collected and processed for histopathological evaluation, immunohistochemistry, immunofluorescence, and transmission electron microscopy (TEM). Immunohistochemistry was performed to detect SARS-CoV-2 nucleoprotein, macrophages (Iba-1), AT2 cells (pro-surfactant protein C), ADI cells (cytokeratin 8), airway basal cells (cytokeratin 14), club cells (secretoglobin 1A1), and M2 macrophages (CD204). Immunofluorescence was used for double labeling to visualize cellular transitions. TEM was used to examine alveolar cell ultrastructure. Digital image analysis using QuPath software was conducted for quantitative assessment of cellular populations and fibrotic areas. Additionally, single-cell RNA sequencing (scRNA-seq) data from a previously published study (GSE162208) were re-analyzed to identify alveolar cell populations at 5 and 14 dpi using Seurat software. Module score analysis was performed to assess pathway enrichments in clusters with high ADI gene expression. Statistical analyses (Mann-Whitney U test, Kruskal-Wallis test with Benjamini-Hochberg correction) were used to compare groups. Human lung samples from three lethal COVID-19 ARDS cases and one non-COVID-19 case were also analyzed for comparison.

Successful SARS-CoV-2 infection in hamsters was confirmed by immunohistochemistry. Viral antigen peaked at 3 dpi and cleared by 14 dpi. A significant and transient broncho-interstitial pneumonia was observed, characterized by DAD, epithelial cell necrosis, fibrin exudation, and inflammatory cell infiltration. Epithelial proliferation was prominent at 3, 6, and 14 dpi. Immunohistochemistry revealed a significant increase in CK8+ ADI cells in infected hamsters compared to controls, particularly within affected alveoli. Double immunofluorescence showed a transition from AT2 to ADI cells, with many ADI cells at 6 and 14 dpi expressing nuclear TP53, indicating cell cycle arrest. TEM confirmed the presence of cells with both AT1 and AT2 characteristics, indicating an ongoing ADI to AT1 cell differentiation. Analysis of human lung samples from lethal COVID-19 cases also revealed ADI cells expressing TP53, unlike a non-COVID-19 sample. The study also showed that multipotent CK14+ airway basal cells migrated from terminal bronchioles to contribute to alveolar regeneration. At 6 dpi, many of these cells differentiated into AT2 cells, while at 14 dpi, some differentiated into club cells. At 14 dpi, incomplete alveolar restoration was observed, along with sub-pleural fibrosis in 7/9 animals, accompanied by numerous M2 macrophages. ScRNA-seq analysis of an independent dataset confirmed the presence of ADI cells at both 5 and 14 dpi. Clusters with high ADI gene expression also showed high module scores for pathways involved in p53 signaling, DNA repair, EMT, angiogenesis, and wnt/β-catenin signaling.

This study demonstrates that the Syrian golden hamster model faithfully recapitulates key features of alveolar regeneration observed in human COVID-19, including the presence of ADI cells, airway progenitor cell mobilization, and the development of fibrosis. The findings highlight the prolonged presence of ADI cells, particularly those expressing TP53, which could contribute to incomplete alveolar repair and potentially contribute to persistent respiratory dysfunction in PASC. The observed fibrosis is consistent with human COVID-19 and suggests the involvement of M2 macrophages and an EMT-like process in AT1/ADI cells. The transcriptomic data provides further evidence of the involvement of various pathways, including those involved in cell cycle arrest, DNA repair, and angiogenesis, in the dysregulated regeneration observed in this model. This hamster model offers a valuable preclinical platform for studying the pathogenesis of PASC and for evaluating potential therapeutic strategies targeting alveolar regeneration.

This study establishes the Syrian golden hamster as a robust model for studying post-COVID-19 alveolar regeneration and its potential contribution to PASC. The findings reveal a complex interplay between ADI cells, airway progenitor cells, and inflammatory cells in the protracted regenerative process following SARS-CoV-2 infection. The model successfully recapitulates key features of human COVID-19 pathology, including the persistence of ADI cells and development of fibrosis. Future research using this model should focus on the long-term effects of infection, the precise mechanisms driving ADI cell persistence, and the evaluation of therapeutic interventions targeting these processes. This model also presents a valuable platform for studying similar lung pathologies characterized by DAD and fibrosis.

The study was limited by the inability to definitively quantify AT1 cells using available antibodies, leading to the reliance on TEM for assessing ADI-AT1 transition. While the findings strongly suggest a connection between ADI cell persistence and fibrosis, further studies are needed to directly establish this causal link. The conclusions regarding cell origins and fate are based on immunohistochemical co-localization and gene expression, necessitating confirmation through lineage-tracing experiments. The interpretation of the scRNA-seq data relies on the analysis of an independent dataset and requires further validation with independent experiments. The study did not include a detailed assessment of lung function or gas exchange in hamsters, which would provide a more direct link between the observed pathology and PASC symptoms.