scRNA-seq reveals persistent aberrant differentiation of nasal epithelium driven by TNFα and TGFβ in post-COVID syndrome

Background: Post-COVID syndrome (PCS) currently affects approximately 3-17% of people following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has the potential to become a significant global health burden. While PCS exhibits diverse symptoms, improved assessment methods are being developed to guide therapy. Despite this, there is a lack of mechanistic insights and treatment options. Objective: To elucidate molecular mechanisms underlying PCS by employing single-cell RNA transcriptomics on patients’ nasal tissue and in vitro validation. Methods: Single-cell RNA transcriptomic analysis was conducted on nasal biopsies from 25 PCS patients with moderate or severe PCS symptoms. Identification of cell types, signaling, and cell-cell communication was performed. In vitro, air-liquid interface cultures were employed to validate findings, with a particular focus on the TNFα-TGFβ axis. Results: Severe PCS was associated with reduced numbers of ciliated cells, increased immune cell presence, and inflammatory signaling leading to TGFβ and TNFα upregulation, all without any viral load. This induced an epithelial-mesenchymal transition, increasing basal cell abundance and a mis-stratified nasal epithelium. In vitro experiments validated TGFβ and TNFα as causal cytokines promoting the reduced abundance of ciliated epithelium and expansion of basal epithelial cells. As such, we posit that a sustained severe PCS phenotype was not linked to continued viral load but instead to immune cell presence and persistent cytokine production. Conclusions: Our study reveals a pathway for persistent severe PCS involving immune-mediated nasal tissue damage, suggesting therapeutic potential by targeting the TNFα-TGFβ axis. The restoration of the nasal epithelium may prove to be a possible treatment strategy for PCS, akin to interventions in other chronic inflammatory conditions. Healthy donor nasal epithelial cells (NECs) were subcultured and plated onto collagen IV-coated transwell supports. Cells were differentiated at air-liquid interface for 28 days. Experimental conditions included treatment with TGFβ (1 ng/ml), TNFα (10 ng/ml), or both, added to the basal chamber. PBS was used as control. Media were changed three times weekly, and cells were washed with hanks balanced salt solution (HBSS). Cells across all stimulation conditions were harvested at 28 days post-airlift for single-cell RNA-seq (Singleron).