[Differential Characteristics of Human Airway Organoids at Different Stages of Differentiation After Respiratory Syncytial Virus Infection].

Abstract

Objective: To investigate the differences in pathological changes and immune responses of human airway organoids at different stages of differentiation following respiratory syncytial virus (RSV) infection.

Methods: Models of human fetal lung organoids (FLO) and induced airway organoids (iAO) were established to simulate immature and mature airway epithelium. Immunofluorescence staining, electron microscopy, and quantitative polymerase chain reaction (Q-PCR) were used to confirm the successful construction of the lung organoid models. Human lung organoids were infected with RSV, and samples were collected at 6 and 48 hours post-infection. The immune characteristics of immature and mature RSV-infected organoids were assessed using immunofluorescence staining, droplet digital PCR (DDPCR), and Q-PCR.

Results: We successfully generated FLO expressing both the progenitor markers sex determining region Y-box transcription factor 2 (SOX2) and sex determining region Y-box transcription factor 9 (SOX9), as well as iAO containing basal cells, ciliated cells, club cells, and goblet cells. In addition, organoid models of RSV infection were established. DDPCR results showed that, at the initial stage of RSV infection, the viral load in iAO was significantly higher than that in FLO (P < 0.001). However, at 48 hours post-infection, the viral load in iAO was lower than that in FLO (P < 0.05). Q-PCR results indicated that the expression of RSV infection receptor genes, including epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R), and nucleolin (NCL), was significantly higher in iAO compared to that in FLO (P < 0.001). RSV infection led to an increase in the expression levels of immune factors, including interleukin 6 (IL-6), interleukin 8 (CXCL8), interferon α (IFN-α), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor α (TNF-α), in iAO compared to those in FLO, and the differences were statistically significant (P < 0.05).

Conclusion: The expression of RSV infection receptor proteins increases with airway maturation, and mature airway epithelial cells exhibit a stronger immune response than immature ones do, effectively inhibiting RSV replication.