Tonic and early interferons defend against respiratory viruses in primary human lung organoid-derived air-liquid interface cultures.

Innate defenses of the respiratory epithelium are the first barrier against incoming respiratory viruses. To understand the contribution of both basal (tonic) and induced interferon (IFN) to antiviral defenses in a physiologically relevant system, we established air-liquid interface (ALI) cultures of primary human bronchial epithelium (HBE) and small airway epithelium (HSE). Via an organoid intermediate stage, the limited healthy donor material was expanded while preserving stemness, and subsequently differentiated. Characterization by immunofluorescent profiling and transcriptomic analyses showed that the cellular diversity and architecture of our ALI cultures were comparable to native human lung epithelium. Infection with human rhinovirus (HRV16) induced a strong and early IFN response, whereas human coronavirus (HCoV-229E and HCoV-NL63) infection caused a more subdued and delayed response. HRV16, but not HCoV-229E, infection was eventually cleared from the cultures after more than 30 days. Depletion of tonic or early type I/III IFNs using neutralizing antibodies or scavengers increased infectious HRV production by ~10- or ~1,000-fold, respectively, suggesting a role of IFNs in clearance. Taken together, we present a method for generating primary lung epithelial air-liquid interface cultures that retain effective IFN responses, demonstrate clearance of HRV by innate defenses, and highlight the importance of tonic and early IFN.IMPORTANCEMild respiratory viral infections, for example, with human common cold coronaviruses or rhinoviruses, are a massive cause of human morbidity. The respiratory tract is the primary entry route for these viruses and also the contact site for initial innate immune defenses. Here, we show that primary human lung epithelial cell-derived air-liquid interface cultures mimic the architecture and cell composition of native human lung epithelium, and retain both induced and tonic interferon (IFN) responses. Notably, our data show that the model's innate immune defense, characterized by rapid and robust IFN responses, are sufficient to clear human rhinovirus (HRV) infections but not human coronavirus 229E. Finally, depletion of induced or tonic IFNs led to a marked increase in HRV infection. Thus, our data suggest that tonic low levels of IFNs contribute to the epithelial defense against viruses, maintaining the tissue's immune readiness.