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

Innate defences 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 defences 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. Characterisation by spatial and transcriptomic analyses showed that the cellular diversity and architecture of our ALI cultures were comparable to native human lung epithelium. Upon infection with relevant human respiratory pathogens, such as Human Rhinovirus (HRV16) and human Coronaviruses (229E and NL63), only HRV16 induced a strong and early type I and III IFN response, leading to its eventual clearance from the cultures. Depletion of tonic type I/III IFNs using neutralising antibodies or scavengers reduced expression of levels of IFN-stimulated genes and increased infectious HRV production by [~]7-10-fold. Taken together, we present a method for generating primary lung epithelial cultures that retain their IFN status, demonstrate clearance of HRV by innate defences, and highlight the importance of tonic IFN in early antiviral defences. 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 defences. 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 models innate immune defences are sufficient to clear human Rhinovirus (HRV) infections, which are characterised by rapid and robust IFN responses. Finally, depletion of tonic IFNs led to a marked increase in HRV infection. Thus, our research suggests that tonic low levels of IFNs contribute to the epithelial defence against viruses, maintaining the tissues immune readiness. Failure to maintain these tonic IFN levels increases the susceptibility towards infections.