Long-Term Culture of Distal Airway Epithelial Cells Allows Differentiation Towards Alveolar Epithelial Cells Suited for Influenza Virus Studies

Imai-Matsushima, Aki; Martin-Sancho, Laura; Karlas, Alexander; Imai, Seiichiro; Zoranovic, Tamara; Hocke, Andreas C.; Mollenkopf, Hans-Joachim; Berger, Hilmar; Meyer, Thomas F.

Chickens have been appointed as potential intermediate hosts for the transmission of avian influenza viruses to humans [57, 58] . Establishing models for avian and human primary DAECs enables direct comparison of influenza A virus replication in different relevant host cells, and ultimately allows deeper studies of the underlying biology. Various mutations that improve the replication efficiency of avian IAVs in mammalian hosts have been identified [59, 60] . Of these, the most extensively studied mutation is the single point mutation at influenza Lineage-negative epithelial progenitors (LNEPs) and distal airway stem cells (DASCs) are the specialized tissue stem cells that proliferate upon tissue damage in mice (e.g. influenza infection) [16, 20, 21] . Vaughan et al. proposed that murine LNEPs require Notch signalling to activate the ΔNp63 and CK5 program, and conversely Notch blockade promotes generation of alveolar epithelial cells [21] . Abbreviations: CHIR, CHIR99021; FGF, fibroblast growth factor; IBMX, 3-isobutyl-1-methylxanthine; 8-Br-cAMP, 8-bromoadenosine 3′,5′cyclic monophosphate. polymerase basic 2 (PB2) 627 residue from glutamic acid (E) to lysine (K). This has been linked to greater replication rates of avian viruses in human cells. Accordingly, the avian-like 627E-expressing virus A/Mallard/Germany/439/2004(H3N2) replicated better in chicken than in human DAECs. Although A/England/195/2009(H1N1pdm) also harbours 627E, compensatory mutations that allow for efficient replication in human cells have been identified [61] . Nonetheless, chicken DAECs still replicated A/England/195/2009(H1N1pdm) more productively than human DAECs. One possible explanation is that these viruses were generated in eggs and they might have acquired mutations that enable higher replication rates in chicken cells. For the other strains tested, these harbour the mammalian-like 627 K mutation in PB2 and still replicated equally well or better in chicken DAECs, suggesting that in addition to E627K PB2, other viral factors might affect host range [62] [63] [64] . Human-adapted viruses, such as H1N1pdm or H3N2, were found unable to infect poultry in previous studies. This discrepancy might be due to the absence of additional cell types that are required to mount an efficient immune response against some strains of influenza A virus in our ex vivo model. Along these lines, establishment of lung organoid cultures will benefit further investigations. Interestingly, the innate immune response of chickens against viruses is weak [65] . Chicken cells lack the retinoic acid-inducible gene (RIG-I) receptor, which can sense IAV, resulting in the induction of type-I interferon (IFN) responses. In contrast, strong RIG-I expression is observed in ducks, potentially explaining duck resistance to most influenza virus strains [66] . In vitro replication kinetics of IAVs in DAECs of aquatic birds, the natural reservoir of influenza virus, could help understand the tropism of influenza virus. However, the establishment of such models will depend on the availability of lungs from these species and the development of molecular tools to allow further characterization.