Establishment of human airway organoids to study respiratory viruses

Abstract

We aim to understand the pathogenesis of human respiratory viruses and virus-host interaction. However, current in vitro models, including short-term cultures human lung explant and primary airway epithelial cells, are barely a biologically-relevant, reproducible, and readily-available model for studying the biology and pathology of the human respiratory tract. Recent advances in stem cell biology have allowed the in vitro growth of 3-dimensional organoids that recapitulate essential attributes of their counterpart organs in vivo. We describe the establishment of adult-stem-cell derived human airway organoids (AO), from which we induced further differentiation and generated proximal differentiated airway organoid (PD AO). As a further improvement, we developed 2D PD airway monolayers with an intact epithelial barrier. The PD AOs can morphologically and functionally simulate the human airway epithelium to a near-physiological level. Human adenoviruses (HAdVs) are a group of non-enveloped double-stranded DNA viruses with a broad tissue tropism. HAdV-3 and HAdV-7 from species B1 account for the majority of adenoviral respiratory infections. Notably, HAdV-7 is associated with higher morbidity and mortality. However, no in vitro model can recapitulate the elevated pathogenicity of HAdV-7. We found the abundantly expressed B1 HAdV cellular receptor DSG2, and productive infections of both HAdV-3 and HAdV-7 on 2D AOs. We further demonstrated that HAdV-7 exhibited a higher infection rate and degree of virus replication over HAdV-3. A whole genome RNA sequencing was performed to elucidate the transcriptomic profiles in the HAdV-infected human airway organoids. DNA replication related pathways and host response to viral infection are significantly enriched in both HAdV-3 and HAdV-7 infections. Particularly, host defense genes were upregulated more intensively and extensively in HAdV-7-infected organoids than HAdV-3-infected organoids. Amongst the DNA replication related genes, we have identified a highly up-regulated host factor, Cyclin E2, especially in HAdV-7-infected organoids. Additionally, among the three early genes, E1B was expressed significantly higher in HAdV-7-infected organoids than in HAdV-3-infected organoids. Thus, the higher expression level of E1B in HAdV-7-infected organoids versus that of HAdV-3-infected organoids may induce a stronger enhancement of Cyclin E2, which in turn leads to an earlier entry into the cell cycle S phase and gives a replication advantage to HAdV-7. Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as the causative agent of severe respiratory infection with a case-fatality rate higher than 30%. Besides respiratory syndrome and other systemic complications, lymphopenia was commonly observed in MERS patients. We demonstrated robust MERS-CoV replication in human airway organoids and then investigated the interaction between T cells and MERS-CoV in disease pathogenesis. Our data suggested that human T cells from the peripheral blood and lymphatic organs including the spleen and tonsil were highly susceptible to MERS-CoV infection and MERS-CoV induced apoptosis. We further demonstrated that MERS-CoV-induced apoptosis in T cells involved the activation of both the extrinsic and intrinsic apoptosis pathways. Taken together, our results suggest that the high mortality rate and pathogenicity of MERS-CoV might be attributed to its capacity to infect and induce apoptosis in T lymphocytes.