Tropism, susceptibility, infectivity, and cytokine releases of differentiated human tonsillar epithelial cells by different Influenza viruses

Abstract

Human tonsil epithelium cells (HTEC) are a heterogeneous group of actively differentiating cells comprising stratified squamous epithelial and reticulated crypt cells with abundant keratin expression. We hypothesized that the tonsils are a primary site for influenza infection and sustained viral replication. Primary HTEC were grown using an air-liquid culture and infected apically with different influenza viruses (IVs) to measure viral growth kinetics. These cultures were highly differentiated, with subpopulations of heterogenous surface stratified squamous cells rich with both cilia and microvilli; these cells contained more 2,6-linked sialic acids, those preferentially bound by human IVs, than 2,3-linked avian like sialic acids. The stratified squamous cells were interrupted by patches of reticular epithelial cells rich in 2,3-linked sialic acids. The HTEC were permissive for influenza A and B virus replication. Following infection, a subset of cells, mostly ciliated cells, underwent apoptosis while others remained intact despite being positive for IV nucleoprotein. H3N2 virus antigen colocalized with non-ciliated cells while H1N1 virus antigen was mostly associated with ciliated cells. Exposure of HTECs to IVs triggers an early proinflammatory response that fluctuates between viruses. The H3N2 IV induces an early response that persists, whereas pH1N1 induces a primarily late response in HTECs. Our results implicated HTEC as a site for IV replication. The HTEC differentiated system provides a valuable in vitro model for studying cellular tropism, infectivity, cytokine responses and the pathogenesis of IVs. IMPORTANCETo develop an effective intervention against influenza, it is important to identify host factors affecting transmission, pathogenesis, and immune response. Tonsils are lymphoepithelial organs characterized by infiltration of B and T lymphocytes into the squamous epithelium of tonsillar crypts, beneath which germinal centers play key roles in antigen processing and immune response. The heterogenicity of HTECs as well as the sialic acid distributions supports the replication of IVs and may play a role in IV adaptation. Furthermore, Tonsillectomy is a surgical procedure in which tonsils are fully removed from the human throat and may contribute to the diverse outcomes among infected individuals.