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Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

2016; Elsevier BV; Volume: 138; Issue: 1 Linguagem: Inglês

10.1016/j.jaci.2015.11.016

ISSN

1097-6825

Autores

Yan Yan, Kai Sen Tan, Chunwei Li, Thai Tran, Siew Shuen Chao, Richard J. Sugrue, Li Shi, Vincent Chow, De Yun Wang,

Tópico(s)

Sinusitis and nasal conditions

Resumo

Nasal epithelium is the first line of mechanical and immunologic defense in the upper respiratory tract.1Yan Y. Gordon W.M. Wang D.Y. Nasal epithelial repair and remodeling in physical injury, infection, and inflammatory diseases.Curr Opin Otolaryngol Head Neck Surg. 2013; 21: 263-270Crossref PubMed Scopus (42) Google Scholar Upper respiratory tract infections, including the common cold, influenza, and acute rhinosinusitis, are very common viral diseases affecting millions of persons annually.2Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European position paper on rhinosinusitis and nasal polyps 2012.Rhinol Suppl. 2012; 50: 1-298Crossref Google Scholar Airway epithelial cells (AECs) are the primary target cells of respiratory viruses,3Braciale T.J. Sun J. Kim T.S. Regulating the adaptive immune response to respiratory virus infection.Nat Rev Immunol. 2012; 12: 295-305Crossref PubMed Scopus (252) Google Scholar such as rhinovirus,E1Othumpangat S. Regier M. Piedimonte G. Nerve growth factor modulates human rhinovirus infection in airway epithelial cells by controlling ICAM-1 expression.Am J Physiol Lung Cell Mol Physiol. 2012; 302: L1057-L1066Crossref PubMed Scopus (25) Google Scholar influenza A virus (IAV),E2Kolesnikova L. Heck S. Matrosovich T. Klenk H.D. Becker S. Matrosovich M. Influenza virus budding from the tips of cellular microvilli in differentiated human airway epithelial cells.J Gen Virol. 2013; 94: 971-976Crossref PubMed Scopus (18) Google Scholar parainfluenza virus,E3Zhang L. Bukreyev A. Thompson C.I. Watson B. Peeples M.E. Collins P.L. et al.Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium.J Virol. 2005; 79: 1113-1124Crossref PubMed Scopus (221) Google Scholar and respiratory syncytial virus.4Jumat M.R. Yan Y. Ravi L.I. Wong P.S. Huong T.N. Li C.W. et al.Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia.Virology. 2015; 484: 395-411Crossref PubMed Scopus (19) Google Scholar, E4Zhang L. Peeples M.E. Boucher R.C. Collins P.L. Pickles R.J. Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology.J Virol. 2002; 76: 5654-5666Crossref PubMed Scopus (417) Google Scholar However, the holistic understanding of the viral replication dynamics and immune responses generated in AECs per se at both early and late infection phases is incomplete.5Wang D.Y. Li Y. Yan Y. Li C. Shi L. Upper airway stem cells: understanding the nose and role for future cell therapy.Curr Allergy Asthma Rep. 2015; 15: 490Crossref PubMed Scopus (26) Google Scholar By using in vivoE6Watanabe T. Kiso M. Fukuyama S. Nakajima N. Imai M. Yamada S. et al.Characterization of H7N9 influenza A viruses isolated from humans.Nature. 2013; 501: 551-555Crossref PubMed Scopus (327) Google Scholar, E7Wang Z. Wan Y. Qiu C. Quinones-Parra S. Zhu Z. Loh L. et al.Recovery from severe H7N9 disease is associated with diverse response mechanisms dominated by CD8(+) T cells.Nat Commun. 2015; 6: 6833Crossref PubMed Scopus (184) Google Scholar and in vitroE5Sutejo R. Yeo D.S. Myaing M.Z. Hui C. Xia J. Ko D. et al.Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses.PLoS One. 2012; 7: e33732Crossref PubMed Scopus (50) Google Scholar, E8Wu D. Huang W. Wang Y. Guan W. Li R. Yang Z. et al.Gene silencing of beta-galactosamide alpha-2,6-sialyltransferase 1 inhibits human influenza virus infection of airway epithelial cells.BMC Microbiol. 2014; 14: 78Crossref PubMed Scopus (15) Google Scholar, E9Clay C.C. Reader J.R. Gerriets J.E. Wang T.T. Harrod K.S. Miller L.A. Enhanced viral replication and modulated innate immune responses in infant airway epithelium following H1N1 infection.J Virol. 2014; 88: 7412-7425Crossref PubMed Scopus (13) Google Scholar, E22Wu W. Zhang W. Duggan E.S. Booth J.L. Zou M.H. Metcalf J.P. RIG-I and TLR3 are both required for maximum interferon induction by influenza virus in human lung alveolar epithelial cells.Virology. 2015; 482: 181-188Crossref PubMed Scopus (61) Google Scholar models, the pathogenesis of human IAV has been partially recapitulated. However, most reported host defense mechanisms of the respiratory system to viral infections are based on studies of the lower respiratory tract, particularly initiation and development processes from innate to adaptive immunity generated mainly by the cells within the subepithelium, such as dendritic cells and macrophages.3Braciale T.J. Sun J. Kim T.S. Regulating the adaptive immune response to respiratory virus infection.Nat Rev Immunol. 2012; 12: 295-305Crossref PubMed Scopus (252) Google Scholar Less is known about the upper respiratory mucosa, the first portal of contact of respiratory infections.5Wang D.Y. Li Y. Yan Y. Li C. Shi L. Upper airway stem cells: understanding the nose and role for future cell therapy.Curr Allergy Asthma Rep. 2015; 15: 490Crossref PubMed Scopus (26) Google Scholar Therefore relevant clinical or preclinical studies are necessary to validate findings from in vitro models, virus-host relationships, and subsequent transmissibility between human subjects. We have established adult human nasal epithelial stem/progenitor cells (hNESPCs) from the nasal biopsy specimens of healthy subjects and patients with allergic rhinitis and rhinosinusitis with nasal polyps (for domain-specific review board and institutional review board, see the Methods section in this article's Online Repository at www.jacionline.org). These in vitro–passaged hNESPCs retain the potential of full differentiation into stratified, multilayered, mucociliary airway epithelium composed of both ciliated columnar cells and goblet cells through the air-liquid interface (ALI) culture.4Jumat M.R. Yan Y. Ravi L.I. Wong P.S. Huong T.N. Li C.W. et al.Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia.Virology. 2015; 484: 395-411Crossref PubMed Scopus (19) Google Scholar, 6Li Y.Y. Li C.W. Chao S.S. Yu F.G. Yu X.M. Liu J. et al.Impairment of cilia architecture and ciliogenesis in hyperplastic nasal epithelium from nasal polyps.J Allergy Clin Immunol. 2014; 134: 1282-1292Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 7Wang W. Yan Y. Li C.W. Xia H.M. Chao S.S. Wang D.Y. et al.Live human nasal epithelial cells (hNECs) on chip for in vitro testing of gaseous formaldehyde toxicity via airway delivery.Lab Chip. 2014; 14: 677-680Crossref PubMed Google Scholar, E10Kumar P.A. Hu Y. Yamamoto Y. Hoe N.B. Wei T.S. Mu D. et al.Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection.Cell. 2011; 147: 525-538Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, E11Zhao X.N. Yu F.G. Li C.W. Li Y.Y. Chao S.S. Loh W.S. et al.The use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro.Am J Rhinol Allergy. 2012; 26: 345-350Crossref PubMed Scopus (40) Google Scholar This in vitro model of differentiated human nasal epithelial cells (hNECs) is emerging as a versatile research tool for airway disease-associated studies,6Li Y.Y. Li C.W. Chao S.S. Yu F.G. Yu X.M. Liu J. et al.Impairment of cilia architecture and ciliogenesis in hyperplastic nasal epithelium from nasal polyps.J Allergy Clin Immunol. 2014; 134: 1282-1292Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 8Yu X.M. Li C.W. Chao S.S. Li Y.Y. Yan Y. Zhao X.N. et al.Reduced growth and proliferation dynamics of nasal epithelial stem/progenitor cells in nasal polyps in vitro.Sci Rep. 2014; 4: 4619Crossref PubMed Scopus (27) Google Scholar for respiratory physiology,7Wang W. Yan Y. Li C.W. Xia H.M. Chao S.S. Wang D.Y. et al.Live human nasal epithelial cells (hNECs) on chip for in vitro testing of gaseous formaldehyde toxicity via airway delivery.Lab Chip. 2014; 14: 677-680Crossref PubMed Google Scholar and, most recently, for the process of respiratory syncytial virus maturation and transmission in ciliated hNECs.4Jumat M.R. Yan Y. Ravi L.I. Wong P.S. Huong T.N. Li C.W. et al.Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia.Virology. 2015; 484: 395-411Crossref PubMed Scopus (19) Google Scholar The hNECs facilitate better control of infection compared with animal models in which the challenge virus often needs to be adapted to the animal while the virus might lose its intrinsic biological properties to cause human disease. This was the impetus for us to study viral replication, pathogen-sensing, innate immunity, inflammation, and apoptotic responses of nasal epithelium to IAV by using hNECs derived from multiple subjects. hNECs were derived from 9 subjects and infected with the human IAV Aichi/2/1968 H3N2 strain (for further information, see the Methods section and Table E1 in this article's Online Repository at www.jacionline.org). We designed a time-course study (see Fig E1 in this article's Online Repository at www.jacionline.org) that spanned the early stage (0-8 hpi, 1 viral replication cycle), intermediate stage (16 and 24 hpi, strong host cell responses after 1 viral replication cycle), and late stage (48 and 72 hpi) to mimic clinical upper respiratory tract infection (at a multiplicity of infection [MOI] of 0.1 and 35°C incubation after infection). First, we examined viral replication dynamics in infected hNECs. By using quantitative real-time PCR (qPCR), the transcripts of influenza matrix protein 1 (M1) and nonstructural protein 1 (NS1) were detectable as early as 4 hpi (61.8- and 91.1-fold increase vs mock infection control, respectively) in all 9 subject-derived hNECs, which increased exponentially and peaked at 48 and 72 hpi for NS1 and M1, respectively (median of 21,235- and 130,730-fold increase Fig 1, A and B). By using plaque assay, progeny viruses were detected from 16 hpi (median, 35,000 plaque-forming units [PFU]) onward, progressing up to 72 hpi (median, 157,500, 192,500, and 285,000 PFU at 24, 48, and 72 hpi, respectively; Fig 1, C). Furthermore, we observed interindividual variability in both viral gene transcription (maximum/minimum, 1,367-fold for M1 and 1,214-fold for NS1) and progeny virus generation (maximum/minimum, 23.9-fold at 72 hpi; see Fig E2, A-C, in this article's Online Repository at www.jacionline.org), suggesting varying susceptibility and sustainability of IAV infection in different subjects. By using Western blotting, mature viral proteins were visualized as early as 4 and 16 hpi for M1 and NS1, respectively, whereas mass viral protein production was observed after 16 hpi (Fig 1, D). Second, to elucidate the sequential and spatiotemporal reactions of pathogen sensors to respiratory viruses documented in different experimental models,E13Ivan F.X. Rajapakse J.C. Welsch R.E. Rozen S.G. Narasaraju T. Xiong G.M. et al.Differential pulmonary transcriptomic profiles in murine lungs infected with low and highly virulent influenza H3N2 viruses reveal dysregulation of TREM1 signaling, cytokines, and chemokines.Funct Integr Genomics. 2012; 12: 105-117Crossref PubMed Scopus (24) Google Scholar, E14Ivan F.X. Tan K.S. Phoon M.C. Engelward B.P. Welsch R.E. Rajapakse J.C. et al.Neutrophils infected with highly virulent influenza H3N2 virus exhibit augmented early cell death and rapid induction of type I interferon signaling pathways.Genomics. 2013; 101: 101-112Crossref PubMed Scopus (21) Google Scholar, E15Iwai A. Shiozaki T. Miyazaki T. Relevance of signaling molecules for apoptosis induction on influenza A virus replication.Biochem Biophys Res Commun. 2013; 441: 531-537Crossref PubMed Scopus (29) Google Scholar, E16Masaki T. Kojima T. Okabayashi T. Ogasawara N. Ohkuni T. Obata K. et al.A nuclear factor-kappaB signaling pathway via protein kinase C delta regulates replication of respiratory syncytial virus in polarized normal human nasal epithelial cells.Mol Biol Cell. 2011; 22: 2144-2156Crossref PubMed Scopus (63) Google Scholar, E17Lee S.M. Kok K.H. Jaume M. Cheung T.K. Yip T.F. Lai J.C. et al.Toll-like receptor 10 is involved in induction of innate immune responses to influenza virus infection.Proc Natl Acad Sci U S A. 2014; 111: 3793-3798Crossref PubMed Scopus (115) Google Scholar, E18Pothlichet J. Meunier I. Davis B.K. Ting J.P. Skamene E. von Messling V. et al.Type I IFN triggers RIG-I/TLR3/NLRP3-dependent inflammasome activation in influenza A virus infected cells.PLoS Pathog. 2013; 9: e1003256Crossref PubMed Scopus (174) Google Scholar, E19Seo S.H. Webster R.G. Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells.J Virol. 2002; 76: 1071-1076Crossref PubMed Google Scholar, E20Sun J. Madan R. Karp C.L. Braciale T.J. Effector T cells control lung inflammation during acute influenza virus infection by producing IL-10.Nat Med. 2009; 15: 277-284Crossref PubMed Scopus (458) Google Scholar, E5Sutejo R. Yeo D.S. Myaing M.Z. Hui C. Xia J. Ko D. et al.Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses.PLoS One. 2012; 7: e33732Crossref PubMed Scopus (50) Google Scholar, 9Garcia-Sastre A. Biron C.A. Type 1 interferons and the virus-host relationship: a lesson in detente.Science. 2006; 312: 879-882Crossref PubMed Scopus (707) Google Scholar we performed analyses of (1) IAV receptors possessing terminal α-2,6–linked or α-2,3–linked sialic acid residues (α-2,6-SA or α-2,3-SA) and the rhinovirus receptor intercellular adhesion molecule 1; (2) the extracellular receptor Toll-like receptor (TLR) 4, the orphan human receptor TLR10, and the cytosolic receptors TLR3, TLR7, TLR8, and TLR9 located in the endosome; (3) the cytoplasmic sensors retinoic acid–inducible gene 1 (RIG-I) and NOD-like receptor family, pyrin domain containing 3; and (4) cytoplasmic molecules for sensing, including interferon regulatory factor (IRF) 3 and IRF7 and IFN-β promoter stimulator 1. The tropism of human and avian IAV is an active research area. We found positive α-2,6-SA staining in 5 of 7 subjects and negative α-2,3-SA staining in 5 mock-infected subject–derived hNECs, with upregulation of α-2,6-SA in infected cells at 16 hpi (Fig 1, E). These results confirmed that hNECs express α-2,6-SAs but not α-2,3-SAs and that the viral receptors are recruited onto the cell membrane on infection, thus leading to entry of more viruses.10Brandenburg B. Zhuang X. Virus trafficking—learning from single-virus tracking.Nat Rev Microbiol. 2007; 5: 197-208Crossref PubMed Scopus (333) Google Scholar By using double immunofluorescence (IF), we stained single cells (on cytospin slides) and multilayered cells (on Transwell membranes; Transwell, Corning, NY) using viral nucleoprotein (NP) and cellular markers (Fig 1, F-I).E11Zhao X.N. Yu F.G. Li C.W. Li Y.Y. Chao S.S. Loh W.S. et al.The use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro.Am J Rhinol Allergy. 2012; 26: 345-350Crossref PubMed Scopus (40) Google Scholar, 6Li Y.Y. Li C.W. Chao S.S. Yu F.G. Yu X.M. Liu J. et al.Impairment of cilia architecture and ciliogenesis in hyperplastic nasal epithelium from nasal polyps.J Allergy Clin Immunol. 2014; 134: 1282-1292Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar We showed that both ciliated and goblet cells in hNECs are target cells of human H3N2 virus (Fig 1, G and H, and see Table E2 in this article's Online Repository at www.jacionline.org). Notably, the p63+ cells that reside at the bottom of multilayered hNECsE10Kumar P.A. Hu Y. Yamamoto Y. Hoe N.B. Wei T.S. Mu D. et al.Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection.Cell. 2011; 147: 525-538Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, E11Zhao X.N. Yu F.G. Li C.W. Li Y.Y. Chao S.S. Loh W.S. et al.The use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro.Am J Rhinol Allergy. 2012; 26: 345-350Crossref PubMed Scopus (40) Google Scholar were not infected (Fig 1, I, and see Table E2), whereas the p63-expressing hNESPCs were susceptible to H3N2 virus infection in a monolayer culture environment (see Fig E3 in this article's Online Repository at www.jacionline.org). This finding indicates the importance of mechanical arrangement wherein the virus could not infect its preferred host cell because of mechanical limitations. Third, through qPCR screening of 28 candidate genes and use of the Luminex/ELISA assay (of the 13 most reactive makers shortlisted from qPCR screening), we analyzed expression levels of candidate genes at both the intracellular mRNA and secreted protein levels (for details, see the Methods section in this article's Online Repository). Our study provides novel detailed analyses of the virus-host interactions between IAV and human nasal epithelium. We revealed the following: (1) TLR7 (see Fig E4, B, in this article's Online Repository at www.jacionline.org), IRF7 (see Fig E4, G), and RIG-I (see Fig E4, J) are important pathogen sensors against IAV, as indicated by their high expression among other sensors (Fig 2, A, and see Fig E4); (2) in addition to type I interferons, type III interferons (see Fig E5, A-E, in this article's Online Repository at www.jacionline.org) are highly inducible in innate immunity, as well as a few selected chemokines and transcription factors (Fig 2, A, and see Fig E5, F-J); (3) hNECs per se could express high levels of IL-1α, IL-1β, IL-6, IL-8, IL-10, and TNF-α and moderate levels of TGF-β on IAV infection (Fig 2, A, and see Fig E6 in this article's Online Repository at www.jacionline.org); (4) infected hNECs elicit similar immune responses comparable with known models but varied among subjects, suggesting retention of individual host characteristics (Fig 2, B and C); and (5) similar but weaker responses associated with pathogen sensing, innate immunity, and inflammation were also observed in H3N2-infected hNESPCs, which might imply less developed immune responses in their undifferentiated state (data not shown). Fourth, we quantitatively analyzed virus-induced cell death. There was a gradual decrease in cell viability, integrity, and survival rate (see Fig E7, A-C, in this article's Online Repository at www.jacionline.org). By using the LIVE/DEAD assay (Life Technologies, Grand Island, NY), reduced cell viability, increased dead cells (see Fig E7, D), and fragmented nuclei at late stages were observed (see Fig E7, E). Then we analyzed apoptotic responses by using the terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay (see Fig E7, F), protein expression/abundance of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase-3 bands (see Fig E8, A, in this article's Online Repository at www.jacionline.org), flow cytometry of recombinant Annexin V conjugated to fluorescein (fluorescein isothiocyanate [FITC]–labeled Annexin V) and red fluorescent propidium iodide (PI) staining (see Fig E8, B), and expression of apoptosis genes (see Fig E9 in this article's Online Repository at www.jacionline.org). Notably, 5 of 6 subjects exhibited apoptotic features to varying degrees and onsets, and one of them did not display signs of apoptosis (see sample H01 in Fig E10 in this article's Online Repository at www.jacionline.org). These data suggest that H3N2-induced apoptosis can occur in a subject-dependent manner and might determine the outcome of infection. Fifth, to further elucidate individual differences in immune responses, the 7 most reactive markers (ie, TLR7, IRF7, RIG-I, IFN-β, IL-28A, IL-29, and interferon-inducible protein 10; n = 9) were selected to elucidate their relationship with viral replication (n = 9) and apoptosis (by ratio of cleaved over pro-PARP and caspase-3; n = 5). These markers exhibited a similar trend after infection (see Fig E2, D), whereas cross-correlation between markers yielded correlation scores of greater than 0.8 (data not shown). We then generated a single-factored variable to represent the host immune and apoptotic responses and plotted by individual subjects at different time points to compare between viral titers and host responses (Fig 2, B) and host responses and apoptosis (Fig 2, C), respectively. The spread of the individual points at each time point further highlights the differential responses and cell death, although a general trend of the responses was evident. The differing levels of cell death might provide insights into the extent of IAV transmissibility in different subjects (see Figs E8 and E10). A positive correlation was noted between the protein quantity of dying (floating) cells and progeny virus titer at 16 and 24 hpi (data not shown). We hypothesize that subjects experiencing greater degrees of cell death in the upper respiratory tract can act as stronger IAV spreaders, especially in a closed environment. Because of the limited availability of hNECs derived from each specimen, analyses of hNECs from more subjects are warranted in the future. The availability of a cell-culture model system that permits surveillance of emerging viruses and characterization of interindividual host tropism closely mimicking clinical scenarios is clearly desirable.E23Deeks S. Drosten C. Picker L. Subbarao K. Suzich J. Roadblocks to translational challenges on viral pathogenesis.Nat Med. 2013; 19: 30-34Crossref PubMed Scopus (6) Google Scholar, E24Lakdawala S.S. Subbarao K. The ongoing battle against influenza: the challenge of flu transmission.Nat Med. 2012; 18: 1468-1470Crossref PubMed Scopus (18) Google Scholar Here we demonstrate that the ability to harvest and culture authentic hNECs from different subjects could provide a valuable platform for testing antivirals and other agents and might serve as an epidemiologic tool for assessing the susceptibility of populations in outbreak regions. hNESPCs were derived from 9 patients with septal deviation who underwent septal plastic surgery at the National University Hospital, Singapore. All subjects were free of symptoms of upper respiratory tract infection and had not used corticosteroids and antibiotics within 3 months before surgery. The medical backgrounds of the donors' samples are summarized in Table E1. Cell-culture methods have been described previously.E11Zhao X.N. Yu F.G. Li C.W. Li Y.Y. Chao S.S. Loh W.S. et al.The use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro.Am J Rhinol Allergy. 2012; 26: 345-350Crossref PubMed Scopus (40) Google Scholar, 6Li Y.Y. Li C.W. Chao S.S. Yu F.G. Yu X.M. Liu J. et al.Impairment of cilia architecture and ciliogenesis in hyperplastic nasal epithelium from nasal polyps.J Allergy Clin Immunol. 2014; 134: 1282-1292Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 7Wang W. Yan Y. Li C.W. Xia H.M. Chao S.S. Wang D.Y. et al.Live human nasal epithelial cells (hNECs) on chip for in vitro testing of gaseous formaldehyde toxicity via airway delivery.Lab Chip. 2014; 14: 677-680Crossref PubMed Google Scholar Fully differentiated hNECs, including beating ciliated cells and mucus-producing goblet cells, were obtained after 32 to 35 days of ALI culture. The hNECs were characterized by means of IF staining of ciliated and goblet cell markers (ie, mouse anti-human βIV-tubulin; clone ONS.1A6, ab11315; Abcam, Cambridge, Mass)E11Zhao X.N. Yu F.G. Li C.W. Li Y.Y. Chao S.S. Loh W.S. et al.The use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro.Am J Rhinol Allergy. 2012; 26: 345-350Crossref PubMed Scopus (40) Google Scholar, 6Li Y.Y. Li C.W. Chao S.S. Yu F.G. Yu X.M. Liu J. et al.Impairment of cilia architecture and ciliogenesis in hyperplastic nasal epithelium from nasal polyps.J Allergy Clin Immunol. 2014; 134: 1282-1292Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar and rabbit anti-human mucin5AC (sc-20118; Santa Cruz Biotechnology, Dallas, Tex).7Wang W. Yan Y. Li C.W. Xia H.M. Chao S.S. Wang D.Y. et al.Live human nasal epithelial cells (hNECs) on chip for in vitro testing of gaseous formaldehyde toxicity via airway delivery.Lab Chip. 2014; 14: 677-680Crossref PubMed Google Scholar We also demonstrated the 3-dimensional structure of in vitro–differentiated hNECs (ie, the ciliated columnar cells and mucus-producing goblet cells with mouse anti-human βIV-tubulin and rabbit anti-human mucin5AC antibodies costained with 4′-6-diamidino-2-phenylindole dihydrochloride [DAPI]), followed by Z-stack confocal imaging. To analyze the infection rate and tropism, we conducted double IF staining by using combinations of one viral marker, mouse anti-influenza NP antibody (clone IVF8, ab8261, Abcam), with 3 cellular markers, rabbit anti-human acetyl-α-tubulin (#5335; Cell Signaling, Boston, Mass), rabbit anti-human mucin5AC, and rabbit-anti-human p63 (ab124762, Abcam), respectively, on Transwell membranes and cytospin slides. The colocalization of βIV-tubulin and α-tubulin signals on the cilia structure was shown to be greater than 95%, as previously tested.6Li Y.Y. Li C.W. Chao S.S. Yu F.G. Yu X.M. Liu J. et al.Impairment of cilia architecture and ciliogenesis in hyperplastic nasal epithelium from nasal polyps.J Allergy Clin Immunol. 2014; 134: 1282-1292Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar The human IAV Aichi/2/1968 H3N2 strain was purchased from the American Type Culture Collection (Manassas, Va), propagated in eggs, and titrated by using the plaque assay with Madin-Darby canine kidney (MDCK; NBL-2) cells. The virus was thawed on ice and immediately diluted in 100 μL of B-ALI differentiation medium (Lonza, Walkersville, Md) at MOIs of 0.01, 0.1, and 1.0; inoculated into the apical chamber of Transwells; and incubated at 35°C for 1 hour in our initial experiments. B-ALI differentiation medium was added into the "mock infection control" well without the virus. Finally, an MOI of 0.1 was chosen for all the assays in terms of viral fitness and virus-induced cell death. For calculation of the MOI, the total cell number was derived by means of trypsinization of hNECs, and cells were counted with a hemocytometer. After inoculation of virus, the Transwells were transferred into a new 24-well plate, with the basal chamber of each well containing 350 μL of B-ALI differentiation medium. The H3N2- and mock-infected hNECs were then incubated at 35°C with 5% CO2 up to 72 hours after infection (hpi). The hNESPCs derived from the same and other subjects were also infected with H3N2 for comparison with the host responses of differentiated hNECs. The flow chart of this study is summarized in Fig E1. Three cellular markers (ie, rabbit anti-human p63 mAb [ab124762, Abcam], rabbit anti-human acetyl α-tubulin [#5335, Cell Signaling], and rabbit anti-human mucin5AC [sc-20118, Santa Cruz Biotechnology]) were used at dilutions of 1:200, 1:800, and 1:400 to stain for basal, ciliated, and goblet cells, respectively. Anti-influenza antibodies (ie, mouse anti-influenza A NP antibody [ab20343, Abcam] and rabbit anti-influenza NS1 antibody [PA5-32243, Thermo Fisher Scientific, Waltham, Mass]) were used at dilutions of 1:200 for IF staining to visualize viral replication in host cells; rabbit anti-influenza NS1 antibody and rabbit anti-influenza M1 antibody (PA5-32253, Thermo Fisher Scientific) were used at dilutions of 1:1000 for Western blotting to analyze viral protein expression. Alexa Fluor 488 (anti-rabbit) and Alexa Fluor 594 (anti-mouse)–labeled IgG (H+L) secondary antibodies (Life Technologies) were used at 1:500 dilution for IF staining. ProLong AntiFade mounting medium with DAPI (Life Technologies) was used to mount the cytospin slides and paraformaldehyde-fixed Transwell membranes. Two typical apoptosis markers were used to detect virus-induced apoptosis. Rabbit anti-PARP antibody (#9542, full-length, Cell Signaling) and rabbit anti–caspase-3 antibody (#9665, full-length, Cell Signaling) were used at 1:1000 dilution for Western blotting to detect the cleaved and pro-forms of PARP and Caspase-3 in both the adherent and floating (detached) hNECs after H3N2 infection. At serial time points, 100 μL of culture medium was added and incubated in the apical chamber at 35°C for 10 minutes to recover progeny viruses, together with the virus load control (aliquot of inoculated virus) was stored at −80°C until titration by using a plaque assay. MDCK cells at 85% to 95% confluence in 24-well plates were incubated with 100 μL of serial dilutions (from 10−1 to 10−4) of virus from infected hNECs at 35°C for 1 hour. The plates were rocked every 15 minutes to ensure equal distribution of virus. The inocula were removed and replaced with 1 mL of Avicel (FMC BioPolymer, Philadelphia, Pa) overlay to each well and incubated at 35°C with 5% CO2 for 65 to 72 hours. Avicel overlay was then removed, and cells were fixed with 4% formaldehyde in 1× PBS for 1 hour. Formaldehyde were removed, and cells were washed with 1× PBS. The fixed cells were stained with 1% crystal violet for 15 minutes and washed. PFU values were calculated as follows: Number of plaques × Dilution factor = Number of PFU per 100 μL. The final data were presented as PFU per 350 μL (sample collection volume). At each time point, single-cell suspensions (1-2 × 105 cells) were dissociated from Transwells by using 0.5× Trypsin/EDTA solution (Gibco, Carlsbad, Calif) at 37°C. The dissociation times were recorded for each time point to evaluate the integrity of multilayered hNECs af

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