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• W2007032789 abstract "The novel pandemic influenza H1N1 (H1N1pdm) virus of swine origin causes mild disease but occasionally leads to acute respiratory distress syndrome and death. It is important to understand the pathogenesis of this new disease in humans. We compared the virus tropism and host-responses elicited by pandemic H1N1pdm and seasonal H1N1 influenza viruses in ex vivo cultures of human conjunctiva, nasopharynx, bronchus, and lung, as well as in vitro cultures of human nasopharyngeal, bronchial, and alveolar epithelial cells. We found comparable replication and host-responses in seasonal and pandemic H1N1 viruses. However, pandemic H1N1pdm virus differs from seasonal H1N1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in its receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection with H1N1pdm. A greater viral replication competence in bronchial epithelium at 33°C may also contribute to the slight increase in virulence of the pandemic influenza virus. In contrast with highly pathogenic influenza H5N1 virus, pandemic H1N1pdm does not differ from seasonal influenza virus in its intrinsic capacity for cytokine dysregulation. Collectively, these results suggest that pandemic H1N1pdm virus differs in modest but subtle ways from seasonal H1N1 virus in its intrinsic virulence for humans, which is in accord with the epidemiology of the pandemic to date. These findings are therefore relevant for understanding transmission and therapy. The novel pandemic influenza H1N1 (H1N1pdm) virus of swine origin causes mild disease but occasionally leads to acute respiratory distress syndrome and death. It is important to understand the pathogenesis of this new disease in humans. We compared the virus tropism and host-responses elicited by pandemic H1N1pdm and seasonal H1N1 influenza viruses in ex vivo cultures of human conjunctiva, nasopharynx, bronchus, and lung, as well as in vitro cultures of human nasopharyngeal, bronchial, and alveolar epithelial cells. We found comparable replication and host-responses in seasonal and pandemic H1N1 viruses. However, pandemic H1N1pdm virus differs from seasonal H1N1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in its receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection with H1N1pdm. A greater viral replication competence in bronchial epithelium at 33°C may also contribute to the slight increase in virulence of the pandemic influenza virus. In contrast with highly pathogenic influenza H5N1 virus, pandemic H1N1pdm does not differ from seasonal influenza virus in its intrinsic capacity for cytokine dysregulation. Collectively, these results suggest that pandemic H1N1pdm virus differs in modest but subtle ways from seasonal H1N1 virus in its intrinsic virulence for humans, which is in accord with the epidemiology of the pandemic to date. These findings are therefore relevant for understanding transmission and therapy. The recent pandemic caused by a novel H1N1 virus (H1N1pdm) arose from the reassortment of three or more viruses of swine origin, including the North American triple reassortant H3N2 and H1N2 viruses, classical swine H1N1, and European swine H1N1/H3N2 viruses.1Smith GJ Vijaykrishna D Bahl J Lycett SJ Worobey M Pybus OG Ma SK Cheung CL Raghwani J Bhatt S Peiris JS Guan Y Rambaut A Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic.Nature. 2009; 459: 1122-1125Crossref PubMed Scopus (1611) Google Scholar, 2Neumann G Noda T Kawaoka Y Emergence and pandemic potential of swine-origin H1N1 influenza virus.Nature. 2009; 459: 931-939Crossref PubMed Scopus (1213) Google Scholar Most patients with pandemic H1N1pdm have mild influenza-like illness, but a minority of patients develop a primary viral pneumonia, sometimes leading to acute respiratory distress syndrome and death.3Dawood FS Jain S Finelli L Shaw MW Lindstrom S Garten RJ Gubareva LV Xu X Bridges CB Uyeki TM Emergence of a novel swine-origin influenza A (H1N1) virus in humans.N Engl J Med. 2009; 360: 2605-2615Crossref PubMed Scopus (2687) Google Scholar, 4Kumar A Zarychanski R Pinto R Cook DJ Marshall J Lacroix J Stelfox T Bagshaw S Choong K Lamontagne F Turgeon AF Lapinsky S Ahern SP Smith O Siddiqui F Jouvet P Khwaja K McIntyre L Menon K Hutchison J Hornstein D Joffe A Lauzier F Singh J Karachi T Wiebe K Olafson K Ramsey C Sharma S Dodek P Meade M Hall R Fowler R Critically ill patients with 2009 influenza A(H1N1) infection in Canada.JAMA. 2009; 302: 1872-1879Crossref PubMed Scopus (1127) Google Scholar Many, but not all, patients with severe disease have pregnancy, obesity, or underlying disease states such as asthma, obstructive airways disease, diabetes, and chronic cardiovascular or renal disease. The disease associated with H1N1pdm so far appears to be comparable with that of seasonal influenza and less severe than that seen in the 1918 pandemic or in zoonotic disease caused by highly pathogenic avian influenza (HPAI) H5N1. However, unlike seasonal influenza where morbidity and mortality are mainly seen in the elderly, pandemic H1N1pdm appears to spare this age-group, possibly because of the presence of cross-neutralizing antibody generated by prior repeated seasonal H1N1 infection.5Hancock K Veguilla V Lu X Zhong W Butler EN Sun H Liu F Dong L Devos JR Gargiullo PM Brammer TL Cox NJ Tumpey TM Katz JM Cross-reactive antibody responses to the 2009 pandemic H1N1 influenza virus.N Engl J Med. 2009; 361: 1945-1952Crossref PubMed Scopus (1097) Google Scholar In California, the median age of all cases was 17 years, of hospitalized cases 26 years, and for fatal cases was 45 years. It is therefore important to understand how the pathogenesis and tissue tropism of H1N1pdm virus in humans differs from seasonal influenza viruses. However, there is so far limited information in this regard. The H1N1pdm virus does not possess the genetic motifs of virulence associated with either the HPAI H5N1 or 1918 H1N1 viruses.2Neumann G Noda T Kawaoka Y Emergence and pandemic potential of swine-origin H1N1 influenza virus.Nature. 2009; 459: 931-939Crossref PubMed Scopus (1213) Google Scholar In experimentally infected ferrets, macaques, and mice, H1N1pdm causes moderately more severe illness compared with seasonal influenza although being much less virulent than HPAI H5N1 or the 1918 pandemic Spanish flu virus.6Itoh Y Shinya K Kiso M Watanabe T Sakoda Y Hatta M Muramoto Y Tamura D Sakai-Tagawa Y Noda T Sakabe S Imai M Hatta Y Watanabe S Li C Yamada S Fujii K Murakami S Imai H Kakugawa S Ito M Takano R Iwatsuki-Horimoto K Shimojima M Horimoto T Goto H Takahashi K Makino A Ishigaki H Nakayama M Okamatsu M Warshauer D Shult PA Saito R Suzuki H Furuta Y Yamashita M Mitamura K Nakano K Nakamura M Brockman-Schneider R Mitamura H Yamazaki M Sugaya N Suresh M Ozawa M Neumann G Gern J Kida H Ogasawara K Kawaoka Y In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.Nature. 2009; 460: 1021-1025PubMed Google Scholar, 7Munster VJ de Wit E van den Brand JM Herfst S Schrauwen EJ Bestebroer TM van de Vijver D Boucher CA Koopmans M Rimmelzwaan GF Kuiken T Osterhaus AD Fouchier RA Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets.Science. 2009; 325: 481-483PubMed Google Scholar, 8Maines TR Jayaraman A Belser JA Wadford DA Pappas C Zeng H Gustin KM Pearce MB Viswanathan K Shriver ZH Raman R Cox NJ Sasisekharan R Katz JM Tumpey TM Transmission and Pathogenesis of Swine-Origin 2009 A(H1N1) Influenza Viruses in Ferrets and Mice.Science. 2009; 325: 484-487Crossref PubMed Scopus (557) Google Scholar In these animal models, H1N1pdm virus was able to infect the alveolar epithelium more readily than seasonal H1N1 virus, but whether this holds true for humans is not known.7Munster VJ de Wit E van den Brand JM Herfst S Schrauwen EJ Bestebroer TM van de Vijver D Boucher CA Koopmans M Rimmelzwaan GF Kuiken T Osterhaus AD Fouchier RA Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets.Science. 2009; 325: 481-483PubMed Google Scholar Though H1N1pdm was initially reported to have a predominantly α2-6 sialic acid (Sia) receptor binding preference8Maines TR Jayaraman A Belser JA Wadford DA Pappas C Zeng H Gustin KM Pearce MB Viswanathan K Shriver ZH Raman R Cox NJ Sasisekharan R Katz JM Tumpey TM Transmission and Pathogenesis of Swine-Origin 2009 A(H1N1) Influenza Viruses in Ferrets and Mice.Science. 2009; 325: 484-487Crossref PubMed Scopus (557) Google Scholar similar to human seasonal influenza viruses, recent glycan array data indicates that there is binding to both “human” Sia α2-6 and “avian” Sia α2-3.9Childs RA Palma AS Wharton S Matrosovich T Liu Y Chai W Campanero-Rhodes MA Zhang Y Eickmann M Kiso M Hay A Matrosovich M Feizi T Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray.Nature Biotechnol. 2009; 27: 797-799Crossref Scopus (281) Google Scholar H1N1pdm virus differs from seasonal influenza viruses in their ability to infect and cause illness in mice without prior adaptation. As the mouse respiratory tract has a predominance of Sia α2-3, rather than Sia α2-6 receptors, these findings support the contention that H1N1pdm viruses have a broader Sia receptor binding profile.8Maines TR Jayaraman A Belser JA Wadford DA Pappas C Zeng H Gustin KM Pearce MB Viswanathan K Shriver ZH Raman R Cox NJ Sasisekharan R Katz JM Tumpey TM Transmission and Pathogenesis of Swine-Origin 2009 A(H1N1) Influenza Viruses in Ferrets and Mice.Science. 2009; 325: 484-487Crossref PubMed Scopus (557) Google Scholar Taken together, these observations suggest that H1N1pdm virus differs in subtle but important ways from seasonal influenza viruses in receptor usage and tissue tropism, and this may be important in its pathogenesis and transmission. Cytokine dysregulation is believed to contribute to the pathogenesis of human disease caused by HPAI H5N1 as well as the 1918 pandemic H1N1 viruses.10Chan MC Cheung CY Chui WH Tsao SW Nicholls JM Chan YO Chan RW Long HT Poon LL Guan Y Peiris JS Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells.Respir Res. 2005; 6: 135Crossref PubMed Scopus (419) Google Scholar, 11Cheung CY Poon LL Lau AS Luk W Lau YL Shortridge KF Gordon S Guan Y Peiris JS Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?.Lancet. 2002; 360: 1831-1837Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar, 12Kash JC Tumpey TM Proll SC Carter V Perwitasari O Thomas MJ Basler CF Palese P Taubenberger JK Garcia-Sastre A Swayne DE Katze MG Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus.Nature. 2006; 443: 578-581Crossref PubMed Scopus (466) Google Scholar, 13Perrone LA Plowden JK Garcia-Sastre A Katz JM Tumpey TM H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice.PLoS Pathog. 2008; 4: e1000115Crossref PubMed Scopus (509) Google Scholar, 14Chan MC Chan RW Yu WC Ho CC Chui WH Lo CK Yuen KM Guan Y Nicholls JM Peiris JM Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells.Respir Res. 2009; 10: 102Crossref PubMed Scopus (87) Google Scholar It is not known whether the H1N1pdm virus differs from seasonal influenza in the induction of proinflammatory host responses in human tissues. The lungs of H1N1pdm-infected mice had a markedly different cytokine profile when compared with seasonal influenza infected animals with elevated levels of interleukin (IL)-4, IL-10, and interferon (IFN)-γ. The lungs of H1N1pdm-infected macaques also had higher levels of chemokines MCP-1, MIP-1α, IL-6, and IL-18.6Itoh Y Shinya K Kiso M Watanabe T Sakoda Y Hatta M Muramoto Y Tamura D Sakai-Tagawa Y Noda T Sakabe S Imai M Hatta Y Watanabe S Li C Yamada S Fujii K Murakami S Imai H Kakugawa S Ito M Takano R Iwatsuki-Horimoto K Shimojima M Horimoto T Goto H Takahashi K Makino A Ishigaki H Nakayama M Okamatsu M Warshauer D Shult PA Saito R Suzuki H Furuta Y Yamashita M Mitamura K Nakano K Nakamura M Brockman-Schneider R Mitamura H Yamazaki M Sugaya N Suresh M Ozawa M Neumann G Gern J Kida H Ogasawara K Kawaoka Y In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.Nature. 2009; 460: 1021-1025PubMed Google Scholar However, it is not known whether these host responses simply reflect the greater or more extensive replication of the H1N1pdm virus in the lung when compared with seasonal influenza viruses or are attributable to intrinsic differences in the virus itself being able to induce a more potent innate host response as occurs with the highly pathogenic avian influenza H5N1 virus. When primary human cells (macrophages and type I-like pneumocytes) are infected with seasonal and HPAI H5N1 influenza viruses of comparable infectious titers, the HPAI H5N1 viruses differentially hyperinduce a range of proinflammatory responses over a single virus replication cycle.10Chan MC Cheung CY Chui WH Tsao SW Nicholls JM Chan YO Chan RW Long HT Poon LL Guan Y Peiris JS Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells.Respir Res. 2005; 6: 135Crossref PubMed Scopus (419) Google Scholar, 11Cheung CY Poon LL Lau AS Luk W Lau YL Shortridge KF Gordon S Guan Y Peiris JS Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?.Lancet. 2002; 360: 1831-1837Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar, 14Chan MC Chan RW Yu WC Ho CC Chui WH Lo CK Yuen KM Guan Y Nicholls JM Peiris JM Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells.Respir Res. 2009; 10: 102Crossref PubMed Scopus (87) Google Scholar Thus it is clear that the H5N1 virus has inherent properties that lead to an exaggerated innate immune response. It is relevant to use a similar approach to investigate the host innate immune responses induced by pandemic H1N1pdm compared with that of seasonal influenza H1N1 virus in primary human respiratory epithelium. We have previously used ex vivo cultures of nasopharynx, tonsillar tissue, and lung for investigating virus tropism.15Nicholls JM Chan MC Chan WY Wong HK Cheung CY Kwong DL Wong MP Chui WH Poon LL Tsao SW Guan Y Peiris JS Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract.Nat Med. 2007; 13: 147-149Crossref PubMed Scopus (281) Google Scholar We have also established in vitro cultures of polarized primary human respiratory epithelial cells, including type I–like alveolar epithelial cells, nasopharyngeal epithelial cells, and differentiated bronchial epithelial cells for investigating tissue tropism and innate immune host responses elicited by influenza viruses.10Chan MC Cheung CY Chui WH Tsao SW Nicholls JM Chan YO Chan RW Long HT Poon LL Guan Y Peiris JS Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells.Respir Res. 2005; 6: 135Crossref PubMed Scopus (419) Google Scholar, 14Chan MC Chan RW Yu WC Ho CC Chui WH Lo CK Yuen KM Guan Y Nicholls JM Peiris JM Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells.Respir Res. 2009; 10: 102Crossref PubMed Scopus (87) Google Scholar, 15Nicholls JM Chan MC Chan WY Wong HK Cheung CY Kwong DL Wong MP Chui WH Poon LL Tsao SW Guan Y Peiris JS Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract.Nat Med. 2007; 13: 147-149Crossref PubMed Scopus (281) Google Scholar These in vitro cultures of bronchial epithelium differentiated at an air–liquid interface (ALI) provide a good representation of the human bronchial epithelium and have a ciliated epithelium as well as mucus producing goblet cells. We have also recently established ex vivo tissue culture models of human conjunctival epithelium. We now use these ex vivo human tissue cultures as well as the primary human respiratory epithelial cell cultures to compare the virus replication competence, cell tropism, and host innate immune responses of the pandemic H1N1pdm virus with that of seasonal influenza H1N1 viruses and, where relevant, avian HPAI H5N1 and H7N7 viruses. We demonstrate that whereas seasonal H1N1 and pandemic H1N1pdm viruses replicate comparably in ex vivo cultures of human nasopharynx and lung tissues, the human conjunctiva is preferentially infected by H1N1pdm rather than seasonal influenza H1N1 or H3N2 viruses. Pandemic H1N1pdm replicates more efficiently than seasonal H1N1 virus in differentiated bronchial epithelial cells in vitro at 33°C, but the two viruses replicate comparably at 37°C. We also demonstrate that the pandemic H1N1pdm virus does not differ from the human seasonal influenza viruses in their ability to induce proinflammatory cytokines and therefore does not appear to have the same potential to induce cytokine dysregulation as that manifested by HPAI H5N1 or the 1918 H1N1 virus. The viruses used in these studies were the pandemic virus A/HongKong/415742/09 (H1N1pdm), seasonal viruses A/HongKong/54/98 (H1N1), A/HongKong/403721/2009 (H1N1) and A/Oklahoma/1992/05 (H3N2), and two avian influenza viruses isolated from humans, A/Vietnam/3046/04 (H5N1) and A/Netherland/33/03 (H7N7). A/Oklahoma/1992/05 (H3N2) was chosen because of the availability of glycan array data demonstrating a restricted Sia α2-6Gal–binding profile.16Kumari K Gulati S Smith DF Gulati U Cummings RD Air GM Receptor binding specificity of recent human H3N2 influenza viruses.Virol J. 2007; 4: 42Crossref PubMed Scopus (133) Google Scholar The viruses were initially isolated and passaged in Madin-Darby canine kidney (MDCK) cells. The virus stock was aliquoted and then titrated to determine tissue culture infection dose 50% (TCID50) in MDCK cells. The experiments were performed in a Bio-safety level 3 (BSL-3) facility at the Department of Microbiology, The University of Hong Kong. Table 1 summarizes all of the influenza A viruses used in this study and their abbreviations.Table 1List of Influenza A Viruses Used in this StudySubtypeNameAbbreviationH1N1A/Hong Kong/54/1998HK98/H1N1H1N1A/Hong Kong/463721/2009HK09/H1N1H1N1A/Hong Kong/415742/2009HK09/H1N1pdmH3N2A/Oklahoma/1992/2005OK05/H3N2H7N7A/Netherland/33/2003NL03/H7N7H5N1A/Vietnam/3046/2004VN04/H5N1 Open table in a new tab Primary human alveolar type I–like pneumocytes, well-differentiated bronchial epithelial cells and nasopharyngeal epithelial cells were derived and cultured as previously described10Chan MC Cheung CY Chui WH Tsao SW Nicholls JM Chan YO Chan RW Long HT Poon LL Guan Y Peiris JS Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells.Respir Res. 2005; 6: 135Crossref PubMed Scopus (419) Google Scholar, 14Chan MC Chan RW Yu WC Ho CC Chui WH Lo CK Yuen KM Guan Y Nicholls JM Peiris JM Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells.Respir Res. 2009; 10: 102Crossref PubMed Scopus (87) Google Scholar, 15Nicholls JM Chan MC Chan WY Wong HK Cheung CY Kwong DL Wong MP Chui WH Poon LL Tsao SW Guan Y Peiris JS Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract.Nat Med. 2007; 13: 147-149Crossref PubMed Scopus (281) Google Scholar, 17Chan RW Chan MC Wong AC Karamanska R Dell A Haslam SM Sihoe AD Chui WH Triana-Baltzer G Li Q Peiris JS Fang F Nicholls JM DAS181 inhibits H5N1 influenza virus infection of human lung tissues.Antimicrob Agents Chemother. 2009; 53: 3935-3941Crossref PubMed Scopus (59) Google Scholar, 18Gray TE Guzman K Davis CW Abdullah LH Nettesheim P Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells.Am J Respir Cell Mol Biol. 1996; 14: 104-112Crossref PubMed Scopus (412) Google Scholar, 19Zhen G Park SW Nguyenvu LT Rodriguez MW Barbeau R Paquet AC Erle DJ IL-13 and epidermal growth factor receptor have critical but distinct roles in epithelial cell mucin production.Am J Respir Cell Mol Biol. 2007; 36: 244-253Crossref PubMed Scopus (202) Google Scholar, 20Tsao SW Wang X Liu Y Cheung YC Feng H Zheng Z Wong N Yuen PW Lo AK Wong YC Huang DP Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes.Biochim Biophys Acta. 2002; 1590: 150-158Crossref PubMed Scopus (160) Google Scholar with modifications. Primary type I–like pneumocytes were isolated using human nonmalignant lung tissue obtained from patients undergoing lung resection in the Department of Cardiothoracic Surgery, Queen Mary Hospital, Hong Kong SAR, under a study approved by the Institutional Review Board of the University of Hong Kong and Hospital Authority Hong Kong West Cluster, and written informed consent was provided by each patient. Briefly, after removing visible bronchi, the lung tissue was minced into pieces of >0.5 mm thickness using a tissue chopper and washed with BSS containing Hanks balanced salt solution (Gibco, Grand Island, NY) with 0.7 mmol/L sodium bicarbonate (Gibco) at pH 7.4 for 3 times to partially remove macrophages and blood cells. The tissue was digested using a combination of 0.5% trypsin (GIBCO BRL, Gaithersburg, MD) and 4 U/ml elastase (Worthington Biochemical Corporation, Lakewood, NJ) for 40 minutes at 37°C in a shaking water-bath. The digestion was stopped by adding DMEM/F12 medium (Gibco) with 40% FBS in and DNase I (350 U/ml) (Sigma-Aldrich, St Louis, MO). Cell clumps were dispersed by repeatedly pipetting the cell suspension for 10 minutes. A disposable cell strainer (gauze size of 50 μm; BD Science, Palto-Alto, CA) was used to separate large undigested tissue fragments. The single cell suspension in the flow-through was centrifuged and resuspended in a 1:1 mixture of DMEM/F12 medium and small airway basal medium (Lonza, Walkersville, MD) supplemented with 0.5 ng/ml epidermal growth factor (hEGF), 500 ng/ml epinephrine, 10 μg/ml transferrin, 5 μg/ml insulin, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine, 0.5 μg/ml hydrocortisone, 30 μg/ml bovine pituitary extract, and 0.5 mg/ml BSA together with 5% FBS and 350 U/ml DNase I. The cell suspension was plated on tissue culture–grade plastic flask (Corning Inc., Corning, NY) and incubated in a 37°C water-jacketed incubator with 5% CO2 supply for 90 minutes. The nonadherent cells were layered on a discontinuous cold Percoll density gradient (densities 1.089 and 1.040 g/ml) and centrifuged at 25g for 20 minutes without brake. The cell layer at the interface of the two gradients was collected and washed four times with BSS to remove the Percoll. The cell suspension was incubated with magnetic beads coated with anti-CD14 antibodies at room temperature for 20 minutes under constant mixing. After the removal of the beads using a magnet (MACS CD14 MicroBeads, Miltenyi Biotech GmbH, Gladbach, Germany), cell viability was assessed by trypan-blue exclusion. The purified pneumocyte suspension was resuspended in small airway growth medium (Lonza) supplemented with 1% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin, and plated at a cell density of 3 × 105 cells/cm2. The cells were maintained in a humidified atmosphere (5% CO2, 37°C) under liquid-covered conditions, and growth medium was changed daily starting from 60 hours after plating the cells. When the cell layer approached 75% confluence, the pneumocytes were detached Hanks buffered saline solution trypsin/EDTA and subcultured for the experiments. Human bronchial epithelial (NHBE) cells were purchased as cryopreserved vials (Lonza). NHBE cells were plated into a T175 tissue culture grade culture flask in a density of 500 cells/cm2 for cell proliferation. Bronchial epithelial basal medium (Lonza Walkersville, Inc.) was supplemented with growth factor and hormones as stated in the suppliers instructions. After subculture, they were plated to a cell density of 2.5 × 105 cells/cm2 on human collagen IV (BD Science)–coated transwell inserts (Corning). Bronchial epithelial growth medium (Lonza Walkersville, Inc.) medium was supplemented with the retinoic acid concentration adjusted to 10−7 mol/L. Medium was changed every 48 hours until the cell layer reached confluence. An ALI was then established by removing the culture medium from the apical compartment. Thereafter, medium was changed in the basolateral compartment every 48 hours until day 21 of ALI culture. The apical compartment was gently washed with phosphate-buffered saline (PBS) once a week to remove accumulated mucus and debris. The transepithelial resistance was measured by epithelial voltohmeter (World Precision Instruments, Sarasota, Fla.). At day 21 of ALI culture, the NHBE cells became well differentiated and ready for use. The transwell cultures were collected and fixed with 10% formalin, paraffin embedded, and then cross-sectioned for histological examination. Slides were stained using hematoxylin-eosin. Ciliated cells were further identified by FITC-conjugated β-tubulin antibody (Sigma, Saint Louis, MO) and goblet cells were identified by biotinylated MUC5AC antibody (Invitrogen, San Francisco, CA). The nasopharyngeal biopsy was cut into 2- to 3-mm fragments and placed onto a 6-cm-sized Falcon culture dish (BD Biosciences, Palo Alto, CA) containing 2 ml of RPMI 1640 culture medium (Gibco) supplemented with 1% dialyzed fetal bovine serum (FBS), 0.25 μmol/L transferrin, 3.32 nmol/L hEGF, 0.7 μmol/L insulin, 0.5 μmol/L phosphoethanolamine, 0.5 μmol/L ethanolamine, 10 nmol/L tiodo-L-thyronine, 2 μmol/L hydrocortisone, 2.7 μmol/L L-epinephrine, 2 mmol/L l-glutamine, 0.03 μmol/L sodium selenium, 1 nmol/L molybdic acid, 0.5 nmol/L stannous chloride, and 0.75 nmol/L nickel sulfate. The medium had 0.4 mmol/L calcium, which facilitated the attachment of explants to the culture surfaces. After 7 to 10 days of culture, the proliferative epithelial outgrowth from the explants were trypsinized and propagated at a splitting-ratio of 3 in a 1:1 mixture of Defined Keratinocyte-SFM (Gibco) and EpiLife medium with full supplements (Sigma-Aldrich). The cells were maintained at 37°C with 5% CO2 in a water-jacketed incubator. The calcium concentration in this mixed medium formulation was below 0.08 mmol/L to stimulate the proliferation of the primary nasopharyngeal epithelial cells and suppress the growth of contaminating fibroblasts. Fresh conjunctiva tissues were obtained from 20 individuals who were undergoing excision for pterygium during surgical management. Biopsies of nasopharyngeal tissue (n = 6) were obtained from patients undergoing elective nasopharyngoscopy as detailed earlier.15Nicholls JM Chan MC Chan WY Wong HK Cheung CY Kwong DL Wong MP Chui WH Poon LL Tsao SW Guan Y Peiris JS Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract.Nat Med. 2007; 13: 147-149Crossref PubMed Scopus (281) Google Scholar Bronchi (n = 18) and lung tissues (n = 18) were obtained from lung carcinoma patients having surgical resection of lung tissue. The biopsies or tissue fragments of normal nonmalignant tissue that was excess to the requirements of clinical diagnosis were used. All of the studies were approved by the Institutional Review Board of the University of Hong Kong and Hospital Authority Hong Kong West Cluster, and a written informed consent was provided by each patients. The lung tissue fragments were placed into culture medium (F-12K nutrient mixture with l-glutamine, and antibiotics) in 24-well tissue culture plates incubated at 37°C for viral infection experiments. The conjunctival, nasopharyngeal, and bronchial biopsies or tissue fragments were placed into culture medium (F-12K nutrient mixture with l-glutamine, and antibiotics) incubated at 33°C (except the bronchial biopsy, which was incubated at 37°C) with a sterile surgical pathology sponge to establish an ALI condition in 24-well culture plates for both conjunctiva, nasopharynx and bronchi ex vivo culture (Corning, New York, NY) for viral infection experiments. The experimental conditions and multiplicity of infection (MOI) used in the ex vivo and in vitro experiments is summarized in Table 2.Table 2Summary Table Showing the Experimental Conditions, MOIs, and Virus Titer Used in the ex Vivo and in Vitro ExperimentsExperiment modelRegionIncubation temperatureMOIEx vivoConjunctiva*Conjunctiva tissues were infected with all the viruses listed in Table 1, while the other respiratory tissue and epithelial cells were infected with HK98/H1N1 and HK09/H1N1pdm.33°CUDNasopharynx33°CUDBronchi37°CUDLung37°CUDIn vitroNasopharyngeal epithelial cells33°C and 37°C0.01†MOI used for replication kinetics study and and 2‡MOI used for cytokine study. The MOI used in the ex vivo model cannot be determined (UD). Influenza viruses were used at a titer of 1 × 106 50% tissue culture infectious doses (TCID50)/ml (a similar titer as used previously15,21) for infecting the ex vivo cultures.Bronchial epithelial cells33°C and 37°C0.01†MOI used for replication kinetics study and and 2‡MOI used for cytokine study. The MOI used in the ex vivo model cannot be determined (UD). Influenza viruses were used at a titer of 1 × 106 50% tissue culture infectious doses (TCID50)/ml (a similar titer as used previously15,21) for infecting the ex vivo cultures.Type I-like" @default.
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• W2007032789 title "Tropism and Innate Host Responses of the 2009 Pandemic H1N1 Influenza Virus in ex Vivo and in Vitro Cultures of Human Conjunctiva and Respiratory Tract" @default.
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• W2007032789 doi "https://doi.org/10.2353/ajpath.2010.091087" @default.
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