Abstract
Dogs are susceptible to infectious diseases that occur primarily in the respiratory tract. The airway epithelium acts as a first line of defense and is constantly exposed to microorganisms present in the environment. Respiratory epithelial cells have recently gained wide use as a cell model for studying the pathogenesis of human, murine or swine respiratory pathogen infections. However, studies of the pathogenic mechanisms of canine pathogens have been hindered by the lack of reliable respiratory cell lines. Here, we cultured primary canine bronchiolar epithelial cells (CBECs), whose characteristics were confirmed by their expression of the epithelial cell-specific marker cytokeratin 18, and have provided protocols for their isolation and ex vivo expansion. Further, we established immortalized CBECs containing the human telomerase reverse transcriptase (hTERT) gene via transfection of primary CBECs with the recombinant plasmid pEGFP-hTERT. Immortalized bronchiolar epithelial cells (hTERT-CBECs) retain the morphological and functional features of primary CBECs, as indicated by reverse transcriptase polymerase chain reaction, proliferation assays, karyotype analysis, telomerase activity assay, and Western blotting, which demonstrate that hTERT-CBECs have higher telomerase activity, an extended proliferative lifespan, and a diploid complement of chromosomes, even after Passage 50. Moreover, this cell line is not transformed, as evaluated using soft agar assays and tumorigenicity analysis in nude mice, and can therefore be safely used in future studies. The isolation and establishment of stable hTERT-CBECs is of great importance for use as an in vitro model for mechanistic studies of canine pathogenic infections.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Explore related subjects
Discover the latest articles and news from researchers in related subjects, suggested using machine learning.References
Abraham G, Zizzadoro C, Kacza J, Ellenberger C, Abs V, Franke J, Schoon HA, Seeger J, Tesfaigzi Y, Ungemach FR (2011) Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions. BMC Vet Res 7:26. doi:10.1186/1746-6148- 7-26
Anno K, Hayashi A, Takahashi T, Mitsui Y, Ide T, Tahara H (2007) Telomerase activation induces elongation of the telomeric single-stranded overhang, but does not prevent chromosome aberrations in human vascular endothelial cells. Biochem Biophys Res Commun 353:926–932
Bals R, Hiemstra PS (2004) Innate immunity in the lung: how epithelial cells fight against respiratory pathogens. Eur Respir J 23:327–333
Blackburn EH (1991) Structure and function of telomeres. Nature 350:569–573
Calaf GM, Roy D (2008) Cancer genes induced by malathion and parathion in the presence of estrogen in breast cells. Int J Mol Med 21:261–268
Carvalho OV, Botelho CV, Ferreira CG, Scherer PO, Soares-Martins JA, Almeida MR, Silva Junior A (2012) Immunopathogenic and neurological mechanisms of canine distemper virus. Adv Virol 2012:163860
Chen Z, Xu P, Salyards GW, Harvey SB, Rada B, Fu ZF, He B (2012) Evaluating a parainfluenza virus 5-based vaccine in a host with pre-existing immunity against parainfluenza virus 5. PLoS One 7:e50144
Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S (1995) Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 85:2315–2320
Counter CM, Meyerson M, Eaton EN, Ellisen LW, Caddle SD, Haber DA, Weinberg RA (1998) Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase. Oncogene 16:1217–1222
Crawford PC, Dubovi EJ, Castleman WL, Stephenson I, Gibbs EP, Chen L, Smith C, Hill RC, Ferro P, Pompey J, Bright RA, Medina MJ, Johnson CM, Olsen CW, Cox NJ, Klimov AI, Katz JM, Donis RO (2005) Transmission of equine influenza virus to dogs. Science 310:482–485
Decaro N, Pinto P, Mari V, Elia G, Larocca V, Camero M, Terio V, Losurdo M, Martella V, Buonavoglia C (2014) Full-genome analysis of a canine pneumovirus causing acute respiratory disease in dogs, Italy. PLoS One 9:e85220
Deng R, Hua X, Li J, Chi W, Zhang Z, Lu F, Zhang L, Pflugfelder SC, Li DQ (2015) Oxidative stress markers induced by hyperosmolarity in primary human corneal epithelial cells. PLoS One 10:e0126561
Ellis J, Rhodes C, Lacoste S, Krakowka S (2014) Antibody responses to Bordetella bronchiseptica in vaccinated and infected dogs. Can Vet J 55:857–864
Fulcher ML, Gabriel S, Burns KA, Yankaskas JR, Randell SH (2005) Well-differentiated human airway epithelial cell cultures. Methods Mol Med 107:183–206
Gericota B, Anderson JS, Mitchell G, Borjesson DL, Sturges BK, Nolta JA, Sieber-Blum M (2014) Canine epidermal neural crest stem cells: characterization and potential as therapy candidate for a large animal model of spinal cord injury. Stem Cells Transl Med 3:334–345
Gorissen SH, Hristova M, Habibovic A, Sipsey LM, Spiess PC, Janssen-Heininger YM, van der Vliet A (2013) Dual oxidase-1 is required for airway epithelial cell migration and bronchiolar reepithelialization after injury. Am J Respir Cell Mol Biol 48:337–345
Guilleret I, Yan P, Grange F, Braunschweig R, Bosman FT, Benhattar J (2002) Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity. Int J Cancer 101:335–341
Ha Thi BM, Campolmi N, He Z, Pipparelli A, Manissolle C, Thuret JY, Piselli S, Forest F, Peoc'h M, Garraud O, Gain P, Thuret G (2014) Microarray analysis of cell cycle gene expression in adult human corneal endothelial cells. PLoS One 9:e94349
Hahn WC (2002) Immortalization and transformation of human cells. Mol Cells 13:351–361
Harkey MA, Asano A, Zoulas ME, Torok-Storb B, Nagashima J, Travis A (2013) Isolation, genetic manipulation, and transplantation of canine spermatogonial stem cells: progress toward transgenesis through the male germ-line. Reproduction 146:75–90
He YL, Wu YH, He XN, Liu FJ, He XY, Zhang Y (2009) An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer. Theriogenology 71:1417–1424
Hong HX, Zhang YM, Xu H, Su ZY, Sun P (2007) Immortalization of swine umbilical vein endothelial cells with human telomerase reverse transcriptase. Mol Cells 24:358–363
Jaeger G, Skogmo HK, Kolbjornsen O, Larsen HJ, Bergsjo B, Sorum H (2013) Haemorrhagic pneumonia in sled dogs caused by Streptococcus equi subsp. zooepidemicus—one fatality and two full recoveries: a case report. Acta Vet Scand 55:67. doi:10.1186/1751-0147-55-67
Jiang M, Min Y, Debusk L, Fernandez S, Strand DW, Hayward SW, Lin PC (2010) Spontaneous immortalization of human dermal microvascular endothelial cells. World J Stem Cells 2:114–120
Jung K, Lee CS, Kang BK, Park BK, Oh JS, Song DS (2010) Pathology in dogs with experimental canine H3N2 influenza virus infection. Res Vet Sci 88:523–527
Kahn CR, Young E, Lee IH, Rhim JS (1993) Human corneal epithelial primary cultures and cell lines with extended life span: in vitro model for ocular studies. Invest Ophthalmol Vis Sci 34:3429–3441
Kang YM, Kim HM, Ku KB, Park EH, Yum J, Seo SH (2013) H3N2 canine influenza virus causes severe morbidity in dogs with induction of genes related to inflammation and apoptosis. Vet Res 44:92. doi:10.1186/1297-9716-44-92
Lam HC, Choi AM, Ryter SW (2011) Isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface. J Vis Exp 48:2513. doi:10.3791/2513
Lange AW, Keiser AR, Wells JM, Zorn AM, Whitsett JA (2009) Sox17 promotes cell cycle progression and inhibits TGF-beta/Smad3 signaling to initiate progenitor cell behavior in the respiratory epithelium. PLoS One 4:e5711
Liu S, Hatton MP, Khandelwal P, Sullivan DA (2010) Culture, immortalization, and characterization of human meibomian gland epithelial cells. Invest Ophthalmol Vis Sci 51:3993–4005
Massin P, Kuntz-Simon G, Barbezange C, Deblanc C, Oger A, Marquet-Blouin E, Bougeard S, van der Werf S, Jestin V (2010) Temperature sensitivity on growth and/or replication of H1N1, H1N2 and H3N2 influenza A viruses isolated from pigs and birds in mammalian cells. Vet Microbiol 142:232–241
Mitchell JA, Brownlie J (2015) The challenges in developing effective canine infectious respiratory disease vaccines. J Pharm Pharmacol 67:372–381
Mitchell JA, Cardwell JM, Renshaw RW, Dubovi EJ, Brownlie J (2013) Detection of canine pneumovirus in dogs with canine infectious respiratory disease. J Clin Microbiol 51:4112–4119
Miyazawa K, Hondo T, Kanaya T, Tanaka S, Takakura I, Itani W, Rose MT, Kitazawa H, Yamaguchi T, Aso H (2010) Characterization of newly established bovine intestinal epithelial cell line. Histochem Cell Biol 133:125–134
Morales CP, Gandia KG, Ramirez RD, Wright WE, Shay JW, Spechler SJ (2003) Characterisation of telomerase immortalised normal human oesophageal squamous cells. Gut 52:327–333
Nguyen AV, Wu YY, Liu Q, Wang D, Nguyen S, Loh R, Pang J, Friedman K, Orlofsky A, Augenlicht L, Pollard JW, Lin EY (2013) STAT3 in epithelial cells regulates inflammation and tumor progression to malignant state in colon. Neoplasia 15:998–1008
Parikh N, Nagarajan P, Sei-ichi M, Sinha S, Garrett-Sinha LA (2008) Isolation and characterization of an immortalized oral keratinocyte cell line of mouse origin. Arch Oral Biol 53:1091–1100
Piper SC, Ferguson J, Kay L, Parker LC, Sabroe I, Sleeman MA, Briend E, Finch DK (2013) The role of interleukin-1 and interleukin-18 in pro-inflammatory and anti-viral responses to rhinovirus in primary bronchial epithelial cells. PLoS One 8:e63365
Puttmann S, Senner V, Braune S, Hillmann B, Exeler R, Rickert CH, Paulus W (2005) Establishment of a benign meningioma cell line by hTERT-mediated immortalization. Lab Invest 85:1163–1171
Randell SH, Walstad L, Schwab UE, Grubb BR, Yankaskas JR (2001) Isolation and culture of airway epithelial cells from chronically infected human lungs. In Vitro Cell Dev Biol Anim 37:480–489
Robertson DM, Li L, Fisher S, Pearce VP, Shay JW, Wright WE, Cavanagh HD, Jester JV (2005) Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line. Invest Ophthalmol Vis Sci 46:470–478
Roxas BA, Li Q (2008) Significance analysis of microarray for relative quantitation of LC/MS data in proteomics. BMC Bioinf 9:187. doi:10.1186/1471-2105-9-187
Santha S, Bommareddy A, Rule B, Guillermo R, Kaushik RS, Young A, Dwivedi C (2013) Antineoplastic effects of alpha-santalol on estrogen receptor-positive and estrogen receptor-negative breast cancer cells through cell cycle arrest at G2/M phase and induction of apoptosis. PLoS One 8:e56982
Schierack P, Nordhoff M, Pollmann M, Weyrauch KD, Amasheh S, Lodemann U, Jores J, Tachu B, Kleta S, Blikslager A, Tedin K, Wieler LH (2006) Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine. Histochem Cell Biol 125:293–305
Shao G, Balajee AS, Hei TK, Zhao Y (2008) p16INK4a downregulation is involved in immortalization of primary human prostate epithelial cells induced by telomerase. Mol Carcinog 47:775–783
Shi L, Ramaswamy M, Manzel LJ, Look DC (2007) Inhibition of Jak1-dependent signal transduction in airway epithelial cells infected with adenovirus. Am J Respir Cell Mol Biol 37:720–728
Song D, Kang B, Lee C, Jung K, Ha G, Kang D, Park S, Park B, Oh J (2008) Transmission of avian influenza virus (H3N2) to dogs. Emerg Infect Dis 14:741–746
Su F, Liu X, Liu G, Yu Y, Wang Y, Jin Y, Hu G, Hua S, Zhang Y (2013) Establishment and evaluation of a stable cattle type II alveolar epithelial cell line. PLoS One 8:e76036
Tompkins DH, Besnard V, Lange AW, Keiser AR, Wert SE, Bruno MD, Whitsett JA (2011) Sox2 activates cell proliferation and differentiation in the respiratory epithelium. Am J Respir Cell Mol Biol 45:101–110
Traversa D, Di Cesare A, Conboy G (2010) Canine and feline cardiopulmonary parasitic nematodes in Europe: emerging and underestimated. Parasit Vectors 3:62. doi:10.1186/1756-3305-3-62
Ulrich R, Puff C, Wewetzer K, Kalkuhl A, Deschl U, Baumgartner W (2014) Transcriptional changes in canine distemper virus-induced demyelinating leukoencephalitis favor a biphasic mode of demyelination. PLoS One 9:e95917
Vaziri H, Benchimol S (1998) Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol 8:279–282
Vos JB, van Sterkenburg MA, Rabe KF, Schalkwijk J, Hiemstra PS, Datson NA (2005) Transcriptional response of bronchial epithelial cells to Pseudomonas aeruginosa: identification of early mediators of host defense. Physiol Genomics 21:324–336
Wang J, Hu G, Lin Z, He L, Xu L, Zhang Y (2014) Characteristic and functional analysis of a newly established porcine small intestinal epithelial cell line. PLoS One 9:e110916
Weese JS, Stull J (2013) Respiratory disease outbreak in a veterinary hospital associated with canine parainfluenza virus infection. Can Vet J 54:79–82
Yang G, Li S, Blackmon S, Ye J, Bradley KC, Cooley J, Smith D, Hanson L, Cardona C, Steinhauer DA, Webby R, Liao M, Wan XF (2013) Mutation tryptophan to leucine at position 222 of haemagglutinin could facilitate H3N2 influenza A virus infection in dogs. J Gen Virol 94:2599–2608
You S, Moon JH, Kim TK, Kim SC, Kim JW, Yoon DH, Kwak S, Hong KC, Choi YJ, Kim H (2004) Cellular characteristics of primary and immortal canine embryonic fibroblast cells. Exp Mol Med 36:325–335
Zeng XJ, Lin Y, Zhao YB, Lu CP, Liu YJ (2013) Experimental infection of dogs with H3N2 canine influenza virus from China. Epidemiol Infect 141:2595–2603
Acknowledgments
This work was supported by the International S&T Cooperation Program of China (ISTCP 2014DFG32770), Graduate Research and Innovation Program of Jiangsu Province (KYLX15_0561) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Ethics statement
All animal experiments were carried out according to animal welfare standards and were approved by the Ethical Committee for Animal Experiments of Nanjing Agricultural University, China.
Competing interests
The authors declare that they have no conflict of interest.
Authors’ contributions
XX carried out most of the experiments described in the manuscript and wrote the article; MDP, SL participated in the animal experiments; LY helped with construction of recombinant plasmid; YBZ, KM, DHK, CPL were involved in analysis, interpretation of data and provided critical evaluation of the manuscript. YJL conceived the study and contributed in its design and coordination. All authors read and approved the final manuscript.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Table S1
(PDF 99 kb)
Rights and permissions
About this article
Cite this article
Xie, X., Pang, M., Liang, S. et al. Establishment and characterization of a telomerase-immortalized canine bronchiolar epithelial cell line. Appl Microbiol Biotechnol 99, 9135–9146 (2015). https://doi.org/10.1007/s00253-015-6794-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-015-6794-8