A novel chicken lung epithelial cell line: Characterization and response to low pathogenicity avian influenza virus
Highlights
► Establishment of CLEC213, a chicken (Gallus gallus) lung epithelial cell line. ► CLEC213 resemble mammalian pneumocytes type II. ► Innate immune response of chicken lung epithelial cells to avian influenza virus. ► CLEC213 support efficient avian influenza virus growth in vitro.
Introduction
A number of avian respiratory pathogens, including the highly pathogenic influenza viruses (Neumann et al., 2010), are of major importance for animal and public health. Birds are prone to infections with these pathogens, and frequently the infection becomes systemic leading to fatal disease patterns. However, birds are also able to raise an immune response against these respiratory pathogens. Studies on the trachea and lung in the chicken have indeed proven that these organs are able to counter influenza virus infections through induction of immune gene transcription and leukocyte accumulation (Daviet et al., 2009, Munier et al., 2010, Reemers et al., 2009a, Reemers et al., 2009b).
The avian respiratory system differs in many aspects from that of mammals. It is characterized by a semi-rigid structure and the presence of air sacs. Contrary to the situation in mammals, birds lack dead-end air alveoles where exchanges between air and blood take place. Instead, bronchi and parabronchi end up in air capillaries and the air flow is continuously circulating in the same direction during inspiration and expiration, due to air sac functioning (Maina, 2002, Reese et al., 2006). This particularity also has consequences on the distribution of pathogens in the lung (Reemers et al., 2009b). Regarding the immune system, chickens have no organized lymph node stricto sensu although an organized immune system is present and functional in the bird respiratory tract. Lymphoid nodules are not present at day 1 after hatching, but have been observed in the lung mainly near the openings of the bronchus system by 8 weeks of age (Reese et al., 2006). Macrophages are generally not detected at the external surface of the airway epitheliums in birds without pathogen stimulation (Nganpiep and Maina, 2002, Reese et al., 2006). This is a major difference to the mammalian system where this type of cells belongs to the first line of non specific immune defence (Chaudhuri and Sabroe, 2008). However, macrophages are quickly recruited upon infection, presumably coming from underneath the respiratory epithelia or from the blood (Maina, 2002, Toth, 2000). Accumulation of other subpopulations of leukocytes can also be observed, demonstrating the presence of the cellular immune response in the avian respiratory tract or the lung parenchyma (Reese et al., 2006). Altogether, by comparison with what is known in mammals, our current knowledge about the functional aspects of the immune response in the bird airway tract and lung is only limited and therefore needs to be improved.
Avian influenza viruses are orthomyxoviruses that infect different types of epithelia expressing the haemagglutinin receptor. Transmission of avian influenza viruses (AIVs) can occur through various routes of infection, depending on the bird species. For example, chickens can readily be infected with AIVs via the respiratory route, where virus excretion also occurs, while ducks are predominantly infected via the digestive tract route (Jeong et al., 2009, Swayne and Slemons, 1994, van der Goot et al., 2003). A recent study has demonstrated that SAα2,3Gal-terminated sialylglycoconjugates, acting as haemagglutinin receptors, are highly expressed in both species in trachea and intestine. These receptors are also highly expressed in the bronchi of chickens, but not of ducks (Pillai and Lee, 2010). As the first cells encountered by AIVs in the chicken, respiratory epithelia may thus be of major importance in the host defence.
In birds, the air is first circulating across turbinates and their circonvulated epithelium in the nasal cavities (Majo et al., 1996). The trachea exhibits a ciliated epithelium with goblet cells and mucus cells (Abd El Rahman et al., 2009, Henning et al., 2008, Purcell, 1971). Ciliated epithelium is also observed in primary bronchi and the initial part of secondary bronchi (Klika et al., 1998). As in mammals, the major role of the mucous and the cilia is to prevent the access of particles or pathogens to the lung parenchyma. The epithelium becomes non ciliated in parabronchi, in atria and in air capillaries (Klika et al., 1998, Scheuermann et al., 1997). Cells with a granular morphology, containing cytoplasmic lamellar bodies, have been identified mostly in the atrium scattered among squamous cells that cover infundibula and air capillaries (Scheuermann et al., 1997). A lipoproteinaceous substance, specific to birds, covers the capillary epithelium. The bird gas-blood barrier is composed of airway epithelial cells with elongated junctional dendrites and of endothelial cells, and constitutes therefore a much thinner barrier than that of mammals (Scheuermann et al., 1997, Watson et al., 2007). The granular cells could be an equivalent to mammalian type II pneumocytes, which are capable of multiplication and differentiation to type I pneumocytes to reconstitute the respiratory epithelium when damaged. However, in birds, the possible analogy of squamous cells with mammalian type I pneumocytes remains to be formally demonstrated (Scheuermann et al., 1997).
In mammals, epithelia participate actively to the host defence against respiratory pathogens. They express various pathogen recognition receptors (PRR), which can recognize specific pathogen molecular motifs. In particular they express at different levels several Toll-like receptors (TLRs), which are stimulated by viruses, bacteria and parasites (Bauer et al., 2009, Hippenstiel et al., 2006). As a consequence, epithelial cells are activated to secrete molecules such as defensins and chemokines, which in turn are responsible for recruiting a panel of immune cells and for inducing the production of a variety of cytokines. In addition to their role as a physical barrier, they are thus part of the immune system (Schleimer et al., 2007).
In birds, specific studies of the function of the different types of avian respiratory epithelia are scarce due to the lack of a standardized method to obtain primary cultures and to the quasi absence of specific cell lines. Beside a turkey turbinate cell line that was recently described (Kong et al., 2007), epithelium lining trachea explants or trachea cells in primary culture have been characterized: they are ciliated, can be infected by viruses including avian influenza viruses and can mount an immune response (Reemers et al., 2009a, Zaffuto et al., 2008). However, these cells are very difficult to maintain in vitro and quickly lose their differentiated phenotype upon repeated passage in culture. Epithelia from bird bronchi, parabronchi and air capillaries have never been isolated ex vivo, nor have permanent cell lines been established so far.
Our aim was thus to prepare and maintain in culture epithelial cells from chicken lung parenchyma and to compare their properties with those of mammalian type II pneumocytes. The latter are of major importance in the lung defence since, in addition to the production of anti-microbial lung surfactant, they are able to secrete various cytokines and chemokines during the lung pro-inflammatory response (Hippenstiel et al., 2006). Here, we describe an avian cell line named CLEC213 that exhibits a number of biochemical and morphological characteristics of pneumocytes. This cell line is permissive to the replication of a panel of low pathogenicity avian influenza viruses, to which it responds through an increased expression of several cytokine mRNAs. Our results thus suggest that chicken lung epithelial cells might participate in the immune response in a way similar to what is observed in mammals.
Section snippets
Viruses
Low-pathogenicity avian influenza viruses A/Duck/France/05057a/2005 (H6N2) and A/Turkey/France/03295/2003 (H9N9) were a kind gift of Dr. Veronique Jestin (ANSeS, Ploufragan, France). Virus A/Mallard/Marquenterre/Z237/83 (H1N1), also referred to as “MZ”, was a kind gift of Dr. Nadia Naffakh (Institut Pasteur, Paris, France). Virus A/ck/It/22A/98PD (H5N9) was a kind gift of Dr. Ilaria Capua (Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy). Viral stocks were prepared by
Lung cell characterization
The CLEC213 cells were derived from a lung explant of a 5 week-old chicken and were maintained in culture for more than 140 passages without apparent changes in their phenotype. CLEC213 cells grew as a monolayer and after 96 passages they were checked for the expression of several epithelial markers. They scored positive with a pan cyto-keratin antibody and expressed E-cadherin at the cell surface (Fig. 1), but not vimentin (data not shown). On cytological preparations, cells were cohesive,
Discussion
Respiratory epithelia are essential cellular compartments participating in the defence against influenza viruses and in particular in inducing a protective or in some cases detrimental immune/inflammatory response, and thus complementary to the function of alveolar macrophages in mammals (Chan et al., 2005, Deng et al., 2008, Peiris et al., 2009). It is not yet known whether avian respiratory epithelia are crucially involved in the initiation of the immune response in a way similar to what is
Acknowledgements
We are grateful to Dr. Nathalie Chanteloup for lively and stimulating discussion, and to Dr. Sascha Trapp for reviewing the manuscript. This work was supported by INRA Young Team funding and a Grant from the French Federative Research Institute 136.
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