Aim of this study is to analyze the interaction of influenza A viruses and airway epithelial cells by using influenza A viruses and two primary cell culture systems:
porcine precision-cut lung slices and air-liquid interface culture.
The first defence line of the respiratory tract is the mucociliary clearance system derived from specialized cells: mucus-producing cells and ciliated cells, the former release mucins which can entrap foreign material and the latter transport mucus out of the respiratory tract via ciliary beating to protect the host. We were interested whether cilia possess a role in impeding virus infection in addition to their transport function.
The culture system of interest is porcine PCLS, as in this ex vivo model the ciliary beating can be observed conveniently under the light microscope. As a respiratory pathogen we chose the H3N2 subtype of swine influenza A viruses, as pigs are an important host for influenza A viruses, and the H3N2 virus is one of the subtypes circulating in pig populations.
Swine-origin influenza A virus of the H1N1 subtype emerged in 2009 and was transmitted from pigs to humans causing a pandemic. To evaluate whether the virulence of strains from human and swine isolated at the beginning year and the years following 2009 changed, porcine air-liquid interface cultures were chosen as an in vitro model to be infected by these strains. ALI culture systems of differentiated airway epithelial cells possess some parameters derived from mucociliary clearance system and intercellular junction complexes that can be used to reflect viral effects in the airway epithelium. In addition, amino acid sequences of proteins from different isolates are available and can be compared. The data of my in vitro-experiments, should be compared to animal experiments that have been performed by our collaboration partner Ralf Dürrwald (summarized in Introduction, section 7). All these data together should provide a better understanding of the viral virulence.
MATERIAL AND METHODS 1. Cells and viruses
Madin-Darby canine kidney (MDCK) cells (Friedrich-Loeffler-Institut, Greifswald, Insel Riems, Germany) were cultured with Eagle’s minimal essential medium (EMEM) containing 10% fetal bovine serum (Sigma, USA), and incubated at 37oC under a humidified atmosphere containing 5% CO2.
A swine influenza A virus of H3N2 subtype (A/sw/Herford/IDT5932/2007) was used to analyze the importance of the ciliary activity of airway epithelial cells in preventing virus infection. Five A(H1N1)pdm09 viruses were used to analyze the changes of virulence, four of which were isolated from patients: A/Germany/1580/2009 (HA09, access number of viral genes: EPI296157, EPI296174-EPI296179, EPI296981), A/Germany/5258/2009 (JE09, access number of viral genes: KJ549775-KJ549782), A/Germany/2688/2010 (JE10) and A/Germany/18909686/2015 (KI15). Virus A/sw/Germany/19989/2014 (SC14, access number of viral genes:
KX013010-KX013017) was isolated from a pig. The acc.nos of the sequences of JE10 and KI15 will be provided as soon as they are available. The five isolates of A(H1N1)pdm09 viruses were randomly selected from the collections of viruses available of that time.
All viruses were propagated in MDCK cells to prepare viral stocks. Following a period of two hours for virus adsorption, infected cells were incubated in EMEM supplemented with acetylated trypsin (Sigma-Aldrich, Munich) 1 μg/mL. After 72 h, the supernatants were harvested and, after removal of cell debris by centrifugation at 2000 × g for 10 min, viruses were stored at −80 °C. Viral titers were determined in MDCK cells with a 50% tissue culture infectious dose (TCID50) assay.
2. Preparation of porcine differentiated airway epithelial cells 2.1 Precision-cut lung slices
All pigs used for preparation of porcine precision-cut lung slices were crossbreeds and raised and kept in the Clinic for Swine and Small Ruminants and forensic Medicine and Ambulatory Service, University of Veterinary Medicine Hannover, Foundation.
The pigs were raised and kept in accordance with the recommendations of the
European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, European Treaty Series, nos. 123 /170 (http://
conventions.coe.int/Treaty/en/Treaties/Html/123.htm and http://conventions.coe.int/
Treaty/en/Treaties/Html/170.htm). Study design, including all measures, and housing of the animals were approved by a local, independent committee on ethics (Commission for ethical estimation of animal research studies of the Lower Saxonian State Office for Consumer Protection and Food Safety (approval number 33.9-42502-05-09A627) and were in accordance with the requirements of the national animal welfare law. Compliance with animal welfare law and ethics were regularly controlled by the local authorities.
Porcine precision-cut lung slices for differentiated airway epithelial cells were prepared as reported previously (Goris et al., 2009; Punyadarsaniya et al., 2011).
Briefly, the cranial, middle, and intermediate lobes of fresh lungs of three-month-old clinically healthy pigs were obtained from the Clinics for Swine and Small Ruminants and forensic Medicine and Ambulatory Service at the University of Veterinary Medicine, Foundation, Hannover, Germany. Prior to removal of the lobes, the pigs were euthanized by intravenous application of 80mg/kg pentobarbital (Euthadorm®, Co. CP Pharma GmbH, Burgdorf, Germany). The lobes were filled with 37 °C warm low-melting agarose (AGAROSE LM; GERBU, Heidelberg, Germany). After solidification, cylindrical portions containing a bronchial airway were stamped out by using an 8-mm tissue coring tool. Using the Krumdieck tissue slicer (TSE systems, model MD4000-01, Bad Homburg, Germany), slices of about 250 μm thickness were generated. PCLS were transferred to 24-well plates (one slice/well) and maintained in 1 mL of RPMI 1640 medium (Genaxxon, Germany) supplemented with antibiotics and antimycotics. Slices were monitored under the light microscope (Zeiss Axiovert 35) for ciliary activity. For experiments, only slices were chosen in which the ciliary activity was retained on the whole luminal surface of the airway visible under the microscope (designated as 100% activity).
2.2 Air-liquid interface cultures
Porcine lungs used in this study were obtained from a local slaughterhouse where pigs were being processed as part of the normal work. Air-liquid interface cultures of
porcine airway epithelial cells were prepared as previously described (Fulcher et al., 2005; Meng et al., 2016). Briefly, primary porcine airway epithelial cells were isolated from bronchi of healthy pigs that had no clinical lung lesions. The cells were cultured in collagen type I-coated flasks with Bronchial Epithelial Cell Basal Medium (BEBM, Lonza) supplemented with the required additives (Meng et al., 2016; Wu et al., 2016). After cells had reached 80-90% confluence, they were digested with trypsin (Life Technologies) and seeded on collagen type IV coated polyester transwell filters (Corning Costar) at a density of 2.5×105 cells per filter in 250μl ALI medium which is a mix of BEBM and Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with the required additives (Meng et al., 2016; Wu et al., 2016). Cells were incubated at 37°C and maintained under ALI conditions for 4-5 weeks for differentiation. During the time period of differentiation, culture medium was replaced every second day and cells were washed with Hanks’ balanced salt solution (HBSS, Life Technologies) once a week.
3. Effects of hypertonic salt on ciliary activity of PCLS
Hypertonic salt concentrations were used to induce ciliostasis (Boek et al., 1999).
Porcine PCLS were treated with RPMI1640 medium containing NaCl at the indicated concentrations (2%, 5%, 7%, 9%, or 11%). When cilia had stopped beating after incubation at 37 °C for the times indicated, the hypertonic medium was replaced by fresh medium immediately. Then the recovery of the ciliary activity was monitored under the light microscope to determine which concentration is the best condition to induce a reversible ciliostasis.
To analyze for how long slices can tolerate the presence of 2% NaCl without affecting the recovery of the ciliary activity, PCLS were treated with that salt concentration for 30 min, 60 min and 90 min. After the incubation, the medium was replaced by fresh medium for the times indicated. The slices were monitored under the light microscope to determine after which time period of salt treatment, ciliary activity can recover completely.
4. Effect of 2% NaCl on the infectivity of H3N2 type virus
To analyze whether treatment with high salt affects the infectivity of influenza viruses,
the H3N2 subtype of swine influenza A virus was incubated with medium in the presence or absence of 2% NaCl for 30 min. Afterwards, the infectivity was determined by performing a plaque assay with MDCK cells as described previously (Vietmeier et al., 2007; Yang et al., 2017).
5. Virus infection and titration
Porcine PCLS were washed three times with phosphate-buffered saline (PBS) and then infected with an H3N2 strain of swine influenza virus at an infectious dose of 104 TCID50 per slice in the presence or absence of the ciliary activity. After incubation at 37oC for 20 min, infectious medium was removed and slices were washed three times with PBS to remove unbound virions. Supernatants were collected at 24h and 48h post-infection and stored at -80oC.
Well-differentiated porcine bronchial epithelial cells (PBEC) grown under air-liquid interface conditions were washed three times with PBS and then infected by either of the five A(H1N1)pdm09 viruses at an infectious dose of 104 TCID50 per filter in 100 μl ALI medium at 37oC for 1h. After the adsorption period, cells were washed three times with PBS to remove unbound virions. Finally, 600μl ALI medium were added to the basal compartment to maintain the cells. Supernatants were collected at 24h, 48h, 72h, 96h and 120h post-infection and stored at -80oC. The samples of supernatants were prepared by adding 100µl ALI medium to the apical compartment at the indicated time points and incubated at 37°C for 30 min.
Viral titers of samples collected from infection of PCLS and PBEC were used to determine the viral infectivity by endpoint dilution titration on MDCK cells as described previously (Yang et al., 2017).
6. Effects of A(H1N1)pdm09 viruses on ALI cultures
Well-differentiated PBEC resemble the structure of the epithelial cells in the host, which are characterized by junctional complexes and a mucociliary clearance system.
To investigate the effects of the viruses on airway epithelial cells, the following parameters were determined: (i) Trans-epithelial electrical resistance (TEER) was measured before and after infection (at 8 days post-infection) by using the Millicell ERS-2 (Millipore) equipment; prior to measurement, well-differentiated PBEC were
washed with PBS three times, then 200μl and 500μl PBS, respectively, were added to the apical and basal compartment; finally TEER was measured according to the manufacturer’s instructions. (ii) Tissue tropism of the viruses was analyzed at 24h post-infection by immunofluorescence assay. (iii) Relative cilia coverage of the cell layer and the thickness of cultures were investigated at 8 days post-infection by immunostaining.
7. Immunofluorescence assay
Primary antibodies were used in this assay including Cy3-labeled antibodies against β-tubulin at a dilution of 1:500 (Sigma), antibodies against mucin-5 AC at a dilution of 1:250 (Santa Cruz Biotechnology), antibodies recognizing the NP protein of influenza A virus at a dilution of 1:750 (AbDSeroTec) and antibodies against ZO-1 at a dilution of 1:250 (Life Technologies). Secondary antibodies used in this assay contained the following fluoresecent chromophores: Alexa Fluor® 488 (green fluorescence) and 568 (red fluorescence). The conjugated antibodies were applied at a dilution of 1:1000 (Life Technologies).
The immunofluorescence assay was performed as follows: At the indicated time points, samples of infected and mock-infected cultures were washed three times with PBS and then fixed with 3% paraformaldehyde (PFA) for 20min. After the removal of PFA, 0.1 M glycine was added for 5min to neutralize the residual PFA. Samples were washed with PBS three times, and permeabilized with 0.2% Triton X-100 for 20min.
After three washes with PBS, samples were incubated with 1% bovine serum albumin (BSA) in PBS for 30min to block nonspecific binding sites. First and secondary antibodies were diluted with dilution buffer containing 1% BSA in PBS and incubated with cultures for 1h at room temperature. To visualize nuclei, samples were washed three times with PBS and subjected to staining with DAPI (4′,6-diamidino-2-phenylindole) for 20min. Finally, samples were taken out and embedded in Prolong Gold Antifade Reagent (Life Technologies), and stored at 4°C for further analysis.
An inverse immunofluorescence microscope Nikon Eclipse Ti-S (Nikon) equipped with a 10x/0.30 and 40x/0.60 Plan Fluor objectives and TCS SP5 confocal laser scanning
microscope (Leica) equipped with a 63x/1.30 NA glycerin HC PL APO objective and a 63x/1.40 oil HCX PL APO objective were used to analyze samples. NIS-Elements Viewer 4.20 software (Nikon), LAS AF Lite sofware (Leica) and ImageJ/Fuji software were used to analyze images.
8. Statistical analyses
All in vitro-experiments were performed at least three times and data were analyzed with Tukey multiple comparison test by using the GraphPad Prism 5 software, results were shown as means with standard deviations. The data observed in the animal experiments were analyzed with Mann–Whitney-U-test by using the program SPSS 15.0. Statistical difference of data were shown in the figures by asterisks (*, p≤0.05 = significant; **, p≤0.01 = highly significant; ***, p≤0.001 = very highly significant;
n.s. = not significant).
RESULTS
1. Ciliostasis of airway epithelial cells facilitates influenza A virus infection (Results described in this paragraph have been published (Fu et al., 2018)) 1.1 Reversible ciliostasis induced by hypertonic salt
Sodium chloride has been described in other cell systems to induce reversible ciliostasis (Boek et al., 1999). We wanted to find out under which conditions NaCl affects the ciliary activity of swine airway cells. Medium containing NaCl at different concentrations (2-11%) were used to treat porcine PCLS, and each of the concentrations induced complete ciliostasis within 5 min (Fig. 9). After replacement of hypertonic salt by fresh medium, cells resumed the ciliary activity. The efficiency of this effect was evaluated by determining whether ciliary beating was detectable along the whole luminal surface of the respective airway (bronchioli) visible under the light microscope (100%) or only on a portion of the epithelial surface. How efficient the epithelial cells resumed the ciliary activitiy, was dependent to some extent on the hypertonic salt concentrations that had been used to induce ciliostasis. Complete recovery of the ciliary activity was observed only when PCLS had been treated with 2%
NaCl (Fig. 9). If the slices were incubated with higher concentrations of NaCl, there was only a partial recovery of the cilia beating which ranged from about 80% (5%
NaCl) to less than 5% (11% NaCl).
After having shown that incubation of PCLS with 2% NaCl results in reversible ciliostasis, we were interested to know for which time period the cells tolerate the salt treatment. For this purpose, slices were incubated 30 min, 60 min and 90 min with medium containing 2% NaCl and then monitored for the ciliary beating. As shown in Fig. 10, ciliary activity was recovered completely only when slices were treated for 30 min with 2% NaCl. If the incubation time was extended to 60 or 90 min, ciliary activity was observed only on about 95 or 90% of the luminal surface, respectively.
Fig. 9 Effect of hypertonic NaCl on ciliary avtivity of PCLS
PCLS were treated with sodium chloride at different concentrations. When cilia had stopped beating, the culture medium was replaced by fresh medium without hypertonic sodium chloride. PCLS were monitored under the microscope for the recovery of the ciliary activity.
Fig. 10 Treatment of PCLS with 2% NaCl for different time periods
PCLS were treated with 2% NaCl for 30min, 60min and 90min. At the indicated times, medium containing 2% NaCl was replaced by fresh medium without hypertonic salt conditions. The ciliary activity was determined by microscopic inspection of the slices.
1.2 Effect of 2% NaCl on the infectivity of influenza A virus
After having established conditions for reversible ciliostasis, we analyzed whether these conditions affect the infectivity of influenza A viruses. Strain A/sw/Herford/IDT5932/2007, a swine influenza A virus of the H3N2 subtype was treated with RPMI 1640 medium in the presence or absence of 2% NaCl at 37oC for 30min. To investigate the effects of the hypertonic salt treatment on the infectivity of the virus a plaque assay with MDCK cells was performed. As shown in Fig. 11, there is no difference detectable between the two samples in their efficiency of plaque formation, i.e. both contain the same amount of infectious viruses. This result indicates treatment with 2% NaCl for 30 min does not affect the viral infectivity.
Fig. 11 Effect of 2% NaCl on H3N2 subtype of swine influenza virus
Swine influenza virus strain of the H3N2 subtype was treated with 2% NaCl at 37oC for 30 min prior to infection of MDCK cells. To analyze the effect of 2% NaCl on influenza virus, a plaque assay was performed at 72h post infection and plaques were counted to determine the infectivity.
1.3 Infection of PCLS under ciliostatic condition
Porcine PCLS were infected with the swine influenza virus of the H3N2 subtype in the presence or absence of the ciliary activity, i.e they had been pretreated with or without NaCl. Supernatants were collected at different time points post-infection and the infectivity was determined by TCID50 assay. The yield of infectious virus under ciliostatic condition at 24h and 48h post-infection was twofold or threefold, respectively, higher than after infection under physiological conditions (Fig. 12). The differences are statistically significant indicating that infection by this swine influenza A virus is more efficient under ciliostatic conditions.
Fig. 12 Comparative analysis of the effect of hypertonic salt conditions on the viral titer of H3N2 virus released into the supernatant collected at 24h and 48h post-infection.
PCLS were infected with swine influenza virus (H3N2) in the presence or absence of 2% NaCl during the attachment step. Supernatants were collected at different times pi and analyzed for infectivity by TCID50 assay.
2. In vitro studies in porcine differentiated airway epithelial cells reveal an evolution of human-derived A(H1N1)pdm09 viruses towards lower virulence between 2009 and 2015
2.1 Infection of well-differentiated airway epithelial cells
Recently it has been shown that infection of differentiated swine airway epithelial cells by influenza viruses results in characteristic changes of the epithelial cell layer (Wu et al., 2016). These effects are evident in the loss of ciliated cells and in a reduction of the thickness of the epithelial cell layer. Nevertheless, the barrier function of the epithelium is maintained as indicated by the trans-epithelial electrical resistance (TEER). These effects are more pronounced after infection by virulent viruses than they are after infection by low-virulent viruses (Wu et al., 2016). We wanted to know whether H1N1 influenza viruses isolated in the years following the 2009 pandemic differ in their virulence properties. Therefore I performed a comparative infection study with differentiated porcine bronchial epithelial cells (PBEC) grown under air-liquid-interface (ALI) conditions. For this study we selected the following strains:
A/Germany/1580/2009 (HA09), A/Germany/5258/2009 (JE09), A/Germany/2688/2010 (JE10) and A/Germany/18909686/2015 (KI15) that had been isolated from patients. Virus A/sw/Germany/19989/2014 (SC14) was isolated from a pig.
2.2 Tissue tropism of the viruses
Well-differentiated PBEC were infected with either of the five A(H1N1)pdm09 viruses, respectively, and analyzed by immunostaining assay at 24h post-infection.
Staining for β-tubulin and mucin-5AC was used to visualize ciliated cells and mucus-producing cells, respectively. Staining for the viral NP protein was applied to detect infected cells. The co-localization of β-tubulin and NP protein as well as mucin-5AC and NP protein indicated that all isolates possess the ability to infect not only ciliated cells but also mucus-producing cells (Fig. 13). There is no difference among the viruses in their tissue tropism.
Fig. 13 Tissue tropism of the five viruses
Well-differentiated PBEC cultures were apically infected with HA09, JE09, JE10, SC14 or KI15 strain at an infection dose of 104TCID50/filter. At 24h post-infection, samples were subjected to antibodies against NP protein (green) and β-tubulin (red) as well as NP protein and mucin (red) to analyze the tissue tropism of these viruses.
DAPI was used to visualize nuclei (blue). Staining of the nuclei with DAPI (inserts at bottom left) shows the monolayers are intact. The experiments were performed three times independently, and each time with three replicates. Scale bar, 20 μm.
2.3 Effect of virus-infection on trans-epithelial electrical resistance
TEER of infected and mock-infected well-differentiated PBEC cultures were measured at the indicated times. The values of infected samples did not differ from each other significantly and they were slightly higher than those of the mock-infected samples, as shown in Fig. 14A. This result indicates that the TEER is not lost after
TEER of infected and mock-infected well-differentiated PBEC cultures were measured at the indicated times. The values of infected samples did not differ from each other significantly and they were slightly higher than those of the mock-infected samples, as shown in Fig. 14A. This result indicates that the TEER is not lost after