Experimental animals
A snatch-farrowed porcine-colostrum-deprived (SF-pCD) piglet (seven-week-old male, large Yorkshire) with neither signs of cardio-pulmonary disorders nor under any drug treatment or vaccinated, was purchased from Nanjing Zhoubang Bio-Tech Co. Ltd. A total of nine four-week-old female BALB/c-nu/nu mice were purchased from the Animal Experiment Center of Yangzhou University. Before sacrificing the piglet, an antibody test based on a serological analysis showed that all the following pathogens were negative, including Mhp, Mhr, MHS, MF, pseudorabies virus, classical swine fever virus, type O foot and mouth disease virus, porcine respiratory and reproductive syndrome virus (PRRSV), swine influenza virus of H1 and H3 subtype and porcine circovirus type 2 (PCV2).
Swine-origin mycoplasma strains and culturing
Swine-origin mycoplasma strains are those strains isolated from swine respiratory tract, including Mhp, Mhr, MF and MHS strains. Here, all mycoplasma strains were thawed from frozen stocks to subculture for three passages before use. Mhp strain J (ATCC 25934), MF strain (ATCC 27716) and MHS strain (ATCC 27095) were passaged once to establish frozen stocks from the ATCC. Mhp Strain 168L was gradually attenuated by continuous passage to the 350th passage from Mhp isolate 168, which was isolated from a piglet displaying typical features of mycoplasmal pneumonia in swine (MPS) in Gansu Province, China [18]. Thus, the Mhp 168L used herein was passage 353. Mhp Strain JS is a virulent strain that can induce typical characteristics of MPS with a lung lesion score of approximately 15, as described previously [19]. Mhp LH is a high virulent clinical strains that was isolated in our lab. Mhr HEF16 and 100928 are representative clinical strains that were isolated in our lab and that can induce polyserositis and arthritis, among which Mhr HEF16 has been stored as several stocks in the China Center for Type Culture Collection under CCTCC NO. M2017462. The two Mhp strains and Mhr strains were subcultured for 10 passages to establish frozen stocks after strain identification.
All Mhp and Mhr strains were cultured in modified Friis medium designated KM2 cell free medium containing 20% (v/v) swine serum (produced in our lab from a clean sF-PCD piglet after irradiation sterilization) at 37°C in humidified incubator [19]. The MF strain was cultured in Friis medium, and the MHS strain was grown in a modified Hayflick’s medium containing horse serum (Solarbio, Cat No. S9050) and arginine/mucin, as previously described [20]. Titers of all swine-origin mycoplasmas listed were quantified using a 50% color change unit (CCU50) assay [21], which was modified based on the CCU assay [22] in culture medium and confirmed by quantitative PCR [18].
Isolation and culture of primary PTECs
Isolation of primary PTECs was performed in accordance and modified from the method for mouse respiratory epithelial cells [23] and porcine bronchial epithelial cells [8]. An overdose forelimb intravenous injection of 20% sodium pentobarbital (3 mL/kg; Sigma-Aldrich, Cat No. P3761) for piglet euthanasia was applied to guarantee experience of the least pain. Then, piglet epidermal tissue disinfection around thoracic skin was performed. After the rib cage was removed upon chest exposure with a cut made from the throat of the piglet to the last rib, the skin around the trachea was removed in case of throat contamination. As soon as the upper trachea was fastened with the help of a hemostatic clamp, the trachea far from the lung was immediately cut. The remaining trachea connected to the lung tissue was removed from chest and placed in a sterilized glass plate covered with ice-cold Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F12, Gibco, Cat No. 11330032) before being transferred to a biosafety cabinet.
Subsequently, isolated trachea tissues were placed in a 50-mL tube (Thermo Fisher, Cat No. 339652), inverted 20 times, and washed with ice-cold DMEM/F12 until the liquid became clear and no mucus was produced. The tracheal tissues were manually cut into approximately 1 mm3 pieces with the help of microscopic scissors before mincing the tissues digested in digestion solution in a 37°C water bath for 50 min. The digestion solution consisted of a total of 25 mL DMEM/F12 medium (Gibco), including 5 mL of 0.0005 M EDTA (diluted 1000 times, Invitrogen, Cat No. AM9260G), 10 mL of collagenase type I (5 mg/mL; Sigma-Aldrich, Cat No. SCR103) and 10 mL of 0.25% trypsin (Gibco, Cat No. 25200056). Digestion was stopped by adding a total of 2.5 mL fetal bovine serum (FBS; Gibco, Cat No.10099141) directly after separation and rounding of the cells. After gentle pipetting through 200 mesh filters, the crude cell-containing digestion suspension after filtering was collected in a 50-mL tube for centrifugation for 10 min at 1500 rpm. The cells were resuspended in 40 mL of 10% DMEM/F12 medium and separately slowly added to two T75 cell flasks (20 mL per flask) before placing them into a 37°C cell incubator with a 5% CO2 atmosphere for cell attachment purification. After 2.5 h, the supernatant was carefully aspirated, followed by another centrifugation before the cell precipitate was resuspended in DMEM/F12 medium containing 10% FBS together with 100 U/mL penicillin (Gibco, Cat No.15070063), 0.1 mg/mL streptomycin (Gibco, Cat No. 11860038), 5 μg/mL amphotericin (Gibco, Cat No. 15290026) and 15 μg/mL enrofloxacin (Sigma-Aldrich, Cat No. PHR1513). The cells were gently pipetted and mixed before being cultured in four 100-mm cell culture dishes biocoated with Collagen I (Corning, Cat No. 354450).
Culture medium was altered with DMEM/F12 containing 3% FBS after 18 h for cell attachment to inhibit the outgrowth of other cells, including smooth muscle cells and other nonepithelial cells. The PTEC cultures were supplemented with the above antibiotics and epithelial cell growth factors (LONZA, Cat No. CC-4175). Every other day, the culture medium was changed once, supplemented with an additional 20 ng/mL hEGF (Sigma-Aldrich, Cat No. E9644) as well as 0.2 ng/mL retinoic acid (Sigma-Aldrich, Cat No. 307-79-4). The morphological characteristics of the primary cells were assessed every three days by light microscopy (Zeiss). Primary PTECs were passaged upon reaching 85% confluence after culturing for 6 days and digested with 0.05% trypsin (Gibco, Cat No.25300062). Primary cultures were analyzed for mycoplasmas using a Mycoplasma PCR Detection Kit (GeneCopoeia, MD, Cat No. MP004-GC) to ensure that the primary PTECs were free from mycoplasma contamination.
Cytokeratin 18 analysis of primary cell cultures
To confirm our isolated primary cells were of epithelial cell origin, immunostaining with the epithelial cell-origin marker cytokeratin 18 was performed as previously described with some modifications [7, 23]. Specifically, primary PTECs of passage 2 were cultured in a 24-well cell plate before being fixed with 4% paraformaldehyde at room temperature (RT) for 15 min. Among them, three wells of primary PTECs were cultured in 25% FBS with no epithelial cell regulation factors to guarantee that nonepithelial cells such as smooth muscle cells become the predominant cells (set as the negative control). The 0.1% Triton X-100 in PBS was added to cells for 3 min at RT, followed by blocking with 1% bovine serum albumin (BSA, Beyotime, Cat No. ST023) in phosphate buffer saline (PBS) for 2 h before incubation with cytokeratin 18 primary monoclonal antibody (Abcam, Cat No. ab668) at a 1:500 dilution at 4°C overnight. After three times PBS washing, the cells were incubated with anti-mouse IgG H&L (Texas Red ®) (Abcam, Cat No. ab6787) at a dilution of 1:1000 for 1 h at 37°C. Cell nuclei were visualized using DAPI staining solution for 3 min before observation by fluorescence microscopy (Zeiss). To evaluate the percentage of PTECs from primary cells, six microscopic fields were randomly selected in each three well in a 24-well plate at 100 × magnification. The same cutoff values and manual exposure settings were used to count the cell numbers. Moreover, cytokeratin 18 on smooth muscle cells was used as a negative control and also observed.
Construction of immortalized hTERT-PTECs
After culturing for two passages, primary PTECs were seeded at a density of 2×105 cells/well into 24-well cell plates, and the culture medium was replaced with fresh medium containing the abovementioned 3% FBS and antibiotics prior to transfection. When the primary PTECs reached approximately 85% confluence, the cells were transfected with our previously established recombinant plasmid pEGFP-hTERT [7] with Lipofectamine® 3000 (Invitrogen, Cat No. L3000015). The pIRES2-EGFP vector was applied as a transfection control. Cells were observed using fluorescence microscopy (Zeiss) after stationary culture for 70 h. Because pEGFP-hTERT contains green fluorescent protein (EGFP) and the neo gene, it could be applied as a marker to guarantee a successful transfection rate by with pEGFP-hTERT (final extracted endotoxin-free plasmid concentration should be more than 1 µg) into primary cells and conferred G418 antibiotic resistance. Selected positive cell clones were obtained in culture medium with 300 µg/mL G418 (Gibco, Cat No. 15710072), and monoclonal cells appeared after approximately 7 d of selection. Reliable hTERT overexpression cell lines were constructed from several healthy as well as large colonies with G418 resistance. Positive cells were finally obtained after three rounds G418 antibiotic selection. The newly established cell line, hTERT-PTECs, was stored after being confirmed as mycoplasma negative.
Cell growth kinetics assessment and replicative lifespan determination
Primary PTECs and immortalized hTERT-PTECs used in this study are primary PTECs at passage 4 and hTERT-PTECs at passage 60 unless specifically stated. Both cells were seeded at a density of 2.5×104 cells/well cultured in a 48-well plate, and medium changed once every two days. Five wells of cells were collected with trypsin, and the cell numbers were calculated every day until day 8 for cell growth kinetics analysis according to a previous study [8, 16].
To compare the proliferate ability between primary and immortalized cells, cell cycle analysis was performed. Both primary and immortalized cells were harvested with 0.25% trypsin without EDTA, fixed with 10 mL 75% ice-cold ethanol at 4°C overnight before washing twice with ice-cold PBS. The supernatant was discarded, and both cells were treated with 5 µg/mL propidium iodide in a volume of 0.3 mL (BD Biosciences, Cat No. 550825) for 30 min in the dark at RT after washing twice with ice-cold PBS. Cell numbers for cell cycle analysis should be no less than 1×106. Flow cytometry on a BD Accuri C6 (BD Biosciences) was applied, and the cell-cycle distribution was determined with FlowJo software [7, 16] as described previously. The percentages of gated cells in the cell cycle process included the G1/G0, S and G2/M phases.
Western blotting analysis of hTERT
As described previously [24], more than 5×106 primary PTECs, hTERT-PTECs and the positive control HEp-2 cells (CLS Cat No.300397/p694_Hep-2) were harvested after twice PBS washing before the cells were extracted with total protein extraction kits (Vazyme, Cat No. E211). Cellular proteins were stored at -80°C directly after rapid liquid nitrogen frozen for 30 s. Approximately 20 µg of each total protein was resolved by 10% SDS-PAGE before being transferred to a nitrocellulose filter membrane (Thermo Fisher, Cat No.77010). The membranes were blocked with 5% nonfat milk in Tris Buffered saline (TBS), which contains 0.5% Tween 20 (TBST) and were put in a 37°C incubator with gentle shaking for complete reaction for 2 h. Then the membrane was followed by incubation with anti-hTERT (Santa Cruz Biotechnology, Cat No. sc-377511) or anti-β-actin (Bioworld, Cat No. BS6007MH) at 1:600 with 5% nonfat milk in TBST in a 37°C incubator for 1.5 h. After three times TBST washing, the membranes were incubated with secondary antibody (HRP-labeled goat anti-mouse IgG (H+L) (Boster, Cat No.BA1051) at 1:5000 with 5% nonfat milk in TBST for 1 h. Incubation with electrochemiluminescence substrate reagents (Thermo Fisher, Cat No. 32109) was conducted after three times TBST washing, before the membrane was finally observed with a ChemiDoc XRS+ system (Bio-Rad).
Telomerase activity assay
Primary cells, hTERT-PTECs and positive control HEp-2 cells were subjected to a telomerase activity assay using a TeloTAGGG Telomerase PCR ELISAPLUS detection kit (Roche, Cat No. 11854666001), as described previously [8, 14]. Positive control HEp-2 cells were subjected to 85°C for more than 10 min (named H-HEp-2, set as negative control) to heat-inactivate telomerase protein. Primary cells, positive control and our constructed immortal cells were adjusted to the same concentration. Absorbance determination was performed at 450 nm with a reference wavelength of 690 nm. Every test was performed in five replicates. An absorbance less than 0.2 indicated that there was no telomerase activity (negative).
Karyotype determination
The chromosomes number determination in immortalized hTERT-PTECs was analyzed and modified as described previously [7, 16]. HTERT-PTECs were seeded in a T25 flask (Corning, Cat No. 354401) for approximately 24 h before cells being treated with colcemid (Gibco, Cat No.15212012) at 0.25 μg/mL (final concentration) and put in a 37°C incubator for 5 h. The cells were then treated with trypsin, centrifuged at 1300 rpm for 10 min, suspended and incubated in a total of 5 mL 0.075 M KCl in a 15-mL tube for 20 min in a 37°C water bath. Fresh ice-cold acetic acid/methanol (1:3, v/v) solution was made, and the cells were fixed and subjected to blowing and careful pipetting with a dropper before centrifugation at 1300 rpm and collection. This procedure was repeated once, and the remaining 0.3 mL was re-suspended, and every two to four drops of the dispersed cell suspension was smeared on every ice-cold glass slide from a height of at least more than 0.6 meters before air drying. The number of chromosomes in metaphase (n = 100 cells) was observed microscopically after the chromosomes were stained with 4% Giemsa solution for 15 min (Gibco, Cat No.10582013).
Relative quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay
Total RNA was extracted from primary and immortalized PTECs immediately after cell lysis using a Total RNA Extraction Kit (Omega, Cat No. R6834). With the HiScript® II QRT kit (+gDNA wiper) (Vazyme, Cat No. R223), more than 1.5 μg of extracted RNA was reverse transcribed before being subjected to the HiScript® II One Step qRT-PCR SYBR® Green Kit (Vazyme, Cat No. Q221) and run on an ABI 7500 Real Time PCR System. Sixteen genes were selected, including genes that participate in the cell-cycle process (sox17, sox2, ccnb1, ccnb2, ccna1 and ccna2) [25-28], response to oxidative stress (GPX1, DUOX1, DUOX2 and SOD1) [25, 29] and host innate immune response (IL-6, CSF2, IL-8, CXCL2, STAT3 and JNK1) [30-32]. Internal controls were β-actin and GAPDH. Primers are listed in Supplementary Table S1. The fold change in mRNA expression between primary and immortalized PTECs was analyzed using the 2-ΔΔCT method.
In vivo nude mouse tumorigenicity assay
A tumorigenicity assay was performed to assess the functional tumorigenicity of immortalized cells. Nine nude mice were randomly separated into three groups, which were inoculated with hTERT-PTECs, HEp-2 cells (positive control) and primary PTECs (negative control). The above cells were subcutaneously injected into the left flank of each of three nude mice (1 site/mouse at 1.5 ×106 cells). All nude mice were housed in a specific pathogen-free environment, and the appearance of tumors was determined once a week until two months followed by sacrifice.
Soft agar analysis in vitro
The soft agar in vitro assay was conducted to check tumorigenicity in vitro in accordance with previous studies [8, 14]. The 0.5% agar in the bottom layer with 0.75 mL medium was seeded in one 24-well cell plate before being placed at 4°C overnight to solidify the gel. The next day, hTERT-PTECs, HEp-2 cells (positive control) and primary PTECs (negative control) were treated with trypsin and resuspended at four concentrations: 5×103, 1×104, 2×104 and 4×104 cells per mL. Then, 0.5 mL of the cell suspension was prepared using 0.5% agar (final agar concentration: 0.33%, Bactoagar, Thermo Fisher, Cat No. DF0140010). The cells were spread over the lower solidified layer before being placed in a cell incubator. Colony observation was performed with a microscope once a week for at least six weeks. Even the appearance of a single colony suggested that the cells were able to undergo anchorage-independent growth.
Susceptibility of hTERT-PTECs to four species swine-origin mycoplasmas
Briefly, 2×105 cells/well were cultured in 24-well cell plates to perform CCU50 experiments to calculate the adhesion rate for various strains of the four mycoplasma species listed above. For the representative Mhp, to confirm whether immortal cells could be used as an adhesion model, 2×105 cells/well were additionally cultured in 24-well cell plates for the indirect immunofluorescence assay (IFA), and 5×105 cells/well were cultured in 12-well cell plates for western blotting and quantitative real-time PCR (qRT-PCR).
For the IFA assay, hTERT-PTECs in a 24-well cell plate after Mhp infection were washed twice with PBS before fixation and blocking as described for the cytokeratin 18 immunostaining above method part, with the exception of the primary antibody, which was the mouse monoclonal antibody (anti-P97R1, made in our lab) at a dilution of 1:400. Cell visualization was conducted after staining the nuclei with DAPI for 2 min at RT with a fluorescence microscope (Zeiss). All Mhp-infected cells were analyzed in triplicate.
hTERT-PTECs after Mhp infection were washed twice in PBS and collected by 0.25% trypsin (without EDTA, Gibco, Cat No.15050065) digestion before western blotting analysis. The detailed protocol is exactly the same as the method described above in the hTERT protein western blotting section, with the exception of the primary antibody, which was the mouse monoclonal antibody anti-P97R1 (produced in our lab) or the loading control anti-β-actin (Bioworld, Cat No. BS6007MH) at the dilution of 1:500.
Moreover, DNA was extracted from the Mhp-infected cells in 12-well cell plates (Axygen, Cat No. AP-MN-BF-VNA-250). DNA was quantified by qRT-PCR using the TaqMan system, and primers and TaqMan-BHQ probes were designed based on the conserved sequence of the P97 gene of Mhp. A standard curve was constructed using a 10-fold diluted standard plasmid, PMD-T-P97, before the QuantStudio R5 Real-Time PCR System was used to determine the DNA copies as described previously [18]. All Mhp-infected cells were run in triplicate. In addition, hTERT-PTECs seeded in 24-well plates were infected with the swine-origin mycoplasma strains isolated from four species for 12 h after three times PBS washing. The cells were then digested with 0.25% trypsin (without EDTA), and collected after the digestion being stopped. The concentration of each strain of swine-origin mycoplasmas before and after infection was calculated with the CCU50 test. Before inoculation, the DMEM/F12 cell culture medium was diluted (Mhp and Mhr strains were diluted 10 and 100 times) or resuspended (MF and MHS strains were resuspended in DMEM/F12 cell culture medium) after centrifugation for 20 min at 11000 rpm to ensure that each strain of the four mycoplasma species was approximately set to 107 CCU50/mL. Every strain of each species was assessed in five independent replicates.
Statistical analysis
Data from quantitative real-time PCR analysis and the cell-cycle test were analyzed using SPSS Statistics software v20.0 and FlowJo software v7.6. Cell numbers for the cell growth curve between primary and immortalized PTECs from day 6 to day 8 were analyzed by the multiple t test, and telomerase activity between primary and immortalized PTECs were assessed via multiple comparisons of analysis of variance (ANOVA). p < 0.05 was considered a significant difference, and p < 0.01 was considered an extremely significant difference.