Expression profiles of the immune genes CD4, CD8β, IFNγ, IL-4, IL-6 and IL-10 in mitogen-stimulated koala lymphocytes (Phascolarctos cinereus) by qRT-PCR

Ethics statement

This study was carried out with the approval of the ethics committee of Taronga Conservation Society Australia/University of Sydney (permit no 4a/01/11) and the New South Wales Government (Scientific licence S10418). All efforts were made to minimise animal suffering. The project was funded by the Australian Research Council Grant LP0989701.

Sample collection and PBMC stimulation

Study animals were 4 male and 3 female koalas from a captive, chlamydia-free, KoRV-A positive collection, with no history of disease within the past 6 months. Blood (4–8 ml) was collected, in February 2012, from the cephalic vein using manual restraint. It was placed immediately into sodium heparin vials (Vacutainer; Becton Dickinson and Co, NJ, USA) and then transported at room temperature for processing within 3 h of collection. The blood was centrifuged at 800 × g for 10 min, the plasma aspirated, and cells resuspended to 2 times the original blood volume in sterile 1 × PBS. Cells (4 - 5 ml) were overlaid on a double-density gradient consisting of 2 ml Percoll 1.056 g∕ml (72.2% PBS, 25% Percoll PLUS, 2.8% 1.5 M NaCl) above 3 ml of Percoll 1.082 g∕ml (35% PBS, 58.5% Percoll PLUS, 6.5% 1.5 M NaCl) (GE Healthcare Bio-sciences Corp, NJ, USA), and then centrifuged at 200 × g for 30 min with no brake. PBMCs were aspirated from the interface, transferred to a new tube, and then washed twice with warm PBS supplemented with 0.2% BSA. Cells were then counted on an automated haematology analyser (Sysmex K4500; TOA Medical Electronics Co, Japan), and suspended at 1 × 10⁶ cells∕ml in warm plain media comprised of RPMI 10% FCS (F9423; Sigma-Aldrich, Castle Hill, NSW, Australia). Cell cultures of 1 ml were incubated in plain media (unstimulated) or in media containing PMA 50 ng∕ml, Ionomycin 1 μg∕ml (Higgins, Hemsley & Canfield, 2004); PMA 25 ng∕ml, PHA 2 μg∕ml (Sullivan et al., 2000); or Con A 5 μg∕ml (Lau et al., 2012) in a sealed tube at 37°C for 5 h. Following incubation, cells were washed twice in warm PBS, resuspended in 1 ml RNAlater (Applied Biosystems, Carlsbad, CA, USA), stored at room temperature overnight and then frozen at −20°C until RNA extraction.

RNA and cDNA preparation

For RNA extraction, PBMC samples stored in RNAlater (approximately 1 × 10⁶ cells) were centrifuged at 2300 × g for 15 min. The supernatant of RNAlater was removed, and remaining cells were homogenised and extracted using the RNeasy Mini Kit (Qiagen, Doncaster, VIC, Australia) following manufacturer’s instructions. To remove contaminating DNA, RNA preparations were treated with DNase I following manufacturer’s instructions (Applied Biosystems, Carslbad, CA, USA). The concentration and purity of RNA was assessed using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). RNA was immediately processed for cDNA synthesis or stored at −80°C.

Reverse transcription was performed using the Revertaid First Strand cDNA Synthesis kit (Thermo Scientific, Lithuania) according to the manufacturer’s instructions, using oligo dT primers. Due to biological variation, 70–700 ng of RNA was used (in 10 μl) in each reaction. To control for contamination for genomic DNA, ‘no reverse transcriptase’ (NRT) controls were made using the same protocol omitting reverse transcriptase. cDNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and then cDNA was stored at −20°C until use in qPCR.

Generation of partial sequence for koala immune genes

Conserved regions of the genes IL-4, IL-10, IFNγ, IL-6, CD4, CD8β and GAPDH in marsupials, including grey short-tailed opossum Monodelphis domestica, tammar wallaby Macropus eugenii, Tasmanian devil Sarcophilus harrisii, common brushtail possum Trichosurus vulpecula (Table 1) were identified using ClustalW (Thompson, Higgins & Gibson, 1994) in BioEdit (Hall, 1999). PCR amplifications were carried out in an MJ Mini^TM thermal cycler (Bio-Rad Laboratories Inc., CA, USA) in 25 μl reactions containing 0.4 μM each primer (Sigma-Aldrich, Castle Hill, NSW, Australia), 1 × High Fidelity PCR Buffer, 1.5 mM MgSO₄, 0.2 mM dNTPs, and 0.75 units of Platinum® Taq DNA Polymerase High Fidelity (Invitrogen, Mulgrave, VIC, Australia). cDNA (10–30 ng) was used for all PCR reactions with the exception of IL-6, where genomic DNA was used as primers designed were positioned within introns due to a scarcity of suitable conserved primer sites within the exons. Cycle conditions for touchdown PCR were: an initial activation step of 95°C for 5 min, then cycles of 94°C for 30 s (denaturation), highest annealing temperature for 30 s (annealing), 72°C for 30 s (extension) with the annealing temperature reducing −1°C per cycle until lowest annealing temperature was reached, followed by 20–25 cycles at the lowest annealing temperature, and a final extension at 72°C for 10 min. Amplicons were visualised by electrophoresis on a 1% agarose gel, and those of expected length were excised, purified using UltraClean GelSpin DNA Extraction Kit (Mo Bio, Calsbad, CA, USA) and sequenced commercially in forward and reverse strands (Macrogen, South Korea). Returned sequence was trimmed to remove low quality sequences and aligned to create a contig using Sequencher® 4.10.1 (Gene Codes, Ann Arbor, MI, USA); and database searches were performed using BLAST (Altschul et al., 1990).

Real-time qPCR of koala immune genes

Partial immune gene sequences obtained (Figs. 1 and 2, Supplementary Material A) were used to design qPCR primers by following recommendations for general primer design for qPCR (Wang & Seed, 2006) and using the primer analysis programs Primer 3 (Rozen & Skaletsky, 2000) and UNAfold (Markham & Zuker, 2008) (Table 1). All qPCR primers designed are located within exons, including IL-6, and annealing temperatures were optimised by gradient PCR. Subsequent qPCR was performed with the following conditions: triplicate qPCR reactions comprised 10 μl of 2 × SsoAdvanced SYBR Green Supermix (Bio-Rad Laboratories Inc., CA, USA), 1 μl each primer (Table 2), 2 μl of cDNA template, and PCR grade water in a final volume of 20 μl. Cycling conditions (CFX96 Touch; Bio-Rad Laboratories Inc., CA, USA) were 95°C for 10 min (activation) followed by 40 cycles of 95°C for 15 s (denaturation), and 60°C (55°C for IL-4, IL-10 and GAPDH) for 60 s (annealing and extension). The melting profile was obtained by 90 s pre-melt conditioning at 55°C and then heating the reactions in one-degree increments from 52°C to 95°C with 5 s stops. Primers were titrated to remove primer dimers from no template controls and qPCR products were confirmed by gel electrophoresis, followed by sequencing and alignment, using BLAST searches as previously described. Primers for the candidate reference genes 28 s rRNA (28 s) and koala β actin were obtained from the literature (Daly et al., 2009; Markey et al., 2007).

Efficiency of each reaction, dynamic range, optimal cDNA concentration, y-intercept, slope and r² calibration curve were determined using a triplicate 10 × serial dilution of a stimulated sample, covering 8 orders of magnitude. Efficiency was calculated as 10^{(−1∕slope)} − 1. Standard curves were also constructed using purified PCR product, quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and then serially diluted (1: 1 × 10³–1: 1 × 10¹²) until the curve was no longer linear (limit of quantification; LOQ) and no sample was detected (limit of detection; LOD). DNA concentration was converted to DNA molecule copy number using the concentration and molecular weight of the amplicon (copy number = [ng DNA × 6.022 × 10²³]∕[length (bps) × 1 × 10⁹ ng g⁻¹ × 650 g mol⁻¹ bps⁻¹]).

Expression of cytokine genes by mitogen stimulated PBMC

Samples of the three types of stimulated cells, and the non-stimulated cells, were assayed in triplicate using each set of cytokine primers and the potential reference gene primers. cDNA (200 ng in 2 μl) was used in each reaction, based on the calculated dynamic range of the assay. No template (NT) and no reverse transcriptase (NRT) control samples were run in triplicate with each primer set.

Raw Cq values were transformed to 2^−Cq for analysis. The 2^−ΔCq method was used to assess difference between unstimulated samples as well as between mitogen stimulation protocols (Schmittgen & Livak, 2008) and to calculate reference gene relative expression (Dheda et al., 2004). The 2^−ΔΔCq method was used to assess cytokine expression fold change (Schmittgen & Livak, 2008). Normality was assessed using a Shapiro–Wilk test (GraphPad Prism 6.03; GraphPad Software Inc., CA, USA). As data were not normally distributed, Mann–Whitney U test and Freidman’s tests were performed, using Minitab 16 (Minitab Inc., PA, USA). A P value of <0.05 was considered significant.

Partial sequence of koala immune genes

Partial koala sequences obtained and used for primer design showed >58% amino acid homology with relevant genes of opossum species, and >71% and 78% with available tammar wallaby and Tasmanian devil amino acid sequences, respectively. GenBank/Ensembl sequences for alignment, forward and reverse primers, optimum annealing temperature and amplified fragment size are all shown in Table 1. All sequences possessed key conserved residues, confirming their identity (Figs. 1 and 2, Supplementary Material A). Sequences for IL-10, and IFNγ were consistent with those of Mathew et al., (2013a; 2013b). Sequences have been lodged in GenBank under the accession numbers: PhciCD4, KF731654; PhciCD8b^∗01, KF731655; PhciCD8b^∗02, KF731656; PhciIFNγ, KF731657; PhciIL4, KF731658; PhciIL10, KF731659; PhciGAPDH, KF731660; and PhciIL6, KF731661.

qPCR optimization and validation

All assays were highly sensitive, had a linear dynamic range greater than 6 orders of magnitude and had an optimal amplification efficiency of 92% or greater (Table 2). The relative expression results for reference genes are shown in Fig. 3. Of the three candidate reference genes, 28 s and GAPDH were considered suitable for all treatments, with a mean relative expression usually <2, and a maximum relative expression <3.5 (Dheda et al., 2004). β actin expression was significantly up-regulated by Con A and was significantly down-regulated by PMA-ionomycin treatment, and was therefore not considered a suitable reference gene for this experiment, although it appears appropriate for use in experiments using PMA–PHA stimulation. Results for all cytokines and all mitogen stimulation protocols were highly similar when calculated as 2^−Cq values (not-normalised) or 2^−ΔCq values (normalised against the geometric mean of the two reference genes, 28 s and GAPDH) further supporting suitability of GAPDH and 28 s as reference genes (Schmittgen & Livak, 2008).

Cytokine and CD4/CD8β expression in unstimulated samples

The 2^−ΔCq cytokine expression in un-stimulated samples varied markedly among individuals, the median × 10⁻⁴ (min −max)2^−ΔCq values are shown in Fig. 4. The expression levels of CD4 and CD8β are much higher than the cytokine levels. The individual with the highest IL-4 and IL-10 expression also had low IFNγ expression (0.9 × 10^-4), consistent with a Th2 dominated profile. The CD4∕CD8β expression ratios for the unstimulated samples varied from 0.1 to 6.3 with a median value of 2.1.

Response of PBMC to mitogen stimulation

Based on ΔΔCq results (Fig. 5), the most potent up-regulation of IFNγ (median fold-change 523) and IL-4 (median fold-change 96) was induced by PMA-ionomycin, though all protocols increased expression of IFNγ. IL-4 was also induced by Con A (median fold-change 5) but not PMA–PHA. IL-10 was also induced but less dramatically; PMA-ionomycin induced a median fold-change of 4.0, while PMA–PHA and Con A did not measurably increase its expression. There was no significant change in IL-6 expression overall with any protocol, though two individuals exhibited 2.8 and 5.3 fold changes in response to PMA–PHA. Expression of CD4 was significantly down-regulated with PMA–PHA stimulation and CD8β was down-regulated with all mitogen stimulation protocols. There was no apparent sex difference in expression of any of the cytokines in the unstimulated or mitogen-stimulated samples, though the sample size was too small for statistical analysis (data not shown). Individual responses to PMA-ionomycin stimulation varied markedly, ranging from fold-changes of 11 to 189 for IL-4, 0.3 to 7 for IL-10 and 25 to 2985 for IFNγ.