Cell culture media and reagents
Phosphate-buffered saline (PBS), Eagle’s minimum essential media (MEM) GlutaMAX, gentamicin/amphotericin B solution were obtained from Invitrogen (Lofer, Austria). RPMI-1640, MEM non-essential amino acid, human male AB serum (sterile-filtered), cycloheximide, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), fetal bovine serum (FBS), and lipopolysaccharide (LPS) from E. coli (055:B5, purified by gel filtration) were purchased from Sigma-Aldrich (St Louis, MO, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD45 monoclonal antibody (mAb), R-phycoerythrin (PE)-conjugated CD14 mAb and the respective IgG control antibodies were from Becton Dickinson (Vienna, Austria).
Propagation of C. pneumoniae
C. pneumoniae strain CWL-029 was obtained from the American Type Culture Collection (ATCC, VR-1310) and propagated in HEp-2 cells (ATCC, CCL23) as previously reported ,,. In brief, HEp-2 cells were passaged in Eagle’s MEM GlutaMAX supplemented with 10 μg/ml gentamicin, 0.25 μg/ml amphotericin B, 1 vol% MEM non-essential amino acids and 10 vol% heat-inactivated FBS. Confluent monolayers were infected with C. pneumoniae and grown in the medium described above containing 1 μg/mL cycloheximide, but lacking antibiotics. Cells were spun at 1700 g for 1 h at 35°C to enhance infectivity. At 72 h post infection (hpi) at 35°C and 5% CO2, the cell monolayer was disrupted using a cell scraper and zirconium dioxide beads. Chlamydial EBs were obtained by sequential centrifugation of the lysates at 600 g (10 min) and at 30,000 g (1 h; 4°C). The pelleted EBs were suspended in sucrose-phosphate-glutamic acid buffer (0.2 M sucrose, 3.8 mM KH2PO4, 7.2 mM Na2HPO4, 5 mM L-glutamic acid, pH 7.4) and stored at −80°C. The number of chlamydial inclusion forming units (IFU) per mL was determined by infectivity titration of EBs in HEp-2 cells for 48 h at 35°C, followed by immunofluorescence staining as described below. To exclude mycoplasma contamination, cell culture and chlamydial stocks were regularly tested using the Venor™GeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany).
Isolation and culture of monocytes
PBMCs were isolated from leukocyte reduction system (LRS) chambers of a TrimaAccel® blood collector after approval by the ethics committee of the Medical University Vienna and written informed consent were obtained from all participants (ECS2177/2013). Blood from LRS chambers was diluted 1:8 (vol/vol) with PBS containing 2 mM ethylene diamine tetraacetic acid (PBS/EDTA), and PBMCs were enriched by Ficoll gradient centrifugation. Monocytes were isolated by negative depletion with the monocyte isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) yielding >80% CD14 positive cells as confirmed by flow cytometry. Viability was >95% as determined by exclusion of 7-ADD.
Monocytes were resuspended at a concentration of 4 × 105/mL in serum-free RPMI-1640 supplemented with 20 mM HEPES and cultured as described . Aliquots of 0.5 mL/well of the monocyte suspension were seeded onto 24 well flat-bottomed tissue culture plates (Corning Incorporated, NY, USA). After 3 h at 37°C, the monocyte monolayer was gently washed with serum-free RPMI-1640 to remove non-adherent cells. Adherent monocytes were kept in RPMI-1640 medium complete containing 20 mM HEPES and 10 vol% human AB serum for an additional 24 h at 37°C.
Infection of adherent monocytes
Adherent monocytes were inoculated with 2 × 103 or 2 × 104 chlamydial IFU/well, respectively, or with 1 ng/mL LPS (positive control) in a final volume of 0.5 mL medium complete. For infection, cells were centrifuged 30 min at 600 g and incubated at 37°C in 5% CO2. Cumulative culture supernatants were collected after 3, 6, 24 and 48 h, respectively, without replacing with fresh media, centrifuged at 600 g for 5 min at 4°C, and stored at −80°C until quantification of cytokines. Mock controls were prepared following the propagation, harvest and purification procedure for EBs ,, but in the absence of chlamydial infection.
The monocyte monolayer exposed to C. pneumoniae for 6 and 48 h was washed with PBS, and cells were scraped and vortexed with zirconium dioxide beads. EBs were obtained from the lysates as described above and passaged onto HEp-2 cells. At 48 hpi, HEp-2 cells were fixed and stained for immunofluorescence as described.
DNA isolation and quantification
Adherent monocytes infected with C. pneumoniae for 6 and 48 h or uninfected cells were washed with PBS and cells were counted on plates prior to DNA isolation. Total genomic DNA was isolated and purified using the QIAmp Mini DNA kit (Qiagen, Hilden, Germany). Purified DNA was quantified at 485/530 nm using the Quant-iTdsDNA HS assay and the Qubit™ fluorometer (Invitrogen, Lofer, Austria).
Real-time quantitative PCR
C. pneumoniae genomes were quantified by real-time quantitative PCR, targeting a 222 bp sequence present on Chlamydia 16S rDNA. The oligonucleotide primers and TaqMan probes were synthesized by Microsynth AG (Balgach, Switzerland) and used as described in detail previously . RT-PCR was performed in a final volume of 20 μL including 1x Master Mix and Taq polymerase (Mastermix 16S, Molzym, Bremen, Germany), forward primer (0.75 μM), reverse primer (0.75 μM), FAM-TAMRA probe (0.75 μM) and 2 ng of DNA. Amplification and detection was performed for 10 min at 95°C, followed by 50 cycles of 10 s at 95°C and 65 s at 60°C. Standards of known concentration (101, 102, 103, 104, 105 and 106 copies) were prepared for the 16S rDNA target gene from PCR amplified C. pneumoniae DNA by conventional PCR, and purified with a QIAmp Mini DNA kit, according to the manufacturer’s instructions. Samples were run in triplicate and all reactions were carried out using the iCycler IQ system (BioRad, Vienna, Austria).
Detection of intracellular pathogens by DNA microarray
In addition to RT-PCR, a prototype oligonucleotide microarray was developed. DNA amplification and labeling was carried out with one universal primer pair targeting a specific region of the 16S rRNA gene. Hybridization was performed with novel probes for Bartonella, Bordetella, Chlamydia and Mycoplasma, which were designed and modified as described ,. Four replicates of each probe at a concentration of 50 μM were printed onto silylated glass slides with reactive aldehyde groups (CSS-100 Silylated Slides; CEL Associates, Texas, USA) by the contact arrayer Omnigrid from GeneMachines (San Carlos, CA, USA) with MP 3 pins (TeleChem, Sunnyvale, CA, USA) leading to spot size of 100 μm. A hybridization control probe (5’ –TTA AAA CGA CGG CCA GTG AGC) was spotted on the array applying the same conditions as used for the target capture probes. DNA amplification and primer extension were performed according to  with modifications as described in detail in Additional file 1. Slides were scanned using an Axon Genepix 4000A microarray scanner (Axon, Union City, California) and data were analyzed as described .
Monocytes infected with C. pneumoniae (2 × 104 IFU) were cultured for 6 and 48 h on glass bottom μ-slides (170 μm thickness; ibidi GmbH, Munich, Germany). Samples were fixed with 4% paraformaldehyde for 4 min and washed 3 times with PBS. Raman spectroscopy was performed by CellTool (Bernried, Germany) using the Bio-Ram® system and the Bio-Ram® software. Raman spectra of 60 cells per assay were recorded with a 785 nm laser (80 mW), applying an accumulated time of 3 × 10 sec. Data of the biologically relevant region (700–3000 cm−1) were pre-treated with a median filter for noise reduction, unit vector normalisation and subsequent multivariate data analysis were done with the statistical software the Unscrambler X 10.3 (Camo Software, Oslo, Norway). We performed Principle Component Analysis (PCA) using the NIPALS algorithm and cross validation, which is a common procedure for spectral data analysis.
Quantification of cytokines and chemokines
The levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-12p70, IL-12p40, IL-10, monocyte chemotactic protein (MCP)-1 (CCL-2), MCP-3 (CCL-7), macrophage inflammatory protein (MIP)-1α (CCL-3), MIP-1β (CCL-4), and IL-8 (CXCL-8) were determined in culture supernatants using the Bio-Plex 200 system (Bio-Rad, Vienna, Austria).
Infected HEp-2 cells or monocytes were cultured on μ-slides (ibidi GmbH, Munich, Germany) at 6 or 48 hpi, washed with PBS and fixed in 0.5 mL of methanol for 10 min. To visualize chlamydial inclusions, cells were stained with FITC-conjugated anti-Chlamydia-LPS mAb and human cells were counterstained with Evans Blue (Pathfinder, Chlamydia Culture Confirmation System, Bio-Rad, Vienna, Austria). The cells were mounted in Fluoromount-GTM containing DAPI (Southern Biotech, Birmingham, UK) and fluorescence images were acquired with a Zeiss LSM 700 laser scanning confocal microscope (Carl Zeiss SAS, Jena, Germany) using a 40x oil objective/1.3 NA or 63x oil objective/1.4 NA.
Purity and viability of monocytes were examined by determination of CD14 positive cells and 7-aminoactinomycin D (7-AAD) exclusion. Monocytes were stained with unconjugated 7-AAD (BioLegend, Fell, Germany), FITC-conjugated anti-CD45 and PE-conjugated CD14 or with the respective IgG control antibody in PBS supplemented with 2 vol% FBS, 0.1 w% sodium azide at 4°C for 30 min. After one washing step, cells were analyzed on a Beckman Coulter FC 500 flow cytometer (Beckman Coulter, Vienna, Austria) and data were analyzed using the FlowJo software (Tree Star Inc, Ashland, OR).
Statistical analysis was performed using the software package SPSS Statistics for Windows, version 18.0 (SPSS Inc., Chicago, Illinois, USA). When comparing two groups, data were analyzed by the nonparametric Wilcoxon rank sum test. Data are expressed as means ± SD. Significance was accepted at P ≤ 0.05.