3.3.1 Chlamydophila pneumoniae propagation and isolation
Since C. pneumoniae can infect humans via aerosols, C. pneumoniae propagation as well as all following studies were carried out in safety cabinets and using personal air filters. C.
pneumoniae HK isolate (a clinical respiratory isolate, generously provided by Prof. E.
Straube, National Consultative Laboratory for Chlamydia, Institute of Medical Microbiology, University of Jena, Germany) was used in this study. Cultivation of C. pneumoniae was performed by intracellular growth of the bacteria in the human epithelial cell line HEp-2 (ATCC CCL-23) (227), as described by Rodel et al. (228). The multiplication was monitored using the Kallestad Pathfinder system (Sanofi Diagnostics Pasteur, Redmond, WA, USA). In order to exclude Mycoplasma contamination, cell cultures and chlamydial stocks were routinely tested by Mycoplasma PCR ELISA (Roche Diagnostics GmbH, Mannheim, Germany). The number of C. pneumoniae was quantified by real-time PCR (see below). For this purpose, C. pneumoniae infected HEp-2 cells were harvested, the cell debris was separated by low speed centrifugation (10 min x 500g) and DNA was prepared with the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
3.3.2 Infection of mice and organ preparation
Four week old, specified pathogen-free female Swiss Webster mice, C3H/HeN mice, C3H/HeJ mice (Charles River Laboratories, Sulzfeld, Germany), TLR2 wild-type mice (TLR2+/+) and TLR2-/- mice, generated by homologous recombination by Deltagen (Menlo Park, CA, USA) and kindly provided by Tularik (South San Francisco, CA, USA) were fed ad libitum with Altromin 1314 (Altromin, Lage an der Lippe, Germany) and kept at 20-22°C, 50-60% air humidity and at a 12 hour day-night cycle. The genetic background of the TLR2 mice was B57BL/6. All TLR2 mice were bred in the animal facility of the University of Konstanz, Germany. They were inoculated intranasally with 106 or 107 EB of C.
pneumoniae HK under light ether anesthesia to induce hyperventilation. One drop (50 µl) of inoculum was delivered onto the nostrils.
3.3.3 Kinetics
At 15 min as well as on days two, four, 11, 18, 33 and 95 after infection, Swiss Webster mice (5 mice per group) were anesthetized (Trapanal i.p. (100 mg/kg), ALTANA Pharma, Konstanz, Germany), opened along the midline of the abdomen and the abdominal aorta was cannulized and perfused thoroughly for 1 min with approximately 5 ml sterile PBS (PAA Laboratories, Cölbe, Germany) with 1,6 mg/ml EDTA (Sarstedt, Nümbrecht, Germany) to remove blood from the organs. The blood was collected from the vena cava and the respective dilution was calculated by determination of the hemoglobin content (Hemoglobin test kit, Sigma, Munich, Germany). BAL fluids were obtained by transtracheal lavage technique. The BAL was immediately stored at -70°C. Samples of lung, aorta, brain, bladder, spleen and liver were obtained. All organs were weighed and homogenized (except in case of the lung where a specimen for histopathology was removed) in a sterile homogeniser (Medicon, 50 µM Dako Cytometation, Hamburg, Germany) using a Medimachine (Dako Cytometation) with 1 ml sterile ice cold PBS. After homogenization the tissue was filtrated through a 50 µM mesh Filcon sterile syringe (Dako Cytometation) to avoid clotting and stored at –70°C.
3.3.4 Antibiotic treatment
Swiss Webster mice (n=5) were inoculated intranasally with C. pneumoniae HK as described above. Antibiotic treatment was started two days after inoculation with C.
pneumoniae. A mixture of 20 mg x kg-1 rifampicin (Sigma) and 20 mg x kg-1 azithromycin (Pfizer, Karlsruhe, Germany) were administered by lavage thrice at 7 a.m., 1 p.m. and 7 p.m. for four days. All substances were dissolved in 1% tylose (Merck, Darmstadt, Germany) and applied in a volume of 25 ml x kg-1. Mice were killed at day seven after infection and serum, BAL and lung were obtained for further analysis.
3.3.5 Role of TLR
Swiss Webster mice, C3H/HeN mice, C3H/HeJ mice, TLR2+/+ and TLR2-/- mice (n=5) were inoculated intranasally with C. pneumoniae HK as described above. At day 2 or 18 after infection mice were killed and BAL and lung were obtained for further analysis.
3.3.6 Histopathology
Histopathology was performed blinded to the infection conditions at the laboratory of Dr. K.
Tuch (ALTANA Pharma, Hamburg, Germany). Lung tissue from infected and control mice was removed as described above and immediately fixed in 8% MOPS-buffered formalin, pH 7.4. Specimens were embedded in paraffin, sectioned and stained with hematoxylin and eosin.
3.3.7 DNA preparation
DNA extraction was carried out using the DNeasy Tissue Kit (Qiagen) according to the manufacturer’s protocol with slight modifications: 40 µl of proteinase K were incubated with 260 µl buffer ATL and 100 µl organ homogenate over-night at 55°C. Then 400 µg RNAse and 400 µl buffer AL were added and the sample was incubated at 70°C for 10 min. After addition of 400 µl 100% ethanol, 650 µl of the sample were loaded onto the column in two steps and centrifuged. After two wash steps the DNA was eluted using 2x 100 µl AE buffer. For extraction of DNA from BAL, 210 µl ATL buffer, 150 µl BAL and 10 µg carrier RNA (Qiagen), the latter of which was used to improve the DNA yield, were mixed and incubated for 3 h at 55°C. No RNAse digestion was performed and the DNA was eluted using 100 µl AE.
3.3.8 Real-time PCR
Real-time PCR was performed using a LightCycler rapid thermal cycler system (Roche).
Primer and probe sequences were selected from the 16S rRNA of C. pneumoniae (Accession no. U68426). The primers resulted in an amplification product of 640 bp.
Primer sequences were for the forward primer 5’-ATGTGGATGGTCTCAACCCCAT-3’ and for the reverse primer 5’-GGCGCCTCTCTCCTATAAATAGG-3’ (Thermo Hybaid/Interactiva Division, Ulm, Germany). Probe sequences were 5’-ACCTCACGGCACGAGCTGACGA-3’ for the fluorescein labelled probe and 5’-AGCCATGCAGCACCTGTGTATCTGTCC-3’ for the LC Red640 labelled probe (TIB MOLBIOL Syntheselabor, Berlin, Germany). Real-time PCR was done in 20 µl with 0.5 µM of each primer, 0.1 µM of each probe, 2 mM MgCl2, 2 µl FastStart DNA Master Hybridization Probes (Roche) and 5 µl DNA (40 ng) template. Thermal cycling was
cycles) with an annealing temperature of 65°C and an elongation time of 26 sec.
Fluorescence was measured at the end of each annealing phase.
Comparison of the selected primers (BLAST search) with the GenBank sequences (National Center for Biotechnology Information, NCBI, Microbial Genome Database, MBGD)(257) revealed no cross-binding activity. We also tested different bacterial strains including Salmonella typhimurium (ATCC 15277), Staphylococcus aureus (ATCC strain 12598), Escherichia coli (E. coli, K-12 strain JM-109, a kind gift from Dr. G. Grütz, Charité, Berlin, Germany), Borrelia burgdorferi strain sensu stricto (a kind gift from R. Oehme, LGA Stuttgart, Germany), Eubacterium acidaminophilum (DSM strain 3595), Methanospirillum hungatei (a kind gift from Prof. B. Schink, University of Konstanz, Germany), and samples of murine DNA prepared from BAL, blood, lung, aorta, brain, bladder, spleen and liver to exclude cross-reactivity in the assay. Furthermore, to control for negative and false-positive results, a false-positive and a negative control were included in each run. The amount of EB in the samples was determined using chlamydial DNA as standard. The concentration of the standard DNA was calculated on the basis that the chlamydial genome consists of 1.2 million base pairs weighing 1.3 fg.
3.3.9 Isolation and stimulation of murine bone marrow cells
C3H/HeN mice, C3H/HeJ mice, TLR2+/+ and TLR2-/- mice were put under terminal pentobarbital anaesthesia (Narcoren, Merial, Halbergmoos, Germany). Bone marrow cells were isolated from the femurs by rinsing with 10 ml ice-cold PBS and were transferred to siliconized glass tubes. After centrifugation, cells were resuspended in medium (RPMI 1640, PAA Laboratories) containing 10% FCS (Roche) and transferred to 96-well cell culture plates (5x 105 cells/well, Greiner, Frickenhausen, Germany). Murine bone marrow cells were then stimulated with C. pneumoniae, LPS from Salmonella abortus equi (Sigma) or LTA from Staphylococcus aureus (prepared in house according to Morath et al. (185)) and incubated for 24 h at 37°C and 5% CO2.
3.3.10 Serology
Serology was performed at the laboratory of Dr. U. Brunner (Konstanz, Germany). Serum antibodies were detected by MIF using glass slides with EBs from the C. pneumoniae isolate TWAR-183 (Labsystems, Helsinki, Finnland, purchased from Merlin, Bornheim, Germany). Murine Ig were detected with fluorescein-conjugated goat antibodies, anti-murine IgM was purchased from Biozol (Eching, Germany) and anti-anti-murine IgG from Sifin
(Berlin, Germany). Titers of <1:8 were regarded as negative because of non-specific background reactions.
3.3.11 ELISA
The cytokines tumour necrosis factor α (TNFα), interleukin 6 6) and interleukin 10 (IL-10) were determined in BAL, blood and supernatants of lung homogenates by ELISA based on commercial antibody pairs and standards (TNFα antibodies were purchased from R&D, Wiesbaden, Germany, IL-6 antibodies and IL-6 and TNFα standards from Pharmingen, Hamburg, Germany). IL-10 was measured by OPT-EIATM mouse IL-10 Set (BD Biosciences, San Diego, CA, USA). Serum amyloid A (SAA) was determined in blood using a kit from Biosource (Solingen, Germany). The detection limits of the ELISAs were 39 pg/ml, 13 pg/ml, 10 pg/ml and 120 ng/ml for TNFα, IL-6, IL-10 and SAA, respectively.
3.3.12 Statistics
Statistical analysis was performed using the GraphPad InStat program 3.0 (GraphPad Software, San Diego, USA). Differences between two groups were assessed by unpaired t test. Unpaired samples were assessed by one-way analysis of variance followed by Dunnett’s test. Data were log-transformed to achieve Gaussian distribution. In the figures
*, ** and *** represent p values <0.05, <0.01 and <0.001, respectively.