The authors are most grateful of the skillful technical help from A. Haas, M. Kreuer-Ullmann and I. Seuffert. The help from T. Rubic, Dr. S. English and Prof. Dr. R.D. Hesch in establishing the PCR is thankfully appreciated. We thank Dr. K. Tuch for performing the histopathology, Dr. U. Brunner for the adaptation of the human MIF to the mouse and performing of the serology and S. von Aulock for her help with the manuscript.
4 Investigation of immune modulation by
Chlamydophila pneumoniae and characterization of its immune stimulatory principle
Manuscripts in preparation:
I. The NOD2 3020insC polymorphism enhances cytokine induction by Chlamydophila pneumoniae and represents a risk factor for atherosclerosis
1Katja Gueinzius, 2Lutz Hamann, 2Ralf R. Schumann, and 1Corinna Hermann
II. Immune modulation by Chlamydophila pneumoniae - a mechanism of persistence?
1Katja Gueinzius, 1Oliver Dehus, and 1Corinna Hermann
III. Isolation of the immune active components of Chlamydophila pneumoniae
1Katja Gueinzius, 3Siegfried Morath, 4Matthias Maass, and 1Corinna Hermann
1Biochemical Pharmacology, University of Konstanz, Germany
2Institute for Microbiology and Hygiene, University Medical Center Charité, Berlin, Germany
3ECVAM, EU Joint Research Centre, IHCP, Ispra, Italy
4Institute for Medical Microbiology and Hygiene, University of Lübeck, Germany
4.1 Introduction
The obligate intracellular Gram-negative pathogen Chlamydophila pneumoniae belongs to the rare bacteria which are capable of persisting in the human organism (99, 116). To clarify, how C. pneumoniae are able to circumvent host defence, it is necessary to understand the mechanisms and consequences of host-pathogen interactions with Chlamydia. Several in vitro studies have shown that C. pneumoniae are able to infect various cell types like endothelial cells, smooth muscle cells, and monocyte/macrophages, leading to their activation, i.e. expression of adhesion molecules and release of
pro-responsible for the recognition by the innate immune system and the trigger of the release of inflammatory molecules remain to be defined. For the initial attachment of C.
pneumoniae to the host cells, heparan sulfate-like glycosaminoglycan has been suggested as receptor for C. pneumoniae on human epithelial cells (276), but it is not clear whether that also holds true for cells of the immune system. Furthermore, there is some evidence for a role of the mannose receptor in the attachment of Chlamydia species to murine macrophages (150).
A few years ago, the toll-like receptors (TLR) have been identified as key molecules for recognition of pathogen associated molecular patterns by immune cells, for review see Lien et al. (159). Activation of the TLR leads to signal transduction and release of cytokines. To date, 10 members of the TLR family have been identified which recognize different specific microbial patterns. While TLR4 has been described to recognize lipopolysaccharide (LPS) of Gram-negative bacteria, TLR2 mediates the recognition of lipoteichoic acid (LTA) of Gram-positive bacteria and bacterial lipoproteins. Since C.
pneumoniae are Gram-negative bacteria, it is assumed that they activate immune cells via TLR4. In general, in Gram-negative bacteria, the LPS of the outer surface membrane represents the major immune stimulatory principle which induces comparable inflammatory responses as whole bacteria. This does not seem to hold true for C.
pneumoniae. Netea et al. have reported (197) that sonicated C. pneumoniae induce the release of pro-inflammatory cytokines from peripheral blood mononuclear cells (PBMC) through TLR2 but not TLR4. The recognition of C. pneumoniae as well as chlamydial heat shock protein 60 (chsp60) by murine bone marrow derived dendritic cells seems to depend largely on TLR2 and only to a minor extent on TLR4 (44, 47, 217). In contrast, chsp60 has also been shown to induce inflammatory responses in endothelial cells and macrophages and to stimulate the proliferation of smooth muscle vascular cells via TLR4 (25, 240).
Nevertheless, there are also hints for TLR independent target cell activation (198). Since C. pneumoniae are obligate intracellular bacteria, the recently identified cytoplasmatic NOD proteins, NOD1 and NOD2, which are implicated in recognition of peptidoglycan components such as muropeptides (35, 124), also qualify as candidates for the recognition of C. pneumoniae. Only recently, it was shown that NOD1, which is expressed in virtually all tissues (123), plays a dominant role in C. pneumoniae-induced interleukin (IL)-8 release in endothelial cells (203). For NOD2, which is so far only described for monocytes/macrophages (94) and Paneth cells of the ileum (155), a frame shift (3020insC) and two missense (2104 and 2722) variants were found and reported to be
independently associated with Crohn’s disease (119). The best studied polymorphism is the 3020insC, which results in the truncation of the ligand-binding region at the C-terminus and seems to be a loss of function phenotype. In Crohn’s patients this may result in the inability to control bacterial infections in the intestinal mucosa, leading to chronic inflammation (81, 82).
The chlamydial LPS was shown to harbour a specific epitope, which has not been detected in other bacteria and is therefore suggested to be family-specific (55). Until now, only the LPS of C. trachomatis was isolated and its structure has been elucidated (106, 233). Chlamydial LPS is a so-called “rough type” LPS that has lost parts of the O-chain. Its lipid A contains five untypical long fatty acids (14 to 21 carbons), which result in a high hydrophobicity (220). However, the TLR-dependence of C. trachomatis LPS is controversially discussed, since it was first described to be TLR4 dependent (216), but a recent study by Erridge et al. demonstrated that it is TLR2 dependent (64). In addition, its immune stimulatory potential was shown to be 100-fold weaker compared to LPS from Salmonella minnesota or from Neisseria gonorrhoeae (121). This might be due to the structural differences of the fatty acid chains of the lipid A moiety (142).
The following studies were performed to investigate immune recognition and immune modulation by C. pneumoniae and to clarify the nature of its immune stimulatory principle.
For this purpose, we investigated the activation of the innate immune system by C.
pneumoniae using human whole blood incubation model (103). In addition, the isolation of immune active components of C. pneumoniae has been carried out by butanol extraction and hydrophobic interaction chromatography, initially established for the isolation of LTA (185), which was now adapted to C. pneumoniae.
4.2 Materials and Methods
4.2.1 Propagation and isolation of C. pneumoniae
C. pneumoniae strains TW-183 (generously provided by Prof. Jens Kuipers, Division of Rheumatology, Medical School Hannover, Germany) and CV6 (which was isolated from a coronary artery plaque and generously provided by Prof. Matthias Maass, Institute of Medical Microbiology and Hygiene, Medical University of Lübeck, Germany) were used in this study. Cultivation of C. pneumoniae in HEp2-cells (ATCC CCL-23) (227) was
mycoplasma cell cultures and chlamydial stocks were routinely tested with the VenorGeM® mycoplasma detection kit by conventional PCR (Minerva Biolabs GmbH, Germany).
Isolation of C. pneumoniae from infected HEp-2 cultures and purification of the chlamydial elementary bodies (EB) was performed according to a protocol described by Maass et al.
(166). The chlamydial stock, containing 3x109 purified EB per ml, was stored at -80°C.
4.2.2 Propagation and isolation of C. trachomatis
C. trachomatis EB (serovar K; generously provided by Prof. Jens Kuipers) were cultured in HEp-2 cells as described by Schmitz et. al. (242). EB were purified in a discontinuous gradient of Urograffin (Schering, Berlin, Germany) by ultracentrifugation, as described (242). Then, EB were resuspended in 1 ml sucrose phosphate buffer (0.01 M sodium phosphate, 0.25 M sucrose, 5 M L-glutamic acid pH 7.2; Sigma, St. Louis, USA) and stored at -80oC.
4.2.3 Quantification of C. pneumoniae and C. trachomatis
To determine the yield of purified chlamydial EB, DNA extraction from a sample was carried out using the DNeasy® Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Real-time PCR was performed using a LightCycler rapid thermal cycler system (Roche Diagnostics GmbH, Mannheim, Germany). Primer sequences were selected from the 16S rRNA of C. pneumoniae (Accession no. U68426) and were suitable for C. trachomatis as well (Accesion no. U68443). The primers result in an amplification product of 649 bp. Primer sequences were chosen as follows: forward primer 5’-ATGTGGATGGTCTCAACCCCAT-3’, for the reverse primer 5’-TCACCTTGGGCGCCT-3’
(Thermo Hybaid/Interactiva Division, Ulm, Germany). Real-time PCR was done in 20 µl with 0.5 µM of each primer, 2 mM MgCl2, 2 µl FastStart DNA Master SYBR Green I (Roche) and 10 µl DNA template. Thermal cycling was performed in glass capillary tubes (Roche) according to the manufacturer’s protocol (50 cycles) with an annealing temperature of 65°C and an elongation time of 26 sec. Fluorescence was measured at the end of each elongation phase. Product specificity was checked routinely by means of melting point analysis (specific melting point of product: 89°C.) Quantification of C.
pneumoniae and C. trachomatis genome equivalents (GE) in the samples was performed by using standard chlamydial DNA, consisting of 1.2 million base pairs per GE, weighing 1.3 fg.
4.2.4 Stimuli
The following bacterial stimuli and inhibitors were used for stimulation of human whole blood or murine macrophages: C. pneumoniae, C. trachomatis, Escherichia coli (E. coli K-12 strain JM 109, a kind gift from Dr. Gerald Grütz, University Medical Centre Charité, Berlin, Germany, grown in LB medium and taken from the logarithmic growth phase), LPS from Salmonella abortus equi (Sigma), LTA from Staphylococcus aureus prepared in-house according to Morath et al. (185), phorbol myristrate acid (PMA; Sigma), LPS-specific binding-protein LALF (Limulus anti-LPS factor; a generous gift from F. Jordan, Charles River/Endosafe, Charleston, USA) and anti-CD14-antibody (biG10; Biometec, Greifswald, Germany).
4.2.5 UV-and heat-inactivation of bacteria
UV-inactivation of bacteria was performed on ice for 60 min using a UV-Stratalinker 1800 (Stratagene, Jolla, CA, USA) at 9999 x 100 uJ. Heat-inactivation was carried out at 95°C for 10 min in a heating block (Liebisch, Bielefeld, Germany).
4.2.6 Mice
TLR4 deficient mice (C3H/HeJ) and their corresponding wild types (C3H/HeN) were purchased from Charles River Laboratories (Sulzfeld, Germany). TLR2 knock-out mice (TLR2-/-) were generated by homologous recombination by Deltagen (Menlo Park, CA, USA) and kindly provided by Tularik (South San Francisco, CA, USA). TLR2/4 double-deficient mice (TLR2-/-/ C3H/HeJ) were obtained by crossbreeding of TLR2-/- and C3H/HeJ mice and generously provided by Dr. Carsten Kirschning (Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Germany).
4.2.7 Isolation of murine macrophages
Mice were subjected to terminal pentobarbital anesthesia (Narcoren, Merial, Halbergmoos, Germany). Bone marrow macrophages were isolated from both femurs by rinsing with 10 ml ice-cold PBS and transferred to siliconized glass tubes (Vacutainer®, BD Biosciences).
After centrifugation, cells were resuspended in RPMI 1640 (Bio Whittaker) containing 10%
FCS (Boehringer Mannheim, Germany) and transferred to 96-well cell-culture plates (5x
6
stimuli in the presence of 5% CO2 at 37°C for 24 h. After incubation the cell-free supernatants were frozen in aliquots at –80°C until cytokine measurement. To control the responsiveness of the TLR-defective cells, 10 µg/ml LTA, 10 ng/ml LPS and 100 ng/ml PMA served as control stimuli for bone marrow macrophages of TLR2- , TLR4- and TLR2/4-defective mice, respectively.
4.2.8 Volunteer population and NOD2 genotyping
Blood samples were collected from 160 healthy volunteers from the University of Konstanz within two hours on two consecutive days. The mean age of the volunteers was 24 ± 5.2 years (range 20-70) and the sex ratio was 85 women to 75 men. The volunteers were healthy, according to the results of a questionnaire, and had taken no medication for one week prior to blood withdrawal. Acute infections were ruled out by differential blood cell count (Pentra60, ABX Technologies, Montpellier, France). DNA preparation was done using the QIAmp DNA Blood Mini Kit (Qiagen), according the manufacturer’s protocol, including RNase-free DNase digestion. The detection of the NOD2 3020insC frame shift mutation and the two NOD2 missense mutations 2104 and 2722 was kindly carried out by Dr. Lutz Hamann (Charité, Berlin, Germany).
4.2.9 Whole blood cytokine response
Incubations of human whole blood in the presence of different stimuli were performed as described previously (104). Briefly, heparinized blood freshly taken from healthy volunteers was diluted five-fold with RPMI 1640 (Bio Whittaker, Apen, Germany) and then incubated with the different stimuli in polypropylene tubes (Eppendorf, Hamburg, Germany) in the presence of 5% CO2 at 37°C for 24h. After resuspension, the cells were pelleted by centrifugation (400 g, 2 min) and the cell-free supernatants were frozen in aliquots at – 80°C until cytokine measurement.
4.2.10 ELISA
Cytokines were determined by ELISA based on antibody pairs against human TNF, IFNγ and IL-8 (Endogen, Perbio Science, Bonn, Germany), IL-1β and IL-6 (R&D, Wiesbaden, Germany), IL-10 (BD Biosciences, Heidelberg, Germany) or against murine TNF (R&D) and murine IL-6 (Pharmingen, Hamburg, Germany). Recombinant cytokines serving as
standards were purchased from National Institute for Biological Standards and Controls, London, Great Britain (hu TNF, IL-1β, IL-6), BD Biosciences (IL-10), PeproTech, Tebu, Frankfurt, Germany (IL-8), Thomae, Biberach, Germany (hu IFNγ), R&D (mu TNF) or Pharmingen (mu IL-6). Binding of biotinylated antibody was quantified using streptavidin-peroxidase (Biosource, Camarillo, CA, USA) and the substrate TMB (3,3’,5,5’-tetramethylbenzidine, Sigma).
4.2.11 PBMC isolation
PBMC (peripheral blood mononuclear cells) of healthy volunteers were prepared with CPTTMCell Preparation Tubes (Becton Dickinson, Franklin Lakes, USA). 107 cells were incubated with different stimuli in polypropylene tubes (Eppendorf) in the presence of 5%
CO2 at 37°C for 30 min.
4.2.12 Protein extraction
Proteins were extracted from isolated PBMCs. After incubation, cells were separated from the medium and lysed using 150 µl of a lysis buffer that contained 20 mMol Tris/(HCl), 10 mMol KCl, 1 mM EDTA, 1 mM EGTA, 0,2% NP-40, 10% glycerol, 1 mMol protease inhibitor (Sigma) and 10 mM phosphatase inhibitor (Na3Vo4,). The pH was adjusted to 7.4.
After centrifugation at 400g for 5 min, the supernatant was stored at –80°C. The Bradford assay was performed for protein quantification as described elsewhere (22).
4.2.13 SDS polyacrylamid gel electrophoresis (PAGE) and Western blot
Samples containing 10–50 µg protein were denatured at 95°C for 5 min in sample buffer containing 8% SDS, 2% dithioerythritol, 20% glycerol and 0,01% bromphenol blue.
Samples were loaded onto a 12% polyacrylamide gel and electrophoresis was performed under reducing conditions at 150V for 1.5h in a continuous buffer system (Bio-Rad, Munich, Germany). Subsequent blotting on a Hybond nitrocellulose membrane (Amersham-Buchler, Braunschweig, Germany) was performed in a Bio-Rad semi-dry transfer cell at 15V for 45 min. Membranes were blocked for at least 1h in blocking buffer (5% non-fat dry milk TBS/0.1% Tween), washed with TBS/0.1% Tween and then incubated over night at 4°C with the following monoclonal rabbit antibodies (Cell Signaling Technology, New England Biolabs): phospho p38-MAPK (Thr 180/Tyr 182) and
anti-phospho ERK1/2, diluted 1:1000 in blocking buffer. After washing in TBS/0.1% Tween and incubation with a polyclonal goat-anti-rabbit peroxidase coupled secondary antibody (GARPOX, DIANOVA, Hamburg, Germany), diluted 1:10000 in blocking buffer, the blots were developed by the chemiluminescence method (ECL, Amersham-Buchler, Braunschweig, Germany). Afterwards, the blots were reprobed with a β-actin antibody (Cell Signaling Technology).
4.2.14 Purification of immune stimulatory components of C. pneumoniae
1010 UV-inactivated C. pneumoniae were dissolved in 30 ml of distilled water and disrupted by sonification on ice for 15 min (Branson Sonifier II 250, G.Heinemann, Schwaebisch Gmünd, Germany; standard titanium horn 1/2 `` diameter, with tip, 40% pulse mode and output level over 6). The bacteria lysate (30 ml) was mixed with an equal volume of n-butanol (Merck, Darmstadt, Germany) under stirring for 15 min at room temperature.
Centrifugation at 13.000 g for 10 min resulted in a 2-phase system and the lower aquatic phase was replaced by fresh distilled water for re-extraction. Then, the two aquatic phases were pooled and lyophilized. The sample was resuspended in 35 ml chromatography buffer (15% n-propanol in 0.1 M ammonium acetate, pH 4.7), centrifuged at 26.900 g for 10 min and filtrated (0.2 µm). The supernatant was subjected to hydrophobic interaction chromatography (HIC) on an octyl-sepharose column (2.5 x 11 cm) using a linear gradient from 15% to 60% of n-propanol in 0.1 ammonium acetate, pH 4.7. At a flow rate of 0.5 ml/min, fractions of 8 ml were collected. Aliquots of the fractions of 100µl were evaporated and resuspended in distilled water and were used as stimuli in human whole blood incubations.
4.2.15 Chemical characterization of C. pneumoniae immune active components
Immune active fractions of C. pneumoniae, which were determined by human whole blood incubation, were pooled, dried and the mass was determined. Further purification of the material was carried out using high performance liquid chromatography (HPLC) on a reverse phase C-8 column (250 x 4 mm), injection volume 100 µl, absorbance at 230 nm, flow rate 0.5 ml/min, with a linear isopropanol gradient from 0% to 100% in distilled water.
Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker Avance II spectrometer with CryoProbe™ technology (Bruker, Rheinstetten, Germany) at 600 MHz
(1H) and 300K. The pool was solved in D2O and the measured spectrum was related to sodium 3-trimethylsilyl-3,3,2,2-teradeuteropropanoate as internal standard.
4.2.16 Limulus amoebocyte lysate assay
Endotxin was measured and quantified photometrically by LAL assay (Limulus amoebocyte lysate; Charles River, Charleston, USA) according to the manufacturer’s instructions.
4.2.17 Statistics
Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad software, San Diego, CA, USA). Data groups were compared with a paired two-tailed Student’s t test.
Data were log-transformed to achieve Gaussian distribution. In the figures, *, ** and ***
represent p values < 0.05, < 0.01 and < 0.001, respectively.
4.3 Results and Discussion