Background
Among the bacterial components that trigger macrophage activation, the most widely studied is lipopolysaccharide (LPS) but bacterial lipoproteins have also been implicated in inflammatory processes [
1‐
3]. Bacterial lipoproteins are characterized by a unique amino-terminal lipo-amino acid,
N-acyl-
S-diacylglyceryl cysteine [
4], and this lipid element and its peptide moieties are known to be critical for cell activation through TLR2 [
5]. In
C. trachomatis, one such lipoprotein, the macrophage infectivity potentiator (Mip), has been shown to be present at the bacterial surface [
6] and to stimulate the proinflammatory cytokine response to
C. trachomatis in human macrophages through toll like receptor (TLR)2/TLR1/TLR6 and CD14. The lipid part of Mip has also been shown to be responsible for its proinflammatory activity [
2]. However, when stimulation of PMA-differentiated THP-1 cells was performed in the presence of serum, the Mip-induced TNF-α production was significantly decreased [
2]. Whereas physiological levels of serum lipoproteins: HDL, LDL, and VLDL have been found to inactivate LPS [
7,
8] and bacterial lipoteichoic acid [
9], no study has been reported so far about the potential of serum lipoproteins to neutralize bacterial lipoproteins. Their ability to neutralize Mip proinflammatory activity was therefore investigated. The study was also extended to another lipoprotein, the
Borrelia burgdorferi outer surface protein (Osp)A. The results of the studies included herein showed that total lipoproteins, HDL, LDL, VLDL, as well as different apolipoproteins and triglycerides prevented proinflammatory activity of Mip and OspA through TLR1/2.
Discussion
Different bacteria or bacterial components are known to interact with serum lipoproteins or apolipoproteins. The best characterized interactions are those between
E. coli LPS and HDL [
19,
20] but also LDL, VLDL, and chylomicrons [
21,
22] as well as purified human apoA-I [
22‐
24], apoB [
23], and apoE [
25,
26]. Lipoteichoic acid has also been shown to interact with several plasma lipoproteins [
9,
27] as well as
Staphylococcus aureus α-toxin [
28],
Porphyromonas gingivalis[
29],
Vibrio vulnificus cytolysin [
30], and
Streptococcus pyogenes collagen-like protein Scl1 [
31]. Very few studies have described interactions between bacterial lipopeptides or lipoprotein and serum components with one report about the binding of lipopeptides from
Mycoplasma arthritidis to apoA-I [
32] and another one about the interaction of pH6-Ag, a lipoprotein of
Yersinia pestis, with apoB [
33]. The present study adds new lipoproteins to the list of bacterial components known to bind serum lipoproteins or apolipoproteins. Indeed, human serum was found to markedly inhibit Mip proinflammatory activity. When tested independently, total lipoproteins, HDL, LDL, VLDL or a triglyceride emulsion all had an inhibitory effect, LDL and VLDL being the most potent. When several apolipoproteins were tested without their physiological lipid complement such as purified apoA-I, apoB, apoE2, and apoE3, all showed an inhibitory effect, apoB being the most potent. This common inhibitory effect cannot be attributed to sequence similarity between the different apolipoproteins but rather to similarity in their lipid-associating domains. Indeed, apoA-I and E are characterized by an abundance of amphipathic α-helices [
34] responsible for their lipid binding character [
35] and it has been shown that the α2 and α3 domains in apoB-100, corresponding to the two major apoB-100 lipid-associating domains, are homologous to certain amphipatic helix-containing regions of apoA-I and apoE [
18]. All these amphipatic domains might interact with the acyl chains of the lipid-modified cysteine at the amino-terminus of Mip.
The possible involvement of LBP in Mip inactivation was also investigated but was found to be poor compared to apoB ability. This result agrees with the absence of LBP effect on cytokine production induced by
B. burgdorferi OspA, another lipoprotein [
36,
37]. In addition, human serum was able to decrease TNF-α production induced by synthetic triacylated and diacylated lipopeptides, OspA, and
E. coli LPS and apoB was able to inactivate OspA and
E. coli LPS. Results obtained with HEK-293 cell lines expressing human TLR1/2 showed that serum factors attenuated proinflammatory activity of both Mip and OspA through their binding to TLR1/2. These results allow us to hypothesize that an attenuation of bacterial lipoprotein proinflammatory activity may occur in blood and in all body compartments where serum lipoproteins are present. This observation may have an important impact because bacterial lipoproteins are produced by the complete spectrum of bacterial pathogens [
38] and have been implicated in inflammatory processes and in pathogenesis of several important bacterial infections, including
Leptospira interrogans[
39],
Mycobacterium tuberculosis[
40],
Treponema pallidum[
41],
Listeria monocytogenes[
42], and
Borrelia burgdorferi[
41]. Lipoproteins can be spontaneously released from membranes when cells are lysed [
43,
44] and treatment of bacteria with antibiotics has been shown to significantly enhance bacterial lipoprotein release [
45]. Thus, bacterium-associated lipoproteins have been found in culture supernatants [
45,
46], infected tissues [
47], or the bloodstream in gram-negative sepsis [
47]. For whole bacteria, the serum lipoprotein deposition on the bacterial surface could prevent recognition of the pathogen by the host defense and be detrimental by impairing the antibacterial response but this mechanism might also be of benefit by preventing ongoing excessive inflammation. However, if this mechanism could play a role in whole blood, in extravascular compartments or at mucosal surfaces, where serum lipoprotein concentrations are lower, bacterial lipoproteins can have proinflammatory effects similar to those described in vitro in the absence of serum. For Chlamydiae that are not primarily bloodstream infectious agents but rather bacteria infecting lung, urogenital system or eyes, i.e. serum-free compartments, the recognition and responses would be much more sensitive than in the circulation. However, plasma components can leak into the sites of infection and antagonize the stimulatory effect induced by bacteria. For example, cervico-vaginal fluid [
48,
49] and tears [
50] have been shown to contain apoA-I. As previously described for LPS [
51], two pathways would coexist: one leading to host cell activation and involving bacterial lipoprotein/lipopeptide interaction with CD14 and TLR2/1/6, and another leading to deactivation and involving bacterial lipoprotein/lipopeptide sequestration by serum lipoproteins. Depending on body compartments, bacterial lipoproteins should be viewed as a double-edged sword in host-pathogen interactions: they can serve both as signal recognized by the host to activate its defenses and limit infection and as agents causing excessive host damage by the pathogen in some situations.
Concerning the mechanism involved in bacterial lipoprotein inactivation by apolipoproteins, it is possible to hypothesize that this is effected by the constitution of micelles between the hydrophobic structure of apolipoproteins and the hydrophobic part of bacterial lipoproteins. For the inactivation by triglycerides, it is probably mediated by a lipid-lipid interaction with the fatty acyl chains of the lipid portion of bacterial lipoproteins. The lipid component of bacterial lipoprotein, known to be the moiety causing monocyte/macrophage activation [
2], would be sheltered or sequestered and unable to bind to cell receptors. It has been shown that apoB-containing lipoproteins prevented binding of pH6-Ag to THP-I monocyte-derived macrophages [
33]. The present study has shown that serum lipoproteins inhibited TLR1/2-mediated proinflammatory response to two bacterial lipoproteins, Mip and OspA.
Methods
Reagents
Recombinant Mip lipoprotein was purified as previously described [
52] and subsequently treated by polymyxin B-agarose (Sigma-Aldrich, Buchs, Switzerland) [
2]. Recombinant
Borrelia burgdorferi outer surface protein A (OspA) was purchased from Biodesign International (Milan Analytica, La Roche, Switzerland). Racemic Pam
3CSK
4 (Pam
3-Cys-Ser-Lys
4-OH) and Pam
2CSK
4 (Pam
2-Cys-Ser-Lys
4-OH) used as synthetic triacylated and diacylated control lipopeptides, respectively, were obtained from EMC Microcollections (Tuebingen, Germany). LPS from
Escherichia coli serotype O55:B5 was purchased from Sigma-Aldrich and repurified [
2]. Human serum and FBS was from Invitrogen (Basel, Switzerland). Delipidated FBS was purchased from Sigma-Aldrich. Long- and medium-chain triglyceride emulsion (Lipofundin; 20%, w/v) was from B. Braun (Melsungen, Germany). Human plasma apoA-I and B were from Calbiochem (Merck Biosciences, VWR International, Dietikon, Switzerland). Recombinant apoE2 and E3 were from Invitrogen. Recombinant LBP was obtained from Biometec (Greifswald, Germany).
Preparation of lipoproteins
Total serum lipoproteins were isolated from human serum by salt gradient ultracentrifugation at d < 1.21 gm/cm
2. HDL, LDL, and VLDL were isolated by sequential density ultracentrifugation as described previously [
53,
54] and dialyzed for 48 h against PBS or RPMI. Protein concentrations were determined by using a micro-bicinchoninic acid protein assay kit (Pierce, Perbio Science, Switzerland) using BSA as a standard.
THP-1 cell culture
The method of THP-1 cell culture was previously described [
2]. Briefly, THP-1 cells were grown in RPMI 1640 medium. For monocytic differentiation, they were seeded in 24-well flat-bottom tissue culture plates at a density of 2.5 × 10
5 cells/1 ml per well and allowed to adhere and differentiate 48 h at 37°C in the presence of 10 nM PMA (Sigma-Aldrich). After repeated washing with RPMI 1640, PMA-differentiated THP-1 cells were stimulated at 37°C with indicated stimuli. Cell-free supernatants were harvested after 4 h (or indicated time periods) of incubation and kept at -70°C until cytokine measurements.
Human differentiated macrophage culture
The study protocol was approved by Institutional Ethics Committee (Geneva University Hospital, Switzerland). Informed consent was obtained from all subjects. The method of monocyte/macrophage preparation was previously described [
2]. Briefly, peripheral blood mononuclear cells from healthy blood donors were isolated by density gradient centrifugation with Ficoll-Hypaque (Amersham Biosciences, GE Healthcare Europe GmbH, Otelfingen, Switzerland). Monocytes/macrophages were separated by aggregation, gradient of FBS, and rosetting. Macrophages were generated by differentiation of purified monocytes/macrophages using a 6-day culture in the presence of 2 ng/ml macrophage-colony-stimulating factor (M-CSF) (R&D Systems, Abingdon, UK). Monocytes/macrophages were seeded into 24-well flat-bottom tissue culture plates at a concentration of 5 × 10
5 cells/0.5 ml per well in RPMI 1640 containing 2 mM GlutaMAX I, supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and heat inactivated (30 min at 56°C) endotoxin-free 10% (v/v) human serum (Invitrogen). The medium was refreshed regularly. After 6-day differentiation, cultures were washed five times with medium without serum. Recombinant Mip was exposed for 30 min to human serum, isolated lipoproteins, or triglyceride emulsion before addition to macrophages. After 4 h stimulation at 37°C with indicated stimuli, cultures were centrifuged at 400 ×
g for 10 min at 4°C and cell-free supernatants were collected and stored at -70°C until TNF-α measurements.
Cytokine measurements
Extracellular release of TNF-α and IL-8 was determined by a sandwich ELISA technique using the DuoSet ELISA Development Systems (R&D), according to the manufacturer's instructions. The ELISA detection limits were 2 pg/ml.
Sequence alignments
The sequences of apoA-I, apoB, and apoE were found in the Swiss-Prot/TrEMBL database. To study homologies among the three apo sequences, the CLUSTALW multiple sequence alignment program was used. To study homologies between two sequences, the SIM binary sequence alignment program [
55] (available at
http://www.expasy.org/tools/sim-prot.html) was used with Blosum62 as a comparison matrix.
Response of TLR1/2 cell lines
Nonphagocytic HEK-293 cells stably transfected with either the empty plasmid (293-Null) or human TLR1/2 genes were purchased from InvivoGen (LabForce, Nunningen, Switzerland) and maintained in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 4.5 g/l glucose, 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10 μg/ml blasticidin S (InvivoGen). For stimulation experiments, stable transfected cells were seeded into individual wells of a 48-well flat-bottom tissue culture plate at a concentration of 3 × 105 cells/0.3 ml per well of complete medium and allowed to adhere overnight. The following day, fresh medium was added and the cells were stimulated with indicated stimuli for 24 h. Culture supernatants were collected and IL-8 content was analyzed.
Statistical analysis
Statistical analyses were performed using the Student's t test with the SPSS statistical software (for Macintosh, v.10). Differences were considered significant at p < 0.05.
Acknowledgements
This work was supported by grants from Novartis, Albert-Boeni, de Reuter, SwissLife, and Rheumasearch Foundations as well as by grants 3200B0-107883 (to S.B.) from the Swiss National Science Foundation. We thank Marie-Claude Brulhart, Ursula Spenato, and Madeleine Vuillet for their excellent technical assistance.
Authors' contributions
SB: conception, design, analysis and interpretation of data, drafting of the manuscript and final approval of the manuscript. RWJ: preparation of lipoproteins, final approval of the manuscript. CG has made substantial contributions in revising the manuscript critically. All authors read and approved the final manuscript.