Since the reported results on TLR dependency of C. pneumoniae are controversial, we have stimulated bone marrow macrophages of TLR2-/-, C3H/HeJ, TLR2-/-/C3H/HeJ mice and their respective wild types with C. pneumoniae. As indicated in figure 7, C.
pneumoniae-induced IL-6 release was nearly completely abolished in bone marrow macrophages derived from TLR2-/- and TLR2-/-/C3H/HeJ mice and partially, but also significantly, reduced in those from TLR4 defective mice, indicating that cytokine induction depends primarily on TLR2 but also on TLR4. Similar results were obtained for TNF release (data not shown).
0 25 50 75 100
125 TLR 2 +/+
TLR 2
-/-C3H/HeN C3H/HeJ
TLR 2+/C3H/HeN TLR 2-/C3H/HeJ
C. pneumoniae5x106
***
***
*
% IL-6 [wildtype= 100%]
Figure 7 C. pneumoniae induced cytokine release depends on both TLR2 and TLR4
5x 106 bone marrow macrophages of TLR2-/-, C3H/HeJ, TLR2-/-/C3H/HeJ mice and their respective wild-types were incubated in the presence of 5x 106 C. pneumoniae per ml for 24h. The release of IL-6 was determined in the cell-free supernatants by ELISA. Data are means ± SEM of cells from 16 mice for TLR2 and TLR4 and from 5 mice for TLR2/4. Data are given in percent; wildtype = 100%. The average C. pneumoniae-induced IL-6 release in ng/ml was TLR2: 0.91 ± 0.18 vs. 0.05 ± 0.03; TLR4: 1.01 ± 0.12 vs. 0.61 ± 0.08; TLR2/4: 0.57
± 0.14 vs. 0.06 ± 0.06, respectively. *,*** significant vs. wild-type.
Currently, the intracellular NOD proteins are also under discussion as PRR for Chlamydia.
Since the group of Prof. Ralf R. Schumann (Charité, Berlin, Germany) identified NOD2 3020 polymorphism as risk factor for the development of atherosclerosis in patients who are seropositive for C. pneumoniae (personal communication), we examined its influence on LPS from S.a.e.- and C. pneumoniae-induced cytokine release in human whole blood.
For this purpose, DNA samples of 160 healthy volunteers were genotyped for the NOD2 3020insC frame shift mutation and two further known NOD2 missense mutations 2104 and 2722. Of the 160 subjects, we found 12 subjects carrying the heterozygous and one subject with homozygous 3020 polymorphism, 12 subjects carrying the heterozygous 2104 polymorphism and seven 2722 heterozygous polymorphic subjects. When C. pneumoniae-induced cytokine release was compared between the subjects with the homozygous wild-type allele or carriers of the heterozygous NOD2 3020 polymorphism, a significantly higher release of cytokines was observed for polymorphism carriers compared to wild types (figure 8a).
Figure 8 Heterozygous NOD2 3020 but not 2104 polymorphism enhances C. pneumoniae induced cytokine release
Cytokine response of whole blood from 160 volunteers genotyped for the NOD2 3020 or 2104 polymorphism stimulated with 107 C. pneumoniae per ml for 24h. Cytokines were measured in the cell-free supernatants by ELISA. Data are depicted as box and whiskers blots that give the range of values, with the box ranging from the 25% to the 75% quantile and subdivided by the median; 50% of all values lie thus within the box. Data are means ± SE of a) 147= wt; 12= NOD2 3020 +/-; 1= NOD2 3020 -/-; b) 148= wt; 12= NOD2 2104. **
significant vs. wild type.
The individual with the homozygous polymorphism also released more IL-10 than the wild-type individuals, while TNF and IFNγ release was low. However, these data are rather not representative since only one subject with homozygous polymorphism could be studied.
No differences in cytokine release were observed for subjects carrying a heterozygous NOD2 2104 or 2722 polymorphism (figure 8b; data only shown for NOD2 2104). Cytokine induction by LPS and LTA, the most typical TLR4 and TLR2 ligands, respectively, was also not altered in polymorphism carries compared to wild types (data not shown), indicating that none of the NOD2 polymorphisms leads to a general alteration of cytokine release. The observation that the NOD2 3020 polymorphism influenced C. pneumoniae-induced cytokine release suggests that indeed intracellular NOD proteins interact beside TLR2 and TLR4 with C. pneumoniae, which so far was only reported for NOD1 and NOD2 in transfected epithelial cells (203). Since only the 3020 NOD2 polymorphism and not the other two NOD2 mutations influenced C. pneumoniae-induced cytokines, the NOD2 3020 mutation seems to specifically affect the C. pneumoniae binding region. Notably, the
factor for atherosclerosis by R. Schumann’s group. Thus, one might speculate that increased cytokine release by monocytes of NOD2 3020 polymorphic subjects leads to enhanced chronic inflammation of the infected vessel wall and might thereby contribute to the pathogenesis of atherosclerosis.
4.3.2 Immune modulation
So far, we have shown that three pattern recognition receptors (PRR), i.e. TLR2, TLR4 and NOD, are engaged in the induction inflammatory responses induced by C.
pneumoniae. This is rather unusual, since most bacteria are recognized via a “main” PRR.
Perhaps the involvement of more PRR represents a mechanism of immune modulation and enables C. pneumoniae to circumvent host defence. Therefore, we have investigated the immune activating potential of C. pneumoniae in comparison to classical Gram-negative bacteria and to LPS.
To assess the cytokine inducing potential of C. pneumoniae, whole blood from healthy volunteers was incubated in the presence of increasing amounts of live C. pneumoniae or UV-inactivated E. coli and the release of the pro-inflammatory cytokine TNF and the anti-inflammatory cytokine IL-10 was determined. As shown in figure 9, 5x 106 C. pneumoniae per ml were necessary to induce a significant release of TNF and IL-10. Compared to 5x 104 E. coli per ml, 5x 106 C. pneumoniae per ml, i.e. 100 fold more bacteria, induced significantly less TNF and IL-10. Although it has to be kept in mind that E. coli are approximately 40-fold bigger in size (surface area: E. coli ≈ 11.0 µm2 vs. C. pneumoniae EB ≈ 0.3 µm2), these results indicate that in general C. pneumoniae are weak inducers of inflammatory responses, which is a possible prerequisite to escape the immune cells and to establish intracellular persistent infections.
To further characterize the cytokine profile induced by C. pneumoniae, the amount of bacteria (107 C. pneumoniae per ml) was adjusted to induce the same amount of TNF like 1 ng per ml of a prototypic LPS from enterobacteria. In this case, the cytokine profile induced by C.
pneumoniae was characterized by significantly lower release of pro-inflammatory cytokines like IL-1β or IL-6 and no induction of IFNγ (table 1). Remarkably, C. pneumoniae led to a stronger induction of the anti-inflammatory IL-10 and the chemokine IL-8. These results support the hypothesis mentioned above, that a weak induction of inflammatory responses, combined with an unusual strong release of the anti-inflammatory mediator IL-10, might enable C. pneumoniae to stay rather undetected until they enter host cells and replicate intracellulary.
TNF IFNγ IL-1β IL-6 IL-8 IL-10
LPS 1 ng 1.41±0.34 3.35±0.58 11.17±1.02 12.67±1.21 4.27±0.30 0.61±0.09 C.p. 107 1.32±0.26 0.04±0.02 1.33±0.19 4.04±0.45 5.66±0.48 1.07±0.13
p ns 0.0001 <0.0001 0.0035 0.016 0.011
Table 1 Comparison of the cytokine-inducing potential of LPS from S.a.e. and C. pneumoniae Data are means ± SEM in ng/ml of n=4 for IL-6 and n=8 for all other cytokines; ns, not significant.
The following experiments were performed to determine the role of LPS for C.
pneumoniae-induced cytokine release. The use of a blocking anti-CD14 antibody led to a significant reduction of C. pneumoniae-induced TNF release, while IL-10 release was not affected (figure 10). Moreover, the protein LALF from Limulus polyphemus, which has a
Figure 9 C. pneumoniae are weak cytokine inducers compared to E. coli
Cytokine response of whole blood from healthy volunteers stimulated with life C.
highly specific LPS-neutralizing activity (115), was used. As shown in figure 4, the induction of TNF by C. pneumoniae was significantly reduced in the presence of LALF, while IL-10 release was not affected. Control experiments confirmed that LPS (10 ng/ml)-induced cytokine release was completely blocked in the presence of the anti-CD14 antibody or LALF (data not shown). For both, the anti-CD14 antibody and LALF, similar results as for TNF were obtained for IFNγ and IL-1β, while IL-8, like IL-10, remained unaffected, indicating that the pro-inflammatory cytokines were most probably induced by the chlamydial LPS, while another component seems to be responsible for IL-8 and IL-10 release.
Figure 10 Effect of LPS inhibitors on C. pneumoniae-induced cytokine release
Whole blood from healthy volunteers was incubated in the presence of 5x 106 C. pneumoniae/ml ± 10 µg/ml anti-CD14 antibody or 10 µg/ml LALF for 24h. TNF and IL-10 were measured in the cell-free supernatants by ELISA. Data are means ± SEM, of n = 8 for TNF and n = 5 for IL-10. Data are given in percent, C.
pneumoniae alone = 100%. The average C. pneumoniae-induced TNF- or IL-10 release was 0.56 ± 0.18 and 0.25 ± 0.03 ng/ ml, respectively. ** significant vs. C. pneumoniae alone.
To investigate whether cytokine induction by a classical LPS from enterobacteria, which is a clear TLR4 agonist, would be influenced by the presence of C. pneumoniae, LPS from S.a.e. was co-incubated with C. pneumoniae. Spikes with C. pneumoniae, which only induced minor cytokine release alone, significantly suppressed LPS-induced TNF, IFNγ and IL-1β release, while IL-8 and IL-10 release remained unaffected (figure 11, data shown for TNF, IFNγ and IL-10). Co-incubation of LTA from S. aureus, which is a TLR2 agonist, with C. pneumoniae did not result in any alteration of cytokine release (data not shown). Furthermore, the immune modulating effects of C. pneumoniae on enterococcal LPS were not seen for C. trachomatis (data not shown), indicating that the immune modulatory effects are a specific for C. pneumoniae.
/ml
Figure 11 C. pneumoniae is a partial agonist of LPS responses
Cytokine induction of whole blood from healthy volunteers stimulated with 1 ng LPS or 5x106 C. pneumoniae per ml or both for 24h. TNF, IFNγ and IL-10 were measured in the cell-free supernatants by ELISA. Data are means ± SEM of n = 8; *significant vs. LPS 1 ng/ml.
Taken together, the modulation of immune responses is restricted to TLR4 agonists and only affects the release of pro-inflammatory mediators and not the release of IL-8 or IL-10, which is in line with the effects of anti-CD14 antibody and LALF protein. This further supports the hypothesis mentioned above, that at least two different immune active components are present on the chlamydial surface. One, most probably the chlamydial LPS, that acts as agonist to the enerococcal LPS and thereby weakens the enterococcal LPS-induced immune response and a second, so far unknown component, which is responsible for IL-10 and IL-8 induction and which does not interfere with responses initiated by enterococcal LPS in TLR4.
It is now challenging to elucidate the molecular mechanisms that underlie these immune modulatory effects. It might be that C. pneumoniae acts as an agonist directly at the TLR4, or that it interferes intracellularly with the TLR4 induced signalling cascade. Members of the mitogen activated protein kinase (MAPK) family, which are ubiquitously expressed and activated by a variety of stimuli, have been demonstrated to be key players, mediating the LPS-induced signal transduction. To clarify which signalling pathways of LPS are affected by co-incubation with C. pneumoniae, human PBMCs were stimulated with LPS and/or C.
pneumoniae, protein was prepared and the phosphorylation of p38-MAPkinase and ERK1/2 was followed by Western blot (figure 12 a). Stimulation with LPS or C.
pneumoniae resulted in specific bands (43 kD for p-p38 and 42/44 kD for p-ERK1/2), which were more pronounced for LPS. However, as confirmed by densitometrical analysis, co-incubation of LPS and C. pneumoniae did not lead to an abrogation of LPS-induced phosphorylation of either p38 or ERK1/2 (figure 12 b).
0 1 2 3
LPS C.p. LPS+C.p. LPS C.p. LPS+C.p. LPS C.p. LPS+C.p.
0.0 0.2 0.4 0.6
*
*
TNF IFNγγγγ IL-10
TNF, IFNγγγγ [ng/ml] IL-10 [ng/ml]
Figure 12 C. pneumoniae does not inhibit LPS signalling pathways
107 PBMCs prepared from healthy volunteers were stimulated with 1 ng LPS or 107 C. pneumoniae per ml or both for 30 min and protein was extracted. The phosphorylation of p38-MAPkinase and ERK1/2 was assessed by SDS PAGE followed by Western blot. The Western blot was reprobed for β-actin (a).
Densitometrical analysis was carried out and p-p38 or p-ERK1/2 results were normalized to β-actin and LPS was set to 100% (b). Data are means ± SEM of n = 2.
Control experiments confirmed that the TNF and IFNγ release was impaired in PBMC after co-incubation of LPS with C. pneumoniae. It was rather surprising that none of the LPS-activated major pro-inflammatory kinases is affected by C. pneumoniae, however, several other kinases are involved into the process of induction of cytokine release. In the next steps, a Western blot for JNK shall be established as well as the influence of C.
pneumoniae on NF-κB activation and translocation will be followed.
4.3.3 Immune active compound
So far, our data indicate that C. pneumoniae interact with several PRR including TLR2, TLR4 and NOD, but are weak inducers of inflammatory responses. The chlamydial LPS is most probably responsible for the induction of pro-inflammatory cytokines, while a further immune active component induces the release of IL-8 and IL-10. To clarify the role of these different structures for immune activation, their isolation and biological and chemical characterization is required.
C. pneumoniae and C. trachomatis are suggested to carry with regard to the sugar moiety, the same family-specific LPS. When human whole blood was treated either with 107 C.
pneumoniae or 107 C. trachomatis per ml (figure 13), the two Chlamydia species induced a distinct cytokine profile.
Figure 13 C. pneumoniae and C. trachomatis induce distinct cytokine profiles
Whole blood from healthy volunteers was incubated in the presence of 107 C. pneumoniae or 107 C.
trachomatis per ml for 24h. Cytokines were measured in the cell-free supernatants by ELISA. Data are means ± SEM of n = 8.
Compared to C. trachomatis, stimulation with C. pneumoniae led to a lower production of pro-inflammatory cytokines and only very little amounts of IFNγ, but led to a stronger release of IL-8 and anti-inflammatory IL-10.
These results in part reflect the clinical picture, where infections with C. trachomatis, in contrast to C. pneumoniae, are characterized by pronounced inflammation, while the property to escape the immune system and to establish chronic infections, is more typical for C. pneumoniae. With regard to the chlamydial LPS, these different cytokine profiles indicate, that either the LPS of C. pneumoniae and C. trachomatis are structurally different, or that additional immune stimulatory principles that modulate the LPS-induced responses, are involved. The following experiments were performed to clarify the nature of the immune stimulatory compounds of C. pneumoniae. UV-inactivated C. pneumoniae, which were not able to replicate in HEp-2 cells, still induced the same amounts of TNF, IFNγ, IL-1β, IL-6, IL-8 and IL-10 as live C. pneumoniae, indicating that the bacteria do not need to be alive to induce cytokine release (data not shown). In addition, heat-inactivated C.
pneumoniae still induced cytokine release comparable to live bacteria (TNF: 0.31 ± 0.05
0 3 6 9 12 15
18 TNF IFNγγγγ IL-1ββββ IL-8 IL-10
0 1 2 3 4 pro-inflammatory anti-inflammatory
C. pneumoniae107/ml C. trachomatis107/ml
IFNγγγγ, IL-1ββββ, IL-8 [ng/ml] TNF, IL-10 [ng/ml]
vs. 0.56 ± 0.09; IFNγ: 0.06 ± 0.01 vs. 0.10 ± 0.02; IL-1β: 2.22 ± 0.77 vs. 2.35 ± 0.71; IL-8:
8.00 ± 1.24 vs. 5.82 ± 0.82 and IL-10: 0.26 ± 0.08 vs. 0.48 ± 0.13; data are ng/ml, n= 12 for live C. pneumoniae vs. heat-inactivated C. pneumoniae, respectively), indicating that the immune-active compound is not heat-labile and is therefore not likely to be a pure protein.
Traditionally extraction procedures based on hot phenol/chloroform are used to isolate LPS from Gram-negative bacteria. However, butanol extraction performed at room temperature (185) was shown to be more appropriate for the isolation of LTA, which is more hydrophobic and more labile than LPS and therefore is prone to degradation during the extraction and purification process. Since the C. trachomatis LPS was shown to exert weak endotoxic activity and its lipid anchor was more hydrophobic compared to typical enterococcal LPS, butanol extractions of 1010 C. pneumoniae have been performed to isolate their immune active compound. The water phases were subjected to hydrophobic interaction chromatography (HIC) and the obtained fractions were tested as to their immune stimulatory potential in human whole blood. The elution profile was assessed with 100 µl of every second fraction (figure 14).
Figure 14 Elution profile of C. pneumoniae extracts after HIC
Cytokine induction of whole blood from healthy volunteers stimulated with 100 µl of evaporated fractions of an extract of 1010 C. pneumoniae for 24h. TNF, IFNγ and IL-10 were measured in the cell-free supernatants by ELISA. Data are means ± SEM of n = 3. Fractions 24-33 were pooled dried and resulted in 2.9 mg of immune active material.
The cytokine inducing property of the fractions 24 to 33 was comparable to that of whole bacteria with regard to the release of TNF, IFNγ and IL-10. These fractions were pooled,
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
IFNγγγγ IL10 TNF
C.pneumoniae-fractions
cytokines [ng/ml]
pool 24-33: 2.9 mg
evaporated and the mass was determined. The extraction of 1010 bacteria led to 2.9 mg of immune active material.
In the next steps, the biological activity of the extracted material was further analyzed.
Firstly, to compare the TLR dependence of C. pneumoniae and the fraction pool, bone marrow macrophages of TLR2-/- and C3H/HeJ mice and their appropriate wild types were stimulated and IL-6 release was measured (figure 15).
Like whole C. pneumoniae, the isolated material was completely TLR2- and to a minor extent TLR4-dependent. Similar results were obtained for TNF release (data not shown).
Next, as found in whole blood incubations, the presence of a blocking anti-CD14 antibody and the LPS inhibitor LALF completely abrogated TNF release induced by the pooled material, but in contrast not in response to whole C. pneumoniae, IL-10 release was also significantly diminished (figure 16). This indicates that probably the extracted material contained chlamydial LPS. In fact, 120 endotoxin units (EU) i.e. 12 ng endotoxin per mg (1 EU = 100 pg) could be detected by LAL. Co-incubation of the fraction pool with enterococcal LPS did not abrogate LPS-induced TNF release (TNF ng/ml: 1.04 ± 0.35 vs.
2.29 ± 0.75; n = 4 for 1 ng LPS vs. 1 ng LPS + 1 µg F 24-33, respectively). The same results were observed for IFNγ (data not shown).
C.p. 5x 106 F-Pool 1 µg
Figure 16 Effect of LPS-specific inhibitors on C. pneumoniae fraction pool 24-33
Whole blood from healthy volunteers was incubated in the presence of 1 µg of pooled fractions 24 to 33 (F 24-33) per ml ± 10 µg/ml anti-CD14 antibody or 10 µg/ml LALF for 24h. TNF and IL-10 were measured in the cell-free supernatants by ELISA. Data are means ± SEM, of n = 8 for TNF and n = 4 for IL-10, ** significant vs. F 24-33 alone.
To clarify the structure of the obtained C. pneumoniae component, a nuclear magnetic resonance (NMR)–spectrum was performed (Figure 17). For probe application CryoProbe technology was used, which allows more efficient detection of weak signals in the presence of very strong signals.
Figure 17 NMR-spectrum of the C. pneumoniae component does not suffice for complete structural analysis
1H NMR spectrum (600 MHz, 300K) of the C. pneumoniae component after butanol extraction. Areas of signal intensities of aromatic amino acids and saccharide are indicated by arrows and fatty acids can be detected from 1 to 3 ppm.
F24-33 1µg/ml
+ aCD14
+ LALF
F24-33 1µg/ml
+ aCD14
+ LALF 0.0
0.2 0.4 0.6 0.8
1.0 TNF IL-10
0.0 0.1 0.2 0.3 0.4 0.5
*** ***
n= 8
*** ***
n= 4
TNF [ng/ml] IL-10 [ng/ml]
saccharide
fatty acids
ppm
aromatic amino acids
As indicated in figure 17, protons of fatty acids and at least six aromatic amino acids could be identified, but on the basis of this spectrum, the precise chemical composition could not be defined. Also, the signal that occurred at the position, where usually the sugars are retained, was too small to allow any interpretation of the respective saccharides.
Nevertheless, the isolated component appears to be a lipopeptide.
All further attempts to determine the nature of the fatty acids and amino acids (performed by PD Dr. R. Süßmuth, Institute of Organic Chemistry, University of Tübingen, Germany) did not lead to a result, most probably because of inhomogeneity of the material.
Therefore, the remaining 100 µg were further purified by high performance liquid chromatography (HPLC) (figure 18a). The peak at 46’ was collected and dried, but the amount was too little to determine its mass. However, as indicated in figure 18b, the peak fraction still induced TNF release from human whole blood. Since the detection limit for mass determination is 10 µg per cup, this result indicates that the material of the peak fraction obtained by HPLC possesses strong TNF inducing activity. In order to confirm these results an upscale of the isolation process is necessary.
[?] [1 µg/ml]
Figure 18 Purification of the C. pneumoniae pool by HPLC
a) 100 µg of the C. pneumoniae component was subjected to HPLC. The component eluted at a retention time of 46 min, but was not weighable after evaporation.
b) The peak component obtained by HPLC was dissolved in 200 µl of distilled water and whole blood
b) The peak component obtained by HPLC was dissolved in 200 µl of distilled water and whole blood