Bone marrow derived cells from C3H/HeJ and TLR2-/- mice as well as from their respective wild-types were incubated in the presence of 5x106 EB of C. pneumoniae/ml and IL-6 release was measured. As shown in figure 1, cells from TLR2-/- mice were not able to respond to stimulation with C. pneumoniae, while cells from C3H/HeJ mice were responsive, but cytokine secretion was significantly reduced compared to C3H/HeN cells.
The responsiveness of the C3H/HeJ and of the TLR2-/- cells was confirmed by stimulation with 1 µg/ml LPS and 10 µg/ml LTA, respectively (data not shown). These in vitro data
TLR2 and TLR4 are involved. In order to investigate the role of TLR2 and TLR4 for pathogen recognition and for the course of infection in vivo, we established a murine C.
pneumoniae infection model.
Figure 1 C. pneumoniae inducible cytokine release by TLR-deficient murine bone marrow cells 5x105 bone marrow cells from TLR2+/+, TLR2-/-, C3H/HeN and C3H/HeJ mice were incubated in the presence of 5x106 C. pneumoniae per ml for 24 h. The release of IL-6 was determined in the cell-free supernatants by ELISA. ***represents significance versus wild-type cells. Data are means ± SEM.
To reflect the physiological situation, where most C. pneumoniae infections are asymptomatic, mice were infected intranasally with a very low infectious inoculum of 106 EB of C. pneumoniae (corresponding to 104-105 inclusion forming units) and the course of infection was monitored by real time PCR for 95 days. At 15 min and on days two, four, 11, 18, 33 and 95 of infection the C. pneumoniae load of lung and BAL was determined by real-time PCR, the lung was analyzed by histopathology and the cytokine levels of TNFα, 6 and 10 were determined in BAL and lung supernatants, as well as TNFα, 6, IL-10 and the acute phase protein SAA in blood. Furthermore, the development of anti-C.
pneumoniae antibodies was monitored by MIF in the serum of the mice. The MIF analysis revealed that the animals produced anti-C. pneumoniae antibodies of the IgM and later the IgG class, indicating a specific immune response (figure 2). IgM was detectable in two animals on day 11 and three animals on day 18, but had vanished in all by day 33.
Accordingly, IgG was detectable first on day 18 in four animals but increased further and was present in all five animals on day 33.
0
Overall, no apparent symptoms of infection were visible. Indeed, neither the cytokines TNFα, IL-6 or IL-10 nor SAA were found at significant levels in serum during the whole course of infection. The levels of these cytokines in BAL and lung supernatants were also below the detection limit at all time points. Histopathology of lung slices revealed that there were no significant signs of interstitial pneumonitis or infiltration of polymorphonuclear leukocytes at any time point after infection. However, C. pneumoniae could be detected in the BAL and lung of all animals for 11 days, in three animals on day 18 and in two animals on day 33 by real-time PCR. Employing a quantitative real-time PCR, the highest numbers of C. pneumoniae were retrieved from the BAL 15 min after infection, which represents the initial inoculum, from the lung two days after infection. C. pneumoniae load then declined steadily, until by day 95, no more C. pneumoniae were detectable (figure 3 a+b).
Figure 3 Time course of C. pneumoniae burden in BAL and lungs of C. pneumoniae-infected mice a) BAL and b) lung were obtained from Swiss Webster mice at 15 min and on day two, four, 11, 18, 33 and 95 after infection with 106 C. pneumoniae EB. DNA was extracted and the bacterial load was determined by real-time PCR at each time-point. Bacterial load of BAL was significantly different (p ≤ 0.01) from non-infected controls at 15 min, on day two and day 11 after infection and for the lung at 15 min, on day two and day four. Bacterial load of the lung was significantly different from day two at day 11-95. Data are means ±
SEM of samples from five mice. determined by MIF. Data are means
± SEM of serum from five mice.
To examine the role of TLR2 and TLR4 for C. pneumoniae infection in vivo, C3H/HeJ, TLR2-/- as well as their respective wild-types were infected intranasally with 106 or 107 C.
pneumoniae EB and sacrificed at day 18. Swiss Webster mice served as positive controls for the C. pneumoniae infection. Day 18 was chosen since at this time-point bacteria were still detectable in the lung and BAL, but eradication had already started and effectiveness of eradication could be compared. Surprisingly, under both conditions, i.e. infection with 106 EB or 107 EB, the bacterial load of C. pneumoniae in the lung (figure 4a) or BAL (figure 4b) was not significantly different between the groups and the antibody levels were also comparable (figure 4c). It is thus concluded that eradication of C. pneumoniae takes place independent of recognition via TLR2 or TLR4. After infection with 107 EB a significant amount of IL-6 could be detected in the lung supernatants of all mice and a trend towards reduction of IL-6-release was observed in samples from the TLR2-/- compared to the TLR2+/+ mice. Since this difference was not statistically significant the experiment was repeated and the mice were sacrificed already at day two after infection. The bacterial burden of the lungs of all mice was again at the same level as confirmed by PCR.
HeN HeJ 2+/+ 2-/- SW
c) Serum was collected from mice at day 18 after infection with 107 C. pneumoniae EB. Anti-C. pneumoniae IgG antibodies were determined by MIF.
Data are medians plus individual values of samples from five mice.
a) b)
c)
Again, the IL-6 levels of the lung supernatants of the TLR2-/- mice showed lower IL-6 levels compared to TLR2+/+, while IL-6 levels were similar in C3H/HeN and HeJ mice (figure 5a).
Similar results were obtained for TNFα release (figure 5b). The levels of the anti-inflammatory cytokine IL-10 were not significantly different between wild-type and TLR-deficient mice (TLR2+/+: 424 ± 51 vs. TLR2-/-: 539 ± 55 pg IL-10/ml; C3H/HeN: 357 ± 75 vs.
C3H/HeJ: 377 ± 79 pg IL-10/ml).
Figure 5 Cytokine levels of lung supernatants of C. pneumoniae-infected mice
Supernatants of lung homogenates were obtained from mice at day two after infection with 107 C.
pneumoniae EB. IL-6 (a) and TNF (b) were determined by ELISA. Data are medians plus individual values of samples from five mice.
The lack of major symptoms further raised the question, whether C. pneumoniae had infected the murine tissue or were simply deposited and eliminated by time. To prove that C. pneumoniae actually infect and replicate intracellularly, we studied the effects of a previously reported effective combination of rifampicin and azithromycin (168). C.
pneumoniae are known to be sensitive to antibiotics only during replication. Treatment started at day two post infection and was carried out p.o. for four days three times daily.
On day seven, the bacterial burden was determined in BAL and lung (figure 6). Compared to untreated controls, bacterial numbers decreased significantly due to antibiotic treatment indicating that intracellular replication rendered them sensitive to antibiotics.
TLR2+/+ TLR2-/- C3H/HeN C3H/HeJ 0
20 40 60 80
TNF [pg/ml]
p= 0.05
TLR2+/+ TLR2-/- C3H/HeN C3H/HeJ 0.0
0.3 0.6 0.9 1.2
p= 0.05
IL-6 [ng/ml]
a) b)
untreated Rif./Az.
In this study a murine model was established in order to investigate the role of TLR2 and TLR4 for C. pneumoniae infection in vivo. This was initiated with a very low infectious inoculum of 106 C. pneumoniae EB, i.e. 104-105 inclusion forming units, via the respiratory tract and the course of infection was monitored both by serology and by bacterial burden of organs. The mild or asymptomatic course of infection in our model appears to reflect the clinic of the human infection. The low potency of C. pneumoniae to elicit an inflammatory cytokine response represents a possible explanation for the often asymptomatic course.
Furthermore, in this model, C. pneumoniae infection in mice appears to occur only in the lungs, with no obvious persistence or spread of bacteria to other organs. Swiss Webster mice were chosen for the establishment of the model, because a homogeneous susceptibility towards intranasal C. pneumoniae infection with less individual variation compared to other mouse strains had been reported(278). The infection-kinetics, self-limitation of infection and development of antibodies as described in the present study were similar to what has been published by others(135, 207, 278). We found that the bacterial load of lung, BAL and antibody development at day 18 after infection was not significantly different between the Swiss Webster and the wild-type mice C3H/HeN and
Figure 6 Effect of antibiotic treatment on C. pneumoniae burden in lung and BAL of C. pneumoniae-infected mice
a) BAL and b) lung were obtained from mice at day seven after infection with C.
pneumoniae. In addition, the different groups of mice were either vehicle treated or received rifampicin/azithromycin. DNA was extracted and the bacterial load was determined by real-time PCR for each time-point. Data are means ± SEM of samples from five mice.