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Infection and Immunity, July 2005, p. 4323-4326, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4323-4326.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Effect of Chlamydia pneumoniae on Cellular ATP Content in Mouse Macrophages: Role of Toll-Like Receptor 2

Kambiz Yaraei,¹ Lee Ann Campbell,¹ Xiaodong Zhu,² W. Conrad Liles,² Cho-chou Kuo,¹ and Michael E. Rosenfeld^1*

Departments of Pathobiology,¹ Medicine, University of Washington, Seattle, Washington²

Received 9 December 2004/ Returned for modification 3 February 2005/ Accepted 28 February 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chlamydiae are obligate intracellular gram-negative bacteria and are dependent on the host cell for ATP. Thus, chlamydial infection may alter the intracellular levels of ATP and affect all energy-dependent processes within the cell. We have shown that both live C. pneumoniae and inactivated C. pneumoniae induce markers of cell death prior to completion of the bacterial growth cycle. As depletion of ATP could account for the observed increase in cell death, the effects of C. pneumoniae on ATP concentrations within mouse macrophages were investigated. Live, heat-killed, and UV-inactivated C. pneumoniae cultures (at multiplicities of infection [MOIs] of 0.01, 0.1, and 1.0) were incubated with mouse bone marrow macrophages isolated from C57BL/6J mice and mice deficient in Toll-like receptors. Treatment of the macrophages with both live and inactivated C. pneumoniae increased the ATP content of the cells. In cells infected with live C. pneumoniae, the increase was inversely proportional to the MOI. In cells treated with inactivated C. pneumoniae, the increase in ATP content was smaller than that induced by infection with live organisms and was proportional to the MOI. The increase in ATP content early in the developmental cycle was independent of the growth of C. pneumoniae, while sustained induction required live organisms. The capacity of C. pneumoniae to increase the ATP content was ablated in macrophages deficient in expression of either Toll-like receptor 2 or the Toll-like receptor accessory protein MyD88. In contrast, no effect was observed in macrophages lacking expression of Toll-like receptor 4.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chlamydia pneumoniae is a human respiratory pathogen that causes a wide spectrum of respiratory diseases (13) and may be a risk factor for immunoreactive disorders, such as adult onset asthma (6), reactive airway disease in children (5), and arthritis (2). C. pneumoniae infection has also been associated with an increased risk of cardiovascular disease, and the organism is found within macrophage- and smooth muscle cell-derived foam cells in atherosclerotic lesions (10). The association between C. pneumoniae and cardiovascular disease has been further supported by studies with animal models (for a review see reference 3). In previous studies, we investigated the hypothesis that C. pneumoniae infection of foam cells could increase atherosclerotic plaque instability by contributing to foam cell death. These studies demonstrated that C. pneumoniae induced cell death in mouse macrophages by a caspase-independent mechanism (K.Yaraei, L. A. Campbell, C.-C. Kuo, and M. E. Rosenfeld, Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. B-1668, 2003). C. pneumoniae infection induced neither formation of the mitochondrial transition pore nor DNA fragmentation. Because chlamydiae are obligate intracellular parasites that are dependent on the host cell for obtaining ATP, one possible mechanism by which C. pneumoniae could induce cell death is by depletion of intracellular ATP. Interestingly, several reports have demonstrated that intracellular levels of ATP can regulate the mode of cell death (4, 14, 24). At high levels of ATP, cells undergo apoptosis, while lower levels of ATP shift cell death toward necrosis or a non-caspase-mediated mechanism. To address the hypothesis that C. pneumoniae-induced cell death is due to decreased host cell ATP levels, different multiplicities of infection (MOIs) were used to evaluate the effect of C. pneumoniae infection on ATP content in mouse macrophages. Furthermore, to determine whether signal transduction through binding to the Toll-like pattern recognition receptors plays a role in the regulation of cellular ATP by C. pneumoniae, primary bone marrow macrophages from Toll-like receptor-deficient mice and inactivated forms of C. pneumoniae were employed in this study.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation of bone marrow cells. Twenty-week-old C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Four mice were kept in each filter top cage in a modified specific-pathogen-free facility. The mice were fed a regular chow diet. Mice were sacrificed by exsanguination, and the femora were aseptically removed and dissected free of adhering tissues. The bone marrow cells were flushed out by injection of RPMI 1640 medium (Invitrogen, Grand Island, NY) at one end of the bone using a sterile needle. The bone marrow cells collected were incubated in a bacteriologic plate in medium containing 50% RPMI 1640 medium, 20% fetal bovine serum, 1% L-glutamine, 1% HEPES, 0.5% penicillin-streptomycin, and 50% L929 cell-conditioned medium. After 7 to 10 days of incubation, cells were harvested and used for the experiments. TLR-2^–/–, TLR-4^–/–, and MyD88 mice with a C57BL/6J background were kindly provided by Thomas Hawn of the Institute for Systems Biology, Seattle, WA.

Preparation of C. pneumoniae. C. pneumoniae strain AR-39 was propagated in HL cells and purified by density gradient centrifugation with Hypaque-76 (Nycomed Inc., Princeton, NJ) (12). Organisms purified by this method contain less than 0.1% host cell material (11). Purified organisms (1 x 10⁸ inclusion-forming units/ml) were resuspended in a chlamydial transport medium, sucrose-phosphate-glutamic acid buffer (0.2 M sucrose, 3.8 mM KH₂PO₄, 6.7 mM Na₂HPO₄, 5 mM L-glutamic acid; pH 7.4), and stored at –70°C in small aliquots for later use.

Heat-killed C. pneumoniae was prepared by incubating C. pneumoniae at 56°C for 30 min in a water bath (11). For UV inactivation, C. pneumoniae was exposed to UV irradiation for 30 min at a distance of 15 cm from a 30-W UV germicidal light source.

Infection of cells. Primary mouse bone marrow macrophages were seeded at a density of 30,000 cells per well in 96-well plates, cultured for 24 h without antibiotics, and inoculated with C. pneumoniae at MOIs of 0.01, 0.1, and 1.0. After inoculation, the cells were incubated at 37°C in a 5% CO₂ atmosphere for 2 h on a rocker platform at 5 amplitudes per min. Following adsorption, the inoculated cells were incubated with culture medium containing no antibiotics at 37°C in 5% CO₂ for up to 72 h postinfection. As a control for bacterial growth, cells were maintained in culture medium containing 20 µg/ml chloramphenicol.

Analysis of cellular ATP content. Cellular ATP concentrations were assayed with an ATP determination kit (Molecular Probes, Inc., Eugene, OR) used according to the directions of the manufacturer. This sensitive bioluminescence assay is based on the requirement of luciferase for ATP for emission of light (maximum emission at 560 nm at pH 7.8) and permits quantitative determination of ATP concentrations. ATP concentrations (nM) were normalized to the level of cell protein and the total cell count.

Statistical analysis. Differences between mean ATP concentrations for groups were compared by the two-tailed t test assuming equal variances. A P value of <0.05 was considered significant. All experiments were performed multiple times with triplicate determinations, and representative results of individual experiments are presented below.

Top Abstract Introduction Materials and Methods Results Discussion References

 
C. pneumoniae infection increases the ATP content. Infection of mouse bone marrow macrophages with live or inactivated C. pneumoniae at different MOIs increased the cellular ATP content (Fig. 1). In cells infected with live C. pneumoniae, the increase was inversely proportional to the MOI. In contrast, in cells treated with inactivated C. pneumoniae, the ATP content was proportional to the MOI and was significantly lower at MOIs of 0.01 and 0.1 than the ATP content induced by live C. pneumoniae. At an MOI of 0.01, the effect of C. pneumoniae infection on the ATP content for up to 48 h postinfection was the same whether the cells were treated with live or inactivated bacteria (Fig. 2). At 72 h postinfection, live C. pneumoniae induced significantly more ATP than inactivated bacteria induced, suggesting that a heat-resistant component, such as lipopolysaccharide (LPS), was responsible for the initial induction but bacterial growth was required for sustained induction (Fig. 2). To confirm this finding, chloramphenicol, which inhibits chlamydial protein synthesis, was added to the growth medium. As shown in Fig. 3, treatment with chloramphenicol had no effect on C. pneumoniae induction of ATP at 24 h postinfection, slightly decreased ATP concentrations at 48 h postinfection, and significantly decreased induction of ATP at 72 h postinfection.

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FIG. 1. C. pneumoniae stimulates ATP production by mouse macrophages. Mouse bone marrow macrophages either were not treated (No Tx) or were treated with live C. pneumoniae (Live C.p.) or inactivated forms of C. pneumoniae (Heat-Killed, U.V. Inactivated) at different MOIs for 72 h, and the cellular content of ATP was measured by the bioluminescence assay. An asterisk indicates that the P value is <0.05 for a comparison with the no-treatment control group. A dagger indicates that the P value is <0.05 for a comparison with cells treated with heat-killed and UV-inactivated C. pneumoniae.

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FIG. 2. Time course of increases in cellular ATP content in C. pneumoniae-treated mouse macrophages. Mouse bone marrow macrophages were treated with live C. pneumoniae (Live C.p.) and inactivated forms of C. pneumoniae at an MOI of 0.01 for up to 72 h. The cellular content of ATP was measured by the bioluminescence assay. An asterisk indicates that the P value is <0.05 for a comparison with the no-treatment control group. A dagger indicates that the P value is <0.05 for a comparison with cells treated with heat-killed C. pneumoniae (H.K.C.p.) and UV-inactivated C. pneumoniae (U.V. Inact. C.p.).

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FIG. 3. Effect of chloramphenicol on the C. pneumoniae-induced increase in the ATP content of mouse macrophages. Mouse bone marrow macrophages were either not treated (No Treatment), treated with 20 µg/ml chloramphenicol alone [Control (Chloramphenicol)], infected with live C. pneumoniae at an MOI of 0.01 (Live C.p.), or treated with chloramphenicol and infected with live C. pneumoniae (Live C.p. + Chloramphenicol) for up to 72 h. The cellular content of ATP was measured by the bioluminescence assay. An asterisk indicates that the P value is <0.05 for a comparison with the no-treatment control group and the group treated with chloramphenicol alone. A dagger indicates that the P value is <0.05 for a comparison with cells treated with both C. pneumoniae and chloramphenicol.

Effect of C. pneumoniae on the ATP content in Toll-like receptor-deficient macrophages. To determine whether the increase in ATP content in the presence of C. pneumoniae occurred via activation of Toll-like receptors, bone marrow macrophages from wild-type, TLR-2^–/–, TLR-4^–/–, and MyD88^–/– mice were infected with C. pneumoniae (MOI, 0.01). As shown in Fig. 4, the induction of ATP by C. pneumoniae was abrogated in macrophages isolated from TLR-2^–/– and MyD88^–/– mice at 72 h postinfection. In contrast, the absence of Toll-like receptor 4 (TLR-4) did not inhibit the induction of ATP by C. pneumoniae.

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FIG. 4. Effects of C. pneumoniae on the ATP content of bone marrow macrophages devoid of Toll-like receptors and MyD88. Mouse bone marrow macrophages (MBMM) were isolated from C57BL/6 mice (Wild Type) and from mice devoid of TLR-2 (TLR-2–/–), TLR-4 (TLR-4–/–),or the accessory protein MyD88 (MyD88–/–). Wild-type cells were not treated [MBMM (No Tx)] or along with the cells from the mice devoid of Toll-like receptors were treated with live C. pneumoniae at different MOIs for 72 h. The cellular content of ATP was measured by the bioluminescence assay. An asterisk indicates that the P value is <0.05 for a comparison with the MBMM (No Tx) control group and TLR-2–/– and MyD88–/– cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chlamydia species are obligate intracellular bacteria that proliferate only in eukaryotic cells. In 1962, Moulder proposed that members of the genus Chlamydia were energy parasites and that host cell metabolites were necessary for their survival (16). Subsequently, Hatch et al. demonstrated that Chlamydia psittaci could transport ATP and hydrolyze it (7). Genomic evidence for a chlamydial ATPase supports these previous findings but also suggests putative pathways for ATP biosynthesis by chlamydiae (21). However, the effect that chlamydial infection has on energy metabolism in the host is not well defined. In a study of Wang et al., infection with either C. pneumoniae or Chlamydia trachomatis was shown to decrease ATP synthesis in myocytes, which was accompanied by other indicators of chlamydial cell damage (25). In contrast, Ojcius et al. observed a marked increase in several metabolites involved in energy metabolism, including ATP, and the peak ATP levels occurred midway in the infectious cycle (18). In the current study we demonstrated that C. pneumoniae increases the content of ATP in mouse macrophages. Interestingly, this response was shown to be dependent on the MOI, with a lower MOI stimulating a higher level of ATP than a larger MOI when the cells were treated with live organisms. One explanation for this observation is that a higher MOI induces more cell stress and ultimately cell death and concomitantly decreases host ATP synthesis. This hypothesis is supported by the finding that the increased ATP content inversely parallels the induction of markers of cell death (annexin V and propidium iodide) by C. pneumoniae (Yaraei et al., 43rd ICAAC, abstr. B-1668). Both live and killed bacteria induced an increase in ATP for up to 48 h postinfection. After 48 h, the intracellular ATP concentration rapidly decreased in cells infected with inactivated C. pneumoniae, indicating that bacterial multiplication was required for sustained production of ATP. This was confirmed by treating cells with chloramphenicol after adsorption to inhibit chlamydial growth. The ATP content in treated cultures paralleled that of cells treated with heat-killed or UV-inactivated C. pneumoniae. Collectively, these results indicate that there was a biphasic response to C. pneumoniae, in which the initial ATP content was dependent on a heat-resistant bacterial component, such as LPS, while the ATP content at 48 h and 72 h was partially contingent on a sustained infection. The slightly decreased levels of ATP in macrophages infected with live C. pneumoniae at 72 h postinfection compared to the levels at 48 h postinfection most likely represented maturation and release of elementary bodies from the host.

The inverse effect observed with increasing MOIs with the live organisms on the ATP content may also help explain differences in ATP production reported in previous studies, although these studies also differed in the species of Chlamydia and the host cells used (18, 25). In the study of Wang et al., in which an MOI of 2 was used, decreased ATP levels were observed in myocytes infected with either C. trachomatis L2 or C. pneumoniae AR39 at various times postinfection (25). In the study of Ojcius et al., in which a lower MOI (0.3) of C. psittaci was used in HeLa cells, enhancement of ATP production was reported (18).

The Toll-like receptors play an important role in the recognition of pathogen-derived pattern ligands. Intracellular signal transduction pathways for both TLR-2 and TLR-4 merge at the level of the adaptor molecule MyD88, and MyD88-deficient mice are unable to recognize the bacterial components (8, 15, 22-24). Paradoxically, although chlamydial LPS and heat shock protein 60 appear to be able to signal through TLR-4 (1, 9), recognition of C. pneumoniae is primarily dependent on TLR-2 as C. pneumoniae-induced production of proinflammatory proteins is mediated primarily by TLR-2 and not TLR-4 (17, 20). Likewise, the C. pneumoniae-induced increase in ATP was shown to occur through TLR-2 as this effect was negated in TLR-2- or MyD88-deficient macrophages. These results suggest that the induction of ATP by C. pneumoniae is mediated by heat-stable components of the bacteria acting through TLR-2.

In conclusion, our results demonstrate that C. pneumoniae-induced cell death of mouse bone marrow macrophages is not due to depletion of ATP by C. pneumoniae. Previous studies by Peeling et al. on how chlamydiae were able to acquire host metabolites to support protein synthesis, which is detected early after entry, demonstrated that the infectious metabolically inert EBs contain intrinsic ATPase activity (19). This activity was detected by reduction of disulfide-linked complexes, which maintain the structural rigidity of the outer membrane. These investigators proposed that upon entry of the organism into the host, the disulfide bonds were reduced, rendering EBs permeable to host ATP could be hydrolyzed by organism ATPase (19). The result from this present study showing the augmentation of ATP levels by C. pneumoniae signaling through the TLR-2 receptor ensures ATP availability for intracellular survival. To our knowledge, this is the first report demonstrating that signaling through TLR-2 results in increased cellular ATP levels. The finding that C. pneumoniae increased levels of ATP induced by C. pneumoniae is ablated in bone marrow macrophages from TLR-2 and MyD88 knockout mice coupled with preliminary evidence indicating decreased growth in these macrophages (unpublished observations) suggests that signaling through TLR-2 is key to intracellular survival of this energy parasite.

 
This study was supported by Public Health Service grants R01 HL6615 (to M.E.R.), R01 HL62995 (to W.C.L.), and R01 HL56036 (to C.-C.K.) from the National Heart, Lung, and Blood Institute.

We gratefully thank our colleagues Jerry Ricks and Amy Lee for their expert technical assistance.

 
* Corresponding author. Mailing address: Department of Pathobiology, Box 353410, University of Washington, Seattle, WA 98195. Phone: (206) 543-1738. Fax: (206) 616-1245. E-mail: ssmjm{at}u.washington.edu.

Editor: J. N. Weiser

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Infection and Immunity, July 2005, p. 4323-4326, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4323-4326.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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