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Cell death and inflammation during infection with the obligate intracellular pathogen, Chlamydia
Biochimie 85 (2003) 763–769 www.elsevier.com/locate/biochi
Cell death and inflammation during infection with the obligate intracellular pathogen, Chlamydia Jean-Luc Perfettini a, Véronique Hospital b, Lynn Stahl c, Thomas Jungas c, Philippe Verbeke c, David M. Ojcius c,* a
Laboratoire Apoptose, Cancer et Immunité, CNRS UMR 1599, Institut Gustave Roussy, 39, rue Camille-Desmoulins, 94805 Villejuif cedex, France b Laboratoire de Signalisation Immunoparasitaire, Département de Parasitologie, CNRS URA 2581, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris cedex 15, France c Université Paris 7, Institut Jacques Monod, CNRS UMR 7592, 2, place Jussieu, 75251 Paris cedex 5, France Received 31 March 2003; accepted 29 August 2003
Abstract Infections by Chlamydia are followed by a strong inflammatory response, which is necessary to eliminate the infection, but at the same time is responsible for the pathology of infection. Resistance of infected cells against apoptosis induced by external ligands, together with the effects of IFNc secreted during infection, would be expected to contribute to persistence of infection. Secretion of TNFa plays an important role during clearance of the chlamydiae, but also triggers apoptosis of uninfected cells in infected tissues. Apoptosis of infected host-cells towards the end of the infection cycle is thought to participate in the release of chlamydiae from infected cells and propagation of the infection. Dysregulation of the apoptotic program during infection leads to a less efficient infection, but paradoxically, results in a higher inflammatory response and more severe pathology. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Apoptosis; Chlamydia; BAX, TNFa; IFNc; Inflammation
1. Introduction Programmed cell death (apoptosis), in contrast to accidental cell death (necrosis), takes place physiologically during embryonic development and cellular homeostasis [1,2]. However, apoptosis can also contribute to different human pathologies, including auto-immune disease, cancer, and neurodegenerative disease [3]. In addition, microbial pathogens such as viruses, bacteria and protozoan parasites can modulate apoptosis of the infected host-cell. Hence, a large number of bacterial pathogens such as Bordetella pertussis, Salmonella typhimurium, Shigella flexneri, Escherichia coli and Staphylococcus aureus induce apoptosis of their infected host-cells [4,5]. Similarly, the protozoan parasites Toxoplasma gondii and Cryptosporidium parvum, and the herpes simplex virus 1 (HSV-1), can both protect infected host-cells against apoptosis induced by immune effector cells or nonphysiological external ligands and induce apoptosis at the * Corresponding author. Tel.: +33-1-44-27-98-58. E-mail address: [email protected] (D.M. Ojcius). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2003.08.006
end of the infection cycle [6–9]. Probably the best characterized among these pathogens is HSV-1, whose genes responsible for resistance against apoptosis and induction of caspase-independent apoptosis have been identified [9]. Thus, pathogens can activate inhibition mechanisms that protect infected cells against the cytotoxic mechanisms of the immune system, while host-cell death at the end of the infection cycle could contribute to propagation of the pathogen in the host organism. Apoptosis could, therefore, be considered as either part of a host defense response that attempts to prevent viral or bacterial propagation, or an intrinsic event in the pathogen’s infection cycle that contributes to establishment of the infection. Regulation of host-cell death thus represents a critical stage in the interaction between a pathogen and its host organism.
2. Infections by Chlamydia Bacteria of the genus Chlamydia are obligate intracellular pathogens that infect mainly epithelial mucosa [10]. Differ-
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ent strains of Chlamydia trachomatis are responsible for infections of ocular tissue and sexually transmitted diseases in humans [11,12]. Chlamydia psittaci elicits different symptoms, depending on its host [13]. It causes intestinal infections in birds and abortion in pregnant ewes, but in humans it manifests itself as pneumonia. Chlamydia pneumoniae is responsible for about a tenth of all pneumonias in humans and has recently been linked to a higher risk for developing atherosclerosis [14]. Chlamydia pecorum is associated with infectious pneumonitis in domestic animals [13]. The bacteria exist in two developmental forms: the infectious extracellular elementary bodies (EB) attach to the hostcell and are internalized into an entry vacuole that avoids fusion with host-cell lysosomes. Within 8–10 h, the EB differentiate into multiplicative intracellular reticulate bodies (RB), which proliferate within the same membrane-bound vacuole. After several divisions, the RB differentiate back into EB, and after 2–3 days, depending on the Chlamydia strain, the EB are released from the infected cell through unknown mechanisms and begin a new cycle of infection [15–18]. Host-cell death observed at the end of the infection cycle could thus be involved in release of EB from the host-cell and could partially contribute to the inflammatory response of the host, since macrophages undergoing apoptosis secrete inflammatory cytokines [19] and cells dying necrotically stimulate inflammation [20]. An inflammatory response is required for the resolution of primary C. trachomatis infection, but chronic inflammation is also responsible for the scarring process observed in trachoma and chlamydial sexually transmitted disease. Thus, a number of inflammatory mediators are present during infection, including interleukin-1b (IL-1b) and tumor necrosis factor (TNFa). TNFa and other inflammatory cytokines may aid in eradicating Chlamydia infection, but also promote long-term tissue damage [21]. In addition, the ability of chlamydiae to persist in the host could be an important factor in exacerbating pathogenesis. The immune mechanisms responsible for chronic inflammation are not fully understood, but it is believed that repeated exposure to chlamydial antigens contributes to pathogenesis and that bacteria in persistently infected cells may serve as a source of long-lasting pathology [22]. Chlamydia muridarum (also known as the mouse pneumonitis strain of C. trachomatis) [23,24] infects the genital tracts of mice and has been used to characterize the host immune response to chlamydial genital tract infection. In C. muridarum infections of mice, a major role for CD4+ T cells secreting interferon-c (IFNc) during clearance of the infection has been described [25–28]. Thus, studies with IFNc deficient mice have shown that IFNc is required for prevention of dissemination of genital tract infection [27,29,30]. Similarly, administration of anti-IFNc antibodies or recombinant IFNc prolongs or resolves the infection, respectively [31]. A persistent state of chlamydiae can be induced in vitro by IFNc treatment, which leads to altered bacterial forms and an antigen profile that is different from that observed during
active infection [22,32]. In IFNc-treated infected cells, the chlamydiae are noninfectious, but the bacteria are viable and can revert to the infectious state following removal of IFNc.
3. Why should Chlamydia modulate apoptosis of infected cells in two opposing directions? On first glance, it may seem odd for a pathogen to have opposing effects on host-cell survival. However, as demonstrated for other pathogens such as HSV-1, a single intracellular microorganism can have different effects at different stages of the infection cycle. Thus, in the case of Chlamydia infections, we propose that resistance of infected cells against apoptosis triggered by external ligands may contribute to persistence of Chlamydia infection, while cell death at the end of the infection cycle would allow chlamydiae to exit from infected cells and initiate a new round of infection (Fig. 1). In vivo studies have revealed that mice that are deficient for perforin, Fas, the Fas ligand, or both perforin and the Fas ligand are able to resolve C. muridarum infection with the same time-course and immune response as wildtype controls, suggesting that an anti-apoptotic activity developed by Chlamydia could protect infected cells against cytotoxic mechanisms of the immune system [33]. Conversely, hostcell lysis associated with the end of the replication cycle could contribute to propagation of the bacteria, as suggested by decreased chlamydial shedding measured in mice whose apoptotic pathways are impaired [34].
4. Protection of infected host-cells against apoptosis due to external ligands Several strains of C. trachomatis and C. pneumoniae protect infected cells against apoptosis due to external ligands, including TNFa, antibodies against Fas, and the kinase inhibitor staurosporine [35–38]. Protection is observable as early as 4 h after infection, suggesting that it could be operative in vivo during early stages of the infection cycle. The anti-apoptotic activity is due to inhibition of cytochrome c release from mitochondria, which thus results in inhibition of the “apoptosome” pathway leading to caspase-9 and subsequently caspase-3 activation. The anti-apoptotic activity is dependent on chlamydial but not host protein synthesis [35], suggesting that chlamydial virulence factors, perhaps secreted via the type III secretion apparatus [39,40], could be responsible for resistance against apoptosis. Cytochrome c release from mitochondria has never been observed at any stage of the infection cycle, suggesting that, once the apoptosome pathway has been turned off, it remains off throughout the cycle. Host-cell death at the end of the cycle would thus have to involve mediators other than caspases. Conversely, any mediators that trigger caspase-independent
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Fig. 1. Infection cycle of Chlamydia. Infectious but metabolically inert EB are internalized by epithelial cells or macrophages via entry vacuoles that avoid fusion with host-cell lysosomes. Within 10 h, EB differentiate into metabolically-active RB, which proliferate within the same membrane-bound vacuole. In the presence of IFNc, chlamydiae can differentiate reversibly into a persistent, atypical form (aRB) that does not proliferate. Persistently-infected cells are resistant to apoptosis. In the absence of IFNc, chlamydiae revert to typical RB, which redifferentiate into EB. Apoptosis at the end of the infection cycle allows chlamydiae to exit from infected cells and initiate a new round of infection.
death at the end of infection cycle (see below) would have to be inhibited until the bacteria are ready to leave the infected cell. Interestingly, cells infected with C. trachomatis in the presence of IFNc are also resistant to apoptosis due to external ligands, via inhibition of the apoptosome pathway [37], suggesting that the resistance against apoptosis could contribute to persistence of infection. 5. Host-cell death and effects on inflammation Cytotoxicity due to infection by human and nonhuman strains of Chlamydia had been observed for many years [41–47], but the mechanisms of cell death had not been investigated. More recently, it was shown that epithelial cells, the preferential target of Chlamydia infection, display characteristic features of apoptosis during infection with the “guinea pig inclusion conjunctivitis” (GPIC) serovar of C. psittaci [48]. Morphological changes associated with apoptosis such as chromatin condensation, nuclear and cytoplasmic segmentation, and blebbing have been observed in infected cell samples by electron microscopy. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method also showed DNA fragmentation in infected cells [48], confirming the apoptotic nature of host-cell death. In addition, phosphatidylserine (PS) is exposed on the surface of infected cells with a time-course similar to host-
cell death [49], consistent with an apoptotic-type mechanism of death during C. psittaci infection. No host-cell death was measured during the first day of infection by either C. psittaci or C. trachomatis, but nuclear condensation, a hallmark of apoptosis, became prominent towards the end of the infection cycle [48,50,51]. As in the case of protection against apoptosis during early stages of the infection cycle, cell death at the end of the infection cycle requires chlamydial but not hostcell protein synthesis [48]. Human monocytes infected with Chlamydia secrete IL1b. Since caspase-1 plays a role in the maturation of IL-1b and IL-18 [52], cells infected with Chlamydia were treated with an irreversible inhibitor of caspase-1. While this inhibitor blocked IL-1b secretion during infection, it had no effect on Chlamydia-induced cell death, suggesting that caspase-1 is activated during infection, but does not participate in cell death [48]. To determine whether other known caspases may be involved in apoptosis, the effects of specific caspase-3 and broad-spectrum caspase inhibitors were tested on host-cell death. These inhibitors had no effect on cell death during infection with C. psittaci or C. trachomatis [48,53]. In addition, caspase-3 is not activated in infected cells, suggesting that known caspases do not participate in apoptosis during Chlamydia infection. Although caspases have long been considered the pivotal executioners of apoptosis, a growing number of recent studies show that apoptosis can take place in the absence of caspase activation [54]. Significantly, overexpression of
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BAX, a pro-apoptotic member of the BCL-2 family, can induce cell death in the absence of caspase activation [55]. BAX normally resides in the cytosol, but translocates to mitochondria after stimulation with pro-apoptotic ligands [56]. During infection with C. psittaci or C. trachomatis, BAX becomes activated and translocates to mitochondria in infected cells. BAX activation appears to play at least a partial role in Chlamydia-induced apoptosis, since overexpression of BAX Inhibitor-1 (BI-1) or the anti-apoptotic protein BCL-2 inhibits apoptosis during infection [53]. Finally, primary fibroblasts lacking BAX die through apoptosis less efficiently than wildtype fibroblasts, demonstrating clearly that BAX activation participates in apoptosis during infection (Fig. 2).
Thus, BAX can contribute to at least some of the death observed during Chlamydia infection, but two unresolved issues still deserve further attention. First, since infected cells do not begin to die until halfway through the infection cycle, it would be worthwhile determining which host-cell factors prevent BAX activation at the beginning of the cycle. One such candidate is humanin, a 24 amino-acid peptide that blocks BAX activation and translocation to mitochondria [57]. However, these studies will be complicated by the possibility that additional factors besides humanin may also inhibit the activity of BAX. Secondly, BAX activation is known to lead to either caspase-dependent or caspaseindependent cell death [55,58,59], but the caspaseindependent mediators responsible for Chlamydia-induced
Fig. 2. Anti- and pro-apoptotic activities during Chlamydia infection. Infected cells are resistant to external pro-apoptotic ligands that trigger cell death via the apoptosome pathway. Thus, cytochrome c release from mitochondria is inhibited, which prevents Apaf-1 and cytochrome c from cleaving pro-caspase 9 into the active caspase-9 (C9a), which would normally then activate caspase-3 (C3a). At the end of the infection cycle, pre-existing BAX is activated, translocates to the mitochondria, and induces cell death in the absence of caspase activation.
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death have yet to be identified. One could propose that caspase-independent factors such as apoptosis-inducing factor (AIF) [60] or endonuclease G [61] may be released selectively from mitochondria, but this would require cytochrome c to remain associated with the mitochondrial intermembrane space under the same conditions. An alternative mechanism is suggested by the observation that lysosomes burst in cells infected with Chlamydia [43], as lysosomal permeabilization can also lead to caspase-independent death [62]. To identify a biological role for BAX-dependent apoptosis, wildtype and BAX-deficient fibroblasts were infected with C. muridarum during at least two infection cycles. The bacterial yield recovered from BAX-deficient cells was significantly reduced, compared to infected wildtype cells, suggesting that chlamydiae may use apoptosis to exit from infected cells and begin a new infection cycle [34]. The PS receptor, which is used for phagocytosis of apoptotic bodies and cells, is expressed on fibroblasts and epithelial cells [63], and we propose that the PS receptor, or similar receptors, may be used to internalize Chlamydia-containing apoptotic bodies by neighboring cells. To confirm the relevance of the in vitro results for a physiological infection, wildtype and BAX-deficient mice were infected intravaginally with C. muridarum. The BAXdeficient mice are a convenient model for studying Chlamydia infection, since the mice are healthy and do not display alterations of the immune system [64]. The infection with C. muridarum was less efficient and disappeared more quickly from the BAX-deficient mice than from the control wildtype mice, suggesting that BAX-dependent apoptosis may contribute to propagation of Chlamydia in vivo [34]. Unexpectedly, the inflammatory response was higher in the BAX-deficient mice than in the wildtype mice, despite the lower intensity of infection in the BAX-deficient mice. These results could be explained if one assumes that there is more necrosis in the BAX-deficient mice than in the wildtype controls. In fact, cells that are prevented from dying through apoptosis usually still manage to die, but they do so later, and they die through necrosis. Consistent with this possibility, BAX-deficient cells infected in vitro die through necrosis more often than wildtype cells [34]. Taken together, these results suggest that Chlamydia-induced apoptosis via BAX contributes to chlamydial propagation and decreases inflammation. Concomitant with the higher levels of cytokine secretion, the BAX-deficient mice contained large granulomas in the genital tract. Thus, paradoxically, BAX-deficiency results in less-intense infection but more pathology. 6. Effect of inflammatory cytokines on cell death in vitro and in vivo Analysis of the infection in BAX-deficient mice demonstrated that dysregulation of cell death can have profound effects on the inflammatory response and host pathology. Conversely, many of the cytokines secreted during Chlamy-
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dia infection, notably TNFa and IFNc, could also modulate apoptosis. To study the possible relationship between TNFa secretion and cell death, mice were infected with C. muridarum and the number of apoptotic cells was measured by the TUNEL assay at different times after infection [51]. There was a large increase in the number of apoptotic cells in the Fallopian tubes and oviducts after 2 or 7 days of infection, compared to uninfected controls. Depletion of TNFa with anti-TNFa antibodies led to a significant decrease in the level of apoptosis in the upper genital tract of mice infected for 7 days, suggesting that TNFa may exert part of its pathological effect by inducing apoptosis of cells in infected tissues. Finally, most of the cells dying as a result of the inflammatory response do not appear to be infected and thus should not be protected against TNFa-mediated apoptosis [51]. IFNc, which is used to induce a persistent phenotype of Chlamydia in vitro, is also known to affect apoptosis and cell survival [65]. IFNc concentrations that induce a persistent state of C. muridarum also inhibit apoptosis due to infection. The inhibitory effect of IFNc is due in part to expression of host-cell indoleamine 2,3-dioxygenase activity, which leads to tryptophan catabolism, as inhibition of apoptosis could be reversed by incubating infected cells with exogenous tryptophan. In addition, a significantly larger number of apoptotic cells were observed in the genital tract of infected IFNc-deficient mice, compared to wildtype mice [66]. These results suggest that IFNc could contribute to the persistence of Chlamydia infections in vivo by inhibiting apoptosis of infected cells. Given the role that BAX-dependent apoptosis plays in propagation of the infection in vitro and in vivo, IFNc would also be expected to inhibit propagation of chlamydiae via apoptotic bodies in vivo. 7. Concluding remarks Apoptosis during Chlamydia infection is affected in two opposing directions, depending on the stage of the infection cycle. The type of cell death taking place at the end of the infection cycle has a large effect on secretion of proinflammatory cytokines by the host, but the host inflammatory response can also modulate apoptosis. Resistance of infected cells against apoptosis triggered by outside ligands probably contributes to persistence of Chlamydia infection. IFNc, a traditional inducer of persistence in vitro, also inhibits Chlamydia-induced death, and thereby, could enhance persistence via its anti-apoptotic effect. On the other hand, the TNFa that is required for elimination of infection promotes apoptosis of uninfected cells, which may be an unintended consequence of the host immune response. Acknowledgements This work was supported by the National Institutes of Health (grant R01 AI054624), Université Paris 7, Centre
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National pour la Recherche Scientifique, the Fondation pour la Recherche Médicale, and the Institut Pasteur (PTR 60).
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Cell death and inflammation during infection with the obligate intracellular pathogen, Chlamydia Jean-Luc Perfettini a, Véronique Hospital b, Lynn Stahl c, Thomas Jungas c, Philippe Verbeke c, David M. Ojcius c,* a
Laboratoire Apoptose, Cancer et Immunité, CNRS UMR 1599, Institut Gustave Roussy, 39, rue Camille-Desmoulins, 94805 Villejuif cedex, France b Laboratoire de Signalisation Immunoparasitaire, Département de Parasitologie, CNRS URA 2581, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris cedex 15, France c Université Paris 7, Institut Jacques Monod, CNRS UMR 7592, 2, place Jussieu, 75251 Paris cedex 5, France Received 31 March 2003; accepted 29 August 2003
Abstract Infections by Chlamydia are followed by a strong inflammatory response, which is necessary to eliminate the infection, but at the same time is responsible for the pathology of infection. Resistance of infected cells against apoptosis induced by external ligands, together with the effects of IFNc secreted during infection, would be expected to contribute to persistence of infection. Secretion of TNFa plays an important role during clearance of the chlamydiae, but also triggers apoptosis of uninfected cells in infected tissues. Apoptosis of infected host-cells towards the end of the infection cycle is thought to participate in the release of chlamydiae from infected cells and propagation of the infection. Dysregulation of the apoptotic program during infection leads to a less efficient infection, but paradoxically, results in a higher inflammatory response and more severe pathology. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Apoptosis; Chlamydia; BAX, TNFa; IFNc; Inflammation
1. Introduction Programmed cell death (apoptosis), in contrast to accidental cell death (necrosis), takes place physiologically during embryonic development and cellular homeostasis [1,2]. However, apoptosis can also contribute to different human pathologies, including auto-immune disease, cancer, and neurodegenerative disease [3]. In addition, microbial pathogens such as viruses, bacteria and protozoan parasites can modulate apoptosis of the infected host-cell. Hence, a large number of bacterial pathogens such as Bordetella pertussis, Salmonella typhimurium, Shigella flexneri, Escherichia coli and Staphylococcus aureus induce apoptosis of their infected host-cells [4,5]. Similarly, the protozoan parasites Toxoplasma gondii and Cryptosporidium parvum, and the herpes simplex virus 1 (HSV-1), can both protect infected host-cells against apoptosis induced by immune effector cells or nonphysiological external ligands and induce apoptosis at the * Corresponding author. Tel.: +33-1-44-27-98-58. E-mail address: [email protected] (D.M. Ojcius). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2003.08.006
end of the infection cycle [6–9]. Probably the best characterized among these pathogens is HSV-1, whose genes responsible for resistance against apoptosis and induction of caspase-independent apoptosis have been identified [9]. Thus, pathogens can activate inhibition mechanisms that protect infected cells against the cytotoxic mechanisms of the immune system, while host-cell death at the end of the infection cycle could contribute to propagation of the pathogen in the host organism. Apoptosis could, therefore, be considered as either part of a host defense response that attempts to prevent viral or bacterial propagation, or an intrinsic event in the pathogen’s infection cycle that contributes to establishment of the infection. Regulation of host-cell death thus represents a critical stage in the interaction between a pathogen and its host organism.
2. Infections by Chlamydia Bacteria of the genus Chlamydia are obligate intracellular pathogens that infect mainly epithelial mucosa [10]. Differ-
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ent strains of Chlamydia trachomatis are responsible for infections of ocular tissue and sexually transmitted diseases in humans [11,12]. Chlamydia psittaci elicits different symptoms, depending on its host [13]. It causes intestinal infections in birds and abortion in pregnant ewes, but in humans it manifests itself as pneumonia. Chlamydia pneumoniae is responsible for about a tenth of all pneumonias in humans and has recently been linked to a higher risk for developing atherosclerosis [14]. Chlamydia pecorum is associated with infectious pneumonitis in domestic animals [13]. The bacteria exist in two developmental forms: the infectious extracellular elementary bodies (EB) attach to the hostcell and are internalized into an entry vacuole that avoids fusion with host-cell lysosomes. Within 8–10 h, the EB differentiate into multiplicative intracellular reticulate bodies (RB), which proliferate within the same membrane-bound vacuole. After several divisions, the RB differentiate back into EB, and after 2–3 days, depending on the Chlamydia strain, the EB are released from the infected cell through unknown mechanisms and begin a new cycle of infection [15–18]. Host-cell death observed at the end of the infection cycle could thus be involved in release of EB from the host-cell and could partially contribute to the inflammatory response of the host, since macrophages undergoing apoptosis secrete inflammatory cytokines [19] and cells dying necrotically stimulate inflammation [20]. An inflammatory response is required for the resolution of primary C. trachomatis infection, but chronic inflammation is also responsible for the scarring process observed in trachoma and chlamydial sexually transmitted disease. Thus, a number of inflammatory mediators are present during infection, including interleukin-1b (IL-1b) and tumor necrosis factor (TNFa). TNFa and other inflammatory cytokines may aid in eradicating Chlamydia infection, but also promote long-term tissue damage [21]. In addition, the ability of chlamydiae to persist in the host could be an important factor in exacerbating pathogenesis. The immune mechanisms responsible for chronic inflammation are not fully understood, but it is believed that repeated exposure to chlamydial antigens contributes to pathogenesis and that bacteria in persistently infected cells may serve as a source of long-lasting pathology [22]. Chlamydia muridarum (also known as the mouse pneumonitis strain of C. trachomatis) [23,24] infects the genital tracts of mice and has been used to characterize the host immune response to chlamydial genital tract infection. In C. muridarum infections of mice, a major role for CD4+ T cells secreting interferon-c (IFNc) during clearance of the infection has been described [25–28]. Thus, studies with IFNc deficient mice have shown that IFNc is required for prevention of dissemination of genital tract infection [27,29,30]. Similarly, administration of anti-IFNc antibodies or recombinant IFNc prolongs or resolves the infection, respectively [31]. A persistent state of chlamydiae can be induced in vitro by IFNc treatment, which leads to altered bacterial forms and an antigen profile that is different from that observed during
active infection [22,32]. In IFNc-treated infected cells, the chlamydiae are noninfectious, but the bacteria are viable and can revert to the infectious state following removal of IFNc.
3. Why should Chlamydia modulate apoptosis of infected cells in two opposing directions? On first glance, it may seem odd for a pathogen to have opposing effects on host-cell survival. However, as demonstrated for other pathogens such as HSV-1, a single intracellular microorganism can have different effects at different stages of the infection cycle. Thus, in the case of Chlamydia infections, we propose that resistance of infected cells against apoptosis triggered by external ligands may contribute to persistence of Chlamydia infection, while cell death at the end of the infection cycle would allow chlamydiae to exit from infected cells and initiate a new round of infection (Fig. 1). In vivo studies have revealed that mice that are deficient for perforin, Fas, the Fas ligand, or both perforin and the Fas ligand are able to resolve C. muridarum infection with the same time-course and immune response as wildtype controls, suggesting that an anti-apoptotic activity developed by Chlamydia could protect infected cells against cytotoxic mechanisms of the immune system [33]. Conversely, hostcell lysis associated with the end of the replication cycle could contribute to propagation of the bacteria, as suggested by decreased chlamydial shedding measured in mice whose apoptotic pathways are impaired [34].
4. Protection of infected host-cells against apoptosis due to external ligands Several strains of C. trachomatis and C. pneumoniae protect infected cells against apoptosis due to external ligands, including TNFa, antibodies against Fas, and the kinase inhibitor staurosporine [35–38]. Protection is observable as early as 4 h after infection, suggesting that it could be operative in vivo during early stages of the infection cycle. The anti-apoptotic activity is due to inhibition of cytochrome c release from mitochondria, which thus results in inhibition of the “apoptosome” pathway leading to caspase-9 and subsequently caspase-3 activation. The anti-apoptotic activity is dependent on chlamydial but not host protein synthesis [35], suggesting that chlamydial virulence factors, perhaps secreted via the type III secretion apparatus [39,40], could be responsible for resistance against apoptosis. Cytochrome c release from mitochondria has never been observed at any stage of the infection cycle, suggesting that, once the apoptosome pathway has been turned off, it remains off throughout the cycle. Host-cell death at the end of the cycle would thus have to involve mediators other than caspases. Conversely, any mediators that trigger caspase-independent
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Fig. 1. Infection cycle of Chlamydia. Infectious but metabolically inert EB are internalized by epithelial cells or macrophages via entry vacuoles that avoid fusion with host-cell lysosomes. Within 10 h, EB differentiate into metabolically-active RB, which proliferate within the same membrane-bound vacuole. In the presence of IFNc, chlamydiae can differentiate reversibly into a persistent, atypical form (aRB) that does not proliferate. Persistently-infected cells are resistant to apoptosis. In the absence of IFNc, chlamydiae revert to typical RB, which redifferentiate into EB. Apoptosis at the end of the infection cycle allows chlamydiae to exit from infected cells and initiate a new round of infection.
death at the end of infection cycle (see below) would have to be inhibited until the bacteria are ready to leave the infected cell. Interestingly, cells infected with C. trachomatis in the presence of IFNc are also resistant to apoptosis due to external ligands, via inhibition of the apoptosome pathway [37], suggesting that the resistance against apoptosis could contribute to persistence of infection. 5. Host-cell death and effects on inflammation Cytotoxicity due to infection by human and nonhuman strains of Chlamydia had been observed for many years [41–47], but the mechanisms of cell death had not been investigated. More recently, it was shown that epithelial cells, the preferential target of Chlamydia infection, display characteristic features of apoptosis during infection with the “guinea pig inclusion conjunctivitis” (GPIC) serovar of C. psittaci [48]. Morphological changes associated with apoptosis such as chromatin condensation, nuclear and cytoplasmic segmentation, and blebbing have been observed in infected cell samples by electron microscopy. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method also showed DNA fragmentation in infected cells [48], confirming the apoptotic nature of host-cell death. In addition, phosphatidylserine (PS) is exposed on the surface of infected cells with a time-course similar to host-
cell death [49], consistent with an apoptotic-type mechanism of death during C. psittaci infection. No host-cell death was measured during the first day of infection by either C. psittaci or C. trachomatis, but nuclear condensation, a hallmark of apoptosis, became prominent towards the end of the infection cycle [48,50,51]. As in the case of protection against apoptosis during early stages of the infection cycle, cell death at the end of the infection cycle requires chlamydial but not hostcell protein synthesis [48]. Human monocytes infected with Chlamydia secrete IL1b. Since caspase-1 plays a role in the maturation of IL-1b and IL-18 [52], cells infected with Chlamydia were treated with an irreversible inhibitor of caspase-1. While this inhibitor blocked IL-1b secretion during infection, it had no effect on Chlamydia-induced cell death, suggesting that caspase-1 is activated during infection, but does not participate in cell death [48]. To determine whether other known caspases may be involved in apoptosis, the effects of specific caspase-3 and broad-spectrum caspase inhibitors were tested on host-cell death. These inhibitors had no effect on cell death during infection with C. psittaci or C. trachomatis [48,53]. In addition, caspase-3 is not activated in infected cells, suggesting that known caspases do not participate in apoptosis during Chlamydia infection. Although caspases have long been considered the pivotal executioners of apoptosis, a growing number of recent studies show that apoptosis can take place in the absence of caspase activation [54]. Significantly, overexpression of
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BAX, a pro-apoptotic member of the BCL-2 family, can induce cell death in the absence of caspase activation [55]. BAX normally resides in the cytosol, but translocates to mitochondria after stimulation with pro-apoptotic ligands [56]. During infection with C. psittaci or C. trachomatis, BAX becomes activated and translocates to mitochondria in infected cells. BAX activation appears to play at least a partial role in Chlamydia-induced apoptosis, since overexpression of BAX Inhibitor-1 (BI-1) or the anti-apoptotic protein BCL-2 inhibits apoptosis during infection [53]. Finally, primary fibroblasts lacking BAX die through apoptosis less efficiently than wildtype fibroblasts, demonstrating clearly that BAX activation participates in apoptosis during infection (Fig. 2).
Thus, BAX can contribute to at least some of the death observed during Chlamydia infection, but two unresolved issues still deserve further attention. First, since infected cells do not begin to die until halfway through the infection cycle, it would be worthwhile determining which host-cell factors prevent BAX activation at the beginning of the cycle. One such candidate is humanin, a 24 amino-acid peptide that blocks BAX activation and translocation to mitochondria [57]. However, these studies will be complicated by the possibility that additional factors besides humanin may also inhibit the activity of BAX. Secondly, BAX activation is known to lead to either caspase-dependent or caspaseindependent cell death [55,58,59], but the caspaseindependent mediators responsible for Chlamydia-induced
Fig. 2. Anti- and pro-apoptotic activities during Chlamydia infection. Infected cells are resistant to external pro-apoptotic ligands that trigger cell death via the apoptosome pathway. Thus, cytochrome c release from mitochondria is inhibited, which prevents Apaf-1 and cytochrome c from cleaving pro-caspase 9 into the active caspase-9 (C9a), which would normally then activate caspase-3 (C3a). At the end of the infection cycle, pre-existing BAX is activated, translocates to the mitochondria, and induces cell death in the absence of caspase activation.
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death have yet to be identified. One could propose that caspase-independent factors such as apoptosis-inducing factor (AIF) [60] or endonuclease G [61] may be released selectively from mitochondria, but this would require cytochrome c to remain associated with the mitochondrial intermembrane space under the same conditions. An alternative mechanism is suggested by the observation that lysosomes burst in cells infected with Chlamydia [43], as lysosomal permeabilization can also lead to caspase-independent death [62]. To identify a biological role for BAX-dependent apoptosis, wildtype and BAX-deficient fibroblasts were infected with C. muridarum during at least two infection cycles. The bacterial yield recovered from BAX-deficient cells was significantly reduced, compared to infected wildtype cells, suggesting that chlamydiae may use apoptosis to exit from infected cells and begin a new infection cycle [34]. The PS receptor, which is used for phagocytosis of apoptotic bodies and cells, is expressed on fibroblasts and epithelial cells [63], and we propose that the PS receptor, or similar receptors, may be used to internalize Chlamydia-containing apoptotic bodies by neighboring cells. To confirm the relevance of the in vitro results for a physiological infection, wildtype and BAX-deficient mice were infected intravaginally with C. muridarum. The BAXdeficient mice are a convenient model for studying Chlamydia infection, since the mice are healthy and do not display alterations of the immune system [64]. The infection with C. muridarum was less efficient and disappeared more quickly from the BAX-deficient mice than from the control wildtype mice, suggesting that BAX-dependent apoptosis may contribute to propagation of Chlamydia in vivo [34]. Unexpectedly, the inflammatory response was higher in the BAX-deficient mice than in the wildtype mice, despite the lower intensity of infection in the BAX-deficient mice. These results could be explained if one assumes that there is more necrosis in the BAX-deficient mice than in the wildtype controls. In fact, cells that are prevented from dying through apoptosis usually still manage to die, but they do so later, and they die through necrosis. Consistent with this possibility, BAX-deficient cells infected in vitro die through necrosis more often than wildtype cells [34]. Taken together, these results suggest that Chlamydia-induced apoptosis via BAX contributes to chlamydial propagation and decreases inflammation. Concomitant with the higher levels of cytokine secretion, the BAX-deficient mice contained large granulomas in the genital tract. Thus, paradoxically, BAX-deficiency results in less-intense infection but more pathology. 6. Effect of inflammatory cytokines on cell death in vitro and in vivo Analysis of the infection in BAX-deficient mice demonstrated that dysregulation of cell death can have profound effects on the inflammatory response and host pathology. Conversely, many of the cytokines secreted during Chlamy-
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dia infection, notably TNFa and IFNc, could also modulate apoptosis. To study the possible relationship between TNFa secretion and cell death, mice were infected with C. muridarum and the number of apoptotic cells was measured by the TUNEL assay at different times after infection [51]. There was a large increase in the number of apoptotic cells in the Fallopian tubes and oviducts after 2 or 7 days of infection, compared to uninfected controls. Depletion of TNFa with anti-TNFa antibodies led to a significant decrease in the level of apoptosis in the upper genital tract of mice infected for 7 days, suggesting that TNFa may exert part of its pathological effect by inducing apoptosis of cells in infected tissues. Finally, most of the cells dying as a result of the inflammatory response do not appear to be infected and thus should not be protected against TNFa-mediated apoptosis [51]. IFNc, which is used to induce a persistent phenotype of Chlamydia in vitro, is also known to affect apoptosis and cell survival [65]. IFNc concentrations that induce a persistent state of C. muridarum also inhibit apoptosis due to infection. The inhibitory effect of IFNc is due in part to expression of host-cell indoleamine 2,3-dioxygenase activity, which leads to tryptophan catabolism, as inhibition of apoptosis could be reversed by incubating infected cells with exogenous tryptophan. In addition, a significantly larger number of apoptotic cells were observed in the genital tract of infected IFNc-deficient mice, compared to wildtype mice [66]. These results suggest that IFNc could contribute to the persistence of Chlamydia infections in vivo by inhibiting apoptosis of infected cells. Given the role that BAX-dependent apoptosis plays in propagation of the infection in vitro and in vivo, IFNc would also be expected to inhibit propagation of chlamydiae via apoptotic bodies in vivo. 7. Concluding remarks Apoptosis during Chlamydia infection is affected in two opposing directions, depending on the stage of the infection cycle. The type of cell death taking place at the end of the infection cycle has a large effect on secretion of proinflammatory cytokines by the host, but the host inflammatory response can also modulate apoptosis. Resistance of infected cells against apoptosis triggered by outside ligands probably contributes to persistence of Chlamydia infection. IFNc, a traditional inducer of persistence in vitro, also inhibits Chlamydia-induced death, and thereby, could enhance persistence via its anti-apoptotic effect. On the other hand, the TNFa that is required for elimination of infection promotes apoptosis of uninfected cells, which may be an unintended consequence of the host immune response. Acknowledgements This work was supported by the National Institutes of Health (grant R01 AI054624), Université Paris 7, Centre
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National pour la Recherche Scientifique, the Fondation pour la Recherche Médicale, and the Institut Pasteur (PTR 60).
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