Cell death, BAX activation, and HMGB1 release during infection with Chlamydia

Infection by a number of Chlamydia species leads to resistance of the host cell to apoptosis, followed by induction of host-cell death. In a population of infected cells that displays protection against staurosporine-induced apoptosis among the adherent cells, we find that cells that had been recovered from the supernatant share characteristics of both apoptosis and necrosis, as assayed by the propidium iodide (PI)-annexin V double-labeling technique. Cell death was observed in both an epithelial cell line and primary fibroblasts, although the primary cells had a higher propensity to die through apoptosis than the immortalized cell line. Staurosporine-mediated activation of the pro-apoptotic BCL-2 family member, BAX, was inhibited in the epithelial cell line infected for 32 h with the lymphogranuloma venereum (LGV/L2) but not the murine pneumonitis (MoPn) strain of C. trachomatis, but inhibition of staurosporine-mediated BAX activation disappeared after 48 h of infection with the LGV/L2 strain. Conversely, infection with MoPn (C. muridarum) but not LGV/L2 led to BAX activation after 72 h, as previously reported for shorter (48 h) infection with the guinea pig inclusion conjunctivitis (GPIC) serovar of C. psittaci (C. caviae). These results suggest that the ability to inhibit staurosporine-mediated BAX activation or to activate BAX due to the infection itself may vary as a function of the chlamydial strain. Interestingly, both the epithelial cells and the fibroblasts also released high mobility group box 1 protein (HMGB1) during infection, although much less HMGB1 was released from fibroblasts, consistent with the higher level of apoptosis observed in the primary cells. HMGB1 is released preferentially by necrotic or permeabilized viable cells, but not apoptotic cells. In the extracellular space, HMGB1 promotes inflammation through interaction with specific cell-surface receptors. Higher levels of HMGB1 were also measured in the genital-tract secretions of mice infected vaginally with C. trachomatis, compared to uninfected controls. These results suggest that cells infected with Chlamydia release intracellular factors that may contribute to the inflammatory response observed in vivo.     

Type I IFNs enhance susceptibility to Chlamydia muridarum lung infection by enhancing apoptosis of local macrophages

Type I IFNs (IFNIs) have pleiotropic functions in regulating host innate and adaptive immune responses to pathogens. To elucidate the role of IFNIs in host resistance to chlamydial infection in vivo, we compared IFN-alpha/beta receptor knockout (IFNAR(-/-)) and wild-type control mice in susceptibility to Chlamydia trachomatis mouse pneumonitis (Chlamydia muridarum) lung infection. We found that the IFNAR(-/-) mice were significantly more resistant to C. muridarum infection showing less bacterial burden and bodyweight loss, and milder pathological changes. However, IFN-gamma response, which is believed to be critical in host defense against chlamydial infection, was similar between the wild-type and IFNAR(-/-) mice. More importantly, TUNEL analysis showed less macrophage apoptosis in IFNAR(-/-) mice, which was consistent with lower expressions of IFNI-induced apoptotic factors, TRAIL, Daxx, and PKR. Furthermore, depletion of lung macrophages with dichloromethylene diphosphonate-liposome significantly increased the susceptibility of the IFNAR(-/-) mice to C. muridarum, confirming the importance of macrophages. Overall, the data indicate that IFNIs play a promoting role in C. muridarum lung infection, largely through increase of local macrophage apoptosis.     

Stimulation of the cytosolic receptor for peptidoglycan, Nod1, by infection with Chlamydia trachomatis or Chlamydia muridarum

Infection of epithelial cells by the intracellular pathogen, Chlamydia trachomatis, leads to activation of NF-kappaB and secretion of pro-inflammatory cytokines. We find that overexpression of a dominant-negative Nod1 or depletion of Nod1 by RNA interference inhibits partially the activation of NF-kappaB during chlamydial infection in vitro, suggesting that Nod1 can detect the presence of Chlamydia. In parallel, there is a larger increase in the expression of pro-inflammatory genes following Chlamydia infection when primary fibroblasts are isolated from wild-type mice than from Nod1-deficient mice. The Chlamydia genome encodes all the putative enzymes required for proteoglycan synthesis, but proteoglycan from Chlamydia has never been detected biochemically. Since Nod1 is a ubiquitous cytosolic receptor for peptidoglycan from Gram-negative bacteria, our results suggest that C. trachomatis and C. muridarum do in fact produce at least the rudimentary proteoglycan motif recognized by Nod1. Nonetheless, Nod1 deficiency has no effect on the efficiency of infection, the intensity of cytokine secretion, or pathology in vaginally infected mice, compared with wild-type controls. Similarly, Rip2, a downstream mediator of Nod1, Toll-like receptor (TLR)-2, and TLR4, increases only slightly the intensity of chlamydial infection in vivo and has a very mild effect on the immune response and pathology. Thus, Chlamydia may not produce sufficient peptidoglycan to stimulate Nod1-dependent pathways efficiently in infected animals, or other receptors of the innate immune system may compensate for the absence of Nod1 during Chlamydia infection in vivo.     

Enhanced virulence of Chlamydia muridarum respiratory infections in the absence of TLR2 activation

Chlamydia trachomatis is a common sexually transmitted pathogen and is associated with infant pneumonia. Data from the female mouse model of genital tract chlamydia infection suggests a requirement for TLR2-dependent signaling in the induction of inflammation and oviduct pathology. We hypothesized that the role of TLR2 in moderating mucosal inflammation is site specific. In order to investigate this, we infected mice via the intranasal route with C. muridarum and observed that in the absence of TLR2 activation, mice had more severe disease, higher lung cytokine levels, and an exaggerated influx of neutrophils and T-cells into the lungs. This could not be explained by impaired bacterial clearance as TLR2-deficient mice cleared the infection similar to controls. These data suggest that TLR2 has an anti-inflammatory function in the lung during Chlamydia infection, and that the role of TLR2 in mucosal inflammation varies at different mucosal surfaces.     

COX-2 inhibition affects growth rate of Chlamydia muridarum within epithelial cells

Chlamydiae alter apoptosis of host target cells, which regulates their growth. Cyclooxygenase-2 (COX-2), the rate-limiting enzyme for prostaglandin E2 (PGE2) production, modulates epithelial cell survival. We addressed whether endogenous PGE2 alters chlamydial growth or apoptosis of epithelial cells infected with Chlamydia muridarum. PGE2 is secreted by infected host cells in the genital tract (GT). Using immunohistochemical techniques, we found that COX-2 enzyme was localized to epithelial cells in the GT in vivo. Pellets of the COX-2 enzyme inhibitor, NS-398, and placebo were implanted in mice subcutaneously and released a constant amount of these chemicals throughout the infection. NS-398-treated mice were found to exhibit 10-fold lower bacterial load than the placebo group on day 3 post infection, suggesting disruption of the chlamydial developmental cycle. To prove this, the human lung adenocarcinoma cell line A549 was then infected with different MOIs of C. muridarum in the presence of multiple concentrations of NS-398 in vitro. There was no difference in inclusion forming units (IFUs) between NS-389-treated and untreated cells. We also found no alterations in C. muridarum IFUs in A549 cells transfected with a 2.0 kb cDNA fragment of human COX-2 cloned in the sense (S) or anti-sense (AS) orientation. However, the inclusion size was reduced and the number of EB was significantly diminished during reinfection in AS-transfected cells. In addition, the absence of COX-2 did not significantly modify apoptosis in infected cells. In total, COX-2 deficiency reduces the infectious burden in vivo and may modulate transmission of the organism.     

Immunoproteomic discovery of novel T cell antigens from the obligate intracellular pathogen Chlamydia

Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-A(b)) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study.     

Plasmid-deficient Chlamydia muridarum fail to induce immune pathology and protect against oviduct disease

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection in the world. In women, genital infection can cause endometritis and pelvic inflammatory disease with the severe sequelae of ectopic pregnancy or infertility. Chlamydia sp. do not damage tissues directly, but induce an injurious host inflammatory response at the infected site. In the murine model of genital disease with Chlamydia muridarum, TLR2 plays a role in both early production of inflammatory mediators and development of chronic oviduct pathology. We report the results of studies with plasmid-cured C. muridarum mutants that retain the ability to infect the murine genital tract, but fail to cause disease in the oviduct. These mutants do not stimulate TLR2-dependent cytokine production in mice, nor in innate immune cells or epithelial cells in vitro. They induce an effective Th1 immune response, with no evidence for Th1-immune-mediated collateral tissue damage. Furthermore, mice previously infected with the plasmid-deficient strains are protected against oviduct disease upon challenge with virulent C. muridarum. If plasmid-cured derivatives of human C. trachomatis biovars exhibit similar phenotypic characteristics, they have the potential to serve as vaccines to prevent human disease.     

NK T cell activation promotes Chlamydia trachomatis infection in vivo

We used two approaches to examine the role of NK T cells (NKT) in an intracellular bacterial (Chlamydia trachomatis mouse pneumonitis (C. muridarum)) infection. One is to use CD1 gene knockout (KO) mice, which lack NKT, and the other is to activate NKT using alpha-galactosylceramide (alpha-GalCer), a natural ligand of these cells. The data showed a promoting effect of NKT activation on Chlamydia lung infection. Specifically, CD1 KO mice exhibited significantly lower levels of body weight loss, less severe pathological change and lower chlamydial in vivo growth than wild-type mice. Immunological analysis showed that CD1 KO mice exhibited significantly lower C. muridarum-specific IL-4 and serum IgE Ab responses as well as more pronounced delayed-type hypersensitivity response compared with wild-type controls. In line with the finding in KO mice, the in vivo stimulation of NKT using alpha-GalCer enhanced chlamydial growth in vivo, which were correlated with reduced delayed-type hypersensitivity response and increased C. muridarum-driven IL-4/IgE production. Moreover, neutralization of IL-4 activity in the alpha-GalCer-treated BALB/c mice significantly reduced the promoting effect of alpha-GalCer treatment on chlamydial growth in vivo. These data provide in vivo evidence for the involvement of NKT in a bacterial pathogenesis and its role in promoting Th2 responses during infection.     

Chlamydia muridarum infection subverts dendritic cell function to promote Th2 immunity and airways hyperreactivity

There is strong epidemiological evidence that Chlamydia infection can lead to exacerbation of asthma. However, the mechanism(s) whereby chlamydial infection, which normally elicits a strong Th type 1 (Th1) immune response, can exacerbate asthma, a disease characterized by dominant Th type 2 (Th2) immune responses, remains unclear. In the present study, we show that Chlamydia muridarum infection of murine bone marrow-derived dendritic cells (BMDC) modulates the phenotype, cytokine secretion profile, and Ag-presenting capability of these BMDC. Chlamydia-infected BMDC express lower levels of CD80 and increased CD86 compared with noninfected BMDC. When infected with Chlamydia, BMDC secrete increased TNF-alpha, IL-6, IL-10, IL-12, and IL-13. OVA peptide-pulsed infected BMDC induced significant proliferation of transgenic CD4(+) DO11.10 (D10) T cells, strongly inhibited IFN-gamma secretion by D10 cells, and promoted a Th2 phenotype. Intratracheal transfer of infected, but not control noninfected, OVA peptide-pulsed BMDC to naive BALB/c mice, which had been i.v. infused with naive D10 T cells, resulted in increased levels of IL-10 and IL-13 in bronchoalveolar lavage fluid. Recipients of these infected BMDC showed significant increases in airways resistance and decreased airways compliance compared with mice that had received noninfected BMDC, indicative of the development of airways hyperreactivity. Collectively, these data suggest that Chlamydia infection of DCs allows the pathogen to deviate the induced immune response from a protective Th1 to a nonprotective Th2 response that could permit ongoing chronic infection. In the setting of allergic airways inflammation, this infection may then contribute to exacerbation of the asthmatic phenotype.     

TLR2, but not TLR4, is required for effective host defence against Chlamydia respiratory tract infection in early life

Chlamydia pneumoniae commonly causes respiratory tract infections in children, and epidemiological investigations strongly link infection to the pathogenesis of asthma. The immune system in early life is immature and may not respond appropriately to pathogens. Toll-like receptor (TLR)2 and 4 are regarded as the primary pattern recognition receptors that sense bacteria, however their contribution to innate and adaptive immunity in early life remains poorly defined. We investigated the role of TLR2 and 4 in the induction of immune responses to Chlamydia muridarum respiratory infection, in neonatal wild-type (Wt) or TLR2-deficient ((-/-)), 4(-/-) or 2/4(-/-) BALB/c mice. Wt mice had moderate disease and infection. TLR2(-/-) mice had more severe disease and more intense and prolonged infection compared to other groups. TLR4(-/-) mice were asymptomatic. TLR2/4(-/-) mice had severe early disease and persistent infection, which resolved thereafter consistent with the absence of symptoms in TLR4(-/-) mice. Wt mice mounted robust innate and adaptive responses with an influx of natural killer (NK) cells, neutrophils, myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, and activated CD4(+) and CD8(+) T-cells into the lungs. Wt mice also had effective production of interferon (IFN)γ in the lymph nodes and lung, and proliferation of lymph node T-cells. TLR2(-/-) mice had more intense and persistent innate (particularly neutrophil) and adaptive cell responses and IL-17 expression in the lung, however IFNγ responses and T-cell proliferation were reduced. TLR2/4(-/-) mice had reduced innate and adaptive responses. Most importantly, neutrophil phagocytosis was impaired in the absence of TLR2. Thus, TLR2 expression, particularly on neutrophils, is required for effective control of Chlamydia respiratory infection in early life. Loss of control of infection leads to enhanced but ineffective TLR4-mediated inflammatory responses that prolong disease symptoms. This indicates that TLR2 agonists may be beneficial in the treatment of early life Chlamydia infections and associated diseases.     

Recruitment of BAD by the Chlamydia trachomatis vacuole correlates with host-cell survival

Chlamydiae replicate intracellularly in a vacuole called an inclusion. Chlamydial-infected host cells are protected from mitochondrion-dependent apoptosis, partly due to degradation of BH3-only proteins. The host-cell adapter protein 14-3-3beta can interact with host-cell apoptotic signaling pathways in a phosphorylation-dependent manner. In Chlamydia trachomatis-infected cells, 14-3-3beta co-localizes to the inclusion via direct interaction with a C. trachomatis-encoded inclusion membrane protein. We therefore explored the possibility that the phosphatidylinositol-3 kinase (PI3K) pathway may contribute to resistance of infected cells to apoptosis. We found that inhibition of PI3K renders C. trachomatis-infected cells sensitive to staurosporine-induced apoptosis, which is accompanied by mitochondrial cytochrome c release. 14-3-3beta does not associate with the Chlamydia pneumoniae inclusion, and inhibition of PI3K does not affect protection against apoptosis of C. pneumoniae-infected cells. In C. trachomatis-infected cells, the PI3K pathway activates AKT/protein kinase B, which leads to maintenance of the pro-apoptotic protein BAD in a phosphorylated state. Phosphorylated BAD is sequestered via 14-3-3beta to the inclusion, but it is released when PI3K is inhibited. Depletion of AKT through short-interfering RNA reverses the resistance to apoptosis of C. trachomatis-infected cells. BAD phosphorylation is not maintained and it is not recruited to the inclusion of Chlamydia muridarum, which protects poorly against apoptosis. Thus, sequestration of BAD away from mitochondria provides C. trachomatis with a mechanism to protect the host cell from apoptosis via the interaction of a C. trachomatis-encoded inclusion protein with a host-cell phosphoserine-binding protein.     

Distinct NKT cell subsets are induced by different Chlamydia species leading to differential adaptive immunity and host resistance to the infections

We investigated the role of NKT cells in immunity to Chlamydia pneumoniae and Chlamydia muridarum infections using a combination of knockout mice and specific cellular activation approaches. The NKT-deficient mice showed exacerbated susceptibility to C. pneumoniae infection, but more resistance to C. muridarum infection. Activation of NKT reduced C. pneumoniae in vivo growth, but enhanced C. muridarum infection. Cellular analysis of invariant NKT cells revealed distinct cytokine patterns following C. pneumoniae and C. muridarum infections, i.e., predominant IFN-gamma in the former, while predominant IL-4 in the latter. The cytokine patterns of CD4(+) and CD8(+) T cells matched those of NKT cells. Our data provide in vivo evidence for a functionally diverse role of NKT cells in immune response to two intracellular bacterial pathogens. These results suggest that distinct NKT subsets are induced by even biologically closely related pathogens, thus leading to differential adaptive immune response and infection outcomes.     

A role for matrix metalloproteinase-9 in pathogenesis of urogenital Chlamydia muridarum infection in mice

Matrix metalloproteinases (MMPs) are a family of host-derived enzymes involved in the turnover of extracellular matrix (ECM) molecules and the processing of cytokines, chemokines and growth factors. We have previously reported that global inhibition of MMP in Chlamydia muridarum urogenital tract infection of susceptible strains of female mice impeded ascension of C. muridarum into the upper genital tract, blunted acute inflammatory responses and reduced the rate of formation of chronic disease. Because we have also observed that MMP-9 (also known as gelatinase B) is expressed in relatively large quantities in susceptible strains of mice in response to infection during acute phases of infection, we explored this further in a more selected fashion. We infected MMP-9 gene knockout mice and wild type controls intravaginally with C. muridarum. Both groups of mice had similar isolation rates from the lower urogenital tract but the absence of MMP-9 resulted in a slightly lower isolation rate in the upper genital tract, blunted acute inflammatory indices in the affected tissues and a reduced rate of formation of hydrosalpinx-a surrogate marker of infertility. These results imply that MMP-9 is involved in pathogenesis of chlamydial infection in this model possibly by amplifying inflammatory responses.     

Immunology of Chlamydia infection: implications for a Chlamydia trachomatis vaccine

Sexually transmitted Chlamydia trachomatis infections are a serious public-health problem. With more than 90 million new cases occurring annually, C. trachomatis is the most common cause of bacterial sexually transmitted disease worldwide. Recent progress in elucidating the immunobiology of Chlamydia muridarum infection of mice has helped to guide the interpretation of immunological findings in studies of human C. trachomatis infection and has led to the development of a common model of immunity. In this review, we describe our current understanding of the immune response to infection with Chlamydia spp. and how this information is improving the prospects for development of a vaccine against infection with C. trachomatis.     

Requirement of BAX for efficient adenovirus-induced apoptosis

Infection of human epithelial cells with adenoviruses induces an apoptosis paradigm that is efficiently suppressed by the expression of viral E1B-19K protein, which is a functional homolog of the cellular antiapoptosis protein BCL-2. The mechanisms of adenovirus (Ad)-induced apoptosis appear to involve the cellular BCL-2 family proapoptotic proteins. Recent genetic studies with fibroblasts derived from mutant mouse embryos indicate that a class of the BCL-2 family proapoptotic proteins (designated BH-123 or multidomain proteins) such as BAX and BAK constitutes an essential component of the core apoptosis machinery in animal cells. We have examined the role of BAX in Ad-induced apoptosis in human epithelial cells using two colon cancer cell lines, HCT116Bax (Bax(+/-)) and HCT116BaxKO (Bax(-/-)) (L. Zhang, J. Yu, B. H. Park, K. W. Kinzler, and B. Vogelstein, Science 290:989-992, 2000). Infection of Bax(+/-) cells with an Ad type 2 mutant (dl250) defective in expression of the E1B-19K protein resulted in enhanced cytopathic effect, large plaques on cell monolayers, fragmentation of cellular DNA, and enhanced cell death. These mutant phenotypes were not efficiently expressed in Bax(-/-) cells, suggesting that BAX is essential for Ad-induced apoptosis. Infection of Bax(+/-) cells with dl250 induced increased levels of an N-terminally processed form of BAX. Cells infected with the 19K mutant also contained enhanced levels of truncated BAX in membrane-inserted form. Our results suggest that at least a part of the mechanism utilized by E1B-19K to suppress apoptosis during Ad infection may involve modulation of the activities of BAX.     

Effect of the purinergic receptor P2X7 on Chlamydia infection in cervical epithelial cells and vaginally infected mice

Ligation of the purinergic receptor, P2X7R, with its agonist ATP has been previously shown to inhibit intracellular infection by chlamydiae and mycobacteria in macrophages. The effect of P2X7R on chlamydial infection had never been investigated in the preferred target cells of chlamydiae, cervical epithelial cells, nor in vaginally infected mice. In this study, we show that treatment of epithelial cells with P2X7R agonists inhibits partially Chlamydia infection in epithelial cells. Chelation of ATP with magnesium or pretreatment with a P2X7R antagonist blocks the inhibitory effects of ATP. Similarly to previous results obtained with macrophages, ATP-mediated inhibition of infection in epithelial cells requires activation of host-cell phospholipase D. Vaginal infection was also more efficient in P2X7R-deficient mice, which also displayed a higher level of acute inflammation in the endocervix, oviduct, and mesosalpingeal tissues than in infected wild-type mice. However, secretion of IL-1beta, which requires P2X7R ligation during infection by other pathogens, was decreased mildly and only at short times of infection. Taken together, these results suggest that P2X7R affects Chlamydia infection by directly inhibiting infection in epithelial cells, rather than through the ability of P2X7R to modulate IL-1beta secretion.     

The proapoptotic Bcl-2 protein Bax plays an important role in the pathogenesis of reovirus encephalitis

Encephalitis induced by reovirus serotype 3 (T3) strains results from the apoptotic death of infected neurons. Extrinsic apoptotic signaling is activated in reovirus-infected neurons in vitro and in vivo, but the role of intrinsic apoptosis signaling during encephalitis is largely unknown. Bax plays a key role in intrinsic apoptotic signaling in neurons by allowing the release of mitochondrial cytochrome c. We found Bax activation and cytochrome c release in neurons following infection of neonatal mice with T3 reoviruses. Bax(-/-) mice infected with T3 Abney (T3A) have reduced central nervous system (CNS) tissue injury and decreased apoptosis, despite viral replication that is similar to that in wild-type (WT) Bax(+/+) mice. In contrast, in the heart, T3A-infected Bax(-/-) mice have viral growth, caspase activation, and injury comparable to those in WT mice, indicating that the role of Bax in pathogenesis is organ specific. Nonmyocarditic T3 Dearing (T3D)-infected Bax(-/-) mice had delayed disease and enhanced survival compared to WT mice. T3D-infected Bax(-/-) mice had significantly lower viral titers and levels of activated caspase 3 in the brain despite unaffected transneuronal spread of virus. Cytochrome c and Smac release occurred in some reovirus-infected neurons in the absence of Bax; however, this was clearly reduced compared to levels seen in Bax(+/+) wild-type mice, indicating that Bax is necessary for efficient activation of proapoptotic mitochondrial signaling in infected neurons. Our studies suggest that Bax is important for reovirus growth and pathogenesis in neurons and that the intrinsic pathway of apoptosis, mediated by Bax, is important for full expression of disease, CNS tissue injury, apoptosis, and viral growth in the CNS of reovirus-infected mice.     

Interleukin-13 promotes susceptibility to chlamydial infection of the respiratory and genital tracts

Chlamydiae are intracellular bacteria that commonly cause infections of the respiratory and genital tracts, which are major clinical problems. Infections are also linked to the aetiology of diseases such as asthma, emphysema and heart disease. The clinical management of infection is problematic and antibiotic resistance is emerging. Increased understanding of immune processes that are involved in both clearance and immunopathology of chlamydial infection is critical for the development of improved treatment strategies. Here, we show that IL-13 was produced in the lungs of mice rapidly after Chlamydia muridarum (Cmu) infection and promoted susceptibility to infection. Wild-type (WT) mice had increased disease severity, bacterial load and associated inflammation compared to IL-13 deficient (-/-) mice as early as 3 days post infection (p.i.). Intratracheal instillation of IL-13 enhanced bacterial load in IL-13-/- mice. There were no differences in early IFN-g and IL-10 expression between WT and IL-13-/- mice and depletion of CD4+ T cells did not affect infection in IL-13-/- mice. Collectively, these data demonstrate a lack of CD4+ T cell involvement and a novel role for IL-13 in innate responses to infection. We also showed that IL-13 deficiency increased macrophage uptake of Cmu in vitro and in vivo. Moreover, the depletion of IL-13 during infection of lung epithelial cells in vitro decreased the percentage of infected cells and reduced bacterial growth. Our results suggest that enhanced IL-13 responses in the airways, such as that found in asthmatics, may promote susceptibility to chlamydial lung infection. Importantly the role of IL-13 in regulating infection was not limited to the lung as we showed that IL-13 also promoted susceptibility to Cmu genital tract infection. Collectively our findings demonstrate that innate IL-13 release promotes infection that results in enhanced inflammation and have broad implications for the treatment of chlamydial infections and IL-13-associated diseases.     

Chlamydia-specific CD4 T cell clones control Chlamydia muridarum replication in epithelial cells by nitric oxide-dependent and -independent mechanisms

Chlamydia trachomatis serovars D-K are sexually transmitted intracellular bacterial pathogens that replicate in epithelial cells lining the human reproductive tract. It is clear from knockout mice and T cell depletion studies using Chlamydia muridarum that MHC class II and CD4 T cells are critical for clearing bacteria from the murine genital tract. It is not clear how CD4 T cells interact with infected epithelial cells to mediate bacterial clearance in vivo. Previous work using an epithelial tumor cell line showed that a Chlamydia-specific CD4 T cell clone was able to inhibit C. muridarum replication in vitro via induction of epithelial NO production. We have previously shown that Chlamydia-specific CD4 T cell clones can recognize and be activated by infected reproductive tract epithelial cells and block Chlamydia replication in them. We extend those observations by investigating the mechanism used by a panel of CD4 T cell clones to control Chlamydia replication in epithelial cells. We found that Chlamydia-specific CD4 T cell clones were cytolytic, but that cytolysis was not likely critical for controlling C. muridarum replication. For one, CD4 T cell clone-induced epithelial NO production was critical for controlling replication; however, the most potent CD4 T cell clones were dependent on T cell degranulation for replication control with only a minor additional contribution from NO production. We discuss our data as they relate to existing knockout mouse studies addressing mechanisms of T cell-mediated control of Chlamydia replication and their implications for intracellular epithelial pathogens in mouse models.