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Recent studies have identified semaphorin 3E (Sema3E) as a novel mediator of immune responses. However, its function in immunity to infection has yet to be investigated. Using a mouse model of chlamydial lung infection, we show that Sema3E plays a significant role in the host immune response to the infection. We found that Sema3E is induced in the lung after chlamydial infection, and Sema3E deficiency has a detrimental impact on disease course, dendritic cell (DC) function, and T cell responses. Specifically, we found that Sema3E knockout (KO) mice exhibited higher bacterial burden, severe body weight loss, and pathological changes after Chlamydia muridarum lung infection compared with wild-type (WT) mice. The severity of disease in Sema3E KO mice was correlated with reduced Th1/Th17 cytokine responses, increased Th2 response, altered Ab response, and a higher number of regulatory CD4 T cells. Moreover, DCs isolated from Sema3E KO mice showed lower surface expression of costimulatory molecules and production of IL-12, but higher expression of PD-L1, PD-L2, and IL-10 production. Functional DC-T cell coculture studies revealed that DCs from infected Sema3E KO mice failed to induce Th1 and Th17 cell responses compared with DCs from infected WT mice. Upon adoptive transfer, mice receiving DCs from Sema3E KO mice, unlike those receiving DCs from WT mice, were not protected against challenge infection. In conclusion, our data evidenced that Sema3E acts as a critical factor for protective immunity against intracellular bacterial infection by modulating DC functions and T cell subsets., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Understanding the cellular populations and mechanisms responsible for overcoming immune compartmentalization is valuable for designing vaccination strategies targeting distal mucosae. In this study, we show that the human pathogen Chlamydia trachomatis infects the murine respiratory and genital mucosae and that T cells, but not Abs, elicited through intranasal immunization can protect against a subsequent transcervical challenge. Unlike the genital infection where CD8(+) T cells are primed, yet fail to confer protection, we found that intranasal priming engages both CD4(+) and CD8(+) T cells, allowing for protection against genital infection with C. trachomatis. The protection is largely dependent on IFN-γ secretion by T cells. Moreover, different chemokine receptors are critical for C. trachomatis-specific CD4(+) T cells to home to the lung, rather than the CXCR3- and CCR5-dependent migration observed during genital infection. Overall, this study demonstrates that the cross-mucosa protective immunity against genital C. trachomatis infection following intranasal immunization is not dependent on Ab response but is mediated by not only CD4(+) T cells but also by CD8(+) T cells. This study provides insights for the development of vaccines against mucosal pathogens that threaten reproductive health worldwide., (Copyright © 2015 by The American Association of Immunologists, Inc.)

IFN-β has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-β expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-β expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-β expression. We determined that cGAS is required for IFN-β expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-β, coculture of cells depleted for either STING or cGAS rescued IFN-β expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-β expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-β expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis., (Copyright © 2014 by The American Association of Immunologists, Inc.)

There is a need to implement a vaccine to protect against Chlamydia trachomatis infections. To test a new vaccine, mice were immunized with the Chlamydia muridarum native major outer membrane protein (nMOMP) solubilized with either amphipol A8-35 or the detergent Z3-14. OVA was used as a negative control, and mice were inoculated intranasally with C. muridarum as positive controls. Animals vaccinated with nMOMP mounted strong Chlamydia-specific humoral and cell-mediated immune responses. Mice vaccinated with nMOMP/A8-35 had a higher ratio of Abs to denatured elementary bodies (EB) over live EB, recognized more synthetic MOMP peptides and had higher neutralizing titers than sera from mice immunized with nMOMP/Z3-14. T cell lymphoproliferative responses and levels of IFN-γ were also higher in mice vaccinated with nMOMP/A8-35 than with nMOMP/Z3-14. Following immunization, animals were challenged intravaginally with C. muridarum. On the basis of the number of mice with positive vaginal cultures, length of vaginal shedding, total number of positive vaginal cultures, and number of Chlamydia inclusion forming units recovered, nMOMP/A8-35 elicited a more robust protection than nMOMP/Z3-14. By depleting T cells with Abs, we determined that CD4(+) and not CD8(+) T cells mediated the protection elicited by nMOMP/A8-35. Mice were subsequently mated, and based on the number of pregnant mice and number of embryos, animals that were vaccinated with nMOMP/A8-35 or nMOMP/Z3-14 had fertility rates equivalent to the positive control group immunized with live EB and the fertility controls. In conclusion, increased accessibility of epitopes in the nMOMP/A8-35 preparation may account for the very robust protection against infection and disease elicited by this vaccine., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Recent studies have shed light on the connection between elevated erythropoetin production/spleen erythropoiesis and increased susceptibility to Salmonella infection. In this article, we provide another mouse model, the SIRPα-deficient (Sirpα⁻/⁻) mouse, that manifests increased erythropoiesis as well as heightened susceptibility to Salmonella infection. Sirpα⁻/⁻ mice succumbed to systemic infection with attenuated Salmonella, possessing significantly higher bacterial loads in both the spleen and the liver. Moreover, Salmonella-specific Ab production and Ag-specific CD4 T cells were reduced in Sirpα⁻/⁻ mice compared with wild-type controls. To further characterize the potential mechanism underlying SIRPα-dependent Ag-specific CD4 T cell priming, we demonstrate that lack of SIRPα expression on dendritic cells results in less efficient Ag processing and presentation in vitro. Collectively, these findings demonstrate an indispensable role of SIRPα for protective immunity to Salmonella infection.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Chlamydia infections that ascend to the upper genital tract can persist, trigger inflammation, and result in serious sequelae such as infertility. However, mouse models in which the vaginal vault is inoculated with C. trachomatis do not recapitulate the course of human disease. These intravaginal infections of the mouse do not ascend efficiently to the upper genital tract, do not cause persistent infection, do not induce significant inflammation, and do not induce significant CD4⁺ T cell infiltration. In this article, we describe a noninvasive transcervical infection model in which we bypass the cervix and directly inoculate C. trachomatis into the uterus. We show that direct C. trachomatis infection of the murine upper genital tract stimulates a robust Chlamydia-specific CD4⁺ T cell response that is both necessary and sufficient to clear infection and provide protection against reinfection.

IL-1β has been implicated in the development of oviduct pathology during Chlamydia muridarum genital infection in the mouse model. The goal of this study was to characterize the role of IL-1 signaling and the inflammasome-activation pathways during genital chlamydial infection. Compared with control mice, IL-1R-deficient mice displayed delayed clearance and increased chlamydial colonization. Consistent with the role for IL-1 signaling in infection clearance, mice deficient for the IL-1R antagonist cleared infection at a faster rate. Despite increased infection, IL-1R-deficient mice had significantly reduced oviduct pathology, which was associated with decreased numbers of neutrophils, but more macrophages, in the genital tract. IL-1β secretion is dependent on caspase-1 and apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) inflammasome during in vitro infection of primed macrophages with C. muridarum. To investigate the role of inflammasome components during in vivo genital infection, mice lacking NLRP3, NLRC4, and ASC were tested and found to display no reduction in oviduct pathology compared with control mice. Mice deficient for ASC displayed a prolonged course of infection, which was associated with reduced T cell recruitment and proliferation. Further, ASC-deficient mice displayed normal levels of IL-1β in genital secretions. However, a significant decrease in caspase-1-dependent IL-18 was observed in both ASC- and NLRP3-deficient mice. These data demonstrate a major role for IL-1 signaling, but a limited role for the inflammasome pathway, in IL-1β secretion and development of oviduct pathology during genital chlamydial infection. The data also suggest an IL-1-independent role for ASC in adaptive immunity during genital chlamydial infection.

Dendritic cells (DC) play a key role in establishing protective adaptive immunity in intracellular bacterial infections, but the cells influencing DC function in vivo remain unclear. In this study, we investigated the role of NK cells in modulating the function of DC using a murine Chlamydia infection model. We found that the NK cell-depleted mice showed exacerbated disease after respiratory tract Chlamydia muridarum infection, which was correlated with altered T cell cytokine profile. Furthermore, DC from C. muridarum-infected NK-depleted mice (NK(-)DC) exhibited a less mature phenotype compared with that of DC from the infected mice without NK depletion (NK(+)DC). NK(-)DC produced significantly lower levels of both IL-12 and IL-10 than those of NK(+)DC. Moreover, NK(-)DC showed reduced ability to direct primary and established Ag-specific Th1 CD4(+) T cell responses in DC-T coculture systems. More importantly, adoptive transfer of NK(-)DC, in contrast to NK(+)DC, failed to induce type 1 protective immunity in recipients after challenge infection. Finally, NK cells showed strong direct enhancing effect on IL-12 production by DC in an NK-DC coculture system, which was partially reduced by blocking NKG2D receptors signaling and virtually abolished by neutralizing IFN-γ activity. The data demonstrate a critical role of NK cells in modulating DC function in an intracellular bacterial infection.

Epithelial cells lining the murine genital tract act as sentinels for microbial infection, play a major role in the initiation of the early inflammatory response, and can secrete factors that modulate the adaptive immune response when infected with Chlamydia. C. muridarum-infected murine oviduct epithelial cells secrete the inflammatory cytokines IL-6 and GM-CSF in a TLR2-dependent manner. Further, C. muridarum infection induces IFN-β synthesis in the oviduct epithelial cells in a TRIF-dependent manner. Because murine oviduct epithelial cells express TLR3 but not TLRs 4, 7, 8, or 9, we hypothesized that TLR3 or an unknown TRIF-dependent pattern recognition receptor was the critical receptor for IFN-β production. To investigate the role of TLR3 in the Chlamydia-induced IFN-β response in oviduct epithelial cells, we used small interfering RNA, dominant-negative TLR3 mutants, and TLR3-deficient oviduct epithelial cells to show that the IFN-β secreted during C. muridarum infection requires a functional TLR3. Interestingly, we demonstrate that the TLR3 signaling pathway is not required for IFN-β synthesis in C. muridarum-infected macrophages, suggesting that there are alternate and redundant pathways to Chlamydia-induced IFN-β synthesis that seem to be dependent upon the cell type infected. Finally, because there is no obvious dsRNA molecule associated with Chlamydia infection, the requirement for TLR3 in Chlamydia-induced IFN-β synthesis in infected oviduct epithelial cells implicates a novel ligand that binds to and signals through TLR3.

Chlamydia pneumoniae is a common respiratory pathogen associated with atypical pneumonia, and it has been suggested as a trigger or promoter of several chronic inflammatory conditions, such as asthma and atherosclerosis. The beta form of IL-1 (IL-1beta) is a proinflammatory cytokine released by many cell types and is an important mediator of inflammation during infection. IL-1beta production is a tightly controlled process that includes regulation at multiple levels and typically requires two distinct signals for activation and release. In this study, we investigated the ability of C. pneumoniae to induce IL-1beta secretion. We found that C. pneumoniae was unique among the other Chlamydia species tested in its ability to potently induce secretion of mature IL-1beta from unprimed bone marrow-derived macrophages during a productive infection. TLR2 was required for induction of pro-IL-1beta, whereas the NLRP3/ASC was required for caspase-1 activation and pro-IL-1beta cleavage to produce mature IL-1beta. Caspase-1 cleavage was independent of endogenous ATP release, but required potassium flux, lysosomal acidification, and cathepsin B release. We further investigated the role of IL-1 in host defense against C. pneumoniae-induced pneumonia using mice deficient in the type I IL-1R. Although the IL-1R(-/-) mice developed an inflammatory infiltrate, the number of infiltrating neutrophils was lower, whereas there was evidence of increased infiltrating fibroblasts and mesenchymal cells and more lung fibrosis. We conclude that C. pneumoniae directly activates the NLRP3/ASC inflammasome, leading to the release of biologically active IL-1beta, and that concurrent IL-1 signaling is required for optimal host defense against acute bacterial pneumonia.

Neutrophilic asthma is a prevalent, yet recently described phenotype of asthma. It is characterized by neutrophilic rather than eosinophilic airway inflammation and airways hyperresponsiveness (AHR) and may have an infectious origin. Chlamydial respiratory infections are associated with asthma, but how these Th1-inducing bacteria influence Th2-mediated asthma remains unknown. The effects of chlamydial infection on the development of asthma were investigated using a BALB/c mouse model of OVA-induced allergic airways disease (AAD). The effects of current and resolved Chlamydia muridarum infection during OVA sensitization on AAD were assessed and compared with uninfected and nonsensitized controls. Current, but not resolved, infection attenuated hallmark features of AAD: pulmonary eosinophil influx, T cell production of IL-5, mucus-secreting cell hyperplasia, and AHR. Current infection also induced robust OVA-driven neutrophilic inflammation and IFN-gamma release from T cells. The phenotype of suppressed but persistent Th2 responses in association with enhanced neutrophilia is reminiscent of neutrophilic asthma. This phenotype was also characterized by increased pulmonary IL-12 and IL-17 expression and activation of APCs, as well as by reduced thymus- and activation-regulated chemokine. Inhibition of pulmonary neutrophil influx during infection blocked OVA-induced neutrophilic inflammation and T cell IFN-gamma production and reversed the suppressive effects on mucus-secreting cell hyperplasia and AHR during AAD. These changes correlated with decreased IL-12 and IL-17 expression, increased thymus- and activation-regulated chemokine and altered APC activation. Blocking IFN-gamma and IL-17 during OVA challenge had no effect. Thus, active chlamydial respiratory infection during sensitization enhances subsequent neutrophilic inflammation and Th1/Th17 responses during allergen exposure and may have a role in the pathogenesis of neutrophilic asthma.

ICOS ligand (ICOSL) plays an important role in controlling specific aspects of T cell activation, differentiation, and function. Th1-type immune responses have been shown to be critical in host defense against chlamydial infections. To assess the role of ICOSL-ICOS interaction in host defense against chlamydial infection, we compared the immune responses and pathological reactions in ICOSL gene knockout (KO) and wild-type (WT) mice following Chlamydia muridarum lung infection. The results showed that ICOSL KO mice exhibited greater body weight loss, higher pathogen burden, and more severe histopathology in their lung than did WT mice. Cytokine analysis revealed that ICOSL KO mice produced lower levels of Th2 (IL-4 and IL-5) and anti-inflammatory (TGF-beta1 and IL-10) cytokines, but higher Th1-related (IFN-gamma and IL-12p40/IL-23) and proinflammatory (IL-6 and TNF-alpha) cytokines. ICOSL KO mice also showed reduced Chlamydia-specific Ab levels in their sera and lung homogenates. In addition, ICOSL KO mice demonstrated significantly lower ICOS expression in T cells and lower Th17 responses than did WT mice. Finally, we showed that ICOS-ICOSL interaction and cell-cell contact are essential for CD4(+) T cells to inhibit chlamydial growth in the cultured lung fibroblasts. The data suggest that ICOSL plays a significant role in immunoregulation and protective immunity against Chlamydia infections and that the Th1 skew in cytokine responses per se is not sufficient for effective control of Chlamydia infections.

Although their contribution to host defense against extracellular infections has been well defined, IL-17 and Th17 are generally thought to have limited impact on intracellular infections. In this study, we investigated the role and mechanisms of IL-17/Th17 in host defense against Chlamydia muridarum, an obligate intracellular bacterium, lung infection. Our data showed rapid increase in IL-17 production and expansion of Th17 cells following C. muridarum infection and significant detrimental impact of in vivo IL-17 neutralization by anti-IL-17 mAb on disease course, immune response, and dendritic cell (DC) function. Specifically, IL-17-neutralized mice exhibited significantly greater body weight loss, higher organism growth, and much more severe pathological changes in the lung compared with sham-treated control mice. Immunological analysis showed that IL-17 neutralization significantly reduced Chlamydia-specific Th1 responses, but increased Th2 responses. Interestingly, the DC isolated from IL-17-neutralized mice showed lower CD40 and MHC II expression and IL-12 production, but higher IL-10 production compared with those from sham-treated mice. In two DC-T cell coculture systems, DC isolated from IL-17-neutralized mice induced higher IL-4, but lower IFN-gamma production by Ag-specific T cells than those from sham-treated mice in cell priming and reaction settings. Adoptive transfer of DC isolated from IL-17-neutralized mice, unlike those from sham-treated mice, failed to protect the recipients against challenge infection. These findings provide in vivo evidence that IL-17/Th17 plays an important role in host defense against intracellular bacterial infection, and suggest that IL-17/Th17 can promote type 1 T cell immunity through modulating DC function.

A vaccine is likely the most effective strategy for controlling human chlamydial infections. Recent studies have shown immunization with Chlamydia muridarum major outer membrane protein (MOMP) can induce significant protection against infection and disease in mice if its native trimeric structure is preserved (nMOMP). The objective of this study was to investigate the immunogenicity and vaccine efficacy of Chlamydia trachomatis nMOMP in a nonhuman primate trachoma model. Cynomolgus monkeys (Macaca fascicularis) were immunized systemically with nMOMP, and monkeys were challenged ocularly. Immunization induced high serum IgG and IgA ELISA Ab titers, with Abs displaying high strain-specific neutralizing activity. The PBMCs of immunized monkeys produced a broadly cross-reactive, Ag-specific IFN-gamma response equivalent to that induced by experimental infection. Immunized monkeys exhibited a significant decrease in infectious burden during the early peak shedding periods (days 3-14). However, at later time points, they exhibited no difference from control animals in either burden or duration of infection. Immunization had no effect on the progression of ocular disease. These results show that systemically administered nMOMP is highly immunogenic in nonhuman primates and elicits partially protective immunity against ocular chlamydial challenge. This is the first time a subunit vaccine has shown a significant reduction in ocular shedding in nonhuman primates. A partially protective vaccine, particularly one that reduces infectious burden after primary infection of children, could interrupt the natural trachoma reinfection cycle. This would have a beneficial effect on the transmission between children and sensitized adults which drives blinding inflammatory disease.

It is well established that intrauterine infections can pose a threat to pregnancy by gaining access to the placenta and fetus, and clinical studies have strongly linked bacterial infections with preterm labor. Although Chlamydia trachomatis (Ct) can infect the placenta and decidua, little is known about its effects on trophoblast cell immune function. We have demonstrated that Ct infects trophoblast cells to form inclusions and completes the life cycle within these cells by generating infectious elementary bodies. Moreover, infection with Ct leads to differential modulation of the trophoblast cell's production of cytokines and chemokines. Using two human first trimester trophoblast cell lines, Sw.71 and H8, the most striking feature we found was that Ct infection results in a strong induction of IL-1beta secretion and a concomitant reduction in MCP-1 (CCL2) production in both cell lines. In addition, we have found that Ct infection of the trophoblast results in the cleavage and degradation of NF-kappaB p65. These findings suggest that the effect of a Chlamydia infection on trophoblast secretion of chemokines and cytokines involves both activation of innate immune receptors expressed by the trophoblast and virulence factors secreted into the trophoblast by the bacteria. Such altered trophoblast innate immune responses may have a profound impact on the microenvironment of the maternal-fetal interface and this could influence pregnancy outcome.

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.

Chlamydia trachomatis infection in neonates, not adults, has been associated with the development of chronic respiratory sequelae. Adult chlamydial infections induce Th1-type responses that subsequently clear the infection, whereas the neonatal immune milieu in general has been reported to be biased toward Th2-type responses. We examined the protective immune responses against intranasal Chlamydia muridarum challenge in 1-day-old C57BL/6 and BALB/c mice. Infected C57BL/6 pups displayed earlier chlamydial clearance (day 14) compared with BALB/c pups (day 21). However, challenged C57BL/6 pups exhibited prolonged deficits in body weight gain (days 12-30) compared with BALB/c pups (days 9-12), which correlated with continual pulmonary cellular infiltration. Both strains exhibited a robust Th1-type response, including elevated titers of serum antichlamydial IgG2a and IgG2b, not IgG1, and elevated levels of splenic C. muridarum-specific IFN-gamma, not IL-4, production. Additionally, elevated IFN-gamma, not IL-4 expression, was observed locally in the infected lungs of both mouse strains. The immune responses in C57BL/6 pups were significantly greater compared with BALB/c pups after chlamydial challenge. Importantly, infected mice deficient in IFN-gamma or IFN-gamma receptor demonstrated enhanced chlamydial dissemination, and 100% of animals died by 2 wk postchallenge. Collectively, these results indicate that neonatal pulmonary chlamydial infection induces a robust Th1-type response, with elevated pulmonary IFN-gamma production, and that endogenous IFN-gamma is important in protection against this infection. The enhanced IFN-gamma induction in the immature neonatal lung also may be relevant to the development of respiratory sequelae in adult life.

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.

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.

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.

Our previous study has shown that the adoptive transfer of dendritic cells (DCs) freshly isolated from Chlamydia-infected mice (iIDCs), unlike those from control naive mice (iNDCs), can inhibit systemic and cutaneous eosinophilia induced by OVA exposure. In the present study, we examined the mechanism by which iIDC inhibits allergen-specific Th2 cell differentiation in vitro and in vivo. The study revealed that iIDCs exhibited higher surface expression of CD8alpha and the ICOS ligand (ICOS-L), as well as higher IL-10 and IL-12 production than iNDCs. In vitro DC:CD4(+) T cell coculture experiments showed that iIDCs could inhibit allergen-specific Th2 cell differentiation and that the inhibitory effect could be abolished by the blockage of IL-10 or IL-12 activity. More interestingly, the coblockade of IL-10 and the ICOS-L showed synergistic effect in enhancing allergen-driven Th2 cytokine production. Furthermore, adoptive transfer of iIDCs, but not iNDCs, to OVA sensitized mice significantly inhibited airway eosinophilia and mucus overproduction following intranasal challenge with OVA. Overall, the data demonstrate a critical role played by ICOS-L-expressing and IL-10-producing DCs from Chlamydia-infected mice in the infection-mediated inhibition of allergic responses.

The roles of Toll-like receptor (TLR) 2 and TLR4 in the host inflammatory response to infection caused by Chlamydia trachomatis have not been elucidated. We examined production of TNF-alpha and IL-6 in wild-type TLR2 knockout (KO), and TLR4 KO murine peritoneal macrophages infected with the mouse pneumonitis strain of C. trachomatis. Furthermore, we compared the outcomes of genital tract infection in control, TLR2 KO, and TLR4 KO mice. Macrophages lacking TLR2 produced significantly less TNF-alpha and IL6 in response to active infection. In contrast, macrophages from TLR4 KO mice consistently produced higher TNF-alpha and IL-6 responses than those from normal mice on in vitro infection. Infected TLR2-deficient fibroblasts had less mRNA for IL-1, IL-6, and macrophage-inflammatory protein-2, but TLR4-deficient cells had increased mRNA levels for these cytokines compared with controls, suggesting that ligation of TLR4 by whole chlamydiae may down-modulate signaling by other TLRs. In TLR2 KO mice, although the course of genital tract infection was not different from that of controls, significantly lower levels of TNF-alpha and macrophage-inflammatory protein-2 were detected in genital tract secretions during the first week of infection, and there was a significant reduction in oviduct and mesosalpinx pathology at late time points. TLR4 KO mice responded to in vivo infection similarly to wild-type controls and developed similar pathology. TLR2 is an important mediator in the innate immune response to C. trachomatis infection and appears to play a role in both early production of inflammatory mediators and development of chronic inflammatory pathology.

Protection against infections with the intracellular bacterium Chlamydia spp. requires Th1-polarized CD4+ T cell immunity. In BALB/c mouse lung infections, immediate innate and nascent Chlamydia-specific immune responses following intranasal inoculation of Chlamydia psittaci strain B577 were modulated by 7-day i.p. administration of murine rIL-12, the initiation cytokine for Th1 immunity. Treatment with IL-12 reduced the severity of chlamydial pneumonia, abolished mortality (37.5% in untreated mice), and significantly reduced numbers of chlamydial organisms in lungs. On day 4 after inoculation, the neutrophil:macrophage ratio in bronchointerstitial pneumonias was 1.96 in untreated mice and 0.51 in IL-12-treated mice. This immediate, IL-12-mediated shift in innate inflammatory phenotype was correlated with a significant reduction of lung concentrations of the neutrophil chemoattractant macrophage inflammatory protein (MIP)-2 (putative murine homologue of human IL-8), monocyte chemotactic protein-1, and TNF-alpha; and a reduction in MIP-1alpha and IFN-gamma, at high-dose infection only, and IL-12-independent IL-10 levels. Chlamydia-specific Ab titers and Ig isotype ratios indicated an IL-12-dependent Th1 shift. Recall responses of IL-12-primed mice to secondary chlamydial lung infection eliminated chlamydiae more effectively and generated a lung cytokine profile conducive to perpetuation of the Th1 memory population. These data support the hypothesis that genetic differences in endogenous IL-12 production and response pathways could determine disease outcomes characterized by poor chlamydial clearance and a purulent inflammatory infiltrate vs effective elimination of chlamydiae in a macrophage-dominated response.

We previously reported that higher IL-10 production is correlated with lower IFN-gamma production, weaker delayed hypersensitivity (DTH), and slower organism clearance following chlamydial infection in mice. To assess more directly the role of IL-10, we examined protective immunity and pathological reaction in C57BL/6 IL-10 gene knockout (KO) and wild-type mice. The results showed that in the absence of endogenous IL-10, mice had significantly accelerated chlamydial clearance and developed significantly stronger DTH responses, which could be inhibited by local delivery of rIL-10. Consistent with the enhancement of DTH responses, IL-10 KO mice showed stronger and more persistent CD4 T cell-dependent IFN-gamma production and significant elevation of IL-12 and TNF-alpha production. Additionally, wild-type, but not IL-10 KO, mice showed granuloma formation that was correlated with higher levels of Th2 cytokine (IL-5) production at the later stages of infection. Moreover, chlamydial infection, unlike parasitic protozoan infection, did not induce significant acute toxicity in IL-10 KO mice, which may be due to the low (undetectable) levels of systemic release of proinflammatory cytokines. These results suggest that IL-10 inhibits the priming and expansion of Th1-like T cell responses and that IL-10 plays a role in the fibrotic reaction seen with chlamydial infection.

We have characterized the cytotoxic activity of the obligate intracellular bacterium Chlamydia psittaci, which resides within a membrane-bound vacuole during the 2-day infection cycle. We have established that infected epithelial cells and macrophages die through apoptosis, which is measurable within 1 day of infection and requires productive infection by the bacteria. Inhibition of host cell protein synthesis has no effect on cell death, but blocking bacterial entry or bacterial protein synthesis prevents apoptosis, implying that bacterial growth is required for death of the host cell. Apoptosis was confirmed through the use of electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, gel agarose electrophoresis of fragmented DNA, and propidium-iodide labeling of host cell nuclei. Although infected cells died preferentially, both infected and uninfected cells became apoptotic, suggesting that the infected cells may secrete proapoptotic factors. Inhibition of either of two proapoptotic enzymes, caspase-1 or caspase-3, did not significantly affect Chlamydia-induced apoptosis. These results suggest that, as in the case of apoptosis due to Bax expression or oncogene dysregulation, which initiate the apoptotic program within the cell interior, the Chlamydia infection may trigger an apoptotic pathway that is independent of known caspases. As apoptotic cells secrete proinflammatory cytokines, Chlamydia-induced apoptosis may contribute to the inflammatory response of the host.