Oviduct Pathology Research Articles

IL‐23 Induces IL‐22 and IL‐17 Production in Response to Chlamydia muridarum Genital Tract Infection, but the Absence of these Cytokines does not Influence Disease Pathogenesis

Chlamydia trachomatis infections are a significant cause of reproductive tract pathology. Protective and pathological immune mediators must be differentiated to design a safe and effective vaccine. Wild-type mice and mice deficient in IL-22 and IL-23 were infected intravaginally with Chlamydia muridarum, and their course of infection and oviduct pathology were compared. Local genital tract and draining lymph node immune responses were also examined in IL-23-deficient mice. IL-22- and IL-23-deficient mice exhibited normal susceptibility to infection and oviduct pathology. IL-23 was required for the development of a Chlamydia-specific Th17 response in the lymph nodes and for production of IL-22 and IL-17 in the genital tract. However, influx of Th1 and innate immune cells was not compromised in the absence of IL-23. IL-22 and IL-23 play either redundant or minimal roles in the pathogenesis of Chlamydia infection in the mouse model. Induction of Th17-associated cytokines by a Chlamydia vaccine should be avoided as these responses are not central to resolution of infection and have pathologic potential.

The Duration of Chlamydia muridarum Genital Tract Infection and Associated Chronic Pathological Changes Are Reduced in IL-17 Knockout Mice but Protection Is Not Increased Further by Immunization

IL-17 is believed to be important for protection against extracellular pathogens, where clearance is dependent on neutrophil recruitment and local activation of epithelial cell defences. However, the role of IL-17 in protection against intracellular pathogens such as Chlamydia is less clear. We have compared (i) the course of natural genital tract C. muridarum infection, (ii) the development of oviduct pathology and (iii) the development of vaccine-induced immunity against infection in wild type (WT) BALB/c and IL-17 knockout mice (IL-17-/-) to determine if IL-17-mediated immunity is implicated in the development of infection-induced pathology and/or protection. Both the magnitude and duration of genital infection was significantly reduced in IL-17-/- mice compared to BALB/c. Similarly, hydrosalpinx was also greatly reduced in IL-17-/- mice and this correlated with reduced neutrophil and macrophage infiltration of oviduct tissues. Matrix metalloproteinase (MMP) 9 and MMP2 were increased in WT oviducts compared to IL-17-/- animals at day 7 post-infection. In contrast, oviducts from IL-17-/- mice contained higher MMP9 and MMP2 at day 21. Infection also elicited higher levels of Chlamydia -neutralizing antibody in serum of IL-17-/- mice than WT mice. Following intranasal immunization with C. muridarum Major Outer Membrane Protein (MOMP) and cholera toxin plus CpG adjuvants, significantly higher levels of chlamydial MOMP-specific IgG and IgA were found in serum and vaginal washes of IL-17-/- mice. T cell proliferation and IFNγ production by splenocytes was greater in WT animals following in vitro re-stimulation, however vaccination was only effective at reducing infection in WT, not IL-17-/- mice. Intranasal or transcutaneous immunization protected WT but not IL-17-/- mice against hydrosalpinx development. Our data show that in the absence of IL-17, the severity of C. muridarum genital infection and associated oviduct pathology are significantly attenuated, however neither infection or pathology can be reduced further by vaccination protocols that effectively protect WT mice.

CD4+CD25+Foxp3+ Regulatory T Cells Promote Th17 Responses and Genital Tract Inflammation upon Intracellular Chlamydia muridarum Infection

The functional role of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in host responses to intracellular bacterial infection was investigated in an in vitro coculturing system and a murine model of Chlamydia muridarum genital tract infection. Remarkably, C. muridarum infection subverted the immune suppressive role of CD4⁺CD25⁺Foxp3⁺ Tregs; instead of hampering immune responses, Tregs not only promoted Th17 differentiation from conventional CD4⁺ T cells but also themselves converted into proinflammatory Th17 cells in both in vitro and in vivo settings. Anti-CD25 mAb PC61 treatment to deplete ∼50% of pre-existing Tregs prior to C. muridarum genital tract infection markedly reduced the frequency and the total number of Th17 but not Th1 CD4⁺ cells at both immune induction and memory phases. Most importantly, Treg-depleted mice displayed significantly attenuated inflammation, neutrophil infiltration, and reduced severity of oviduct pathology upon C. muridarum genital infection. To our knowledge, this is the first report demonstrating that the level of pre-existing CD4⁺CD25⁺Foxp3⁺ Tregs in Chlamydia-infected hosts has a major impact on the development Chlamydia-associated diseases.

Intranasal Vaccination with Chlamydia pneumoniae Induces Cross-Species Immunity against Genital Chlamydia muridarum Challenge in Mice

Chlamydia trachomatis is the most common bacterial sexually transmitted disease in the world and specifically in the United States, with the highest incidence in age-groups 14–19 years. In a subset of females, the C. trachomatis genital infection leads to serious pathological sequelae including pelvic inflammatory disease, ectopic pregnancy, and infertility. Chlamydia pneumoniae, another member of the same genus, is a common cause of community acquired respiratory infection with significant number of children aged 5–14 yr displaying sero-conversion. Since these bacteriae share several antigenic determinants, we evaluated whether intranasal immunization with live C. pneumoniae (1×106 inclusion forming units; IFU) in 5 week old female C57BL/6 mice would induce cross-species protection against subsequent intravaginal challenge with Chlamydia muridarum (5×104 IFU), which causes a similar genital infection and pathology in mice as C. trachomatis in humans. Mice vaccinated intranasally with live C. pneumoniae, but not mock (PBS) immunized animals, displayed high levels of splenic cellular antigen-specific IFN-γ production and serum antibody response against C. muridarum and C. trachomatis. Mice vaccinated with C. pneumoniae displayed a significant reduction in the vaginal C. muridarum shedding as early as day 12 after secondary i.vag. challenge compared to PBS (mock) immunized mice. At day 19 after C. muridarum challenge, 100% of C. pneumoniae vaccinated mice had cleared the infection compared to none (0%) of the mock immunized mice, which cleared the infection by day 27. At day 80 after C. muridarum challenge, C. pneumoniae vaccinated mice displayed a significant reduction in the incidence (50%) and degree of hydrosalpinx compared to mock immunized animals (100%). These results suggest that respiratory C. pneumoniae infection induces accelerated chlamydial clearance and reduction of oviduct pathology following genital C. muridarum challenge, and may have important implications to the C. trachomatis-induced reproductive disease in humans.

Vaginal chlamydial clearance following primary or secondary infection in mice occurs independently of TNF-α

The role of TNF-α in chlamydial clearance is uncertain. Antibody-mediated depletion of TNF-α in mice and guinea pigs has been shown not to significantly affect chlamydial clearance, whereas production of TNF-α in addition to IFN-γ from T cells has been shown to correlate with enhanced clearance. The aim of our study is to evaluate the mechanistic role of TNF-α in clearance of primary and secondary chlamydial infection from the genital tract (GT) using C57BL/6 TNF-α deficient (TNF-α−/−) and wild type (WT) mice. Chlamydial shedding from the lower GT was evaluated following primary and secondary intravaginal challenge. Also, antibody and antigen specific cytokine responses were analyzed from the infected GT and spleens, and oviduct pathology determined to analyze the role of TNF-α in upper GT pathological sequelae. MHC II−/− mice, known to display muted adaptive immune responses and failure to resolve genital chlamydial infections, were used as a negative control. Following both primary and secondary genital chlamydial infection, TNF-α−/− mice exhibited elevated granzyme B production, but similar IFN-γ and antibody responses. Importantly, absence of TNF-α did not significantly alter the resolution of infection. However, TNF-α−/− mice displayed significantly reduced upper genital tract (UGT) pathology compared to WT mice. This study demonstrates mechanistically that optimal chlamydial clearance following primary and secondary chlamydial genital infection can occur in the complete absence of TNF-α, and considered with the reduction of upper GT pathology in TNF-α−/− mice, suggests that targeted induction of anti-chlamydial TNF-α responses by vaccination may be unnecessary, and moreover could be potentially pathogenic.

The Role of Regulatory T Cells in Determining Outcomes of Chlamydial Genital Tract Infection (164.15)

Abstract Females are at risk for severe oviduct disease after genital tract infection with Chlamydia trachomatis. Previous studies in our laboratory demonstrate Toll-like receptor 2 (TLR2) activation plays an important role in the development of chronic oviduct pathology after murine chlamydial genital tract infection. Clearance of chlamydial infection has been shown in human and murine investigations to depend on IFN-γ-producing CD4+ T cells. However, an overly zealous adaptive T cell response may contribute to the development of chronic tissue pathology. Pathogen specific T effector cell responses can be suppressed by CD4+CD25+Foxp3+regulatory T cells (Tregs), whose suppressive activity may be abrogated by TLR2 activation. We hypothesize Chlamydia specific TLR2 activation abrogates Treg function resulting in an exacerbated adaptive immune response, which contributes to tissue pathology. Here, we successfully depleted Tregs in a Foxp3DTR mouse model by administration of diphtheria toxin prior to intravaginal infection with TLR2-stimulating Chlamydia murdarium. Although bacterial burden was unaltered, conditional ablation of Tregs resulted in increased IFN-γ-producing CD4+ and CD8+ T cells. Additional studies will determine if removal of Treg suppression leads to increased immunopathology.

The Recall Response Induced by Genital Challenge with Chlamydia muridarum Protects the Oviduct from Pathology but Not from Reinfection

The significant morbidities of ectopic pregnancy and infertility observed in women after Chlamydia trachomatis genital infection result from ascension of the bacteria from the endocervix to the oviduct, where an overly aggressive inflammatory response leads to chronic scarring and Fallopian tube obstruction. A vaccine to prevent chlamydia-induced disease is urgently needed. An important question for vaccine development is whether sterilizing immunity at the level of the oviduct is essential for protection because of the possibility that a chlamydial component drives a deleterious anamnestic T cell response upon oviduct reinfection. We show that mice inoculated with attenuated plasmid-cured strains of Chlamydia muridarum are protected from oviduct pathology upon challenge with wild-type C. muridarum Nigg despite induction of a response that did not prevent reinfection of the oviduct. Interestingly, repeated abbreviated infections with Nigg also elicited recall responses that protected the oviduct from pathology despite low-level reinfection of this vulnerable tissue site. Challenged mice displayed significant decreases in tissue infiltration of inflammatory leukocytes with marked reductions in frequencies of neutrophils but significant increases in frequencies of CD4 Th1 and CD8 T cells. An anamnestic antibody response was also detected. These data indicate that exposure to a live attenuated chlamydial vaccine or repeated abbreviated genital infection with virulent chlamydiae promotes anamnestic antibody and T cell responses that protect the oviduct from pathology despite a lack of sterilizing immunity at the site.

Significant Role of IL-1 Signaling, but Limited Role of Inflammasome Activation, in Oviduct Pathology during Chlamydia muridarum Genital Infection

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.

Enhanced neutrophil longevity and recruitment contribute to the severity of oviduct pathology during Chlamydia muridarum infection.

Our previous studies revealed that intravaginal infection of mice with a plasmid-deficient strain of Chlamydia muridarum, CM3.1, does not induce the development of oviduct pathology. In this study, we determined that infection with CM3.1 resulted in a significantly reduced frequency and absolute number of neutrophils in the oviducts during acute infection. This reduction in neutrophils was associated with significantly lower levels of neutrophil chemokines in the oviducts and decreased production of neutrophil chemokines by oviduct epithelial cells infected with CM3.1 in vitro. Infection with CM3.1 also resulted in an increased frequency of late apoptotic/dead neutrophils in the oviduct. Examination of the ability of Chlamydia trachomatis to prevent neutrophil apoptosis in vitro revealed that C. trachomatis strain D/UW-3/Cx exhibited an enhanced ability to prevent neutrophil apoptosis compared to plasmid-deficient CTD153, and this effect was dependent on the presence of CD14(high) monocytes. The presence of monocytes also resulted in enhanced neutrophil cytokine production and increased production of tissue-damaging molecules in response to D/UW-3/Cx relative to results with CTD153. Attempts to use antibody-mediated depletion to discern the specific role of neutrophils in infection control and pathology in vivo revealed that although Ly6G(high) neutrophils were eliminated from the blood and oviducts with this treatment, immature neutrophils and high levels of tissue-damaging molecules were still detectable in the upper genital tract. These data support the role of neutrophils in chlamydia-induced pathology and reveal that novel methods of depletion must be developed before their role can be specifically determined in vivo.

Interferon Regulatory Transcription Factor 3 Protects Mice from Uterine Horn Pathology during Chlamydia muridarum Genital Infection

Mice with the type I interferon (IFN) receptor gene knocked out (IFNAR KO mice) or deficient for alpha/beta IFN (IFN-α/β) signaling clear chlamydial infection earlier than control mice and develop less oviduct pathology. Initiation of host IFN-β transcription during an in vitro chlamydial infection requires interferon regulatory transcription factor 3 (IRF3). The goal of the present study was to characterize the influence of IRF3 on chlamydial genital infection and its relationship to IFN-β expression in the mouse model. IRF3 KO mice were able to resolve infection as well as control mice, overcoming increased chlamydial colonization and tissue burden early during infection. As previously observed for IFNAR KO mice, IRF3 KO mice generated a potent antigen-specific T cell response. However, in contrast to IFNAR KO mice, IRF3 KO mice exhibited unusually severe dilatation and pathology in the uterine horns but normal oviduct pathology after infection. Although IFN-β expression in vivo was dependent on the presence of IRF3 early in infection (before day 4), the IFN-independent function of IRF3 was likely driving this phenotype. Specifically, early during infection, the number of apoptotic cells and the number of inflammatory cells were significantly less in uterine horns from IRF3 KO mice than in those from control mice, despite an increased chlamydial burden. To delineate the effects of IFN-β versus IRF3, neutralizing IFN-β antibody was administered to wild-type (WT) mice during chlamydial infection. IFN-β depletion in WT mice mimicked that in IFNΑR KO mice but not that in IRF3 KO mice with respect to both chlamydial clearance and reduced oviduct pathology. These data suggest that IRF3 has a role in protection from uterine horn pathology that is independent of its function in IFN-β expression.

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.

Tumor Necrosis Factor Alpha Production from CD8+T Cells Mediates Oviduct Pathological Sequelae following Primary Genital Chlamydia muridarum Infection

The immunopathogenesis of Chlamydia trachomatis-induced oviduct pathological sequelae is not well understood. Mice genetically deficient in perforin (perforin(-/-) mice) or tumor necrosis factor alpha (TNF-α) production (TNF-α(-/-) mice) displayed comparable vaginal chlamydial clearance rates but significantly reduced oviduct pathology (hydrosalpinx) compared to that of wild-type mice. Since both perforin and TNF-α are effector mechanisms of CD8(+) T cells, we evaluated the role of CD8(+) T cells during genital Chlamydia muridarum infection and oviduct sequelae. Following vaginal chlamydial challenge, (i) mice deficient in TAP I (and therefore the major histocompatibility complex [MHC] I pathway and CD8(+) T cells), (ii) wild-type mice depleted of CD8(+) T cells, and (iii) mice genetically deficient in CD8 (CD8(-/-) mice) all displayed similar levels of vaginal chlamydial clearance but significantly reduced hydrosalpinx, compared to those of wild-type C57BL/6 mice, suggesting a role for CD8(+) T cells in chlamydial pathogenesis. Repletion of CD8(-/-) mice with wild-type or perforin(-/-), but not TNF-α(-/-), CD8(+) T cells at the time of challenge restored hydrosalpinx to levels observed in wild-type C57BL/6 mice, suggesting that TNF-α production from CD8(+) T cells is important for pathogenesis. Additionally, repletion of TNF-α(-/-) mice with TNF-α(+/+) CD8(+) T cells significantly enhanced the incidence of hydrosalpinx and oviduct dilatation compared to those of TNF-α(-/-) mice but not to the levels found in wild-type mice, suggesting that TNF-α production from CD8(+) T cells and non-CD8(+) cells cooperates to induce optimal oviduct pathology following genital chlamydial infection. These results provide compelling new evidence supporting the contribution of CD8(+) T cells and TNF-α production to Chlamydia-induced reproductive tract sequelae.

Significant role of IL-1 signaling, but limited role of inflammasome activation in oviduct pathology during Chlamydia muridarum genital infection (110.6)

Abstract 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 C. muridarum genital infection. Compared to control mice, IL-1 receptor KO (knockout) mice display delayed clearance and increased infection burden, but very few incidences of oviduct hydrosalpinx. Consistent with this observation, IL-1R antagonist KO mice display an opposite phenotype of rapid infection clearance. Flow cytometry analysis at day 10 and day 20 post infection reveal significantly more macrophages in the oviducts of IL-1R KO mice in comparison to control mice. Despite increased infection and recruited macrophages, IL-1R-KO mice have less tissue fibrosis and dilation, suggesting an anti-inflammatory role for these cells in the absence of IL-1 signaling. To investigate the role of inflammasome components during in vivo genital infection, mice lacking NLRP3, NLRC4 and ASC were tested. Although IL-1β secretion is dependent on caspase-1 during in vitro infection of primed macrophages and requires ASC, it is independent of NLRP3 and NLRC4 activation. Surprisingly, during in vivo genital infection, mice lacking NLRP3, NLRC4, as well as ASC display no reduction in oviduct pathology. These data suggest a major role for IL-1 signaling but a limited role for inflammasome pathway in development of oviduct pathology.

Toll-Like Receptor 2 Activation by Chlamydia trachomatis Is Plasmid Dependent, and Plasmid-Responsive Chromosomal Loci Are Coordinately Regulated in Response to Glucose Limitation by C. trachomatis but Not by C. muridarum

We previously demonstrated that plasmid-deficient Chlamydia muridarum retains the ability to infect the murine genital tract but does not elicit oviduct pathology because it fails to activate Toll-like receptor 2 (TLR2). We derived a plasmid-cured derivative of the human genital isolate Chlamydia trachomatis D/UW-3/Cx, strain CTD153, which also fails to activate TLR2, indicating this virulence phenotype is associated with plasmid loss in both C. trachomatis and C. muridarum. As observed with plasmid-deficient C. muridarum, CTD153 displayed impaired accumulation of glycogen within inclusions. Transcriptional profiling of the plasmid-deficient strains by using custom microarrays identified a conserved group of chromosomal loci, the expression of which was similarly controlled in plasmid-deficient C. muridarum strains CM972 and CM3.1 and plasmid-deficient C. trachomatis CTD153. However, although expression of glycogen synthase, encoded by glgA, was greatly reduced in CTD153, it was unaltered in plasmid-deficient C. muridarum strains. Thus, additional plasmid-associated factors are required for glycogen accumulation by this chlamydial species. Furthermore, in C. trachomatis, glgA and other plasmid-responsive chromosomal loci (PRCLs) were transcriptionally responsive to glucose limitation, indicating that additional regulatory elements may be involved in the coordinated expression of these candidate virulence effectors. Glucose-limited C. trachomatis displayed reduced TLR2 stimulation in an in vitro assay. During human chlamydial infection, glucose limitation may decrease chlamydial virulence through its effects on plasmid-responsive chromosomal genes.

Immunization with Dendritic Cells Pulsed ex vivo with Recombinant Chlamydial Protease-Like Activity Factor Induces Protective Immunity Against Genital Chlamydiamuridarum Challenge

We have shown that immunization with soluble recombinant chlamydial protease-like activity factor (rCPAF) and a T helper 1 type adjuvant can induce significantly enhanced bacterial clearance and protection against Chlamydia-induced pathological sequelae in the genital tract. In this study, we investigated the use of bone marrow derived dendritic cells (BMDCs) pulsed ex vivo with rCPAF + CpG in an adoptive subcutaneous immunization for the ability to induce protective immunity against genital chlamydial infection. We found that BMDCs pulsed with rCPAF + CpG efficiently up-regulated the expression of activation markers CD86, CD80, CD40, and major histocompatibility complex class II (MHC II), and secreted interleukin-12, but not IL-10 and IL-4. Mice adoptively immunized with rCPAF + CpG-pulsed BMDCs or UV-EB + CpG-pulsed BMDCs produced elevated levels of antigen-specific IFN-γ and enhanced IgG1 and IgG2a antibodies. Moreover, mice immunized with rCPAF + CpG-pulsed BMDCs or UV-EB + CpG-pulsed BMDCs exhibited significantly reduced genital Chlamydia shedding, accelerated resolution of infection, and reduced oviduct pathology when compared to infected mock-immunized animals. These results suggest that adoptive subcutaneous immunization with ex vivo rCPAF-pulsed BMDCs is an effective approach, comparable to that induced by UV-EB–BMDCs, for inducing robust anti-Chlamydia immunity.

MyD88 Deficiency Leads to Decreased NK Cell Gamma Interferon Production and T Cell Recruitment duringChlamydia muridarumGenital Tract Infection, but a Predominant Th1 Response and Enhanced Monocytic Inflammation Are Associated with Infection Resolution

We have previously shown that MyD88 knockout (KO) mice exhibit delayed clearance of Chlamydia muridarum genital infection compared to wild-type (WT) mice. A blunted Th1 response and ineffective suppression of the Th2 response were also observed in MyD88 KO mice. The goal of the present study was to investigate specific mechanisms whereby absence of MyD88 leads to these effects and address the compensatory mechanisms in the genital tract that ultimately clear infection in the absence of MyD88. It was observed that NK cells recruited to the genital tract in MyD88 KO mice failed to produce gamma interferon (IFN-γ) mRNA and protein. This defect was associated with decreased local production of interleukin-17 (IL-17), IL-18, and tumor necrosis factor alpha (TNF-α) but normal levels of IL-12p70. Additionally, recruitment of CD4 T cells to the genital tract was reduced in MyD88 KO mice compared to that in WT mice. Although chronic infection in MyD88 KO mice resulted in oviduct pathology comparable to that of WT mice, increased histiocytic inflammation was observed in the uterine horns. This was associated with increased CCL2 levels and recruitment of macrophages as a potential compensatory mechanism. Further deletion of TLR4-TRIF signaling in MyD88 KO mice, using TLR4/MyD88 double-KO mice, did not further compromise host defense against chlamydiae, suggesting that compensatory mechanisms are Toll-like receptor (TLR) independent. Despite some polarization toward a Th2 response, a Th1 response remained predominant in the absence of MyD88, and it provided equivalent protection against a secondary infection as observed in WT mice.

Heat denatured enzymatically inactive recombinant chlamydial protease-like activity factor induces robust protective immunity against genital chlamydial challenge

We have shown previously that vaccination with recombinant chlamydial protease-like activity factor (rCPAF) plus interleukin-12 as an adjuvant induces robust protective immunity against primary genital Chlamydia muridarum challenge in mice. Since CPAF is a protease, we compared the effects of enzymatically active and inactive (heat denatured) rCPAF to determine whether proteolytic activity is expendable for the induction of protective immunity against chlamydial challenge. Active, but not inactive, rCPAF immunization induced high levels of anti-active CPAF antibody, whereas both induced robust splenic CPAF-specific IFN-γ production. Vaccination with active or inactive rCPAF induced enhanced vaginal chlamydial clearance as early as day 6 with complete resolution of infection by day 18, compared to day 30 in mock-vaccinated and challenged animals. Importantly, significant and comparable reductions in oviduct pathology were observed in active and inactive rCPAF-vaccinated mice compared to mock-vaccinated animals. Thus, rCPAF induced anti-chlamydial immunity is largely independent of enzymatic activity and secondary or higher order protein conformation.

Critical Role for Interleukin-1β (IL-1β) duringChlamydia muridarumGenital Infection and Bacterial Replication-Independent Secretion of IL-1β in Mouse Macrophages

Recent findings have implicated interleukin-1beta (IL-1beta) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1beta is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1beta expression during a chlamydial genital infection. In the present study, IL-1beta(-/-) mice exhibited delayed chlamydial clearance and decreased frequency of hydrosalpinx compared to wild-type (WT) mice, implying an important role for IL-1beta both in the clearance of infection and in the mediation of oviduct pathology. At the peak of IL-1beta secretion in WT mice, the major producers of IL-1beta in vivo are F4/80(+) macrophages and GR-1(+) neutrophils, but not CD45(-) epithelial cells. Although elicited mouse macrophages infected with Chlamydia muridarum in vitro secrete minimal IL-1beta, in vitro prestimulation of macrophages by Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) purified from Escherichia coli or C. trachomatis L2 prior to infection greatly enhanced secretion of IL-1beta from these cells. By using LPS-primed macrophages as a model system, it was determined that IL-1beta secretion was dependent on caspase-1, potassium efflux, and the activity of serine proteases. Significantly, chlamydia-induced IL-1beta secretion in macrophages required bacterial viability but not growth. Our findings demonstrate that IL-1beta secreted by macrophages and neutrophils has important effects in vivo during chlamydial infection. Additionally, prestimulation of macrophages by chlamydial TLR ligands may account for the elevated levels of pro-IL-1beta mRNA observed in vivo in this cell type.

Lack of oviduct pathology in mice infected with TLR2-signaling deficient chlamydiae is associated with earlier resolution of oviduct inflammation, and a decreased Th17 response (129.10)

Abstract TLR2 KO mice infected with wild-type C. muridarum strain Nigg and immunologically normal mice infected with plasmid-deficient C. muridarum CM3.1, that fails to activate TLR2, are protected from oviduct pathology during primary and challenge infection. Genital tracts harvested from normal mice sacrificed 7, 14, 21, and 28 days post infection with Nigg or CM3.1 were examined histologically. On day 14, neutrophil infiltrates were significantly decreased in the oviducts from mice infected with CM3.1 versus oviducts from mice infected with Nigg. On days 21 and 28, neutrophils and lymphocytes were significantly decreased in oviducts of mice infected with CM3.1 versus Nigg. Quantitative culture of oviduct homogenates thru 35 days post infection revealed equivalent bacterial burden and resolution of infection in both groups by day 19. Significant amounts of TNF, IL-1α, IL-1β, IL-6, GM-CSF, G-CSF, MIP-2, KC and IFN-γ were detected by bead assay in the oviduct homogenates of Nigg-infected mice on days 9 through 15, but were low or undetectable in mice infected with CM3.1. ELISPOT for IFN-γ and IL-17 producing CD4 T cells in iliac nodes reveal significantly increased Th17 cells on days 7 and 20 post infection in Nigg-infected mice. Chlamydial-induced TLR2 signaling promotes enhanced cytokine production, prolonged oviduct inflammation, and activation of Th17 cells that may contribute to induction of pathology.

Type I Interferon Signaling Exacerbates Chlamydia muridarum Genital Infection in a Murine Model

Type I interferons (IFNs) induced during in vitro chlamydial infection exert bactericidal and immunomodulatory functions. To determine the precise role of type I IFNs during in vivo chlamydial genital infection, we examined the course and outcome of Chlamydia muridarum genital infection in mice genetically deficient in the receptor for type I IFNs (IFNAR(-/-) mice). A significant reduction in chlamydial shedding and duration of lower genital tract infection was observed in IFNAR(-/-) mice in comparison to the level of chlamydial shedding and duration of infection in wild-type (WT) mice. Furthermore, IFNAR(-/-) mice developed less chronic oviduct pathology in comparison to that in WT mice. Compared to the WT, IFNAR(-/-) mice had a greater number of chlamydial-specific T cells in their iliac lymph nodes 21 days postinfection. IFNAR(-/-) mice also exhibited earlier and enhanced CD4 T-cell recruitment to the cervical tissues, which was associated with increased expression of CXCL9 in the genital secretions of IFNAR(-/-) mice, but not with expression of CXCL10, which was reduced in the genital secretions of IFNAR(-/-) mice. These data suggest that type I IFNs exacerbate C. muridarum genital infection through an inhibition of the chlamydial-specific CD4 T-cell response.