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.     

Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection

Chlamydia trachomatis is a pathogen of the genital tract and ocular epithelium. Infection is established by the binding of the metabolically inert elementary body (EB) to epithelial cells. These are taken up by endocytosis into a membrane-bound vesicle termed an inclusion. The inclusion avoids fusion with host lysosomes, and the EBs differentiate into the metabolically active reticulate body (RB), which replicates by binary fission within the protected environment of the inclusion. During the extracellular EB stage of the C. trachomatis life cycle, antibody present in genital tract or ocular secretions can inhibit infection both in vivo and in tissue culture. The RB, residing within the intracellular inclusion, is not accessible to antibody, and resolution of infection at this stage requires a cell-mediated immune response mediated by gamma interferon-secreting Th1 cells. Thus, an ideal vaccine to protect against C. trachomatis genital tract infection should induce both antibody (immunoglobulin A [IgA] and IgG) responses in mucosal secretions to prevent infection by chlamydial EB and a strong Th1 response to limit ascending infection to the uterus and fallopian tubes. In the present study we show that transcutaneous immunization with major outer membrane protein (MOMP) in combination with both cholera toxin and CpG oligodeoxynucleotides elicits MOMP-specific IgG and IgA in vaginal and uterine lavage fluid, MOMP-specific IgG in serum, and gamma interferon-secreting T cells in reproductive tract-draining caudal and lumbar lymph nodes. This immunization protocol resulted in enhanced clearance of C. muridarum (C. trachomatis, mouse pneumonitis strain) following intravaginal challenge of BALB/c mice.     

Vaccination against murine gamma-herpesvirus infection

The gamma-herpesviruses establish life-long latency in the host and are important human pathogens. T cells play a major role in controlling the initial acute infection and subsequently maintaining the virus in a quiescent state. However, the nature of the T-cell response to gamma-herpesvirus infection and the requirements for effective vaccination are poorly understood. The recent development of a murine gamma-herpesvirus (murine herpesvirus-68 [MHV-68]) has made it possible to analyze T-cell responses and test vaccination strategies in a small animal model. Intranasal infection with MHV-68 induces an acute infection in the lung and the subsequent establishment of long-term latency, which is associated with splenomegaly and an infectious mononucleosis-like syndrome. Here we review the T-cell response to different phases of the infection and the impact of vaccination against either lytic-cycle, or latency-associated T-cell epitopes.     

Poly-immunoglobulin receptor-mediated transport of IgA into the male genital tract is important for clearance of Chlamydia muridarum infection

PROBLEM: Chlamydia trachomatis is the most common sexually transmitted infection worldwide. While infection in females requires a Th1 response for clearance, such a response in males may disrupt the immune privileged nature of the male reproductive tract, potentially contributing to infertility. METHOD OF STUDY: We investigated the role of IgA in protection against an intrapenile Chlamydia muridarum infection of C57BL/6 and pIgR-/- mice. RESULTS: Here, we show that the poly immunoglobulin receptor is the main pathway for IgA transport into the male reproductive tract. The high levels of IgA seen in prostatic fluid of wild-type mice correlate with reduction in chlamydial infection both in vitro and in vivo. CONCLUSION: These findings indicate that a Chlamydia vaccine that induces neutralizing IgA in the prostate will aid in the protection against infection in males.     

Prior genital tract infection with a murine or human biovar of Chlamydia trachomatis protects mice against heterotypic challenge infection

We sought to assess the degree of cross-protective immunity in a mouse model of chlamydial genital tract infection. Following resolution of genital infection with the mouse pneumonitis (MoPn) biovar of Chlamydia trachomatis, mice were challenged intravaginally with either MoPn or human serovar E or L2. The majority of animals previously infected with MoPn were solidly immune to challenge with either of the two human biovars. Surprisingly, approximately 50% of animals became reinfected when homologously challenged with MoPn, although the secondary infection yielded significantly lower numbers of the organism isolated over a shorter duration than in the primary infection. Primary infection with serovar E also protected against challenge with MoPn or serovar L2, although the degree of immune protection was lower than that resulting from primary infection with MoPn. Blast transformation and assessment of delayed-type hypersensitivity indicated that mice previously infected with either human or murine biovars produced broadly cross-reactive T cells that recognized epitopes of either murine or human biovars of C. trachomatis. Immunoblotting demonstrated that primary MoPn infection produced immunoglobulin G (IgG) antibody to antigens of MoPn as well as at least three distinct antigenic components of human serovar E, one of which was identical in molecular weight to the major outer membrane protein (MOMP). Primary infection with serovar E produced IgG antibody reactive against serovar E but not MoPn MOMP and against at least one ca. 60-kDa protein of both chlamydial strains. Our results indicate that primary genital infection of mice with murine C. trachomatis induces immunity against challenge with either of two human biovars.     

Differences in innate immune responses correlate with differences in murine susceptibility to Chlamydia muridarum pulmonary infection

We investigated the phenotypic basis for genetically determined differences in susceptibility and resistance to Chlamydia muridarum pulmonary infection using BALB/c and C57BL/6 mice. Following C. muridarum intranasal inoculation, the intensity of infection was very different between BALB/c and C57BL/6 beginning as early as 3 days post‐infection. Intrapulmonary cytokine patterns also differed at early time‐points (days 2 and 4) between these two strains of mice. The early recruitment of neutrophils to lung tissue was greater in BALB/c than in C57BL/6 mice and correlated with a higher number of inclusion forming units (IFU) of C. muridarum. At day 12 post‐infection, BALB/c mice continued to demonstrate a greater burden of infection, significantly higher lung cytokine levels for tumour necrosis factor‐α and interleukin‐17 (IL‐17) and a significantly lower level for interferon‐γ than did C57BL/6 mice. In vitro, bone‐marrow‐derived dendritic cells (BMDCs) from BALB/c mice underwent less functional maturation in response to C. muridarum infection than did BMDCs from C57BL/6 mice. The BMDCs of BALB/c mice expressed lower levels of activation markers (CD80, CD86, CD40 and major histocompatibility complex class II) and secreted less IL‐12 and more IL‐23 than BMDCs from C57BL/6 mice. Overall, the data demonstrate that the differences exhibited by BALB/c and C57BL/6 mice following C. muridarum pulmonary infection are associated with differences in early innate cytokine and cellular responses that are correlated with late differences in T helper type 17 versus type 1 adaptive immune responses.     

A murine model for the study of Chlamydia trachomatis genital infections during pregnancy

Pregnant BALB/c mice were inoculated intravaginally on day 5 of gestation with the Chlamydia trachomatis mouse pneumonitis biovar. Animals that received 10(5), 10(6), or 10(7) inclusion-forming units (IFU) of C. trachomatis delivered prematurely on days 15 to 16 of gestation. A focal inflammatory infiltrate was observed in the wall of the uterus on the day 14 of gestation in animals inoculated with 10(5) IFU. In this group of mice, immunohistochemical analysis showed chlamydial inclusions in the endometrium and fetal membranes.     

B-cell-deficient mice develop complete immune protection against genital tract infection with Chlamydia trachomatis.

We evaluated the ability of mice made genetically deficient for B cells to resolve a primary infection and to develop protective immunity against vaginal challenge with a human isolate of Chlamydia trachomatis bacteria. The B-cell-deficient microMT mice cleared a primary ascending infection with similar or faster kinetics compared with wild-type mice. The presence of chlamydial inclusion bodies and the degree of inflammation in the upper genital tract was comparable and showed similar kinetics in microMT as in wild-type mice. Following resolution of the primary infection the mice were challenged by 100 ID50 of live bacteria and the level of protection and the extent of local inflammation was assessed. Strikingly, all microMT mice, as well as most of the wild-type mice, demonstrated complete immune protection with no bacterial shedding. While high titres of chlamydia-specific antibodies were stimulated locally and systemically in wild-type mice, no antibodies were detected in microMT mice. However, in both strains, immunohistochemical analysis of the upper genital tract demonstrated the presence of large numbers of CD4+ T cells and increased levels of interferon-gamma (IFN-gamma)-producing cells. The results unequivocally demonstrate that antibodies are not required for full protection to develop against ascending infection with a high dose of C. trachomatis in the female genital tract. Our study confirms the notion that cell-mediated immunity, in particular that owing to CD4+ T helper I (Th1)-type cells, is critical for host resistance against C. trachomatis in mice.     

Murine MicroRNA‐214 regulates intracellular adhesion molecule (ICAM1) gene expression in genital Chlamydia muridarum infection

The hallmark of chlamydial infection is the development of upper genital pathology in the form of hydrosalpinx and oviduct and/or tubal dilatation. Although molecular events leading to genital tissue presentation and cellular architectural remodelling are unclear, early-stage host immune responses are believed to contribute to these long-term sequelae. Recently, we reported the contribution of selected infection-associated microRNAs (miRs) in the generation of host immunity at early-stage infection (day 6 after intravaginal Chlamydia muridarum challenge in C57BL/6 mice). In this report, we describe the contribution of an infection-associated microRNA, i.e. miR-214, to host immunity. Chlamydia muridarum infection in the C57BL/6 mouse genital tract significantly down-regulated miR-214 while up-regulating intracellular adhesion molecule 1 (ICAM1) gene expression. These in vivo observations were confirmed by establishing direct regulation of ICAM-1 by miR-214 in ex vivo genital cell cultures in the presence of miR-214 mimic and inhibitor. Because, ICAM-1 contributes to recruitment of neutrophils following infection, we also demonstrated that alteration of ICAM1 by miR-214 in interleukin-17A-deficient (IL-17A^−/−) mice correlated with reduction of neutrophils infiltrating genital tissue at day 6 after challenge. Additionally, these early-stage events resulted in significantly decreased genital pathology in IL-17A^−/− mice compared with C57BL/6 mice. This report provides evidence for early-stage regulation of ICAM1 by microRNAs, resulting in reduction of genital pathology associated with chlamydial infection.     

Experimental infection of the marmoset genital tract with Chlamydia trachomatis.

A strain of Chlamydia trachomatis isolated in McCoy cells from the urethra of a patient suffering from non-gonococcal urethritis was inoculated into the vagina of 8 female marmosets. Chlamydiae were isolated repeatedly for 10-42 days from the lower genital tract of 7 of the marmosets. Six of the infected animals developed an acute inflammatory reaction in the genital tract and chlamydial inclusions in epithelial cells were seen in smears from 2 of them. In addition, each of 6 infected marmosets examined developed humoral antibodies to C. trachomatis. In contrast, 3 control animals inoculated intravaginally with chlamydia-free McCoy cells showed no evidence of chlamydial infection. Since the marmoset is small and easily bred in captivity, it should provide a useful model for studying the mechanisms of chlamydial pathogenicity.     

Immunogenicity and protection against genital Chlamydia infection and its complications by a multisubunit candidate vaccine.

BACKGROUND AND PURPOSE: Genital infections due to Chlamydia trachomatis pose a considerable public health challenge worldwide and a vaccine is urgently needed to protect against these infections. We examined whether a vaccine composed of a combination of the major outer membrane protein (MOMP) and porin B protein (PorB) of C. trachomatis would have a protective advantage over a single subunit construct. METHODS: Single and multisubunit vaccines expressing MOMP and PorB were constructed and evaluated in the mouse model of genital infection. Thus, groups of female C57BL/6 mice were immunized intramuscularly with recombinant Vibrio cholerae ghosts (VCG) expressing the vaccine antigens or VCG alone and humoral and cell-mediated immune responses were evaluated. RESULTS: Significant levels of Chlamydia-specific secretory immunoglobulin A and immunoglobulin G2a were detected in vaginal washes and serum of immunized mice. The multisubunit construct induced a significantly higher level of T-helper Type 1 response than the single subunits as measured by the amount of interferon-gamma produced by immune T cells in response to re-stimulation with ultraviolet-irradiated elementary bodies in vitro. Three weeks after the last immunization, animals were challenged intravaginally with 10(7) inclusion-forming units of C. trachomatis serovar D. There was a significant difference in the intensity and duration of vaginal shedding between the vaccine-immunized mice and controls. All the animals immunized with the multisubunit vaccine had completely resolved the infection 2 weeks post-challenge. Higher numbers of embryos were observed in vaccinated animals than in controls, indicating protection against infertility. CONCLUSION: These results underscore the potential, albeit moderate, vaccine advantage of the multisubunit formulation.     

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.     

Specific-pathogen-free pigs as an animal model for studying Chlamydia trachomatis genital infection

The purpose of the present study was to evaluate pigs as a large-animal model for female genital infection with two Chlamydia trachomatis human serovar E strains. Sixteen-week-old specific-pathogen-free female pigs (gilts) were intravaginally infected with the trachoma type E reference strain Bour or the urogenital serovar E strain 468. Several conclusions can be drawn from our findings on the pathogenicity of a primary C. trachomatis genital infection in gilts. First of all, we demonstrated that the serovar E strains Bour and 468 could ascend in the genital tract of gilts. The serovar E strains could replicate in the superficial columnar cervical epithelium and in the superficial epithelial layer of the uterus, which are known to be the specific target sites for a C. trachomatis genital infection in women. Second, inflammation and pathology occurred at the replication sites. Third, the organisms could trigger a humoral immune response, as demonstrated by the presence of immunoglobulin M (IgM), IgG, and IgA in both serum and genital secretion samples. Our findings imply that the pig model might be useful for studying the pathology, pathogenesis, and immune response to a C. trachomatis infection of the genital system.     

Identifying a role for Toll-like receptor 3 in the innate immune response to Chlamydia muridarum infection in murine oviduct epithelial cells

Because epithelial cells are the major cell type productively infected with Chlamydia during genital tract infections, the overall goal of our research was to understand the contribution of infected epithelial cells to the host defense. We previously showed that Toll-like receptor 3 (TLR3) is the critical pattern recognition receptor in oviduct epithelial (OE) cells that is stimulated during Chlamydia infection, resulting in the synthesis of beta interferon (IFN-β). Here, we present data that implicates TLR3 in the expression of a multitude of other innate-inflammatory immune modulators including interleukin-6 (IL-6), CXCL10, CXCL16, and CCL5. We demonstrate that Chlamydia-induced expression of these cytokines is severely disrupted in TLR3-deficient OE cells, whereas Chlamydia replication in the TLR3-deficient cells is more efficient than in wild-type OE cells. Pretreatment of the TLR3-deficient OE cells with 50 U of IFN-β/ml prior to infection diminished Chlamydia replication and restored the ability of Chlamydia infection to induce IL-6, CXCL10, and CCL5 expression in TLR3-deficient OE cells; however, CXCL16 induction was not restored by IFN-β preincubation. Our findings were corroborated in pathway-focused PCR arrays, which demonstrated a multitude of different inflammatory genes that were defectively regulated during Chlamydia infection of the TLR3-deficient OE cells, and we found that some of these genes were induced only when IFN-β was added prior to infection. Our OE cell data implicate TLR3 as an essential inducer of IFN-β and other inflammatory mediators by epithelial cells during Chlamydia infection and highlight the contribution of TLR3 to the inflammatory cytokine response.     

CXCR3 and CCR5 are both required for T cell-mediated protection against C. trachomatis infection in the murine genital mucosa

Chemokine receptors direct T lymphocytes to the site of an infection by following coordinated chemokine gradients, which allow their recruitment to specific tissues. Although identification of receptors needed for homing to some mucosal sites, such as skin and gut, have been elucidated, the receptors that direct lymphocytes to the genital mucosa remain relatively uncharacterized. In this study we identify that the chemokine receptors CXCR3 (chemokine (C-X-C motif) receptor 3) and CCR5 (chemokine (C-C motif) receptor 5) are pivotal for T-lymphocyte access to the genital tract during Chlamydia trachomatis infection. Chlamydia-specific CD4⁺ transgenic T cells that lack CXCR3 or CCR5 do not accumulate in the genital mucosa following infection. Loss of either CXCR3 or CCR5 impairs the protective capacity of Chlamydia-specific T cells, whereas T cells lacking both receptors are completely nonprotective. These results show that CXCR3 and CCR5 are the predominant chemokine receptors that act cooperatively to promote homing to the genital mucosa during Chlamydia infection.     

Multicenter evaluation of the AntigEnz Chlamydia enzyme immunoassay for diagnosis of Chlamydia trachomatis genital infection.

To evaluate the AntigEnz Chlamydia enzyme immunoassay (EIA) (Baxter/Bartels, Issaquah, Wash.), we studied 320 men and 1,209 women attending clinics for sexually transmitted diseases in Baltimore, San Francisco, and Seattle. At examination, two separate swabs were obtained from each patient, one for chlamydial culture and one for EIA. Cervical samples were collected from women, and urethral samples were collected from men. The prevalence of chlamydial infection by culture was 9% in Baltimore (n = 532), 11% in Seattle (n = 500), and 9% in San Francisco (n = 497). To resolve specimens with discrepant culture and EIA results, the EIA transport buffer was centrifuged and the resuspended pellet was stained by direct immunofluorescence to determine whether elementary bodies were present. Overall sensitivity of the AntigEnz Chlamydia assay compared with culture was 87% in men and 86% in women, and overall specificities were 94 and 97%, respectively. Differences between centers were seen, with sensitivities ranging from 76% among men and 79% among women in Seattle to 100% among men and 95% among women in Baltimore. With a true positive considered to be either a culture-positive or an EIA- and direct immunofluorescence-positive specimen, the revised sensitivity was 91% in men and 88% in women. Overall revised specificity was 99% in both men and women. We conclude that in this high-prevalence population, the sensitivity and specificity of this assay compare favorably with those of other noncultural antigen detection tests for the diagnosis of chlamydial genital infection.     

The infecting dose of Chlamydia muridarum modulates the innate immune response and ascending infection

Murine vaginal infection with the obligate intracellular bacterium Chlamydia muridarum is commonly used as a model for ascending Chlamydia infections of the human female genital tract. Gamma interferon-producing Th1 cells, in concert with other mononuclear infiltrates, primarily mediate antichlamydial immunity. However, many factors modify this response, including the bacterial load. To investigate the manner in which the inoculating dose of C. muridarum modulates a genital infection, we measured innate and adaptive cell numbers, CD4+ lymphocyte cytokine profile, chemokine expression, course of infection, and pathological sequelae in genital tracts of BALB/c mice infected with doses of C. muridarum ranging from 10(4) to 10(7) inclusion-forming units. We found that the influx of both innate and adaptive immune cells responded similarly in the lower genital tract (cervical-vaginal tissues) and upper genital tract (oviduct tissues) to increasing inoculating doses. However, cells expressing the innate markers Gr-1 and CD11c were affected to a greater degree by increasing dose than lymphocytes of the adaptive immune response (Th1, CD4+, CD8+, CD19+), resulting in a change in the balance of innate and adaptive cell numbers to favor innate cells at higher infecting doses. Surprisingly, we detected greater numbers of viable chlamydiae in the oviducts at lower inoculating doses, and the number of organisms appeared to directly correlate with hydrosalpinx formation after both primary infection and repeat infection. Taken together, these data suggest that innate immune cells contribute to control of ascending infection.