Plasmid CDS5 influences infectivity and virulence in a mouse model of Chlamydia trachomatis urogenital infection.

The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene.

Expression of matrix metalloproteinases subsequent to urogenital Chlamydia muridarum infection of mice.

The central hypothesis of this study was that matrix metalloproteinases (MMPs) would be enhanced following murine chlamydial infection and that their expression would vary in mouse strains that differ in their susceptibility to chronic chlamydia-induced disease. To address this hypothesis, female C3H/HeN and C57BL/6 mice were infected intravaginally with Chlamydia muridarum. Uterine and oviduct tissues were assessed for transcription of MMP genes and their tissue inhibitors. An increased activity of MMP genes relative to preinfection tissues was observed in the C3H/HeN mice when compared to C57BL/6 mice. Using gelatin zymography, we detected constitutive MMP-2 activity in both strains of mice but an increase in MMP-9. Casein zymography indicated the presence of two elastase-like activities consistent with MMP-12 and possibly MMP-7. Western blotting and antigen capture enzyme-linked immunoassay also confirmed an increase in MMP-9 but constitutive MMP-2 expression subsequent to the infection in both strains of mice. In C57BL/6 mice, MMP-9 was present in monomer and dimer form throughout the 56-day monitoring period. C3H/HeN mice produced dimeric MMP-9, but increases in the monomer form were also observed through day 14. Post-translational modification of MMP-9 between the two strains also differed. Immunohistochemistry revealed neutrophils as a prominent source for MMP-9 in both strains of mice. We conclude that differences in the relative expression and activity of MMPs, particularly MMP-9, occur in mice differing in their susceptibility to the development of chronic chlamydial disease. These differences may account for disparate outcomes with regard to chronic sequelae of the disease.

Inducible nitric oxide synthase regulates production of isoprostanes in vivo during chlamydial genital infection in mice.

Urinary nitrite and F(2)-isoprostanes, an index of oxidant stress, were elevated during chlamydial genital infection of mice. Enhancement of urinary nitrite and F(2)-isoprostanes was observed in phagocyte oxidase-deficient mice. Inhibition of inducible nitric oxide synthase reduced isoprostane excretion. We conclude that nitrogen radicals induce F(2)-isoprostane production and excretion during murine chlamydial genital infection.

Role for inducible nitric oxide synthase in protection from chronic Chlamydia trachomatis urogenital disease in mice and its regulation by oxygen free radicals.

It has been previously reported that although inducible nitric oxide synthase (iNOS) gene knockout (NOS2(-/-)) mice resolve Chlamydia trachomatis genital infection, the production of reactive nitrogen species (RNS) via iNOS protects a significant proportion of mice from hydrosalpinx formation and infertility. We now report that higher in vivo RNS production correlates with mouse strain-related innate resistance to hydrosalpinx formation. We also show that mice with a deletion of a key component of phagocyte NADPH oxidase (p47(phox-/-)) resolve infection, produce greater amounts of RNS in vivo, and sustain lower rates of hydrosalpinx formation than both wild-type (WT) NOS2(+/+) and NOS2(-/-) controls. When we induced an in vivo chemical block in iNOS activity in p47(phox-/-) mice using N(G)-monomethyl-L-arginine (L-NMMA), a large proportion of these mice eventually succumbed to opportunistic infections, but not before they resolved their chlamydial infections. Interestingly, when compared to WT and untreated p47(phox-/-) controls, L-NMMA-treated p47(phox-/-) mice resolved their infections more rapidly. However, L-NMMA-treated p47(phox-/-) mice lost resistance to chronic chlamydial disease, as evidenced by an increased rate of hydrosalpinx formation that was comparable to that for NOS2(-/-) mice. We conclude that phagocyte oxidase-derived reactive oxygen species (ROS) regulate RNS during chlamydial urogenital infection in the mouse. We further conclude that while neither phagocyte oxidase-derived ROS nor iNOS-derived RNS are essential for resolution of infection, RNS protect from chronic chlamydial disease in this model.

Chlamydia trachomatis persistence in the female mouse genital tract: inducible nitric oxide synthase and infection outcome.

It was previously reported that female mice resolve a primary Chlamydia trachomatis urogenital infection independent of inducible nitric oxide synthase (iNOS). We now report that although iNOS-deficient (NOS2(-/-)) mice resolve culture-apparent infection in a fashion similar to that of normal control (NOS2(+/+)) mice, they sustain significantly increased rates of disease, as assessed by hydrosalpinx formation. PCR amplification of ompA followed by Southern blot detection of amplicands revealed the presence of chlamydial DNA in the lower genital tracts of both NOS2(-/-) and NOS2(+/+) mice at > or =120 days postinfection and in upper genital tract tissues at >120 days postinfection. However, only NOS2(-/-) mice shed low numbers of viable chlamydiae from the lower genital tract after immunosuppressive treatment at 120 days postinfection. When cultured primary murine lung fibroblasts were activated in the presence of gamma interferon (IFN-gamma), inhibition of chlamydial growth occurred in both NOS2(+/+) and NOS2(-/-) cells, but the inhibition was reversible after removal of the cytokine in the NOS2(-/-) primary cell culture only. The iNOS-independent inhibition was microbistatic but was independent of 2,3-indoleamine dioxygenase activity. We conclude that chlamydial DNA and antigens persist in mice subsequent to culture-apparent resolution. In addition, IFN-gamma induces in vivo inhibition of chlamydial growth through microbistatic mechanisms in the absence of iNOS activity, but in the presence of iNOS activity, IFN-gamma is microbicidal and effects eradication.

Disease outcome subsequent to primary and secondary urogenital infection with murine or human biovars of Chlamydia trachomatis.

A susceptible strain of mice infected intravaginally with the mouse pneumonitis biovar of Chlamydia trachomatis became infertile and sustained high rates of hydrosalpinx formation regardless of prior infection with a human serovar. Conversely, susceptible mice infected with human serovars remained fertile unless challenged with a homologous human serovar.

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.

Escherichia coli lipopolysaccharide potentiation and inhibition of rat neonatal microglia superoxide anion generation: correlation with prior lactic dehydrogenase, nitric oxide, tumor necrosis factor-alpha, thromboxane B2, and metalloprotease release.

The effects of lipopolysaccharide (LPS) on the central nervous system, one of the first organs to be affected by sepsis, are still incompletely understood. Rat microglia (BMphi) constitute the main leukocyte-dependent source of reactive oxygen species in the central nervous system. The in vitro effect of LPS on agonist-stimulated superoxide (O2-) generation from BMphi appears controversial. Our purpose was to determine the time- and concentration-dependent effect of Escherichia coil LPS on phorbol-12 myristate 13-acetate-stimulated O2- generation from BMphi. Our results demonstrate that BMphi O2- generation in vitro peaked 17 h after stimulation of with .3 ng/mL LPS. Furthermore, stimulation of BMphi with LPS for 17 h resulted in the following concentration-dependent responses: .1-1 ng/mL LPS induced no prior mediator generation but potently enhanced subsequent phorbol-12 myristate 13-acetate-stimulated O2- generation; 3-10 ng/mL LPS caused nitric oxide, tumor necrosis factor-alpha (TNF-alpha), thromboxane B2 and matrix metalloproteinase-9 release although partially inhibiting ensuing phorbol-12 myristate 13-acetate-stimulated O2- generation; 30-100 ng/mL LPS, maximized nitric oxide, TNF-alpha, thromboxane B2, matrix metalloproteinase-9 generation with concomitant lactic dehydrogenase release although strongly deactivating successive phorbol-12 myristate 13-acetate-stimulated O2 production. Our in vitro studies suggest that enhanced release of these four mediators (nitric oxide, TNF-alpha, thromboxane B2, and matrix metalloproteinase-9) during stimulation of BMphi with LPS might play a critical role in the subsequent ability of BMphi to generate O2- in vivo. Potential clinical implications of our findings are suggested by the fact that LPS levels similar to the ones used in this study have been observed in cerebrospinal fluid both in Gram-negative meningitis and sepsis.

The in vitro antimicrobial capacity of human colostrum against Chlamydia trachomatis.

We sought to assess the antimicrobial capacity of human colostrum against Chlamydia trachomatis. a common agent of ophthalmia neonatorum. Colostrum was collected from 13 post-partum females and tested in an in vitro assay of chlamydial growth inhibition using HeLa 229 cells as the host cell line. All samples significantly inhibited chlamydial growth in a dose-response manner. The percent inhibition ranged from 45.3 to 99.0 (mean=88.1+/-4.1). The chlamydial growth inhibition activity of colostrum was found to be: heat- and freezing-resistant: more concentrated in colostrum than breast milk; was not attributable to interferon or antibody activity; and, could not be attributed to host cell cytotoxicity. Additionally, chlamydial growth inhibition occurred in < or = 15 min and was effective only when colostrum was incubated with chlamydiae prior to addition to HeLa 229 monolayers. Lastly, centrifugal fractionation of the colostrum yielded similar activity in the lipid pellicle and in the lipid-free supernatant. These results indicate that topically applied colostrum may have efficacy in the prophylaxis of ophthalmia neonatorum of chlamydial etiology in the absence of conventional modalities.

Inducible nitric oxide synthase does not affect resolution of murine chlamydial genital tract infections or eradication of chlamydiae in primary murine cell culture.

Mice lacking inducible nitric oxide synthase (iNOS) or treated with iNOS inhibitors resolved chlamydial genital tract infections. Additionally, treatment of primary murine cell cultures with gamma interferon restricted chlamydial growth in the absence of nitric oxide. From these results, iNOS activity is unnecessary for the resolution of chlamydial genital tract infections in mice and inhibition of chlamydial growth in culture.

Reactivation of chlamydial genital tract infection in mice.

A model was developed to study chlamydial quiescence in C3H/HeN (C3H) and C57BL/6N (C57) mice following genital tract infection by Chlamydia trachomatis MoPn. Reactivation of chlamydial shedding following immunosuppression indicated that viable MoPn remained in the genital tract for up to 4 or 5 weeks after the apparent clearance of a primary infection. Either cyclophosphamide or cortisone acetate treatment could cause reactivation, but cyclophosphamide was more effective. However, the frequency of reactivation by either drug diminished with time in both mouse strains. Progesterone treatment prior to infection of C57 mice greatly reduced the frequency of reactivation by cyclophosphamide and also correlated with the development of marked fluid accumulation and distension of the uterine horns in the vast majority of those animals. This pathology was apparent by 5 to 7 weeks postinfection and was consistently seen through 110 days postinfection. Neither of these phenomena was observed in C57 mice that had not been treated with progesterone or in C3H mice under any conditions tested. The infecting dose of MoPn did not clearly influence the frequency of reactivation in either inbred strain as defined by this model.

Dissemination of Chlamydia trachomatis chronic genital tract infection in gamma interferon gene knockout mice.

Mice (C57BL/6), treated with progesterone and infected intravaginally with the mouse pneumonitis strain of Chlamydia trachomatis (MoPn), acquired genital tract disease that ascended from the endocervix to the uterine horns, oviducts, and ovaries in a temporal fashion before the occurrence of spontaneous microbiological resolution by about 28 days after infection. Surprisingly, dissemination of MoPn in small numbers to draining lymph nodes, the peritoneal cavity, spleen, liver, kidneys, and lungs occurred in normal mice during the early stages of disease (7 to 14 days) in a portion of infected animals but resolved from these tissues, by microbiological criteria, prior to resolution of genital tract involvement. In contrast, gamma interferon knockout (IFN-gamma KO) mice exhibited dissemination of infection to a greater extent and for longer periods in a variety of tissues, and a portion of infected IFN-gamma KO mice failed to microbiologically resolve their genital tract disease. By comparison, C57BL/6 SCID mice uniformly failed to resolve their genital tract disease and exhibited high levels of dissemination to all tissues tested for extended (50-day) periods of times. Interestingly, although IFN-gamma KO mice failed to completely clear organisms from their genital tracts, they exhibited an attenuated infection indistinguishable from that of heterozygous littermates when challenged 112 days after primary infection. These data support a role for IFN-gamma in containing dissemination of MoPn from the genital tract to extragenital sites and in the microbiological resolution of infection. Data also indicate that IFN-gamma is not required for modulating reinfections, which normally follow a shorter and less dramatic course.

Inflammatory cytokines produced in response to experimental human gonorrhea.

Inflammatory cytokine production in men was examined after intraurethral challenge of volunteers with Neisseria gonorrhoeae MS11mkA or MS11mkC. Increased interleukin (IL)-8, IL-6, and tumor necrosis factor-alpha (TNF-alpha) were detected in urine before the onset of symptoms and peaked simultaneously with the detection of IL-1 beta at the onset of symptoms. Urine cytokine levels returned to baseline or near baseline within 48 h after antibiotic therapy. In plasma, IL-8, TNF-alpha, IL-1 beta, and IL-6 were elevated at the onset of symptoms in 9, 5, 4, and 3 of 10 subjects, respectively, and returned to near normal within 48 h after treatment. IL-1 alpha and granulocyte-macrophage colony-stimulating factor were not consistently detected in urine or plasma after challenge. Cytokine mRNA transcripts in peripheral blood mononuclear cells were not altered by the infection. The findings suggest that IL-8, IL-6, and possibly TNF-alpha were produced at the local site of infection, whereas IL-1 beta was derived from infiltrating leukocytes.

Resolution of murine chlamydial genital infection by the adoptive transfer of a biovar-specific, Th1 lymphocyte clone.

MoPn-specific T-cell clones were isolated from a T-cell line that was capable of curing chlamydial genital infection by the Chlamydia trachomatis agent of mouse pneumonitis (MoPn) after adoptive transfer. Two clones (designated as 2.14-0 and 2.14-3) were characterized by flow cytometry techniques to be homogenous for L3T4, CD3, and alpha/beta T cell receptor (TcR) T-helper cell markers. The two clones were biovar specific, because they reacted to MoPn but not the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC) or C. trachomatis, serovar type E. Cytokine profile analysis, by a combination of bioassays, ELISA, and slot/Northern blotting for specific cytokine messenger RNAs, further revealed that cultures of antigen-stimulated clone 2.14-0 contained interleukin-2 (IL-2), tumor necrosis factor-alpha, and gamma interferon (a T helper 1 cell [Th1] profile). Clone 2.14-3 was also positive for gamma interferon, a level much lower than that of clone 2.14-0, and negative for IL-4 secretion, suggesting a Th1 profile as well. The ability of these clones to bring about the resolution of the chronic genital chlamydial infection of nude mice was tested by the adoptive transfer of 10(7) cells of each clone into the mice. By 4 weeks after cell transfer of clone 2.14-0, 81% of recipient nude mice (30 of 37) resolved the disease. In contrast, clone 2.14-3 or a control T-cell clone specific for a heterologous antigen were unable to resolve the infection in 20 recipients in each case, even after 100 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of gamma interferon on resolution of murine chlamydial genital infection.

Mice infected in the genital tract with the Chlamydia trachomatis agent of mouse pneumonitis were treated with monoclonal rat anti-gamma interferon (anti-IFN-gamma) antibody to determine whether IFN-gamma participated in the resolution of the infection. In two experiments, anti-IFN-gamma antibody treatment resulted in significantly prolonged infections. In support of these data, passive administration of recombinant IFN-gamma to chronically infected nu/nu mice was able to bring about resolution of the infection in some animals.

Resolution of chlamydial genital infection with antigen-specific T-lymphocyte lines.

To determine cell-mediated immune mechanisms involved in the resolution of chlamydial genital infection of mice, we utilized an established murine model in which it has been demonstrated that resolution of infection occurs independently of the antibody response. Splenic T lymphocytes were obtained from mice that had previously been immunized with viable elementary bodies of the mouse pneumonitis agent (MoPn), a Chlamydia trachomatis biovar. Antigen-reactive T lymphocytes were maintained and expanded in vitro by frequent restimulation with UV light-inactivated MoPn in the presence of antigen-presenting cells and recombinant interleukin-2 (rIL-2). Flow cytometry indicated that this cell line was at least 92% positive for the pan-specific T-cell marker Thy1.2. Stimulation of the cells in the presence of syngeneic antigen-presenting cells plus MoPn antigen and in the absence of exogenous IL-2 induced the cells to produce IL-2 activity in culture supernatants. Following adoptive transfer, this T-lymphocyte line was effective in inducing resolution of an ongoing MoPn genital infection in congenitally athymic nude mice which otherwise maintain chronic unresolved infections. The line was less efficient in resolving the infection after longer periods in culture. An additional T-lymphocyte line was derived from the spleens of athymic mice that had received the first line and had resolved the infection. These T cells were also capable of inducing resolution of the infection. Lastly, this cell line was treated with specific antibody and complement to delete either CD4+ or CD8+ T lymphocytes in an attempt to enrich for T-cell subpopulations prior to transfer into infected athymic mice. The anti-CD4-treated line was essentially depleted of CD4 cells, while the anti-CD8-treated line was only partially enriched for CD4 cells, with a large proportion of CD8 cells still present. Nude mice that received either of the treated T-cell lines or the parental cell line were capable of resolving the infection, although the line with increased numbers of CD4 cells was more efficient than either the parental line or the CD8 line.

Humoral immune response to chlamydial genital infection of mice with the agent of mouse pneumonitis.

The objective of this study was to characterize the humoral immune response to chlamydial genital infection of mice with the mouse pneumonitis agent (MoPn). With an enzyme-linked immunoabsorbent assay, immunoglobulin G antibodies to MoPn were first detected in plasma by day 14. Peak plasma antibody concentrations were reached by day 49, and this response did not decline significantly throughout the 300-day monitoring period. Immunoglobulin A against MoPn could first be detected in pooled vaginal washes by day 21 after infection and had reached peak concentrations by day 28, but anti-MoPn immunoglobulin G was not consistently present in secretions. The antibody response in secretions had declined slightly by day 300. Immunoblot analysis revealed that the early phase of the plasma antibody response to MoPn as a result of genital infection was against lipopolysaccharide, the major outer membrane protein, and a 62-kilodalton (kDa) protein. In secretions, early-phase immunoglobulin A antibodies were directed to the major outer membrane protein and lipopolysaccharide. Late reactions to 15-, 22-, and 83-kDa proteins in plasma were noted. Late reactions to the 62-kDa protein in secretions were also noted. The cause of these late responses remains unexplained. When mice were challenged intravaginally with MoPn at 50-day intervals after the primary infection, it was found that mice inoculated on day 100 or after were susceptible to reinfection. Susceptibility could not be related to a decline in the antibody concentration in plasma or secretions or in the antibody response to specific components of MoPn as measured by immunoblot analysis.

Antibody-mediated modulation of arthritis induced by Chlamydia.

The purpose of this investigation was to determine the role of the humoral immune response in the production of arthritis in mice immunized with the chlamydial agent of mouse pneumonitis (MoPn) (Chlamydia trachomatis biovar). Mice were made B cell deficient (BCD) by treatment with rabbit antiserum to murine IgM. Control mice included animals treated similarly with normal rabbit serum or phosphate-buffered saline. Male mice were immunized with MoPn inactivated with ultraviolet irradiation while female mice were immunized by genital tract infection with viable chlamydiae. Arthritis was elicited in all mice by intra-articular inoculation of inactivated MoPn. When knee joints were examined for pathologic changes at varying times after challenge, a marked enhancement of the arthritis was observed in both male and female BCD mice when compared with controls at all time points. These data indicated that the humoral immune response is not essential for the production of arthritic disease in this model but may have some role in the modulation of the process in immunologically intact animals. Persistence of chlamydial antigen in joint tissue of BCD mice suggested that antibody may play a role in the elimination of antigen, thus decreasing the stimulus for the development of cell-mediated immunologic injury. Regulatory role for T suppressor cells cannot be ruled out however, because B cell deficient mice have been shown to lack certain T suppressor cell subsets.

Resolution of chlamydial genital infection in B-cell-deficient mice and immunity to reinfection.

The purpose of this investigation was to determine the relative roles of the humoral and cell-mediated immune responses in the resolution of chlamydial genital infection of mice and resistance to reinfection. To this end, female BALB/c mice were rendered B cell deficient by treatment with heterologous anti-immunoglobulin M (IgM) serum from birth. Controls were similarly treated with either normal serum or phosphate-buffered saline. Before inclusion in each experiment, anti-IgM-treated mice were screened for the absence of IgM in serum and for the presence of cell-mediated immune responses. In addition, spleen cells from anti-IgM-treated mice responded to concanavalin A and phytohemagglutinin but not to lipopolysaccharide. By these criteria, mice were designated B cell deficient. B-cell-deficient mice and controls were inoculated intravaginally with a suspension of mouse pneumonitis agent (MoPn), a Chlamydia trachomatis biovar. All B-cell-deficient mice resolved the infection. Additionally, no significant difference was seen in the course of the infection in B-cell-deficient mice when compared with controls. In contrast to control mice, B-cell-deficient mice displayed no detectable antibody responses to MoPn in serum or in genital secretions. However, both B-cell-deficient mice and controls developed delayed-type hypersensitivity and T-cell proliferative responses to MoPn. When challenged 53 days after primary infection, no significant difference was seen in the resistance of B-cell-deficient mice to reinfection when compared with that of the controls. These data indicate that cell-mediated immune mechanisms play an important role in the resolution of and resistance to chlamydial genital infection in this model.