Tumor Necrosis Factor (TNF) Receptor Superfamily Member 1b on CD8+ T Cells and TNF Receptor Superfamily Member 1a on Non-CD8+ T Cells Contribute Significantly to Upper Genital Tract Pathology Following Chlamydial Infection.

BACKGROUND: We demonstrated previously that tumor necrosis factor α (TNF-α)-producing Chlamydia-specific CD8(+) T cells cause oviduct pathological sequelae. METHODS: In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8(+) T cells to study chlamydial pathogenesis. RESULTS: TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8(+) T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8(+) T cells but not with TNFR2 KO CD8(+) T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8(+) T cells restored oviduct pathology to WT levels in both KO groups. CONCLUSIONS: Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection.

Time-dependent disruption of oviduct pacemaker cells by Chlamydia infection in mice.

Chlamydia trachomatis is the most commonly reported infectious disease in the United States. In women, this infection can lead to pelvic inflammatory disease and cause ectopic pregnancy and tubal factor infertility. Oviduct interstitial cells of Cajal (ICC-OVI) have been identified as pacemakers, responsible for generating slow waves that underlie myosalpinx contractions that are critical for egg transport. ICC-OVI are damaged in mice by the host inflammatory response to Chlamydia, leading to loss of pacemaker activity and associated contractions. However the inflammatory mediator(s) that causes this damage has not been identified. Mice resolve C. muridarum 3-4 wk postinfection but it remains unexplored whether ICC-OVI and pacemaker activity recovers. We have investigated the time dependence of C. muridarum infection with respect to ICC-OVI loss and examined the inflammatory mediator(s) that may be responsible for this damage. Intracellular recordings from the myosalpinx were made at 1, 2, 4 and 7 wk postinfection with Chlamydia. Immunohistochemistry was performed at similar time points to examine changes in ICC-OVI networks and expression of nitric oxide synthase 2 (NOS2) and prostaglandin synthase 2 (PTGS2). Chlamydia-induced expression of NOS2 occurred in stellate-shaped, macrophage-like cells, and damage to ICC-OVI and pacemaker activity occurred as NOS2 expression increased. Immunohistochemistry revealed that macrophages were in close proximity to ICC-OVI. Changes to ICC-OVI were not correlated with PTGS2 expression. These data suggest that ICC-OVI networks and pacemaker activity may be damaged by nitric oxide produced in NOS2-expressing macrophages in response to C. muridarum infection. As the infection resolves, NOS2 expression decreases, ICC-OVI networks recover, and pacemaker activity resumes.

Strain and virulence diversity in the mouse pathogen Chlamydia muridarum.

The mouse chlamydial pathogen Chlamydia muridarum has been used as a model organism for the study of human Chlamydia trachomatis urogenital and respiratory tract infections. To date, two commonly used C. muridarum isolates have been used interchangeably and are essentially taken to be identical. Herein, we present data that indicate that this is not the case. The C. muridarum Weiss isolate and C. muridarum Nigg isolate varied significantly in their virulences in vivo and possessed different growth characteristics in vitro. Distinct differences were observed in intravaginal 50% infectious doses and in challenge infections, with the Weiss isolate displaying greater virulence. Respiratory infection by the intranasal route also indicated a greater virulence of the Weiss isolate. In vitro, morphometric analysis revealed that the Weiss isolate produced consistently smaller inclusions in human cervical adenocarcinoma cells (HeLa 229) and smaller plaques in monolayers of mouse fibroblasts (L929) than did the Nigg isolate. In addition, the Weiss isolate possessed significantly higher replicative yields in vitro than did the Nigg isolate. In plaque-purified isolates derived from our stocks of these two strains, total genomic sequencing identified several unique nonsynonymous single nucleotide polymorphisms and insertion/deletion mutations when our Weiss (n = 4) and Nigg (n = 5) isolates were compared with the published Nigg sequence. In addition, the two isolates shared 11 mutations compared to the published Nigg sequence. These results prove that there is genotypic and virulence diversity among C. muridarum isolates. These findings can be exploited to determine factors related to chlamydial virulence and immunity.

Elimination of Mycoplasma contamination in Chlamydia stocks as a result of in vivo passage or plaque isolation.

OBJECTIVE: This study aims to eliminate Mycoplasma spp. contamination from laboratory stocks of Chlamydia spp. by in vivo passage or by plaque assay. RESULTS: We have described two methods of eliminating Mycoplasma contamination from Chlamydia laboratory stocks. We conclude that Mycoplasma species commonly contaminating chlamydial stocks do not survive passage in mice. Chlamydia may also be derived Mycoplasma-free by plaque assay.

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

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

Outcome of urogenital infection with Chlamydia muridarum in CD14 gene knockout mice.

BACKGROUND: CD14 has been postulated to play a role in chlamydial immunity and immunopathology. There is evidence to support this role in human infections but its function in a mouse model has not been investigated. METHODS: Female CD14 gene knockout and C57BL/6J wild type mice were infected intravaginally with Chlamydia muridarum. The infection course was monitored by detection of viable chlamydiae from serially collected cervical-vaginal swabs. The sequela of tubal factor infertility was assessed using hydrosalpinx formation as a surrogate marker. RESULTS: A significantly abbreviated infection course was observed in the CD14 gene knockout mice but hydrosalpinx formation occurred at similar rates between the two groups. CONCLUSION: Involvement of CD14 during chlamydial infection impedes infection resolution but this does not affect the sequela of infertility as assessed by hydrosalpinx formation.

Detection of Chlamydia infection in Peromyscus species rodents from sylvatic and laboratory sources.

To determine if Chlamydia muridarum, or other chlamydiae, are enzootic in rodents, we probed a serum bank of wild Peromyscus spp. mice for immunoglobulin G-antibody reactivity to ultraviolet light-inactivated C. muridarum elementary bodies (EBs) using an enzyme-linked immunoassay. Applying a cut-off for a positive reaction of OD(405) nm = 0.1 at a 1:20 dilution, we found titratable antibody reactivity in 190 of 247 specimens surveyed (77%, mean OD(405) = 0.33 ± 0.26, range = 0.11-1.81, median = 0.25). In addition, serum samples were obtained from a colony of specific pathogen-free Peromyscus spp. maintained at the University of South Carolina and six of 12 samples were reactive (50%, mean OD(405) = 0.19 +/- 0.08, range = 0.1-0.32, median = 0.18). Lastly, 40 additional wild Peromyscus spp. were captured in a disparate region of Midwestern USA and 22 serum specimens were reactive (55%, mean OD(405) = 0.22 +/- 0.11, range = 0.1-0.48, median = 0.2). Specificity of selected reactive sera for chlamydial antigen was confirmed on Western blot using resolved purified EBs as the detecting antigen. From tissues removed from several mice at necropsy, the gene for chlamydial 16S ribosomal ribonucleic acid (rRNA) was amplified by polymerase chain reaction (PCR). Positive samples of 16S rRNA were subjected to additional PCR for the major outer membrane protein gene (ompA). The amplicons of three select ompA positive samples were sequenced with ≥99% homology with C. muridarum. Our findings indicate that chlamydial infection is enzootic for Peromyscus spp., and that C. muridarum, or a closely related species or strain, is likely the agent in the tested rodent species.

Genomic variant representation in a Chlamydia population is dynamic and adaptive with dependence on in vitro and in vivo passage.

We have previously shown that Chlamydia muridarum has multiple genomic variants that concomitantly vary in their in vitro and in vivo phenotype. Herein, we used real-time polymerase chain reaction-based genotyping assays to query plaque-cloned isolates of C. muridarum for the frequency of eight selected polymorphisms. These strains had no history of passage in vivo since their original isolation from laboratory mice. There was significant variance in the frequency of two of the eight polymorphisms assessed with the remaining exhibiting a low rate of variance. To determine if any of these polymorphisms were more favorable for in vivo conditions, we blindly passaged non-clonal C. muridarum three times at 7-day intervals through the urogenital tract of mice. Seven of the eight polymorphisms varied in frequency following in vivo passage and four of these varied between C. muridarum strains. Selected isolates displayed variable growth rates and cytopathic effect in vitro. We conclude that multiple genotypic variants are present within the existing known C. muridarum strains and that the frequency of these variants changes upon introduction into the mouse host. These findings lend support to the concept that genotypic proportional representation in a chlamydial population is dynamic and adaptive.

Plasmid deficiency in urogenital isolates of Chlamydia trachomatis reduces infectivity and virulence in a mouse model.

We hypothesized that the plasmid of urogenital isolates of Chlamydia trachomatis would modulate infectivity and virulence in a mouse model. To test this hypothesis, we infected female mice in the respiratory or urogenital tract with graded doses of a human urogenital isolate of C. trachomatis, serovar F, possessing the cognate plasmid. For comparison, we inoculated mice with a plasmid-free serovar F isolate. Following urogenital inoculation, the plasmid-free isolate displayed significantly reduced infectivity compared with the wild-type strain with the latter yielding a 17-fold lower infectious dose to yield 50% infection. When inoculated via the respiratory tract, the plasmid-free isolate exhibited reduced infectivity and virulence (as measured by weight change) when compared to the wild-type isolate. Further, differences in infectivity, but not in virulence were observed in a C. trachomatis, serovar E isolate with a deletion within the plasmid coding sequence 1 when compared to a serovar E isolate with no mutations in the plasmid. We conclude that plasmid loss reduces virulence and infectivity in this mouse model. These findings further support a role for the chlamydial plasmid in infectivity and virulence in vivo.

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. muridarumMajor 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.

A link between neutrophils and chronic disease manifestations of Chlamydia muridarum urogenital infection of mice.

Vigorous acute inflammatory responses accompany Chlamydia muridarum infections in mice and are positively correlated with adverse urogenital and respiratory tract infection outcomes in the mouse model. Thus, we tested the hypothesis that neutrophils induce an acute inflammatory insult that, in the repair phase, leads to the chronic sequelae of hydrosalpinx - a surrogate marker of infertility in the mouse model. To this end, we induced neutropenia in mice using a neutrophil-depleting monoclonal antibody during acute phases of C. muridarum urogenital infection only (days 2-21 postinfection). To prove induced neutropenia, peripheral blood was monitored for neutrophils during the treatment regimen. Neutropenic mice had a similar infection course as control mice, but had significantly reduced levels of certain histopathological parameters, reduced production of matrix metalloproteinase-9 (MMP-9) and reduced rates of hydrosalpinx following resolution of the infection. We conclude that neutrophils are a major source of MMP-9, a previously proved pathological factor in this model. Further, we conclude that acute inflammation in the form of neutrophils and neutrophil activation products are at least partially responsible for inducing the histological changes that ultimately result in fibrosis and infertility in the mouse model of chlamydial upper genital tract disease.

A role for CXC chemokine receptor-2 in the pathogenesis of urogenital Chlamydia muridarum infection in mice.

We tested the hypothesis that a specific chemokine receptor, CXC chemokine receptor-2 (CXCR2), mediates acute inflammatory damage during chlamydial urogenital infection, which ultimately leads to the chronic sequelae of hydrosalpinx - a surrogate marker of infertility. Homozygous CXCR2 genetic knockouts (CXCR2-/-), heterozygous littermates (CXCR2+/-) or homozygous wild-type (wt) controls (CXCR2+/+) were infected intravaginally with Chlamydia muridarum. Although no change was observed in the infection in the lower genital tract based on CXCR zygosity, a delay in the ascension of infection into the upper genital tract was seen in CXCR2-/- mice. Significantly elevated peripheral blood neutrophil counts were observed in CXCR2-/- mice when compared with controls. Reduced rates of acute inflammatory indices were observed in the affected tissue, indicating reduced neutrophil extravasation capacity in the absence of CXCR2. Of note was a reduction in the postinfection development of hydrosalpinx that correlated with CXCR2 zygosity, with both CXCR2-/- (13%) and their CXCR2+/- (35%) littermates displaying significantly lower rates of hydrosalpinx formation than the wt CXCR2-sufficient mice (93%). We conclude that CXCR2 ligands are a major chemotactic signal that induces damaging acute inflammation and the resulting chronic pathology during the repair phase of the host response, but are dispensable for the resolution of infection.

Chlamydia infection causes loss of pacemaker cells and inhibits oocyte transport in the mouse oviduct.

Chlamydia trachomatis is a common sexually transmitted bacterial infection that results in health care costs in the United States that exceed $2 billion per year. Chlamydia infections cause damage to the oviducts, resulting in ectopic pregnancy and tubal factor infertility, but the reasons for defective oviduct function are poorly understood. We have investigated the role of oviduct contractions in egg transport and found that underlying electrical pacemaker activity is responsible for oviduct motility and egg transport. Specialized pacemaker cells, referred to as oviduct interstitial cells of Cajal (ICC-OVI), are responsible for pacemaker activity. The ICC-OVI, labeled with antibodies to KIT protein, form a dense network associated with the smooth muscle cells along the entire length of the oviduct. Selective removal of ICC-OVI with KIT-neutralizing antibody resulted in loss of electrical rhythmicity and loss of propulsive contractions of the oviduct. We tested whether infection might adversely affect the ICC-OVI. Mice infected with Chlamydia muridarum displayed dilation of oviducts, pyosalpinx, and loss of spontaneous contractile activity. Morphological inspection showed disruption of ICC-OVI networks, and electrophysiological recordings showed loss of intrinsic pacemaker activity without change in basal smooth muscle membrane potential. Chlamydia infection also was associated with upregulation of NOS2 (iNOS) and PTGS2 (COX II) in leukocytes. Loss of ICC-OVI and pacemaker activity causes oviduct pseudo-obstruction and loss of propulsive contractions for oocytes. This, accompanied by retention of oviduct secretions, may contribute to the development of tubal factor infertility.

Inhibition of matrix metalloproteinases protects mice from ascending infection and chronic disease manifestations resulting from urogenital Chlamydia muridarum infection.

Matrix metalloproteinases (MMP) are a family of host-derived enzymes involved in the turnover of extracellular matrix molecules. We have previously reported enhanced expression of matrix metalloproteinases in Chlamydia muridarum urogenital tract infection of female mice. Kinetics and patterns of MMP expression as well as enhanced expression in susceptible strains of mice in the prior study implied a role for MMP in pathogenesis. To explore this further, we infected a susceptible strain of mice (C3H/HeN) with C. muridarum and treated two groups of mice with either one of two chemical inhibitors of MMP (MMPi; captopril and a chemically modified tetracycline) and reserved infected sham-treated mice as controls. Neither of the treatments affected shedding of viable chlamydiae from the lower urogenital tract, but the administration of either MMPi protected mice from the formation of hydrosalpinx-a surrogate marker of oviduct occlusion and infertility. Interestingly, the mechanism of protection for mice treated with chemically modified tetracycline 3, appeared to be related to prevention of ascending upper genital tract infection. These results imply that MMP are involved in pathogenesis of chlamydial infection in this model by mediating ascension of the infection into the upper genital tract.

Histopathologic changes related to fibrotic oviduct occlusion after genital tract infection of mice with Chlamydia muridarum.

OBJECTIVES: We sought to determine if intraluminal occluding fibrosis of the oviduct occurs after urogenital Chlamydia muridarum infection in mice. STUDY: Oviduct occlusion was assessed by infusing dye into the distal uterus and tracking the diffusion of the dye into the oviduct. We also conducted histologic assessment of the affected tissues using hematoxylin and eosin (H&E) and Masson trichrome stains. RESULTS: All previously infected susceptible mice had occluded oviducts compared with 17.5% of previously uninfected mice. Oviduct occlusion correlated with hydrosalpinx formation and infertility. Intraluminal oviduct fibrosis was observed in several sections of tissue displaying hydrosalpinx but not in tissues without hydrosalpinx. Fibrosis was localized to the oviduct isthmus and oviduct proper, proximal to the uterus. CONCLUSION: Intralumenal occluding fibrosis of the oviduct is a sequela of infection with C. muridarum in this model. These observations support the use of the murine model to study pathogenesis of chlamydial upper genital tract infection.