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

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection in the world. In women, genital infection can cause endometritis and pelvic inflammatory disease with the severe sequelae of ectopic pregnancy or infertility. Chlamydia sp. do not damage tissues directly, but induce an injurious host inflammatory response at the infected site. In the murine model of genital disease with Chlamydia muridarum, TLR2 plays a role in both early production of inflammatory mediators and development of chronic oviduct pathology. We report the results of studies with plasmid-cured C. muridarum mutants that retain the ability to infect the murine genital tract, but fail to cause disease in the oviduct. These mutants do not stimulate TLR2-dependent cytokine production in mice, nor in innate immune cells or epithelial cells in vitro. They induce an effective Th1 immune response, with no evidence for Th1-immune-mediated collateral tissue damage. Furthermore, mice previously infected with the plasmid-deficient strains are protected against oviduct disease upon challenge with virulent C. muridarum. If plasmid-cured derivatives of human C. trachomatis biovars exhibit similar phenotypic characteristics, they have the potential to serve as vaccines to prevent human disease.

In this study, we expand on the examination of genetically determined differences in host responses that correlate with clearance of Chlamydia trachomatis from the genital tract. We infected C57BL/6, BALB/c, and C3H/HeN mice with the mouse pneumonitis agent of C. trachomatis (MoPn). C57BL/6 mice had the shortest course of infection (22 days) and the lowest incidence of severe hydrosalpinx. BALB/c mice also had a short course of infection (25 days), but all developed hydrosalpinx. C3H/HeN mice had the longest course of infection (38 days), and all developed severe hydrosalpinx. Determination of local cytokine responses by enzyme-linked immunosorbent assay (ELISA) of genital tract secretions revealed that the levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-␣) and interleukin-1␤ (IL-1␤) were significantly increased in the C57BL/6 and BALB/c strains compared to those in the C3H/HeN strain whereas the level of IL-6 was not different. The level of the neutrophil chemokine macrophage inflammatory protein 2 (MIP-2) was increased during the first week of infection in all three strains but was significantly higher in the BALB/c strain, the strain with the most rapid influx of neutrophils into the genital tract. Prolonged detection of MIP-2 in C3H/HeN mice was associated with a protracted presence of neutrophils in the genital tract. Early increases in the levels of the proinflammatory cytokines TNF-␣ and IL-1␤ are associated with earlier eradication of infection in the C57BL/6 and BALB/c strains than in the C3H/HeN strain. Increased levels of MIP-2 and neutrophils in BALB/c and C3H/HeN mice relative to C57BL/6 mice suggest that these responses may contribute to pathology.

Type I IFN signaling has recently been shown to be detrimental to the host during infection with Chlamydia muridarum in both mouse lung and female genital tract. However, the pattern recognition receptor and the signaling pathways involved in chlamydial-induced IFN-β are unclear. Previous studies have demonstrated no role for TLR4 and a partial role for MyD88 in chlamydial-induced IFN-β. In this study, we demonstrate that mouse macrophages lacking TLR3, TRIF, TLR7, or TLR9 individually or both TLR4 and MyD88, still induce IFN-β equivalent to wild type controls, leading to the hypothesis that TLR-independent cytosolic pathogen receptor pathways are crucial for this response. Silencing nucleotide-binding oligomerization domain 1 in HeLa cells partially decreased chlamydial-induced IFN-β. Independently, small interfering RNA-mediated knockdown of the stimulator of IFN gene (STING) protein in HeLa cells and mouse oviduct epithelial cells significantly decreased IFN-β mRNA expression, suggesting a critical role for STING in chlamydial-induced IFN-β induction. Conversely, silencing of mitochondria-associated antiviral signaling proteins and the Rig-I–like receptors, RIG-I, and melanoma differentiation associated protein 5, had no effect. In addition, induction of IFN-β depended on the downstream transcription IFN regulatory factor 3, and on activation of NF-κB and MAPK p38. Finally, STING, an endoplasmic reticulum-resident protein, was found to localize in close proximity to the chlamydial inclusion membrane during infection. These results indicate that C. muridarum induces IFN-β via stimulation of nucleotide-binding oligomerization domain 1 pathway, and TLR- and Rig-I–like receptor-independent pathways that require STING, culminating in activation of IFN regulatory factor 3, NF-κB, and p38 MAPK.

IFN-γ-inducible protein 10 (IP-10) is a chemokine important in the attraction of T cells, which are essential for resolution of chlamydial genital tract infection. During infections with Gram-negative bacteria, the IP-10 response mediated through type I IFNs usually occurs as a result of TLR4 stimulation by bacterial LPS. However, we found that levels of IP-10 in genital tract secretions of Chlamydia trachomatis-infected female wild-type mice were similar to those of infected TLR2- and TLR4-deficient mice but significantly greater than those of infected MyD88-deficient mice. We investigated the mechanism of IP-10 and IFN-β induction during chlamydial infection using mouse macrophages and fibroblasts infected ex vivo. The induction of IP-10 and IFN-β was unchanged in Chlamydia-infected TLR2- and TLR4-deficient cells compared with wild-type cells. However, infection of MyD88-deficient cells resulted in significantly decreased responses. These results suggest a role for MyD88-dependent pathways in induction of IP-10 and IFN-β during chlamydial infection. Furthermore, treatment of infected macrophages with an endosomal maturation inhibitor significantly reduced chlamydial-induced IFN-β. Because endosomal maturation is required for MyD88-dependent intracellular pathogen recognition receptors to function, our data suggest a role for the intracellular pathogen recognition receptor(s) in induction of IFN-β and IP-10 during chlamydial infection. Furthermore, the intracellular pathways that lead to chlamydial-induced IFN-β function through TANK-binding kinase mediated phosphorylation and nuclear translocation of IFN regulatory factor-3.

Recent findings have implicated interleukin-1␤ (IL-1␤

No abstract

Whether there is a pathogenic or protective outcome to chlamydial infection may be defined by the host response. We infected C57BL/6 (C57) and C3H/HeN (C3H) mice with the human biovar of Chlamydia trachomatis, serovar E, and, in select experiments, with the mouse pneumonitis agent of C. trachomatis (MoPn). We compared the courses of infection, histopathology, and host responses that resulted from these infections. The duration of infection with either chlamydial biovar was significantly increased in the C3H strain of mice. The intensity of infection was examined in mice infected with serovar E, and it was significantly increased in the C3H strain. Histopathology revealed the incidence of severe hydrosalpinx to be significantly greater in C3H mice than in C57 mice. In contrast, severe distention of the uterine horns was observed in all infected C57 mice compared to none of the C3H mice infected with serovar E and only 25% of those infected with MoPn. Acute inflammation was significantly increased in the uterine horns of C57 mice compared to that of C3H mice. Examination of antigen-specific responses revealed qualitatively similar responses in the two strains. Determination of gamma interferon-versus interleukin 4-producing cells revealed the predominance of a Th1 response in both strains. Serum enzyme-linked immunosorbent assays for immunoglobulin G1 (IgG1) and IgG2a revealed a predominance of IgG2a antibody in both strains, although the levels of antibody were significantly greater in C3H mice. Lymphocyte proliferation studies revealed increased proliferation in the iliac nodes of both strains at 1 to 3 weeks after infection. Because of the early eradication of infection observed in the C57 strain, we explored the relative production of tumor necrosis factor alpha (TNF-␣) in the two strains. TNF-␣ levels were significantly increased in the genital tract secretions of C57 mice compared to that of C3H mice during the first week of infection. Increased TNF-␣ may be beneficial to the host by leading to earlier eradication of infection, thereby preventing infection of the oviduct and thus the major disease sequelae associated with chlamydial infection of the genital tract.