Differential sensitivity of distinct Chlamydia trachomatis isolates to IFN-gamma-mediated inhibition.

Resistance to the mouse pneumonitis (MoPn) strain of Chlamydia trachomatis has been mapped to MHC class II-restricted, IL-12-dependent CD4+ T cells that secrete a type 1 profile of proinflammatory cytokines, which includes IFN-gamma and TNF-alpha. The relative contribution of IFN-gamma is controversial, however, due to variation in results presented by different laboratories. To determine whether C. trachomatis strain differences contributed to this apparent conflict, the relative resistance of IFN-gamma-deficient mice to murine and human strains of C. trachomatis was compared. All human serovars were much more sensitive to the direct inhibitory actions of IFN-gamma than the MoPn strain. Furthermore, genital clearance of human serovar D in the C57BL/6 mouse was mediated by class II-independent mechanisms that probably involved local production of IFN-gamma by cells of the innate immune system. TNF-alpha also contributed indirectly to host resistance against all strains tested. The differential susceptibility of distinct C. trachomatis strains to effector cytokines such as IFN-gamma could not have been predicted by interstrain biologic variation or by the profile of cytokines stimulated during infection. These findings indicate that strain variation should be considered in situations where related isolates of a given parasite produce conflicting data in models of infection and immunity. They also suggest that stimulation of mucosal IFN-gamma activity is a relevant goal for a human chlamydial vaccine.

Clearance of Chlamydia trachomatis from the murine genital mucosa does not require perforin-mediated cytolysis or Fas-mediated apoptosis.

The molecular mechanisms of resistance to genital infection with the mouse pneumonitis (MoPn) strain of Chlamydia trachomatis are unknown. A role for major histocompatibility complex class II-restricted, interleukin-12-dependent CD4(+) T cells has been established, but the functional activity of these cells does not depend on secretion of gamma interferon. Here we examined the potential contribution of T-cell-mediated cytotoxicity and apoptosis to mucosal clearance of MoPn by using mice deficient in the molecular mediators of target cell lysis. Animals lacking perforin, Fas, Fas ligand, or both perforin and Fas ligand were infected genitally with C. trachomatis MoPn and monitored for expression of immunity to chlamydial antigens and clearance of MoPn from the genital mucosa. In each case, the profile of spleen cytokine production, the magnitude of the host antibody response, and the kinetics of chlamydial clearance were similar to those of genetically intact controls. Compensatory overproduction of tumor necrosis factor alpha, an alternate mediator of apoptosis in certain cell types, did not appear to account for the ability of mutant mice to resolve Chlamydia infections. These results fail to support CD4(+) T-cell-mediated apoptosis or CD8(+) T-cell-mediated cytotoxicity as being critical to the clearance of C. trachomatis MoPn urogenital infections.

Distinct homing pathways direct T lymphocytes to the genital and intestinal mucosae in Chlamydia-infected mice.

Immunity to genital tract infection with Chlamydia trachomatis is mediated by type 1 CD4+ T lymphocytes. To define the signals that govern lymphocyte trafficking to the genital mucosa, integrins expressed by infiltrating T cells and endothelial addressins displayed on local vasculature were characterized during the course of infection. All T cells expressed the alphaLbeta2 heterodimer that binds vascular ICAM-1, and most displayed enhanced levels of the alpha4beta1 integrin that interacts with VCAM-1. AlphaE and beta7(low) integrin chains were detected on approximately 15 and 30% of infiltrating T cells, respectively. Lymphocytes derived from the spleen or draining lymph nodes expressed this same integrin profile, suggesting that cells are recruited to the genital mucosa from the systemic circulation without significant selection pressure for these markers. Immunofluorescent staining for the corresponding vascular addressins revealed intense expression of VCAM-1 on small vessels within Chlamydia-infected genital tracts and up-regulation of ICAM-1 on endothelial, stromal, and epithelial cells. Mucosal addressin cell adhesion molecule-1 was not detected within genital tissues. These results indicate that T lymphocyte homing to the genital mucosa requires the interaction of alphaLbeta2 and alpha4beta1 with endothelial ICAM-1 and VCAM-1, respectively, which is the same pathway that directs lymphocytes to systemic sites of inflammation. Homing pathways defined for the intestinal mucosa and assumed to be relevant to all mucosal sites are not well represented in the genital tract. The identification of T lymphocyte trafficking pathways shared between systemic and mucosal tissues should facilitate vaccine strategies aimed at maximizing immune responses against Chlamydia and other pathogens of the urogenital tract.

Neither interleukin-6 nor inducible nitric oxide synthase is required for clearance of Chlamydia trachomatis from the murine genital tract epithelium.

Female mice bearing targeted mutations in the interleukin-6 or inducible nitric oxide synthase locus mounted effective immune responses following vaginal infection with Chlamydia trachomatis. Chlamydial clearance rates, local Th1 cytokine production, and host antibody responses were similar to those of immunocompetent control mice. Therefore, neither gene product appears to be critical for the resolution of chlamydial infections of the urogenital epithelium.

Chlamydia trachomatis genital tract infection of antibody-deficient gene knockout mice.

The importance of antibody-mediated immunity in primary and secondary Chlamydia trachomatis genital tract infections was examined by using a definitive model of B-cell deficiency, the microMT/microMT gene knockout mouse. Vaginally infected B-cell-deficient microMT/microMT mice developed a self-limiting primary infection that was indistinguishable from infection of control C57BL/6 mice. Sera and vaginal secretions from infected mice were analyzed for anti-Chlamydia antibodies. C57BL/6 mice produced high-titered serum anti-Chlamydia immunoglobulin G2a (IgG2a), IgG2b, and IgA antibodies, and vaginal washes contained predominately anti-Chlamydia IgA. Serum and vaginal washes from infected B-cell-deficient mice were negative for anti-Chlamydia antibody. T-cell proliferation and delayed-type hypersensitivity assays were used as measures of Chlamydia-specific cell-mediated immunity and were found to be comparable for C57BL/6 and B-cell-deficient mice. Seventy days following primary infection, mice were rechallenged to assess acquired immunity. B-cell-deficient mice which lack anti-Chlamydia antibodies were more susceptible to reinfection than immunocompetent C57BL/6 mice. However, acquired immune resistance was evident in both strains of mice and characterized by decreased shedding of chlamydiae and an infection of shorter duration. Thus, this study demonstrates that cell-mediated immune responses alone were capable of resolving chlamydial infection; however, in the absence of specific antibody, mice were more susceptible to reinfection. Therefore, these data suggest that both humoral and cell-mediated immune responses were important mediators of immune protection in this model, though cell-mediated immune responses appear to play a more dominant role.

Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-gamma-dependent and -independent pathways.

Mucosal immunity to Chlamydia trachomatis in a mouse model of female genital tract infection is mediated predominantly by Th1-type cells, as shown by in vivo neutralization of cytokines involved in the Th1 vs Th2 pathways. Neutralization of IL-12 was associated with an apparent decrease in the infiltration of CD4+ T cells into infected tissues, systemic reductions in the production of IFN-gamma, and prolonged shedding of high levels of bacteria. Neutralization of IL-4 had no detectable effect on host immunity or on bacterial clearance. To dissociate the protective role of IL-12 from that of IL-12-induced IFN-gamma, resistance to C. trachomatis was compared in IL-12-depleted and IFN-gamma-deficient animals. IL-12-depleted mice displayed minimal bacterial clearance for 1 mo post-infection but eventually resolved genital tract infections completely. IFN-gamma-deficient mice, on the other hand, cleared 99.9% of genital Chlamydia within the first 3 wk but then developed systemic disease associated with dissemination of bacteria to multiple organs. Animals surviving this stage often maintained low level persistent infections within the urogenital tract. These results indicate that the bulk of chlamydial clearance from the genital mucosa is mediated by an IL-12-dependent, IFN-gamma-independent mechanism, while prevention of disseminated disease requires the action of IFN-gamma.

Gene knockout mice establish a primary protective role for major histocompatibility complex class II-restricted responses in Chlamydia trachomatis genital tract infection.

Mice with disrupted beta 2-microglobulin (beta 2m-/-), I-A (class II-/-), or CD4 (CD4-/-) genes were examined for their capacity to resolve Chlamydia trachomatis genital tract infection. C57BL/6 and beta 2m-/- mice resolved infection similarly and were culture negative by 4 to 5 weeks following infection. Conversely, major histocompatibility complex (MHC) class II-/- mice failed to resolve infection, and CD4-/- mice showed a significant delay (2 weeks). Secondary challenge of C57BL/6, beta 2m-/-, and CD4-/- mice established that acquired protective immunity, which was characterized by an infection of shortened duration and reduced shedding of infectious organisms, developed. Serological analysis of C57BL/6 and beta 2m-/- mice by enzyme-linked immunosorbent assays revealed no striking differences in the immunoglobulin subclass specificity of the anti-Chlamydia response, although some differences were observed in the magnitude of the immunoglobulin G2a (IgG2a) and IgG2b responses. Class II-/- mice produced lower-titered serum anti-Chlamydia antibodies of all isotypes. The serum antibody responses of CD4-/- mice were similar to those of C57BL/6 mice, except that the anti-Chlamydia IgA response was delayed by approximately 3 weeks. Analysis of vaginal washes for Chlamydia-reactive antibodies revealed the presence of IgG2a, IgG2b, and IgA in C57BL/6 and beta 2m-/- mice and primarily of IgA in CD4-/- mice. Vaginal washes from class II-/- mice were consistently antibody negative. Interestingly, the Chlamydia-specific IgA response in the vaginal washes of CD4-/- mice was delayed, but its appearance coincided with decreased shedding of infectious organisms and resolution of infection. Our results demonstrate that MHC class II-restricted T-cell responses are necessary for the development of protective immunity to Chlamydia genital tract infection and that local (vaginal) anti-Chlamydia IgA antibody coincides with the resolution of infection. A substantive role for MHC class I-restricted T-cell responses in protective immunity to Chlamydia genital tract infection was not confirmed.