Chlamydial infection in inducible nitric oxide synthase knockout mice

Abstract

Type 1 CD4+-T-cell-mediated immunity is crucial for the resolution of chlamydial infection of the murine female genital tract. Previous studies demonstrating a correlation between CD4+-T-cell-mediated inhibition of chlamydial growth and gamma interferon (IFN-gamma)-mediated induction of nitric oxide synthase suggested a potential role for the nitric oxide (NO) effector pathway in the clearance of Chlamydia from genital epithelial cells by the immune system. To clarify the role of this pathway, the growth levels of Chlamydia trachomatis organisms in normal (iNOS+/+) mice and in genetically engineered mice lacking the inducible nitric oxide synthase (iNOS) gene (iNOS-/- mice) were compared. There was no significant difference in the course of genital chlamydial infections in iNOS+/+ and iNOS-/- mice as determined by recovery of Chlamydia organisms shed from genital epithelial cells. Dissemination of Chlamydia to the spleen and lungs occurred to a greater extent in iNOS-/- than in iNOS+/+ mice, which correlated with a marginal increase in the susceptibility of macrophages from iNOS-/- mice to chlamydial infection in vitro. However, infections were rapidly cleared from all affected tissues, with no clinical signs of disease. The finding of minimal dissemination in iNOS-/- mice suggested that activation of the iNOS effector pathway was not the primary target of IFN-gamma during CD4+-T-cell-mediated control of chlamydial growth in macrophages because previous reports demonstrated extensive and often fatal dissemination of Chlamydia in mice lacking IFN-gamma. In summary, these results indicate that the iNOS effector pathway is not required for elimination of Chlamydia from epithelial cells lining the female genital tract of mice although it may contribute to the control of dissemination of C. trachomatis by infected macrophages.

FIG. 1

Chlamydial multiplication in PEMs from iNOS^−/− and iNOS^+/+ mice. PEMs were infected at a multiplicity of infection of 1 (MoPn/PEM ratio). At the end of a 48-h incubation period, the productive growth of chlamydiae in PEMs in the presence or absence of cytokines-LPS (CY/LPS) and l-NAME was determined by direct staining of wells with fluorescein-labeled, genus-specific antichlamydial antibodies to detect chlamydial inclusions by indirect immunofluorescence, as described in Materials and Methods. Results are expressed as the mean of values (IFU per milliliter) from at least three separate experiments. The asterisk indicates that the twofold increase in MoPn multiplication in PEMs from iNOS^−/− mice relative to that in PEMs from iNOS^+/+ mice was statistically significant (P < 0.001).

FIG. 2

Dissemination of Chlamydia in iNOS^−/− and iNOS^+/+ mice after genital infection. Tissue homogenates of spleens and lungs were prepared from genitally infected iNOS^−/− and iNOS^+/+ mice at the indicated time points postinfection. Chlamydial burdens in these tissues were assayed as described in Materials and Methods. Results represent the means ± standard errors from two independent experiments, each containing six animals per group.