| Infection with Chlamydia pneumoniae, a human respiratory pathogen, has been implicated as a potential risk factor in atherosclerosis. The role of C. pneumoniae in the pathogenesis of atherosclerosis may occur through a suggested persistent life form. Previous studies in our laboratory have induced C. pneumoniae into an altered life cycle when cell cultures were treated with low levels of interferon-gamma (IFN-γ), presumably through induction of indoleamine 2,3 dioxygenase (IDO) enzyme and degradation of intracellular tryptophan pools. Interferon gamma (IFN-γ) mediated indoleamine 2,3-dioxygenase (IDO) activity, with resulting tryptophan catabolism, induces a C. trachomatis persistent body. The present study investigated if similar cellular mechanisms exist, causing persistence of C. pneumoniae in HEp-2 cells and aortic smooth muscle cells (ASMC), a critical cell in atherogenesis. This study focused on examining the effects of IFN-γ-mediated IDO activity on C. pneumoniae growth, and investigated morphological and ultrastructural differences between altered and normal replicative forms in Hep-2 cells and ASMC. Both cell lines were infected with C. pneumoniae and stimulated with IFN-γ or pre-treated with 1-methyl-DL-tryptophan (1-MT), an IDO competitive inhibitor, followed by infection. In ASMC, IFN-γ induction of IDO mRNA was determined by RT-PCR and IDO activity was measured through percent [H3]-tryptophan catabolism. Infectivity of inclusions in HEp-2 cells was determined by passage of infected monolayers onto a fresh HEp-2 monolayer without IFN-γ The inclusions were analyzed microscopically for morphological differences using immunofluorescence and immunoperoxidase staining. Ultrastructural differences were examined by transmission electron microcopy (TEM). Examination of IFN-γ-mediated IDO activity demonstrated ASMC treated with IFN-γ were able to transcribe and translate an active IDO enzyme, as demonstrated through tryptophan catabolism. The C. pneumoniae replication showed a dose-dependent decrease when treated with increasing concentrations of IFN-γ but never reached zero. When HEp-2 cells and ASMC were pre-treated with 1-MT, C. pneumoniae inclusions were maintained at control levels in the presence of increasing concentrations of IFN-γ. Inclusions examined by immunofluorescence and immunoperoxidase staining using Chlamydia anti-lip polysaccharide (-LPS) or anti-C. pneumoniae major outer membrane protein (-MOMP) antibodies showed a phenotypic switch, with a resulting decrease in typical inclusions when stimulated with IFN-γ and a gradual increase in smaller, less-dense atypical inclusions. Ultrastructural analysis of IFN-γ-treated C. pneumoniae infected HEP-2 cells showed atypical inclusions containing large reticulate-like aberrant bodies (AB) with no evidence of re-differentiation into elementary bodies (EB). These atypical inclusions were generally smaller in diameter and contained fewer bacteria than typical inclusions. These data demonstrate an IFN-γ-mediated induction of atypical C. pneumoniae inclusions, due to IDO enzymatic activity, which differ morphologically and ultrastructurally from typical inclusions. This suggests that an inflammatory cytokine environment may developmentally alter C. pneumoniae replication leading to persistence, and may correlate with the pathogenic process seen with chronic disease such as atherosclerosis. |
