Sandra G. Morrison

Chlamydial Heat Shock Protein 60 Activates Macrophages and Endothelial Cells Through Toll-Like Receptor 4 and MD2 in a MyD88-Dependent Pathway

Active inflammation and NF-κB activation contribute fundamentally to atherogenesis and plaque disruption. Accumulating evidence has implicated specific infectious agents including Chlamydia pneumoniae in the progression of atherogenesis. Chlamydial heat shock protein 60 (cHSP60) has been implicated in the induction of deleterious immune responses in human chlamydial infections and has been found to colocalize with infiltrating macrophages in atheroma lesions. cHSP60 might stimulate, enhance, and maintain innate immune and inflammatory responses and contribute to atherogenesis. In this study, we investigated the signaling mechanism of cHSP60. Recombinant cHSP60 rapidly activated NF-κB in human microvascular endothelial cells (EC) and in mouse macrophages, and induced human IL-8 promoter activity in EC. The inflammatory effect of cHSP60 was heat labile, thus excluding a role of contaminating LPS, and was blocked by specific anti-chlamydial HSP60 mAb. In human vascular EC which express Toll-like receptor 4 (TLR4) mRNA and protein, nonsignaling TLR4 constructs that act as dominant negative blocked cHSP60-mediated NF-κB activation. Furthermore, an anti-TLR4 Ab abolished cHSP60-induced cellular activation, whereas a control Ab had no effect. In 293 cells, cHSP60-mediated NF-κB activation required both TLR4 and MD2. A dominant-negative MyD88 construct also inhibited cHSP60-induced NF-κB activation. Collectively, our results indicate that cHSP60 is a potent inducer of vascular EC and macrophage inflammatory responses, which are very relevant to atherogenesis. The inflammatory effects are mediated through the innate immune receptor complex TLR4-MD2 and proceeds via the MyD88-dependent signaling pathway. These findings may help elucidate the mechanisms by which chronic asymptomatic chlamydial infection contribute to atherogenesis.

Immunity to Murine Chlamydia trachomatis Genital Tract Reinfection Involves B Cells and CD4+ T Cells but Not CD8+ T Cells

ABSTRACT CD4+ T-helper type 1 (Th1) responses are essential for the resolution of a primary Chlamydia trachomatis genital tract infection; however, elements of the immune response that function in resistance to reinfection are poorly understood. Defining the mechanisms of immune resistance to reinfection is important because the elements of protective adaptive immunity are distinguished by immunological memory and high-affinity antigen recognition, both of which are crucial to the development of efficacious vaccines. Using in vivo antibody depletion of CD4+ and CD8+ T cells prior to secondary intravaginal challenge, we identified lymphocyte populations that functioned in resistance to secondary chlamydial infection of the genital tract. Depletion of either CD4+ or CD8+ T cells in immune wild-type C57BL/6 mice had a limited effect on resistance to reinfection. However, depletion of CD4+ T cells, but not CD8+ T cells, in immune B-cell-deficient mice profoundly altered the course of secondary infection. CD4-depleted B-cell-deficient mice were unable to resolve a secondary infection, shed high levels of infectious chlamydiae, and did not resolve the infection until 3 to 4 weeks following the discontinuation of anti-CD4 treatment. These findings substantiated a predominant role for CD4+ T cells in host resistance to chlamydial reinfection of the female genital tract and demonstrated that CD8+ T cells are unnecessary for adaptive immune resistance. More importantly, however, this study establishes a previously unrecognized but very significant role for B cells in resistance to chlamydial reinfection and suggests that B cells and CD4+ T cells may function synergistically in providing immunity in this model of chlamydial infection. Whether CD4+ T cells and B cells function independently or dependently is unknown, but definition of those mechanisms is fundamental to understanding optimum protective immunity and to the development of highly efficacious immunotherapies against chlamydial urogenital infections.

A Predominant Role for Antibody in Acquired Immunity to Chlamydial Genital Tract Reinfection

Acquired immunity to murine Chlamydia trachomatis genital tract reinfection has long been assumed to be solely dependent on cell-mediated immunity. However, in this study, we identify a previously unrecognized protective role for Ab. Immunity develops in Ab-deficient mice following the resolution of primary chlamydial genital infection. Subsequent depletion of CD4+ T cells, but not CD8+ T cells, in those immune Ab-deficient mice before secondary infectious challenge, resulted in an infection that did not resolve. Passive immunization with immune (convalescent) serum conferred a marked level of protective immunity to reinfection, which was characterized by a striking decrease in bacterial shedding, from >100,000 inclusion forming units to fewer than 10 inclusion forming units, and a shortened duration of infection. Furthermore, mAbs to the chlamydial major outer membrane protein and LPS conferred significant levels of immunity to reinfection and reduced chlamydial shedding by >100-fold. Anti-heat shock protein 60 mAb had no protective effect. In contrast to the marked protective efficacy of immune serum on reinfection, the course of primary infection was essentially unaltered by the passive transfer of immune serum. Our results convincingly demonstrate that Abs contribute importantly to immunity to chlamydial genital tract reinfection, and that Ab-mediated protection is highly dependent on CD4+ T cell-mediated adaptive changes that occur in the local genital tract tissues during primary infection. These results impact our understanding of immunity to chlamydial genital infection and may provide important insight into vaccine development.

Cellular Oxidation of Low-Density Lipoprotein by Chlamydia pneumoniae

A spectrum of clinical and epidemiologic studies implicate infectious agents, including Chlamydia pneumoniae, in the pathogenesis of atherosclerosis. The complexity of atherosclerotic disease necessitates examining the role of infection in the context of defined risk factors, such as high levels of native low-density lipoprotein (LDL). Although native LDL does not have atherogenic properties, cellular oxidation of LDL alters the lipoprotein into a highly atherogenic form. In this report, C. pneumoniae and chlamydial hsp60, an inflammatory antigen that was recently localized to atheromas, were found to induce cellular oxidation of LDL. These data provide initial evidence that an infectious agent can render LDL atherogenic and suggest a mechanism whereby C. pneumoniae may promote atheroma development.

Cross-Reactive B-Cell Epitopes of Microbial and Human Heat Shock Protein 60/65 in Atherosclerosis

Objective—Growing evidence suggests that immune reactions to heat shock protein 60 (HSP60) are involved in atherogenesis. Because of the high phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for breaking the tolerance to self-HSP60, which is expressed on the surface of stressed arterial endothelial cells. Methods and Results—We purified serum antibodies to Escherichia coli HSP60 (GroEL), the 60-kD chlamydial HSP, and HSP65 of Mycobacterium tuberculosis by affinity chromatography from clinically healthy subjects with sonographically proven carotid atherosclerosis. Reactivity of the purified antibodies with overlapping human HSP60 peptides was measured, and 8 shared common epitopes, recognized by all anti-bacterial HSP60/65 antibodies, were identified. Antisera specific for these cross-reactive epitopes were produced by immunizing rabbits with peptides derived from human HSP60. By immunohistochemistry, the epitopes were found to be present in the arterial wall of young subjects during the earliest stages of the disease. Conclusions—Antibodies to microbial HSP60/65 recognize specific epitopes on human HSP60. These cross-reactive epitopes were shown to serve as autoimmune targets in incipient atherosclerosis and might provide further insights into the mechanisms of early atherogenesis.

The natural history of untreated Chlamydia trachomatis infection in the interval between screening and returning for treatment.

Background: Studies of the natural history of genital chlamydial infections in humans are sparse and have had study design limitations. An improved understanding of chlamydial natural history may influence recommendations for elements of control efforts such as chlamydia screening frequency or time parameters for partner notification. Methods: Addressing limitations of prior studies in part, we are prospectively studying chlamydial natural history in sexually transmitted diseases clinic patients in the interval between screening and returning for treatment of positive chlamydial tests. Results of repeat chlamydial testing and clinical outcomes and their associated predictors are being evaluated. Results: In the initial 129 subjects, 89% were female, 88% were black, median age was 21 years, and the median interval between screening and treatment was 13 days. Based on nucleic acid amplification testing at treatment, spontaneous resolution of chlamydia occurred in 18%. Resolution was somewhat more common in subjects with longer intervals between screening and treatment. Persisting infections more often progressed to develop clinical signs at the time of treatment (e.g., urethritis or cervicitis). Two women and one man developed chlamydial complications between screening and treatment. Conclusions: Our findings demonstrate that although spontaneous resolution of chlamydia is common, many persons with persisting chlamydia progress to develop signs of infection and some develop complications.

Chlamydial virulence determinants in atherogenesis : The role of chlamydial lipopolysaccharide and heat shock protein 60 in macrophage-lipoprotein interactions

Data from a spectrum of epidemiologic, pathologic, and animal model studies show that Chlamydia pneumoniae infection is associated with coronary artery disease, but it is not clear how the organism may initiate or promote atherosclerosis. It is postulated that C. pneumoniae triggers key atherogenic events through specific virulence determinants. C. pneumoniae induces mononuclear phagocyte foam cell formation by chlamydial lipopolysaccharide (cLPS) and low-density lipoprotein oxidation by chlamydial hsp60 (chsp60). Thus, different chlamydial components may promote distinct events implicated in the development of atherosclerosis. Data implicating cLPS and chsp60 in the pathogenesis of atherosclerosis are discussed and novel approaches are presented for attempting to elucidate how these putative virulence determinants signal mononuclear phagocytes to modulate lipoprotein influx and modification.

Resolution of Secondary Chlamydia trachomatis Genital Tract Infection in Immune Mice with Depletion of Both CD4+ and CD8+ T cells

ABSTRACT The essential role of T cells in the resolution of primary murineChlamydia trachomatis genital tract infection is inarguable; however, much less is known about the mechanisms that confer resistance to reinfection. We previously established that CD4+ T cells and B cells contribute importantly to resistance to reinfection. In our current studies, we demonstrate that immune mice concurrently depleted of both CD4+ T cells and CD8+ T cells resisted reinfection as well as immunocompetent wild-type mice. The in vivo depletion of CD4+ and CD8+ T cells resulted in diminished chlamydia-specific delayed-type hypersensitivity responses, but antichlamydial antibody responses were unaffected. Our data indicate that immunity to chlamydial genital tract reinfection does not rely solely upon immune CD4+ or CD8+ T cells and further substantiate a predominant role for additional effector immune responses, such as B cells, in resistance to chlamydial genital tract reinfection.

In Situ Analysis of the Evolution of the Primary Immune Response in Murine Chlamydia trachomatis Genital Tract Infection

ABSTRACT Adaptive immune responses contribute to the resolution ofChlamydia trachomatis genital tract infection and protect against reinfection, but our understanding of the mechanisms of those protective responses is incomplete. In this study, we analyzed by in situ immunohistochemistry the progression of the inflammatory and cytokine responses in the genital tracts of mice vaginally infected with C. trachomatis strain mouse pneumonitis. The cellular inflammatory response was characterized by an initial elevation in myeloid cells in the vagina (day 3) and uterine horns (day 7), followed by a marked rise in the number of T cells, predominantly CD4+ cells. CD8+ T cells and CD45R+B cells were also detected but were much less numerous. Perivascular clusters of CD4+ T cells, which resembled clusters of T cells seen in delayed-type hypersensitivity responses, were evident by 2 weeks postinfection. Following the resolution of infection, few CD8+ T cells and CD45R+ B cells remained, whereas numerous CD4+ T cells and perivascular clusters of CD4+ T cells persisted in genital tract tissues. Interleukin-12 (IL-12)- and tumor necrosis factor alpha (TNF-α)-producing cells were observed in vaginal tissue by day 3 of infection and in uterine tissues by day 7. Cells producing IL-4 or IL-10 were absent from vaginal tissues at day 3 of infection but were present in uterine tissues by day 7 and were consistently more numerous than IL-12- and TNF-α-producing cells. Thus, the evolution of the local inflammatory response was characterized by the accumulation of CD4+ T cells into perivascular clusters and the presence of cells secreting both Th1- and Th2-type cytokines. The persistence of CD4+-T-cell clusters long after infection had resolved (day 70) may provide for a readily mobilizable T-cell response by which previously infected animals can quickly respond to and control a secondary infectious challenge.

Seroreactivity to Chlamydia trachomatis Hsp10 Correlates with Severity of Human Genital Tract Disease

ABSTRACT We have identified the chlamydial heat shock protein Hsp10 as a potential correlate to the immunopathogenic process in women with tubal factor infertility (TFI). The human serologic response to chlamydial Hsp10, Hsp60, and major outer membrane protein (MOMP) was measured by enzyme-linked immunosorbent assay. Three populations of women were studied: uninfected controls (CU), acutely infected (AI) women, and women with TFI. Sera from women in the AI and TFI groups both recognized Hsp10 more frequently and at a higher overall level than sera from healthy uninfected controls. Moreover, the infertile women had significantly greater Hsp10 seroreactivity than acutely infected women, indicating a concomitant increase of Hsp10 recognition in populations with increasing levels of disease severity. Hsp60 reactivity showed a similar correlation in these populations, while MOMP reactivity peaked at the same level in both AI and TFI populations but did not increase with disease severity. Test populations were standardized by level of reactivity to formalin-fixed Chlamydia trachomatis elementary bodies (EBs) to address whether these associations were reflections of increased overall chlamydial exposure rather than a property specific to Hsp10. Associations between Hsp10 seropositivity and TFI were greater in the EB+ subgroup while associations among the EB− subgroup were diminished. When restricted to the EB+ subgroups, Hsp60 and MOMP responses in the TFI population did not increase significantly over the level of AI group responses. Thus, among women with similar exposure to chlamydiae, the serologic response to Hsp10 exhibited a stronger correlation with TFI than did the response to Hsp60 or MOMP. These findings support the hypothesis that the serological response toC. trachomatis heat shock proteins is associated with the severity of disease and identifies Hsp10 as an antigen recognized by a significant proportion of women with TFI.