Gerald I. Byrne

Induction of Macrophage Foam Cell Formation by Chlamydia pneumoniae

Foam cell formation is the hallmark of early atherosclerosis. It was found that the intracellular bacterium Chlamydia pneumoniae induces foam cell formation by human monocyte-derived macrophages. Exposure of macrophages to C. pneumoniae followed by low-density lipoprotein (LDL) caused a marked increase in the number of foam cells and accumulation of cholesteryl esters. Foam cell formation was not inhibited by the antioxidant butylated hydroxytoluene nor fucoidan, suggesting that lipid accumulation did not involve scavenger receptors. In contrast, addition of heparin, which blocks binding of LDL to the LDL receptor, inhibited C. pneumoniae-induced foam cell formation, suggesting that the pathogen induced lipid accumulation by dysregulating native LDL uptake or metabolism (or both). These data demonstrate that an infectious agent can induce macrophage foam cell formation and implicate C. pneumoniae as a causative factor in atherosclerosis.

Repeated and persistent infection with Chlamydia and the development of chronic inflammation and disease

Chlamydia trachomatis is an important human pathogen that mediates disease processes capable of inflicting permanent damage. Aggressive inflammatory responses to repeated infections, and to a persistent form of this intracellular bacterium, are thought to initiate the pathogenic events that lead to the debilitating sequelae of blinding trachoma and infertility.

Chlamydia and apoptosis: life and death decisions of an intracellular pathogen

The chlamydiae are important obligate intracellular prokaryotic pathogens that, each year, are responsible for millions of human infections involving the eye, genital tract, respiratory tract, vasculature and joints. The chlamydiae grow in cytoplasmic vesicles in susceptible host cells, which include the mucosal epithelium, vascular endothelium, smooth muscle cells, circulating monocytes and recruited or tissue-specific macrophages. One important pathogenic strategy that chlamydiae have evolved to promote their survival is the modulation of programmed cell death pathways in infected host cells. The chlamydiae can elicit the induction of host cell death, or apoptosis, under some circumstances and actively inhibit apoptosis under others. This subtle pathogenic mechanism highlights the manner in which these highly successful pathogens take control of infected cells to promote their own survival — even under the most adverse circumstances.

Interferon-Induced Indoleamine 2,3-Dioxygenase Activity in Human Mononuclear Phagocytes

Interferon (IFN)‐induced tryptophan degradation, catalyzed by indoleamine 2,3‐dioxygenase (IDO), has been shown to mediate antimicrobial activity in epithelial cells. IDO activity has also been augmented in peripheral blood mononuclear cells (PBMC) treated with IFN or interleukin‐2 (IL‐2). The effector cells in this population have now been further characterized. PBMCs were isolated from normal donors, separated into monocyte and lymphocyte populations by plastic adherence, treated with IFN or IL‐2, and cultivated in medium supplemented with [3H]tryptophan. Culture supernatants were collected after a 48‐h incubation and fractionated by high‐performance liquid chromatography; radioactivity was determined in fractions corresponding to tryptophan and its metabolites. IFN‐γ and IFN‐β induced IDO activity only in monocytes (plastic‐adherent, nonspecific esterase‐positive PBMCs). The induction of IDO activity by IL‐2 required both monocytes and lymphocytes. Interaction was required between these populations for induction of IDO by IL‐2, due to production of IFN‐γ by T lymphocytes, with subsequent IFN‐γ‐mediated induction of IDO in monocytes. A number of myeloid cell lines as well as monocyte‐derived macrophages were also tested for their ability to be induced to degrade tryptophan in response to IFN treatment. Monocyte‐derived macrophages were found to retain their capacity to be induced by IFN‐γ and IFN‐β to degrade tryptophan after differentiation, and to possess seven times more IDO activity per cell than IFN‐induced monocytes. However, the presence of lipopolysaccharide (LPS) in the culture medium was required for the maximum induction of IDO activity by IFN‐β. Furthermore, higher concentrations of LPS were sufficient to induce IDO activity in macrophages in the absence of exogenous IFN.

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.

Interferons and indoleamine 2,3-dioxygenase: Role in antimicrobial and antitumor effects

Indoleamine 2,3-dioxygenase (IDO) is an interferon (IFN)-induced protein that initiates the metabolism of tryptophan along the kynurenine pathway. Although IDO can be induced by IFN-γ in many cell types, only mononuclear phagocytes have been shown to be induced to decyclize tryptophan by all three IFN classes. Since tryptophan is an essential amino acid necessary for a variety of metabolic processes, depletion of available tryptophan may be an important mechanism for control of rapidly-dividing microbial pathogens and tumors. The purpose of this review is to present evidence that documents the effects of IFN-induced IDO on prokaryotic and eukaryotic pathogens, as well as on a variety of tumor cell lines.

Chlamydia pneumoniae-Induced Macrophage Foam Cell Formation Is Mediated by Toll-Like Receptor 2

ABSTRACT Chlamydia pneumoniae induces macrophage foam cell formation, a hallmark of early atherosclerosis, in the presence of low-density lipoprotein (LDL). This study examined the role that Toll-like receptor 2 (TLR2) and TLR4 may play in pathogen-induced foam cell formation. Murine macrophage RAW 264.7 cells either infected with C. pneumoniae or treated with the TLR4 ligand E. coli lipopolysaccharide (LPS) or the TLR2 ligand Pam3-Cys-Ala-Gly-OH (Pam) became Oil Red O-stained foam cells and showed increased cholesteryl ester (CE) content when cocultured with LDL. In macrophages from TLR2−/− mice, foam cells were induced by Escherichia coli LPS but not by C. pneumoniae or Pam. Conversely, C. pneumoniae or Pam, but not E. coli LPS, induced foam cells in the TLR4-deficient GG2EE macrophage cell line, suggesting that C. pneumoniae elicits foam cell formation predominantly via TLR2. Enhancing cholesterol efflux using the liver X receptor (LXR) agonist GW3965 significantly decreased the CE content of cells exposed to each of the three TLR ligands (C. pneumoniae, Pam, and E. coli LPS). Overall, our results suggest that activation of the LXR signaling pathway may affect potentially atherogenic processes modulated by the TLR ligands.

Chlamydia pneumoniae expresses genes required for DNA replication but not cytokinesis during persistent infection of HEp-2 cells.

ABSTRACT Chlamydia pneumoniae causes community-acquired pneumonia and is associated with several chronic diseases, including asthma and atherosclerosis. The intracellular growth rate of C. pneumoniae slows dramatically during chronic infection, and such persistence leads to attenuated production of new elementary bodies, appearance of morphologically aberrant reticulate bodies, and altered expression of several chlamydial genes. We used an in vitro system to further characterize persistent C. pneumoniaeinfection, employing both ultrastructural and transcriptional activity measurements. HEp-2 cells were infected with C. pneumoniae (TW-183) at a multiplicity of infection of 3:1, and at 2 h postinfection gamma interferon (IFN-γ) was added to the medium at 0.15 or 0.50 ng/ml. Treated and untreated cultures were harvested at several times postinfection. RNA was isolated and reverse transcribed, and reverse transcription (RT)-PCR analyses targeting primary transcripts from chlamydial rRNA operons as well asdnaA, polA, mutS,minD, ftsK, and ftsW mRNA were done. Some cultures were fixed and stained for electron microscopic analysis, and a real-time PCR assay was used to assess relative chlamydial chromosome accumulation under each culture condition. The latter assays showed that bacterial chromosome copies accumulated severalfold during IFN-γ treatment of infected HEp-2 cells, although less accumulation was observed in cells treated with the higher dose. Electron microscopy demonstrated that high-dose IFN-γ treatment elicited aberrant forms of the bacterium. RT-PCR showed that chlamydial primary rRNA transcripts were present in all IFN-γ-treated and untreated cell cultures, indicating bacterial metabolic activity. Transcripts from dnaA,polA, mutS, and minD, all of which encode products for bacterial chromosome replication and partition, were expressed in IFN-γ-treated and untreated cells. In contrast, ftsK and ftsW, encoding products for bacterial cell division, were expressed in untreated cells, but expression was attenuated in cells treated with low-dose IFN-γ and absent in cells given the high dose of cytokine. Thus, the development of persistence included production of transcripts for DNA replication-related, but not cell division-related, genes. These results provide new insight regarding molecular activities that accompany persistence of C. pneumoniae, as well as suggesting requirements for reactivation from persistent to productive growth.

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.

The p47 GTPases Iigp2 and Irgb10 Regulate Innate Immunity and Inflammation to Murine Chlamydia psittaci Infection

C57BL/6J mice were 105-fold more resistant to Chlamydia psittaci infection than DBA/2J mice by LD100 determinations. Linkage analysis using BXD recombinant inbred strains revealed a single effector locus at a 1.5-Mbp region on chromosome 11 encoding a cluster of three p47 GTPases (Irgb10, Igtp, and Iigp2). Western blots of infected tissue showed that Irgb10 was elevated in resistant mice and one of the two possible Iigp2 protein isoforms was preferentially expressed in susceptible mice. The BXD39 strain, susceptible at Irgb10 and resistant at Iigp2, had an intermediate phenotype implicating the nonredundant role of these p47 GTPases. C57BL/6J and DBA/2J exhibited a difference in IFN-γ-dependent chlamydial control, which was reversible by Iigp2 small interfering RNA knockdown. Microarrays of infected peritoneal lavage revealed >10-fold up-regulation of neutrophil-recruiting chemokines in susceptible mice and >100-fold increase in macrophage differentiation genes in resistant mice, indicating that the susceptibility pattern involves the stimulation of different inflammatory cell-recruiting pathways. Massive neutrophil recruitment was seen in susceptible mice by histology and flow cytometry, and neutrophil chemokine receptor (CXCR2) knockout mice on a susceptible background survived a lethal challenge, confirming that neutrophil recruitment was required for susceptibility. Congenic Igtp knockout mice also susceptible at Irgb10 and Iigp2 on a resistant background recruited neutrophils and succumbed to infection. We conclude that Irgb10 and Iigp2 act together to confer differential susceptibility against murine chlamydial infection. Data indicate that these p47 GTPases have cell-autonomous effects that result in vastly different inflammatory stimulations, leading to either recovery or death.