Andrew H. Lichtman

Chlamydial Heat Shock Protein 60 Localizes in Human Atheroma and Regulates Macrophage Tumor Necrosis Factor-α and Matrix Metalloproteinase Expression

BACKGROUND Recent evidence has implicated Chlamydia pneumoniae in the aggravation of atherosclerosis. However, the mechanisms by which this agent affects atherogenesis remain poorly understood. Chlamydiae produce large amounts of heat shock protein 60 (HSP 60) during chronic, persistent infections, and C pneumoniae localizes predominantly within plaque macrophages. Several studies have furnished evidence that endogenous (human) HSP 60 may play a role in atherogenesis. We tested here the hypothesis that atheroma contains chlamydial HSP 60 and that this bacterial product might stimulate macrophage functions considered relevant to atherosclerosis and its complications, such as production of proinflammatory cytokines as tissue necrosis factor-alpha (TNF-alpha) and matrix-degrading metalloproteinases (MMPs). METHODS AND RESULTS Surgical specimens of human carotid atherosclerotic arteries (n = 19) and normal arterial wall samples (n=7, 2 carotid arteries and 5 aortas) were tested immunohistochemically for the presence of chlamydial HSP 60 and human HSP 60. Macrophage localization of these antigens was assessed by double immunostaining. Murine peritoneal macrophages, maintained in serum-free conditions for 48 hours after harvesting, were incubated with C pneumoniae, chlamydial HSP 60, human HSP 60, or Escherichia coli lipopolysaccharide (LPS). Culture supernatants, collected at 24 hours for concentration-dependence experiments and at up to 72 hours for time-dependence experiments, were analyzed for TNF-alpha by ELISA and for MMP by gelatin zymography. Atherosclerotic lesions showed immunoreactive chlamydial HSP 60 in 47% (9 of 19) of the cases and human HSP 60 in 89% (17 of 19) of the cases. Chlamydial HSP 60 colocalized with human HSP 60 within plaque macrophages in 77% (7 of 9) of the cases. Nonatherosclerotic samples contained neither HSP. Both C pneumoniae and recombinant chlamydial HSP 60 induced TNF-alpha production by mouse macrophages in a concentration- and time-dependent fashion. E coli LPS and human HSP 60 produced similar effects. Similarly, C pneumoniae and HSPs induced MMPs in a concentration- and time-dependent manner. Heat treatment abolished the effect of C pneumoniae and HSPs on both TNF-alpha and MMP production, but it did not alter the ability of E coli LPS to induce these functions. CONCLUSIONS Chlamydial HSP 60 frequently colocalizes with human HSP 60 in plaque macrophages in human atherosclerotic lesions. Chlamydial and human HSP 60 induce TNF-alpha and MMP production by macrophages. Chlamydial HSP 60 might mediate the induction of these effects by C pneumoniae. Induction of such macrophage functions provides potential mechanisms by which chlamydial infections may promote atherogenesis and precipitate acute ischemic events.

Cutting Edge: Heat Shock Protein (HSP) 60 Activates the Innate Immune Response: CD14 Is an Essential Receptor for HSP60 Activation of Mononuclear Cells

Heat shock proteins (HSP), highly conserved across species, are generally viewed as intracellular proteins thought to serve protective functions against infection and cellular stress. Recently, we have reported the surprising finding that human and chlamydial HSP60, both present in human atheroma, can activate vascular cells and macrophages. However, the transmembrane signaling pathways by which extracellular HSP60 may activate cells remains unclear. CD14, the monocyte receptor for LPS, binds numerous microbial products and can mediate activation of monocytes/macrophages and endothelial cells, thus promoting the innate immune response. We show here that human HSP60 activates human PBMC and monocyte-derived macrophages through CD14 signaling and p38 mitogen-activated protein kinase, sharing this pathway with bacterial LPS. These findings provide further insight into the molecular mechanisms by which extracellular HSP may participate in atherosclerosis and other inflammatory disorders by activating the innate immune system.

Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages

Both chlamydial and human heat shock protein 60s (HSP 60), which colocalize in human atheroma, may contribute to inflammation during atherogenesis. We tested the hypothesis that chlamydial or human HSP 60 activates human endothelial cells (ECs), smooth muscle cells (SMCs), and monocyte-derived macrophages. We examined the expression of adhesion molecules such as endothelial-leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), and the production of the proinflammatory cytokine interleukin-6 (IL-6). We also tested whether either HSP 60 induces nuclear factor-kappaB (NF-kappaB), which contributes to the gene expression of these molecules. Either chlamydial or human HSP 60 induced E-selectin, ICAM-1, and VCAM-1 expression on ECs similar to levels induced by Escherichia coli lipopolysaccharide (LPS). Each HSP 60 also significantly induced IL-6 production by ECs, SMCs, and macrophages to an extent similar to that induced by E. coli LPS, as assessed by enzyme-linked immunosorbent assay (ELISA). In ECs, either HSP 60 triggered activation of NF-kappaB complexes containing p65 and p50 Rel proteins. Heat treatment abolished all these effects, but did not alter the ability of E. coli LPS to induce these functions. Chlamydial and human HSP 60s therefore activate human vascular cell functions relevant to atherogenesis and lesional complications. These findings help to elucidate the mechanisms by which a chronic asymptomatic chlamydial infection might contribute to the pathophysiology of atheroma.

Immune Effector Mechanisms Implicated in Atherosclerosis: From Mice to Humans

According to the traditional view, atherosclerosis results from a passive buildup of cholesterol in the artery wall. Yet, burgeoning evidence implicates inflammation and immune effector mechanisms in the pathogenesis of this disease. Both innate and adaptive immunity operate during atherogenesis and link many traditional risk factors to altered arterial functions. Inflammatory pathways have become targets in the quest for novel preventive and therapeutic strategies against cardiovascular disease, a growing contributor to morbidity and mortality worldwide. Here we review current experimental and clinical knowledge of the pathogenesis of atherosclerosis through an immunological lens and how host defense mechanisms essential for survival of the species actually contribute to this chronic disease but also present new opportunities for its mitigation.

Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus

Dendritic cell (DC) presentation of self antigen to thymocytes is essential to the establishment of central tolerance. We show here that circulating DCs were recruited to the thymic medulla through a three-step adhesion cascade involving P-selectin, interactions of the integrin VLA-4 with its ligand VCAM-1, and pertussis toxin–sensitive chemoattractant signaling. Ovalbumin-specific OT-II thymocytes were selectively deleted after intravenous injection of antigen-loaded exogenous DCs. We documented migration of endogenous DCs to the thymus in parabiotic mice and after painting mouse skin with fluorescein isothiocyanate. Antibody to VLA-4 blocked the accumulation of peripheral tissue–derived DCs in the thymus and also inhibited the deletion of OT-II thymocytes in mice expressing membrane-bound ovalbumin in cardiac myocytes. These findings identify a migratory route by which peripheral DCs may contribute to central tolerance.

Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis.

Interactions between CD8+ T cells and endothelial cells are important in both protective and pathologic immune responses. Endothelial cells regulate the recruitment of CD8+ Tcells into tissues, and the activation of CD8+ T cells by antigen presentation and costimulatory signals. PD‐L1 and PD‐L2 are recently described B7‐family molecules which bind to PD‐1 on activated lymphocytes and down‐regulate T cell activation. We found that PD‐L1 is expressed on interferon‐γ stimulated cultured human and mouse endothelial cells, while PD‐L2 was found on stimulated human but not mouse endothelial cells. Expression was further up‐regulated by TNF‐α. Antibody blockade of endothelial cell PD‐L1 and PD‐L2 enhanced endothelial cell costimulation of PHA‐activated human CD8+ T cells. Antibody blockade of mouse endothelial cell PD‐L1 enhanced both IFN‐γ secretion and cytolytic activity of CD8+ T cells in response to endothelial cellantigen presentation. These results show that IFN‐γ activated endothelial cells can inhibit T cell activation via expression of the immunoinhibitory PD‐L1 and PD‐L2 molecules. Endothelial expression of PD‐ligands would allow activation and extravasation of T cells without excessive vessel damage. Our findings highlight a potentially important pathway by which endothelial cells down‐regulate CD8+ T cell‐mediated immune responses.

Mouse Eotaxin expression parallels eosinophil accumulation during lung allergic inflammation but it is not restricted to a Th2-type response

A model of lung eosinophilia based on the repeated exposure of mice to aerosolized OVA has been used to identify C-C chemokine genes expressed at stages of massive eosinophil infiltration. We describe the identification and cloning of a cDNA that encodes a mouse C-C chemokine with 68% amino acid identity to guinea pig Eotaxin. The recombinant protein encoded by this gene displays potent and specific chemotactic activity for eosinophils, both in vivo and in vitro. Its mRNA levels parallel the kinetics of eosinophil accumulation in the lung during the experimentally induced eosinophilia and it is mainly produced by type I alveolar epithelial cells. The mRNA expression of mouse Eotaxin is not restricted to Th2 T cells in vitro and is independent of the development of a Th2-type response during N. brasiliensis infection, in vivo.

Influence of Interferon-γ on the Extent and Phenotype of Diet-Induced Atherosclerosis in the LDLR-Deficient Mouse

Objective—The aim of this study was to investigate the influence of interferon-&ggr; (IFN-&ggr;) on atherosclerosis in low density lipoprotein receptor (LDLR)–null mice. Methods and Results—We cross-bred IFN-&ggr;–deficient mice with LDLR-null mice and analyzed lipoprotein profiles and atherosclerosis in the compound mutant progeny after 8 and 20 weeks on a cholesterol-enriched diet. IFN-&ggr; deficiency did not affect serum cholesterol levels or lipoprotein profiles, but it did affect the extent and phenotype of atherosclerosis. Atherosclerotic lesions in IFN-&ggr;–deficient mice were reduced by 75% in the aortic arch and by 46% in the descending aorta compared with control mice after 8 weeks on the diet. After 20 weeks, arch lesions were reduced by 43%, and descending aorta lesions were reduced by 65% in IFN-&ggr;–deficient mice compared with controls. At 8 weeks, percent lesional macrophage and smooth muscle content was significantly less in the IFN-&ggr;–deficient mice, but not at 20 weeks. Although there were fewer class II major histocompatibility complex–positive cells in the lesions of IFN-&ggr;–deficient animals compared with controls, class II major histocompatibility complex expression on endothelial cells overlying lesions persisted in the absence of IFN-&ggr;. Conclusions—These data provide direct evidence that IFN-&ggr; influences atherosclerosis development and phenotype in the LDLR-deficient mouse, independent of changes in blood lipoprotein profiles.

NF-κB Directs Dynamic Super Enhancer Formation in Inflammation and Atherogenesis

Proinflammatory stimuli elicit rapid transcriptional responses via transduced signals to master regulatory transcription factors. To explore the role of chromatin-dependent signal transduction in the atherogenic inflammatory response, we characterized the dynamics, structure, and function of regulatory elements in the activated endothelial cell epigenome. Stimulation with tumor necrosis factor alpha prompted a dramatic and rapid global redistribution of chromatin activators to massive de novo clustered enhancer domains. Inflammatory super enhancers formed by nuclear factor-kappa B accumulate at the expense of immediately decommissioned, basal endothelial super enhancers, despite persistent histone hyperacetylation. Mass action of enhancer factor redistribution causes momentous swings in transcriptional initiation and elongation. A chemical genetic approach reveals a requirement for BET bromodomains in communicating enhancer remodeling to RNA Polymerase II and orchestrating the transition to the inflammatory cell state, demonstrated in activated endothelium and macrophages. BET bromodomain inhibition abrogates super enhancer-mediated inflammatory transcription, atherogenic endothelial responses, and atherosclerosis in vivo.

Impaired Regulatory T-Cell Response and Enhanced Atherosclerosis in the Absence of Inducible Costimulatory Molecule

Background— T-cell–mediated immunity contributes to the pathogenesis of atherosclerosis, but little is known about how these responses are regulated. We explored the influence of the inducible costimulatory molecule (ICOS) on atherosclerosis and associated immune responses. Methods and Results— Bone morrow chimeras were generated by transplanting ICOS-deficient or wild-type bone marrow into irradiated atherosclerosis-prone, LDR receptor–deficient mice, and the chimeric mice were fed a high-cholesterol diet for 8 weeks. Compared with controls, mice transplanted with ICOS-deficient marrow had a 43% increase in the atherosclerotic burden, and importantly, their lesions had a 3-fold increase in CD4+ T cells, as well as increased macrophage, smooth muscle cell, and collagen content. CD4+ T cells from ICOS-deficient chimeras proliferated more and secreted more interferon-γ and tumor necrosis factor-α than T cells from control mice, which suggests a lack of regulation. FoxP3+ regulatory T cells (Treg) were found to constitutively express high ICOS levels, which suggests a role for ICOS in Treg function. ICOS-deficient mice had decreased numbers of FoxP3+ Treg and impaired in vitro Treg suppressive function compared with control mice. Conclusions— ICOS has a key role in regulation of atherosclerosis, through its effect on regulatory T-cell responses.