De-xiu Bu

Mechanisms for the anti-inflammatory effects of statins.

Purpose of review Statins have diverse effects on the cellular mediators of inflammation and immunity that may be partially responsible for their efficacy in preventing cardiovascular disease, and which have encouraged their use in treating immune/inflammatory diseases. We discuss a selection of recently published studies that provide new insights into the mechanisms by which statins exert anti-inflammatory effects. Recent findings Statins have a variety of direct effects on the gene expression and function of cells of both the innate and adaptive immune systems, including endothelial cells, macrophages, dendritic cells and T cells. Many of these effects are related to statin blockade of GTPase isoprenylation, as has been shown in older literature, although newly identified cell type-specific downstream pathways of GTPase have been described. Recently published analyses of data from clinical trials have also provided further evidence that statin therapy has anti-inflammatory effects and benefits independent of lowering cholesterol. Summary Ongoing research continues to strengthen the case that statins can modulate immune responses by several mechanisms, independent of lowering blood cholesterol. A major challenge for investigators will be to determine how to take advantage of these new mechanistic insights to improve treatment of cardiovascular disease and primary immune/inflammatory disorders.

Dynamic Changes in Regulatory T Cells Are Linked to Levels of Diet-Induced Hypercholesterolemia

Background— Regulatory T cells (Treg) are present in atherosclerotic lesions and can modulate disease. In this study we characterized changes in Treg responses associated with prolonged hypercholesterolemia and lesion progression. Methods and Results— Low-density lipoprotein receptor null mice in which Treg express green fluorescent protein were fed a control or cholesterol-rich diet, and green fluorescent protein–positive cells were enumerated in lymphoid tissues and in aorta. Splenic Treg numbers increased after 4, 8, and 20 weeks in cholesterol-diet–fed mice. However, the number of circulating and lesional Treg peaked at 4 weeks and decreased significantly at 8 and 20 weeks, concomitant with increased numbers of CD4+ effector T cells and increased lesion size over this period. Treg expression of selectin ligands and their ability to bind to aortic endothelium decreased after prolonged hypercholesterolemia, and apoptosis of lesional Treg increased. After 4 weeks of cholesterol-rich diet, a switch to a control diet for 4 weeks reduced serum cholesterol and stopped lesion growth, and the high aortic Treg content was maintained, compared with mice fed a cholesterol diet for 8 weeks. After the diet reversal, the splenic Treg retained the phenotype of Treg after 4 weeks of cholesterol diet. Conclusions— Prolonged hypercholesterolemia impairs Treg but not effector T cell accumulation in lesions, but reversal of hypercholesterolemia can prevent loss of lesional Treg. Therefore, cholesterol-lowering therapies may induce dynamic and beneficial changes in Treg:effector T cell ratios in atherosclerotic lesions.

PD-1 Protects against Inflammation and Myocyte Damage in T Cell-Mediated Myocarditis

PD-1, a member of the CD28 family of immune regulatory molecules, is expressed on activated T cells, interacts with its ligands, PD-L1/B7-H1 and PD-L2/B7-DC, on other cells, and delivers inhibitory signals to the T cell. We studied the role of this pathway in modulating autoreactive T cell responses in two models of myocarditis. In a CD8+ T cell-mediated adoptive transfer model, we found that compared with Pd1+/+ CD8+ T cells, Pd1−/− CD8+ T cells cause enhanced disease, with increased inflammatory infiltrate, particularly rich in neutrophils. Additionally, we show enhanced proliferation in vivo and enhanced cytotoxic activity of PD-1–deficient T lymphocytes against myocardial endothelial cells in vitro. In experimental autoimmune myocarditis, a disease model dependent on CD4+ T cells, we show that mice lacking PD-1 develop enhanced disease compared with wild-type mice. PD-1–deficient mice displayed increased inflammation, enhanced serum markers of myocardial damage, and an increased infiltration of inflammatory cells, including CD8+ T cells. Together, these studies show that PD-1 plays an important role in limiting T cell responses in the heart.

Statin-induced Kruppel-like factor 2 expression in human and mouse T cells reduces inflammatory and pathogenic responses

The transcription factor Krüppel-like factor 2 (KLF2) is required for the quiescent and migratory properties of naive T cells. Statins, a class of HMG-CoA reductase inhibitors, display pleiotropic immunomodulatory effects that are independent of their lipid-lowering capacity and may be beneficial as therapeutic agents for T cell-mediated inflammatory diseases. Statins upregulate KLF2 expression in endothelial cells, and this activity is associated with an antiinflammatory phenotype. We therefore hypothesized that the immunomodulatory effects of statins are due, in part, to their direct effects on T cell KLF2 gene expression. Here we report that lipophilic statin treatment of mouse and human T cells increased expression of KLF2 through a HMG-CoA/prenylation-dependent pathway. Statins also diminished T cell proliferation and IFN-gamma expression. shRNA blockade of KLF2 expression in human T cells increased IFN-gamma expression and prevented statin-induced IFN-gamma reduction. In a mouse model of myocarditis induced by heart antigen-specific CD8+ T cells, both statin treatment of the T cells and retrovirally mediated overexpression of KLF2 in the T cells had similar ameliorating effects on disease induction. We conclude that statins reduce inflammatory functions and pathogenic activity of T cells through KLF2-dependent mechanisms, and this pathway may be a potential therapeutic target for cardiovascular diseases.

Impairment of the Programmed Cell Death-1 Pathway Increases Atherosclerotic Lesion Development and Inflammation

Objective—Programmed cell death-1 (PD-1) is a member of the CD28 superfamily that delivers negative signals on interaction with its 2 ligands, PD-L1 and PD-L2. We studied the contribution of the PD-1 pathway to regulation of T cells that promote atherosclerotic lesion formation and inflammation. Methods and Results—We show that compared with Ldlr−/− control mice, Pd1−/−Ldlr−/− mice developed larger lesions with more abundant CD4+ and CD8+ T cells and macrophages, accompanied by higher levels of serum tumor necrosis factor-&agr;. Iliac lymph node T cells from Pd1−/−Ldlr−/− mice proliferated more to &agr;CD3 or oxidized low-density lipoprotein stimulation compared with controls. CD8+ T cells from Pd1−/−Ldlr−/− mice displayed more cytotoxic activity compared with controls in vivo and in vitro. Administration of a blocking anti-PD-1 antibody increased lesional inflammation in hypercholesterolemic Ldlr−/− mice with more lesional T cells and more activated T cells in paraaortic lymph nodes. The changes in lesional T-cell content when PD-1 was absent or blocked were also observed in bone marrow chimeric Ldlr−/− mice lacking PD-L1 and PD-L2 on hematopoietic cells. Conclusion—PD-1 has an important role in downregulating proatherogenic T-cell responses, and blockade of this molecule for treatment of viral infections or cancer may increase risk of cardiovascular complications.

Foxp3+-inducible regulatory T cells suppress endothelial activation and leukocyte recruitment.

The ability of regulatory T cells (Treg) to traffic to sites of inflammation supports their role in controlling immune responses. This feature supports the idea that adoptive transfer of in vitro expanded human Treg could be used for treatment of immune/inflammatory diseases. However, the migratory behavior of Treg, as well as their direct influence at the site of inflammation, remains poorly understood. To explore the possibility that Treg may have direct anti-inflammatory influences on tissues, independent of their well-established suppressive effects on lymphocytes, we studied the adhesive interactions between mouse Treg and endothelial cells, as well as their influence on endothelial function during acute inflammation. We show that Foxp3+ adaptive/inducible Treg (iTreg), but not naturally occurring Treg, efficiently interact with endothelial selectins and transmigrate through endothelial monolayers in vitro. In response to activation by endothelial Ag presentation or immobilized anti-CD3ε, Foxp3+ iTreg suppressed TNF-α– and IL-1β–mediated endothelial selectin expression and adhesiveness to effector T cells. This suppression was contact independent, rapid acting, and mediated by TGF-β–induced activin receptor-like kinase 5 signaling in endothelial cells. In addition, Foxp3+ iTreg adhered to inflamed endothelium in vivo, and their secretion products blocked acute inflammation in a model of peritonitis. These data support the concept that Foxp3+ iTreg help to regulate inflammation independently of their influence on effector T cells by direct suppression of endothelial activation and leukocyte recruitment.

Difference in Th1 and Th17 Lymphocyte Adhesion to Endothelium

T cell subset-specific migration to inflammatory sites is tightly regulated and involves interaction of the T cells with the endothelium. Th17 cells often appear at different inflammatory sites than Th1 cells, or both subsets appear at the same sites but at different times. Differences in T cell subset adhesion to endothelium may contribute to subset-specific migratory behavior, but this possibility has not been well studied. We examined the adhesion of mouse Th17 cells to endothelial adhesion molecules and endothelium under flow in vitro and to microvessels in vivo and we characterized their migratory phenotype by flow cytometry and quantitative RT-PCR. More Th17 than Th1 cells interacted with E-selectin. Fewer Th17 than Th1 cells bound to TNF-α–activated E-selectin–deficient endothelial cells, and intravital microscopy studies demonstrated that Th17 cells engage in more rolling interactions with TNF-α–treated microvessels than Th1 cells in wild-type mice but not in E-selectin–deficient mice. Th17 adhesion to ICAM-1 was dependent on integrin activation by CCL20, the ligand for CCR6, which is highly expressed by Th17 cells. In an air pouch model of inflammation, CCL20 triggered recruitment of Th17 but not Th1 cells. These data provide evidence that E-selectin– and ICAM-1–dependent adhesion of Th17 and Th1 cells with endothelium are quantitatively different.

T Cells and Blood Vessels Costimulation Turns Up the Pressure

Remarkable advances in our understanding of innate and adaptive immunity have shed light on why inflammation is centrally involved in the pathogenesis of many diseases traditionally not viewed as primarily inflammatory in nature. One key area of progress has been the discovery and study of innate immune receptors, referred to as pattern recognition receptors (PRRs), such as toll-like receptors and nod-like receptors. PRRs recognize molecules commonly produced by microbial pathogens, broadly defined as pathogen associated molecular patterns (PAMPs), but PRRs also recognize self molecules that are indicators of cell injury or death. These self molecules, called damage-associated molecular patterns (DAMPs), include reactive oxygen species, intracellular crystalline deposits, normal molecules in abnormal locations (ATP, heat shock proteins, nuclear proteins), and many others.1,2 DAMPs may be induced or change location because of cell injury or death by any number of causes (genetic, traumatic, toxic, metabolic), and their recognition by innate immune system receptors will initiate responses that include local and systemic inflammation. This is why diseases with just about any underlying cause may be expected to have an inflammatory component. Importantly, the innate immune system has evolved to amplify and modify responses to best combat diverse infections, but these amplified and specialized responses, if inappropriately targeted against self, may themselves become intrinsic to the progression of disease processes. One way this can occur is by innate immune stimulation of adaptive immune responses mediated by T lymphocytes. In fact, normal protective T cell-mediated immunity requires a kick-start by the innate immune system, a condition that ensures that we do not readily mount powerful and potentially harmful adaptive immune responses when there is no real damage or danger present. Article see p 2529 One of major ways in which innate immunity promotes T cell-mediated immunity is through the activation of antigen-presenting …

Dendritic Cell KLF2 Expression Regulates T Cell Activation and Proatherogenic Immune Responses

Dendritic cells (DCs) have been implicated as important regulators of innate and adaptive inflammation in many diseases, including atherosclerosis. However, the molecular mechanisms by which DCs mitigate or promote inflammatory pathogenesis are only partially understood. Previous studies have shown an important anti-inflammatory role for the transcription factor Krüppel-like factor 2 (KLF2) in regulating activation of various cell types that participate in atherosclerotic lesion development, including endothelial cells, macrophages, and T cells. We used a pan-DC, CD11c-specific cre-lox gene knockout mouse model to assess the role of KLF2 in DC activation, function, and control of inflammation in the context of hypercholesterolemia and atherosclerosis. We found that KLF2 deficiency enhanced surface expression of costimulatory molecules CD40 and CD86 in DCs and promoted increased T cell proliferation and apoptosis. Transplant of bone marrow from mice with KLF2-deficient DCs into Ldlr−/− mice aggravated atherosclerosis compared with control mice, most likely due to heightened vascular inflammation evidenced by increased DC presence within lesions, enhanced T cell activation and cytokine production, and increased cell death in atherosclerotic lesions. Taken together, these data indicate that KLF2 governs the degree of DC activation and hence the intensity of proatherogenic T cell responses.