Aaron P. Kithcart

Pregnancy Suppresses Experimental Autoimmune Encephalomyelitis through Immunoregulatory Cytokine Production

Women with multiple sclerosis (MS) often experience a decrease in relapse rate during pregnancy, most notably during the third trimester, with a flare of disease activity 3–6 mo postpartum. Studies in experimental autoimmune encephalomyelitis (EAE), an animal model for MS, have shown that pregnancy delays the onset and decreases the incidence of disease. We investigated the effect of pregnancy and the postpartum period in a remitting-relapsing model of murine EAE. When immunization occurs during pregnancy, mice show a reduction in the incidence of EAE as well as a decrease in clinical severity, while mice immunized during the postpartum period exhibit more severe disease. No differences in lymphocyte proliferation or expression of activation markers were noted when immunization occurred during pregnancy as compared with the nonpregnant controls. Mice immunized during pregnancy produced less TNF-α and IL-17, and showed an increased number of IL-10-secreting cells within the CD11b+, CD11c+, CD19+, and CD4+/CD25+ populations. No differences were noted in the production of IFN-γ, IL-2, IL-4, and IL-5. These results suggest that when an Ag is introduced during pregnancy, an immunoregulatory rather than an immunosuppressive or Th2 environment predominates.

Macrophage Migration Inhibitory Factor Potentiates Autoimmune-Mediated Neuroinflammation

Macrophage migration inhibitory factor (MIF) is a multipotent cytokine that is associated with clinical worsening and relapses in multiple sclerosis (MS) patients. The mechanism through which MIF promotes MS progression remains undefined. In this study, we identify a critical role for MIF in regulating CNS effector mechanisms necessary for the development of inflammatory pathology in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Despite the ability to generate pathogenic myelin-specific immune responses peripherally, MIF-deficient mice have reduced EAE severity and exhibit less CNS inflammatory pathology, with a greater percentage of resting microglia and fewer infiltrating inflammatory macrophages. We demonstrate that MIF is essential for promoting microglial activation and production of the innate soluble mediators IL-1β, IL-6, TNF-α, and inducible NO synthase. We propose a novel role for MIF in inducing microglial C/EBP-β, a transcription factor shown to regulate myeloid cell function and play an important role in neuroinflammation. Intraspinal stereotaxic microinjection of MIF resulted in upregulation of inflammatory mediators in microglia, which was sufficient to restore EAE-mediated inflammatory pathology in MIF-deficient mice. To further implicate a role for MIF, we show that MIF is highly expressed in human active MS lesions. Thus, these results illustrate the ability of MIF to influence the CNS cellular and molecular inflammatory milieu during EAE and point to the therapeutic potential of targeting MIF in MS.

Macrophage migration inhibitory factor is a therapeutic target in treatment of non-insulin-dependent diabetes mellitus

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in the pathogenesis of a variety of autoimmune inflammatory diseases. Here, we investigated the role of MIF in the pathogenesis of non‐insulin‐dependent diabetes mellitus (NIDDM) using MIF−/− mice and a mouse model of streptozotocin (STZ)‐induced NIDDM. Following single injection of STZ, MIF+/+ BALB/c mice showed a significant increase in blood glucose levels, developed polyuria, and succumbed to disease. In contrast, no such increase in blood glucose was observed in MIF −/− BALB/c mice treated with STZ. These mice produced significantly less inflammatory cytokines and resistin as compared with MIF+/+ mice and failed to develop clinical disease. Finally, oral administration of a smallmolecule MIF antagonist, CPSI‐1306, to outbred ICR mice following induction of NIDDM significantly lowered blood glucose levels in the majority of animals, which was also associated with a significant reduction in the levels of the proinflammatory cytokines IL‐6 and TNF‐α in the sera. Taken together, these results demonstrate that MIF is involved in the pathogenesis of NIDDM and is a therapeutic target to treat this disease.—Sanchez‐Zamora, Y., Terrazas, L. I., Vilches‐Flores, A, Leal, E., Jua´rez, I., Whitacre, C, Kithcart, A., Pruitt, J., Sielecki, T., Satoskar, A. R, Rodriguez‐Sosa, M. Macrophage migration inhibitory factor is a therapeutic target in treatment of non‐insulin‐dependent diabetes mellitus. FASEB J. 24, 2583–2590 (2010). www.fasebj.org

A small-molecule inhibitor of macrophage migration inhibitory factor for the treatment of inflammatory disease.

Multiple sclerosis (MS) is a chronic, debilitating disease of the central nervous system (CNS) characterized by demyelination and axon loss. The proinflammatory cytokine macrophage migration inhibitory factor (MIF) has been shown to be elevated in the cerebrospinal fluid of patients during relapses. The purpose of this study was to evaluate a new small‐molecule inhibitor of MIF and its ability to reduce the severity of an animal model of MS, experimental autoimmune encephalomyelitis (EAE). We utilized 2 structurally related isoxazolines, which show in vitro inhibition of MIF tautomerase activity. We found that administration of an inhibitor of MIF to mice with established EAE immediately reduced the severity of clinical signs and expanded a population of regulatory T lymphocytes. We also noted that the inhibitor reduced relapses of disease in a relapsing/remitting model of EAE. An analysis of leukocyte migration into the brain revealed that administration of inhibitor reduced entry of these cells. No effects on inflammatory cytokine production or T‐cell activation in the periphery were noted. From these studies, we conclude that a small‐molecule inhibitor of MIF reduces the severity of EAE and prevents access of immune cells into the CNS, which could be of therapeutic relevance to MS.—Kithcart, A. P., Cox, G. M., Sielecki, T., Short, A., Pruitt, J., Papenfuss, T., Shawler, T., Gienapp, I., Satoskar, A. R., Whitacre, C. C. A small‐molecule inhibitor of macrophage migration inhibitory factor for the treatment of inflammatory disease. FASEB J. 24, 4459–4466 (2010). www.fasebj.org

Memory cells specific for myelin oligodendrocyte glycoprotein (MOG) govern the transfer of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an inflammatory disease of the CNS mediated by CD4(+) T cells directed against myelin antigens. Experimental autoimmune encephalomyelitis (EAE) is induced by immunization with myelin antigens like myelin oligodendrocyte glycoprotein (MOG). We have explored the transfer of EAE using MOG(35-55)-specific TCR transgenic (2D2) T cells. Unsorted 2D2 Th1 cells reliably transferred EAE. Further, we found that CD44(hi)CD62L(lo) effector/memory CD4(+) T cells are likely responsible for the disease transfer due to the up-regulation of CD44. Given the importance of MOG in MS pathogenesis, mechanistic insights into adoptively transferred EAE by MOG-specific Th1 cells could prove valuable in MS research.

Disease-modifying capability of murine Flt3-ligand DCs in experimental autoimmune encephalomyelitis

Dendritic cells (DCs) bridge the innate and adaptive immune response, are uniquely capable of priming naïve T cells, and play a critical role in the initiation and regulation of autoimmune and immune‐mediated disease. At present, in vivo expansion of DC populations is accomplished primarily through the administration of the recombinant human growth factor fms‐like tyrosine kinase 3 ligand (hFL), and in vitro DCs are generated using cytokine cocktails containing GM‐CSF ± IL‐4. Although hFL has traditionally been used in mice, differences in amino acid sequence and biological activity exist between murine FL (mFL) and hFL, and resultant DC populations differ in phenotype and immunoregulatory functional capabilities. This study developed and characterized mFL‐generated DCs and determined the therapeutic capability of mFL DCs in the autoimmune disease experimental autoimmune encephalomyelitis (EAE). Our findings demonstrate that mFL and hFL expand splenic DCs equally in vivo but that mFL‐expanded, splenic DCs more closely resemble normal, resting, splenic DCs. In addition, a novel method for generating mFL‐derived bone marrow‐derived DCs (BM‐DCs) was developed, and comparison of mFL with hFL BM‐DCs found mFL BM‐DCs to be less mature (i.e., lower MHC Class II, CD80, and CD86) than hFL BM‐DCs. These immature mFL DCs up‐regulated costimulatory molecules in response to maturation stimuli LPS and TNF‐α. Mature mFL BM‐DCs were immunogenic and exacerbated the clinical disease course of EAE.

Using Zebrafish for High-Throughput Screening of Novel Cardiovascular Drugs

Summary Cardiovascular diseases remain a major challenge for modern drug discovery. The diseases are chronic, complex, and the result of sophisticated interactions between genetics and environment involving multiple cell types and a host of systemic factors. The clinical events are often abrupt, and the diseases may be asymptomatic until a highly morbid event. Target selection is often based on limited information, and though highly specific agents are often identified in screening, their final efficacy is often compromised by unanticipated systemic responses, a narrow therapeutic index, or substantial toxicities. Our understanding of complexity of cardiovascular disease has grown dramatically over the past 2 decades, and the range of potential disease mechanisms now includes pathways previously thought only tangentially involved in cardiac or vascular disease. Despite these insights, the majority of active cardiovascular agents derive from a remarkably small number of classes of agents and target a very limited number of pathways. These agents have often been used initially for particular indications and then discovered serendipitously to have efficacy in other cardiac disorders or in a manner unrelated to their original mechanism of action. In this review, the rationale for in vivo screening is described, and the utility of the zebrafish for this approach and for complementary work in functional genomics is discussed. Current limitations of the model in this setting and the need for careful validation in new disease areas are also described. An overview is provided of the complex mechanisms underlying most clinical cardiovascular diseases, and insight is offered into the limits of single downstream pathways as drug targets. The zebrafish is introduced as a model organism, in particular for cardiovascular biology. Potential approaches to overcoming the hurdles to drug discovery in the face of complex biology are discussed, including in vivo screening of zebrafish genetic disease models.

Casting NETs to predict cardiovascular outcomes

Cardiovascular disease has become the primary cause of morbidity and mortality in the developed world. Efforts to date to stem this epidemic have focused on risk reduction, including smoking cessation, lipid lowering, blood pressure management, and healthy diet and exercise. There remains, however, an unacceptable residual burden of disease. Identifying patients at risk for recurrence has been one of the emerging themes of the past several years. Markers of inflammation, including C-reactive protein, provide one way to identify higher risk patients beyond traditional risk factors. In this issue of the European Journal of Preventative Cardiology, Langseth et al. suggest yet another marker for risk. Thrombosis contributes causally to the complications of many common cardiovascular conditions including atherosclerosis, venous thromboembolism and atrial fibrillation. Over the past several years, we have learned much about the role neutrophils may play in this process. Polymorphonuclear leukocytes (PMNs) comprise a major component of the circulating immune system. Traditionally thought of as defenders against foreign invaders, they also play roles in thrombosis. We now appreciate that PMNs generate neutrophil extracellular traps (NETs), which participate in venous and arterial thrombosis. NETs consist of strands of DNA, to which primitive defense proteins may attach, derived from the neutrophil itself (e.g. myeloperoxidase), or from the circulation (e.g. tissue factor procoagulant). NETs can also entrap circulating platelets, fibrin strands and von Willebrand factor, forming a scaffold for clot formation. Earlier studies have shown that PMNs, and more specifically NETs, associate with thrombus formation in patients undergoing acute myocardial infarction. PMNs taken from the culprit lesion during an acute coronary syndrome are highly activated and readily undergo NETosis. Our group found that PMNs associate with human atheromata with morphologies characteristic of those complicated by superficial erosion. Studies in vitro suggest that PMNs potentiate endothelial apoptosis and detachment. Antineutrophil cytoplasmic antibodies recognize components of NETs, including proteinase 3 and myeloperoxidase, and are likely to participate causally in forms of vasculitis. These observations implicate neutrophil dysregulation in a variety of vascular diseases, not only coronary artery disease (CAD), but also ischemic stroke, venous thrombosis and vasculitides. Taken together, good evidence now establishes that neutrophils participate importantly in a large swath of cardiovascular diseases (Figure 1). Langseth et al. show that serum markers of NETosis, including double-stranded DNA (dsDNA), can predict cardiovascular events. They began with a cohort of patients with stable CAD and followed them for at least two years. Those with the highest three quartiles of dsDNA had double the risk of experiencing a clinical endpoint, including unstable angina, nonhemorrhagic stroke, myocardial infarction, or death. The authors also show an association of higher dsDNA levels with known cardiovascular risk factors, including male sex, smoking, metabolic syndrome and a history of myocardial infarction. Despite the apparent stability of these CAD patients, the data presented by Langseth et al. suggest the presence of chronic NET activity perhaps related to plaque disruptions that occur below the threshold of clinical detection. Such events appear common. This concept of ongoing subclinical plaque complications could have important implications for secondary prevention as it places additional emphasis on anticoagulation and antiplatelet therapy. Patients with stable CAD should receive a guideline-recommended cocktail of medications, including antiplatelet therapy, beta blockers and statins. The recently published COMPASS trial examined the benefit of low-dose rivaroxaban, a direct oral anticoagulant, in addition to aspirin in patients with stable coronary or peripheral artery

A New Educational Framework to Improve Lifelong Learning for Cardiologists

Lifelong learning is essential for the practicing cardiologist. Present lifelong learning mechanisms are stagnant and at risk for not meeting the needs of currently practicing cardiologists. With the increasing burden of cardiovascular disease, growing complexity of patient care, and ongoing pressures of nonclinical responsibilities, educational programming must evolve to meet the demands of the contemporary cardiovascular professional. A paradigm shift, replete with modern and practical educational tools, is needed in the lifelong learning armamentarium. Emerging evidence of novel educational strategies in graduate medical education supports the promise of broader application of these tools to different stages of professional life. In this commentary from the Fellows-in-Training Section Leadership Council, the authors propose 3 novel educational tools-personalized learning, adaptive learning, and the flipped classroom-to improve lifelong learning to meet the educational needs of fellows-in-training to practicing cardiologists alike.

Treatment of Hypertension in Patients Receiving Cancer Therapy

Hypertension is a common cardiovascular disease associated with high morbidity and mortality when left untreated. As the incidence of hypertension increases in the general population, its prevalence among oncology patients continues to rise. Some of this can be attributed to common risks among hypertension and cancer, including obesity, smoking, and family history; however, some of the therapies used in the pharmacologic treatment of cancer also have direct cardiovascular effects leading to hypertension. The best studied of these drugs include the VEGF and other angiogenesis inhibitors, alkylating agents, and taxanes. Medications commonly used for other diseases, including immunosuppressive agents, are also associated with hypertension. A comprehensive review of the risk factors for hypertension and the medications associated with hypertension is necessary in order to facilitate successful treatment. The contemporary cardio-oncologist has an increasing arsenal of medications, including thiazide-type diuretics, calcium channel blockers, ACE inhibitors, and aldosterone receptor blockers at his or her disposal. The most successful treatment plan allows the continuation of chemotherapy while preventing the secondary damage of untreated hypertension.