UVB Exposure Prevents Atherosclerosis by Regulating Immunoinflammatory Responses.

OBJECTIVE: UVB irradiation is an established treatment for immunoinflammatory cutaneous disorders and has been shown to suppress cutaneous and systemic inflammatory diseases through modulation of the adaptive immune response. However, it remains unknown whether UVB irradiation prevents an immunoinflammatory disease of arteries such as atherosclerosis. APPROACH AND RESULTS: Here, we show that UVB exposure inhibits the development and progression of atherosclerosis in atherosclerosis-prone mice by expanding and enhancing the functional capacity of CD4+ forkhead box P3+ regulatory T cells and regulating proatherogenic T-cell responses. Experimental studies in Langerhans cell-depleted mice revealed that epidermal Langerhans cells play a critical role in UVB-dependent induction of CD4+ forkhead box P3+ regulatory T cells, suppression of proatherogenic T-cell responses, and prevention of atherosclerotic plaque development. CONCLUSIONS: Our findings suggest the skin immune system as a novel therapeutic target for atherosclerosis and provide a novel strategy for the treatment and prevention of atherosclerosis.

Commensal bacteria at the crossroad between cholesterol homeostasis and chronic inflammation in atherosclerosis.

The gut microbiota were shown to play critical roles in the development of atherosclerosis, but the detailed mechanism is limited. The purpose of this study is to clarify the influence of gut microbiota on atherogenesis via lipid metabolism and systemic inflammation. Germ-free or conventionally raised (Conv) ApoE-deficient (ApoE-/-) mice were fed chow diet and euthanized at 20 weeks of age. We found that the lack of gut microbiota in ApoE-/- mice caused a significant increase in the plasma and hepatic cholesterol levels compared with Conv ApoE-/- mice. The absence of gut microbiota changed the bile acid composition in the ileum, which was associated with activation of the enterohepatic fibroblast growth factor 15, fibroblast growth factor receptor 4 axis, and reduction of cholesterol 7α-hydroxylase and hepatic bile acid synthesis, resulting in the accumulation of liver cholesterol content. However, we found that the lack of microbiota caused a significant reduction in atherosclerotic lesion formation compared with Conv ApoE-/- mice, which might be associated with the attenuation of lipopolysaccharide-mediated inflammatory responses. Our findings indicated that the gut microbiota affected both hypercholesterolemia and atherogenesis in mice.

Overexpression of Cytotoxic T-Lymphocyte-Associated Antigen-4 Prevents Atherosclerosis in Mice.

OBJECTIVE: Although T-cell-mediated chronic inflammation contributes to atherosclerosis development, the role of a negative regulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) in atherosclerosis is poorly understood. We investigated the effects of CTLA-4 overexpression on atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice. APPROACH AND RESULTS: We generated CTLA-4 transgenic (CTLA-4-Tg)/Apoe(-/-) mice that display constitutive cell surface and intracellular expression of CTLA-4 in T cells and assessed atherosclerosis at age 16 weeks. CTLA-4 overexpression significantly reduced atherosclerotic lesion formation and intraplaque accumulation of macrophage and CD4(+) T cells in the aortic root compared with controls. CTLA-4-Tg/Apoe(-/-) mice showed decreased numbers of effector CD4(+) T cells and decreased expression of costimulatory molecules CD80 and CD86, ligands for CTLA-4, and a costimulatory molecule CD28, on CD11c(+) dendritic cells compared with controls. Consistent with in vivo findings, in vitro experiments revealed that CD4(+) T cells from CTLA-4-Tg/Apoe(-/-) mice showed decreased proliferative capacity and proinflammatory cytokine production, downregulated CD80 expression on CD11c(+) dendritic cells, and suppressed the proliferation of other T cells by limiting the costimulatory pathway. Moreover, CD11c(+) dendritic cells from CTLA-4-Tg/Apoe(-/-) mice showed reduced proliferative activity of T cells in vitro, suggesting the suppression of dendritic cell maturation in vivo. CONCLUSIONS: CTLA-4 regulates atherosclerosis by suppressing proatherogenic immune responses and could be an attractive therapeutic target for atherosclerosis.

Characterization of gut microbiota profiles in coronary artery disease patients using data mining analysis of terminal restriction fragment length polymorphism: gut microbiota could be a diagnostic marker of coronary artery disease.

The association between atherosclerosis and gut microbiota has been attracting increased attention. We previously demonstrated a possible link between gut microbiota and coronary artery disease. Our aim of this study was to clarify the gut microbiota profiles in coronary artery disease patients using data mining analysis of terminal restriction fragment length polymorphism (T-RFLP). This study included 39 coronary artery disease (CAD) patients and 30 age- and sex- matched no-CAD controls (Ctrls) with coronary risk factors. Bacterial DNA was extracted from their fecal samples and analyzed by T-RFLP and data mining analysis using the classification and regression algorithm. Five additional CAD patients were newly recruited to confirm the reliability of this analysis. Data mining analysis could divide the composition of gut microbiota into 2 characteristic nodes. The CAD group was classified into 4 CAD pattern nodes (35/39 = 90 %), while the Ctrl group was classified into 3 Ctrl pattern nodes (28/30 = 93 %). Five additional CAD samples were applied to the same dividing model, which could validate the accuracy to predict the risk of CAD by data mining analysis. We could demonstrate that operational taxonomic unit 853 (OTU853), OTU657, and OTU990 were determined important both by the data mining method and by the usual statistical comparison. We classified the gut microbiota profiles in coronary artery disease patients using data mining analysis of T-RFLP data and demonstrated the possibility that gut microbiota is a diagnostic marker of suffering from CAD.

Oral administration of the lactic acid bacterium Pediococcus acidilactici attenuates atherosclerosis in mice by inducing tolerogenic dendritic cells.

The intestinal microbiota appears to play an important role in the development of atherosclerosis. We investigated the effect of the probiotic lactic acid bacterium Pediococcus acidilactici R037 on atherosclerosis using apolipoprotein E-deficient (ApoE -/-) mice. Six-week-old ApoE -/- mice were orally administered R037 six times a week. Mice treated with R037 for 12 weeks exhibited markedly attenuated atherosclerotic lesions in the aortic root (2.3 ± 0.15 × 105 µm2 vs. 3.3 ± 0.29 × 105 µm2, respectively; P < 0.01; n = 15-17 each group). The expression of Ki-67 in CD4+ T cells, the population of interferon γ-producing CD4+ T cells in the spleen, and pro-inflammatory cytokine production from splenic lymphocytes were significantly decreased in R037-treated mice. Interestingly, splenic dendritic cells (DCs) isolated from R037-treated mice suppressed CD4+ T-cell proliferation and pro-inflammatory cytokine production ex vivo, suggesting that R037 treatment induced tolerogenic DCs. Programmed cell death ligand 1 expression in DCs was significantly enhanced in R037-treated mice, which might explain the immunosuppressive effect of DCs at least in part. These results indicate that R037 attenuates atherosclerosis by inducing tolerogenic DCs, which suppress Th1-driven inflammation and the proliferative activity of CD4+ T cells. Our findings may provide a novel therapeutic approach for the prevention of atherosclerosis based on dietary supplementation with probiotics.

Regulatory/effector T-cell ratio is reduced in coronary artery disease.

BACKGROUND: The protective function of regulatory T cells (Treg) has been identified in experimental atherosclerosis, but the contribution of Treg to the pathogenesis of human coronary artery disease (CAD) remains poorly understood. We investigated Treg and regulatory T-cell/effector T-cell (Treg/Teff) ratio in peripheral blood samples from CAD patients using a new strategy for precise identification of Treg. METHODS AND RESULTS: Peripheral blood samples were collected from 73 stable CAD patients (55 middle-aged CAD patients and 18 old CAD patients) and 64 controls (47 middle-aged controls and 17 young controls). CD3(+)CD4(+)FoxP3(+)T cells were divided into 3 fractions: CD45RA(+)FoxP3(low)resting Treg(Fr1), CD45RA(-)FoxP3(high)activated Treg(Fr2), and CD45RA(-)FoxP3(low)non-Treg(Fr3). CAD patients had lower percentages of Fr1 and Fr2 and higher percentages of Fr3 and CD45RA(-)Foxp3(-)Teff(Fr4+5) within the CD3(+)CD4(+)T-cell population compared to age-matched controls. Treg/Teff ratio (Fr1+2/Fr3+4+5) in CAD patients was also markedly lower than in controls (middle-aged control, 0.17±0.09 vs. middle-aged CAD, 0.10±0.05; P<0.001). The percentage of CD4(+)CD28(null)T cells within the CD4(+)T-cell population was negatively correlated with Treg/Teff ratio, excluding CD4(+)CD28(null)T cells <0.3% (r=-0.27, P<0.05). High-sensitivity C-reactive protein was also negatively correlated with Treg/Teff ratio (r=-0.22, P<0.05). CONCLUSIONS: CAD patients had reduced Treg and Treg/Teff ratio compared to healthy controls. The present findings may be helpful when developing immunotherapy for the prevention of CAD.

Ultraviolet B Exposure Inhibits Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Mice by Expanding CD4+Foxp3+ Regulatory T Cells.

BACKGROUND: Pathogenic immune responses are known to play an important role in abdominal aortic aneurysm (AAA) development. Ultraviolet B (UVB) irradiation has been demonstrated to have therapeutic potential not only for cutaneous diseases but also for systemic inflammatory diseases in mice by suppressing immunoinflammatory responses. We investigated the effect of UVB irradiation on experimental AAA. METHODS AND RESULTS: We used an angiotensin II-induced AAA model in apolipoprotein E-deficient mice fed a high-cholesterol diet. Mice aged 10 weeks were irradiated with 5 kJ/m2 UVB once weekly for 6 weeks (UVB-irradiated, n=38; nonirradiated, n=42) and were euthanized for evaluation of AAA formation at 16 weeks. Overall, 93% of angiotensin II-infused mice developed AAA, with 60% mortality possibly because of aneurysm rupture. UVB irradiation significantly decreased the incidence (66%) and mortality (29%) of AAA (P=0.004 and P=0.006, respectively). UVB-irradiated mice had significantly smaller diameter AAA (P=0.008) and fewer inflammatory cells in the aortic aneurysm tissue than nonirradiated mice, along with systemic expansion of CD4+Foxp3+ regulatory T cells and decreased effector CD4+CD44highCD62Llow T cells in para-aortic lymph nodes. Genetic depletion of regulatory T cells abrogated these beneficial effects of UVB treatment, demonstrating a critical role of regulatory T cells. CONCLUSIONS: Our data suggest that UVB-dependent expansion of regulatory T cells has beneficial effects on experimental AAA and may provide a novel strategy for the treatment of AAA.

Analysis of Gut Microbiota in Coronary Artery Disease Patients: a Possible Link between Gut Microbiota and Coronary Artery Disease.

AIM: Recent studies have suggested that metabolic disorders such as obesity and type 2 diabetes are associated with gut microbiota. The association between atherosclerosis and gut microbiota has also been attracting increased attention. Our aim was to specify a characteristic trend of gut microbiota in coronary artery disease (CAD). METHODS: This study included 39 CAD patients, 30 age- and sex-matched no-CAD controls (Ctrls) with coronary risk factors and 50 healthy volunteers (HVs) without coronary risk factors. Bacterial DNA was extracted from their fecal samples and analyzed by terminal restriction fragment length polymorphism. RESULTS: A characteristic change of gut microbiota was observed in CAD patients, where the order Lactobacillales was increased (CAD, Ctrl vs. HV; 13.6%±12.0%, 6.2%±7.7% vs. 4.1%±5.9%; p＜0.001) and the phylum Bacteroidetes (Bacteroides＋Prevotella) was decreased (CAD, Ctrl vs. HV;35.5%±11.6%, 43.9%±11.2% vs. 47.4%±11.5%; p＜0.001). The CAD group was over-represented in enterotype "others" (III), compared with the Ctrl or HV group (p＜0.001, chi-squared test), although we could not deny the possibility that some drugs affect the gut flora types. CONCLUSIONS: Although this study had some limitations, we demonstrated that the incidence of CAD was linked with an alteration of gut microbiota. A prospective study is desired to clarify a causal relationship between CAD and gut microbiota.

Foxp3+ regulatory T cells play a protective role in angiotensin II-induced aortic aneurysm formation in mice.

Although regulatory T cells (Tregs) have been shown to play a protective role in abdominal aortic aneurysm (AAA) formation, it remains unclear whether expansion of endogenous Foxp3(+) Tregs prevents AAA. In the current study, we determined the effects of endogenous Foxp3(+) Treg expansion or depletion in an experimental model of AAA. We continuously infused 12-week-old apolipoprotein E-deficient mice fed a high-cholesterol diet with angiotensin II (n=60) or normal saline (n=12) by implanting osmotic mini-pumps and evaluated AAA formation at 16 weeks. The angiotensin II-infused mice received interleukin-2/anti-interleukin-2 monoclonal antibody complex (interleukin-2 complex; n=31) or PBS (n=29). Eighty-one percent of angiotensin II-infused mice developed AAA, with 42% mortality possibly because of aneurysm rupture. Interleukin-2 complex treatment systemically increased the number of Foxp3(+) Tregs and significantly decreased the incidence (52%) and mortality (17%) of AAA. Immunohistochemical analysis showed reduced accumulation of macrophages and increased numbers of Foxp3(+) Tregs in aneurysmal tissues, suggesting that expansion of Tregs may suppress local inflammation in the vessel wall and provide protection against AAA formation. Furthermore, genetic depletion of Foxp3(+) Tregs led to a significant increase in the mortality of AAA, suggesting the protective role of Foxp3(+) Tregs against AAA. Our findings suggest that Foxp3(+) Tregs may play a protective role in AAA formation and that promotion of an endogenous regulatory immune response may be a potentially valuable therapeutic approach for preventing AAA.

Regression of atherosclerosis with anti-CD3 antibody via augmenting a regulatory T-cell response in mice.

AIMS: Although recent animal studies have investigated the cellular and molecular mechanisms underlying the process of atherosclerosis regression, it remains unknown whether adaptive immune responses including T cells are involved in this process. We investigated the role of T cells in atherosclerosis regression. METHODS AND RESULTS: LDL receptor-deficient mice were fed a high-cholesterol diet for 8 weeks to form atherosclerotic lesions and were then changed to a standard diet, and atherosclerosis was assessed 4 weeks later. Just before changing the diet, the mice received an iv injection of anti-CD3 antibody (CD3-Ab) or control immunoglobulin G for 5 consecutive days. CD3-Ab treatment regressed atherosclerosis and decreased the accumulation of macrophages and CD4(+) T cells in the plaques. CD3-Ab treatment also dramatically reduced CD4(+) T cells and increased the proportion of regulatory T cells (Tregs). Depletion of Tregs by anti-CD25 antibody injection abolished the regression of atherosclerosis seen in CD3-Ab-treated mice, indicating the essential role for Tregs in this process. CONCLUSION: CD3-Ab treatment induced rapid regression of established atherosclerosis via reducing CD4(+) T cells and increasing the proportion of Tregs. These findings suggest that therapeutic intervention for T-cell-mediated immune responses may represent a novel strategy to induce atherosclerosis regression in combination with lipid-lowering therapy.

CD3 antibody and IL-2 complex combination therapy inhibits atherosclerosis by augmenting a regulatory immune response.

BACKGROUND: Accumulating evidence suggests that the balance between pathogenic effector T cells (Teffs) and regulatory T cells (Tregs) may be important for controlling atherosclerotic disease. We hypothesized that a combination therapy with anti-CD3 antibody (CD3-Ab) and IL-2/anti-IL-2 monoclonal antibody complex (IL-2 complex) aimed at increasing the ratio of Tregs to Teffs would effectively inhibit atherosclerosis in mice. METHODS AND RESULTS: We treated apolipoprotein E-deficient mice fed a high-cholesterol diet with vehicle, CD3-Ab, IL-2 complex, or their combination. Mice receiving the combination therapy had markedly reduced atherosclerotic lesions than mice treated with CD3-Ab or IL-2 complex alone. In addition, a striking increase in the Treg/Teff ratio of lymphoid organs and atherosclerotic lesions, along with plaque stabilization characterized by decreased macrophage content and increased collagen content was observed. The combination treatment also markedly reduced splenic Ly6C(high) inflammatory monocytes and might induce a favorable macrophage phenotype change in atherosclerotic lesions. CONCLUSIONS: Our results indicate that in addition to suppressing Teff responses, enhancing Treg-mediated immune responses is more efficacious in preventing atherosclerosis, suggesting a novel therapeutic approach for atherosclerosis.

Torsades de Pointes with QT prolongation related to donepezil use.

An 83-year-old female, who had a history of anterior myocardial infarction, was treated for Alzheimer's disease with donepezil. She suffered from repeated diarrhea and vomiting, and experienced syncope. She was admitted to our hospital and was diagnosed with acute colitis and syncope. On admission, her heart rate was 54 beats/min with regular rhythm. Laboratory data showed a low plasma potassium level. Electrocardiogram (ECG) showed poor R progression, ST elevation, negative T in precordial leads, and marked QT prolongation. Transthoracic echocardiogram showed the enlargement of the left atrium and aneurysmal area at the apex. Torsades de Pointes (TdP) with syncope and convulsion were confirmed on ECG monitoring twice after admission. We treated her with potassium chloride and started magnesium sulfate and lidocaine, and then added isoprenaline injection. After these treatments, her heart rate increased and we did not detect TdP again. With the aging population in Japan, prescriptions for donepezil are increasing. We have to be vigilant for syncope in patients taking donepezil, which is possibly related to QT prolongation and TdP.

Usefulness of transesophageal echocardiographic observation during chemotherapy for cardiac metastasis of non-Hodgkin lymphoma complicated with left ventricular diastolic collapse.

A 53-year-old man, who had been treated for penile origin diffuse large B cell type non-Hodgkin lymphoma (NHL), suffered from right femoral pain and dyspnea. Positron emission tomography (PET) revealed abnormal accumulation in his right femur and cardiac segments. Transthoracic echocardiography revealed massive localized pericardial effusion with the collapse of both ventricles and the mass-like echo in the left atrium. We performed emergent pericardiocentesis and diagnosed this case as a recurrence of NHL with cardiac metastasis. With the use of transesophageal echocardiography (TEE), we confirmed the mass-like echo around the inter-atrial septum, which directly invaded to the aortic ring and the right atrial wall. In order to evaluate the effect of chemotherapy, we performed TEE and observed the precise changes of intra-cardiac tumor size. With the use of TEE monitoring, we could select the appropriate chemotherapeutic regimen, and the tumor became smaller and finally diminished. The femoral accumulation detected by PET also disappeared. We experienced a case of cardiac metastasis of NHL complicated with left ventricular diastolic collapse due to the massive localized pericardial effusion. TEE is a useful tool to evaluate precisely the efficacy of chemotherapy for intra-cardiac tumors.

Rates and extents of antifungal activities of amphotericin B, flucytosine, fluconazole, and voriconazole against Candida lusitaniae determined by microdilution, Etest, and time-kill methods.

Amphotericin B, flucytosine, fluconazole, and voriconazole alone and in combination were evaluated against isolates of Candida lusitaniae. MICs were determined by broth microdilution and Etest, and time-kill studies were conducted. Amphotericin B resulted in fungicidal activity against most isolates, whereas fluconazole, voriconazole, and flucytosine produced primarily fungistatic activities. The addition of flucytosine to amphotericin B resulted in a faster rate and greater extent of kill for isolates for which the MICs of amphotericin B were elevated.