Continuous infusion of resolvin D2 in combination with Angiotensin-II show contrary effects on blood pressure and intracardiac artery remodeling.

Specialized pro-resolving mediators (SPMs) are key effectors of resolution of inflammation. This is highly relevant for cardiac and vessel remodeling, where the net inflammatory response contributes to determine disease outcome. Herein, we used a mice model of angiotensin (Ang)-II-induced hypertension to study the effect of the SPM Resolvin D2 (RvD2), on hypertension and cardiac remodeling. By using subcutaneous osmotic minipumps, mice were treated with PBS or Ang-II in combination with or without RvD2 for two weeks. Mice receiving RvD2 gained less blood pressure increase compared to Ang-II alone. Surprisingly, however, examination of intracardiac arteries revealed that RvD2 treatment in combination with Ang-II exacerbated Ang-II-induced fibrosis. Measures of vascular smooth muscle cell dedifferentiation correlated with the level of vascular remodeling, indicating that this dedifferentiation, including increased proliferation and migration, is a contributing factor. RNA sequencing of left ventricle cardiac tissue supported these findings as pathways related to cell proliferation and cell differentiation were upregulated in mice treated with Ang-II in combination with RvD2. Additionally, the RNA sequencing also showed upregulation of pathways related to SPM metabolism. In line with this, Mass spectrometry analysis of lipid mediators showed reduced cardiac levels of the arachidonic acid derived metabolite leukotriene E4 in RvD2 treated mice. Our study suggests that continuous infusion through osmotic minipumps should not be the recommended route of RvD2 administration in future studies.

Chronic HIV Infection Increases Monocyte NLRP3 Inflammasome-Dependent IL-1α and IL-1ß Release.

Antiretroviral treatment (ART) has converted HIV from a lethal disease to a chronic condition, yet co-morbidities persist. Incomplete immune recovery and chronic immune activation, especially in the gut mucosa, contribute to these complications. Inflammasomes, multi-protein complexes activated by innate immune receptors, appear to play a role in these inflammatory responses. In particular, preliminary data indicate the involvement of IFI16 and NLRP3 inflammasomes in chronic HIV infection. This study explores inflammasome function in monocytes from people with HIV (PWH); 22 ART-treated with suppressed viremia and 17 untreated PWH were compared to 33 HIV-negative donors. Monocytes were primed with LPS and inflammasomes activated with ATP in vitro. IFI16 and NLRP3 mRNA expression were examined in a subset of donors. IFI16 and NLRP3 expression in unstimulated monocytes correlated negatively with CD4 T cell counts in untreated PWH. For IFI16, there was also a positive correlation with viral load. Monocytes from untreated PWH exhibit increased release of IL-1α, IL-1ß, and TNF compared to treated PWH and HIV-negative donors. However, circulating monocytes in PWH are not pre-primed for inflammasome activation in vivo. The findings suggest a link between IFI16, NLRP3, and HIV progression, emphasizing their potential role in comorbidities such as cardiovascular disease. The study provides insights into inflammasome regulation in HIV pathogenesis and its implications for therapeutic interventions.

Identification of two tofacitinib subpopulations with different relative risk versus TNF inhibitors: an analysis of the open label, randomised controlled study ORAL Surveillance.

OBJECTIVES: Based on primary results from ORAL Surveillance, an event-driven clinical trial of risk-enriched patients, identify subpopulations with different relative risk (ie, 'high-risk' and 'low-risk') with tofacitinib versus tumour necrosis factor inhibitors (TNFi). METHODS: Patients with rheumatoid arthritis aged ≥50 years with ≥1 additional cardiovascular risk factor received tofacitinib 5 or 10 mg two times a day or TNFi. Prior analyses had identified age and smoking as risk factors of particular interest across safety outcomes. Hazard ratios (HRs) and incidence rates were evaluated by age and smoking individually and in combination. Results were validated across tofacitinib development programmes. RESULTS: 'Age ≥65 years or ever smoker' defined a group ('high-risk') with increased risk of malignancies (excluding non-melanoma skin cancer), major adverse cardiovascular events, myocardial infarction, venous thromboembolism and all-cause death with tofacitinib (combined doses) versus TNFi (HRs 1.41-5.19). In patients 'aged <65 years and never smokers' ('low-risk'), there was no detectable risk increase with tofacitinib versus TNFi (HRs ≈1.0) up to 6 years of follow-up, and absolute risk remained low and was corroborated across tofacitinib rheumatoid arthritis, psoriatic arthritis and ulcerative colitis programmes with up to 10 years of observation. CONCLUSIONS: This posthoc analysis of ORAL Surveillance identified two tofacitinib subpopulations with different relative risk versus TNFi. High risk was confined to patients defined by distinct risk factors age ≥65 years or smoking, and these differentiating risk factors accounted for the excess risk observed with tofacitinib versus TNFi. These findings can guide individualised benefit/risk assessment and clinical decision-making on treatment with tofacitinib. TRIAL REGISTRATION NUMBERS: NCT02092467, NCT01262118, NCT01484561, NCT00147498, NCT00413660, NCT00550446, NCT00603512, NCT00687193, NCT01164579, NCT00976599, NCT01059864, NCT01359150, NCT02147587, NCT00960440, NCT00847613, NCT00814307, NCT00856544, NCT00853385, NCT01039688, NCT02281552, NCT02187055, NCT02831855, NCT00413699, NCT00661661, NCT00787202, NCT01465763, NCT01458951, NCT01458574, NCT01470612, NCT01877668, NCT01882439, NCT01976364.

Risk of major adverse cardiovascular events with tofacitinib versus tumour necrosis factor inhibitors in patients with rheumatoid arthritis with or without a history of atherosclerotic cardiovascular disease: a post hoc analysis from ORAL Surveillance.

OBJECTIVES: Evaluate risk of major adverse cardiovascular events (MACE) with tofacitinib versus tumour necrosis factor inhibitors (TNFi) in patients with rheumatoid arthritis (RA) with or without a history of atherosclerotic cardiovascular disease (ASCVD) in ORAL Surveillance. METHODS: Patients with RA aged ≥50 years with ≥1 additional CV risk factor received tofacitinib 5 mg or 10 mg two times per day or TNFi. Hazard rations (HRs) were evaluated for the overall population and by history of ASCVD (exploratory analysis). RESULTS: Risk of MACE, myocardial infarction and sudden cardiac death were increased with tofacitinib versus TNFi in ORAL Surveillance. In patients with history of ASCVD (14.7%; 640/4362), MACE incidence was higher with tofacitinib 5 mg two times per day (8.3%; 17/204) and 10 mg two times per day (7.7%; 17/222) versus TNFi (4.2%; 9/214). HR (combined tofacitinib doses vs TNFi) was 1.98 (95% confidence interval (CI) 0.95 to 4.14; interaction p values: 0.196 (for HR)/0.059 (for incidence rate difference)). In patients without history of ASCVD, MACE HRs for tofacitinib 5 mg two times per day (2.4%; 30/1251) and 10 mg two times per day (2.8%; 34/1234) versus TNFi (2.3%; 28/1237) were, respectively, 1.03 (0.62 to 1.73) and 1.25 (0.76 to 2.07). CONCLUSIONS: This post hoc analysis observed higher MACE risk with tofacitinib versus TNFi in patients with RA and history of ASCVD. Among patients without history of ASCVD, all with prevalent CV risk factors, MACE risk did not appear different with tofacitinib 5 mg two times per day versus TNFi. Due to the exploratory nature of this analysis and low statistical power, we cannot exclude differential MACE risk for tofacitinib 5 mg two times per day versus TNFi among patients without history of ASCVD, but any absolute risk excess is likely low. TRIAL REGISTRATION NUMBER: NCT02092467.

IL-18 and IL-18 binding protein are related to disease severity and parasitemia during falciparum malaria.

BACKGROUND: Several inflammatory molecules participate in the immune response to malaria. Interleukin (IL)-18 is an inflammatory cytokine activated by NLRP3 inflammasomes. In clinical falciparum malaria, with and without HIV co-infection, data on IL-18 and in particular on its binding protein, IL-18bp, is scarce. METHODS: Clinical data and blood samples were collected from adults in Mozambique with P. falciparum infection, with (n = 70) and without (n = 61) HIV co-infection, from HIV-infected patients with similar symptoms without malaria (n = 58) and from healthy controls (n = 52). In vitro studies were performed in endothelial cells using hemozoin crystals. RESULTS: (i) IL-18 and IL-18bp were markedly up-regulated during falciparum malaria with particular high levels in malaria patients co-infected with HIV and severe malaria disease. (ii) In the malaria group as a whole, both IL-18 and IL-18bp were positively correlated with disease severity, parasitemia, and endothelial cell activation as assessed by vWF in plasma. (iii) Whereas there was no change in IL-18 levels in malaria patients co-infected with HIV during follow-up, the patients with malaria only had slightly increased IL-18 levels. Further, the IL-18pb levels declined and thereby contributed to an increase in IL-18/IL-18bp ratio in all subgroups of malaria patients. (iv) IL-27, previously shown to be up-regulated in this malaria cohort, markedly induced a release of IL-18bp from endothelial cells in vitro, and notably, this presumably anti-inflammatory effect was counteracted by hemozoin. CONCLUSIONS: Our findings suggest that the IL-18 system could be an important mediator in the immune pathogenesis during falciparum malaria, potentially also representing a target for therapy.

Low Cellular NAD+ Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes.

NAD+ is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD+ on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD+ affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD+ was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD+ levels on TLR4-mediated NF-κB activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD+ enhanced IL-1ß release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD+ depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD+ depletion, specifically TAK1, IKKß, IkBα, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD+ affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD+ and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.

Increased Complement Factor B and Bb Levels Are Associated with Mortality in Patients with Severe Aortic Stenosis.

Inflammation is involved in initiation and progression of aortic stenosis (AS). However, the role of the complement system, a crucial component of innate immunity in AS, is unclear. We hypothesized that circulating levels of complement factor B (FB), an important component of the alternative pathway, are upregulated and could predict outcome in patients with severe symptomatic AS. Therefore, plasma levels of FB, Bb, and terminal complement complex were analyzed in three cohorts of patients with severe symptomatic AS and mild-to-moderate or severe asymptomatic AS (population 1, n = 123; population 2, n = 436; population 3, n = 61) and in healthy controls by enzyme immunoassays. Compared with controls, symptomatic AS patients had significantly elevated levels of FB (2.9- and 2.8-fold increase in population 1 and 2, respectively). FB levels in symptomatic and asymptomatic AS patients were comparable (population 2 and 3), and in asymptomatic patients FB correlated inversely with valve area. FB levels in population 1 and 2 correlated with terminal complement complex levels and measures of systemic inflammation (i.e., CRP), cardiac function (i.e., NT-proBNP), and cardiac necrosis (i.e., Troponin T). High FB levels were significantly associated with mortality also after adjusting for clinical and biochemical covariates (hazard ratio 1.37; p = 0.028, population 2). Plasma levels of the Bb fragment showed a similar pattern in relation to mortality. We concluded that elevated levels of FB and Bb are associated with adverse outcome in patients with symptomatic AS. Increased levels of FB in asymptomatic patients suggest the involvement of FB from the early phase of the disease.

Soluble Markers of Interleukin 1 Activation as Predictors of First-Time Myocardial Infarction in HIV-Infected Individuals.

People with human immunodeficiency virus (HIV) infection (PWH) have an increased risk of cardiovascular disease (CVD), compared with the general population. In a nested case-control study of 55 PWH with first-time myocardial infarction (MI; cases) and 182 PWH with no CVD (controls), we measured soluble markers of interleukin 1 (IL-1) activation at 4 different time points before the case's MI. Cases had higher levels of IL-1 receptor antagonist (IL-1Ra) at all time points leading up to first-time MI, and higher levels of IL-1Ra were associated with an approximately 1.5-fold increased risk of MI, supporting the rationale to target IL-1 activation to reduce cardiovascular risk in PWH.

Serum levels of inflammation-related markers and metabolites predict response to neoadjuvant chemotherapy with and without bevacizumab in breast cancers.

Angiogenesis is necessary for tumor growth and has been targeted in breast cancer; however, it is unclear which patients will respond and benefit from antiangiogenic therapy. We report noninvasive monitoring of patient response to neoadjuvant chemotherapy given alone or in combination with anti-vascular endothelial growth factor (bevacizumab) in a randomized clinical trial. At four time points during neoadjuvant chemotherapy ± bevacizumab of receptor tyrosine-protein kinase erbB-2-negative breast cancers, we measured metabolites and inflammation-related markers in patient's serum. We report significant changes in the levels of several molecules induced by bevacizumab, the most prominent being an increase in pentraxin 3 (PTX3) and von Willebrand factor (VWF). Serum levels of AXL, VWF and pulmonary and activation-regulated cytokine (PARC/CCL18) reflected response to chemotherapy alone or in combination with bevacizumab. We further analyzed serum cytokines in relation to tumor characteristics such as gene expression, tumor metabolites and tumor infiltrating leukocytes. We found that VWF and growth-differentiation factor 15 tumor mRNA levels correlated with their respective serum protein levels suggesting that these cytokines may be produced by tumors and outflow to the bloodstream while influencing the tumor microenvironment locally. Finally, we used binomial logistic regression which allowed to predict patient's response using only 10 noninvasive biomarkers. Our study highlights the potential of monitoring circulating levels of cytokines and metabolites during breast cancer therapy.

Complement component C3 and the TLR co-receptor CD14 are not involved in angiotensin II induced cardiac remodelling.

Inflammation is centrally involved in the development of cardiac hypertrophy and the processes of remodelling. The complement system and Toll-like receptor (TLR) family, two upstream arms of the innate immune system, have previously been reported to be involved in cardiac remodelling. However, the role of complement component 3 (C3), TLR co-receptor CD14 and the synergy between them have not been addressed during pressure overload-induced cardiac remodelling. Here, we examined angiotensin II-induced cardiac hypertrophy and remodelling for 7 days in male C57Bl/6 J mice deficient in C3, CD14, or both (C3CD14), and WT controls. Angiotensin II infusion induced a mild concentric hypertrophic phenotype in WT mice with increased left ventricle weight, wall thicknesses and reduced ventricular internal diameter, associated with increased cardiac fibrosis. However, there were no differences between WT mice and mice deficient for C3, CD14 or C3CD14, as systolic blood pressure, cardiac function and structure and levels of fibrosis were comparable between WT mice and the three other genotypes. C5a did not change in angiotensin II treated mice, whereas Mac2 levels were increased in angiotensin II treated mice, but did not differ between genotypes. The inflammatory IL-6 response was comparable between WT and C3 deficient mice, however, it was decreased in CD14 and C3CD14 deficient mice. We conclude that deficiency in C3, CD14 or C3CD14 had no effect on cardiac remodelling following angiotensin II-induced pressure overload. This suggests that C3 and CD14 are not involved in angiotensin II-induced adverse cardiac remodelling.

Plasma levels of interleukin 27 in falciparum malaria is increased independently of co-infection with HIV: potential immune-regulatory role during malaria.

BACKGROUND: The immune response during falciparum malaria mediates both harmful and protective effects on the host; however the participating molecules have not been fully defined. Interleukin (IL)-27 is a pleiotropic cytokine exerting both inflammatory and anti-inflammatory effects, but data on IL-27 in malaria patients are scarce. METHODS: Clinical data and blood samples were collected from adults in Mozambique with P. falciparum infection, with (n = 70) and without (n = 61) HIV-1 co-infection, from HIV-infected patients with similar symptoms without malaria (n = 58) and from healthy controls (n = 52). In vitro studies were performed in endothelial cells and PBMC using hemozoin crystals. Samples were analyzed using enzyme immunoassays and quantitative PCR. RESULTS: (i) IL-27 was markedly up-regulated in malaria patients compared with controls and HIV-infected patients without malaria, showing no relation to HIV co-infection. (ii) IL-27 was correlated with P. falciparum parasitemia and von Willebrand factor as a marker of endothelial activation, but not with disease severity. (iii) In vitro, IL-27 modulated the hemozoin-mediated cytokine response in endothelial cells and PBMC with enhancing effects on IL-6 and attenuating effects on IL-8. CONCLUSION: Our findings show that IL-27 is regulated during falciparum malaria, mediating both inflammatory and anti-inflammatory effects, potentially playing an immune-regulatory role during falciparum malaria.

Caspase-1 induces smooth muscle cell growth in hypoxia-induced pulmonary hypertension.

Lung diseases with hypoxia are complicated by pulmonary hypertension, leading to heart failure and death. No pharmacological treatment exists. Increased proinflammatory cytokines are found in hypoxic patients, suggesting an inflammatory pathogenesis. Caspase-1, the effector of the inflammasome, mediates inflammation through activation of the proinflammatory cytokines interleukin (IL)-18 and IL-1ß. Here, we investigate inflammasome-related mechanisms that can trigger hypoxia-induced pulmonary hypertension. Our aim was to examine whether caspase-1 induces development of hypoxia-related pulmonary hypertension and is a suitable target for therapy. Wild-type (WT) and caspase-1-/- mice were exposed to 10% oxygen for 14 days. Hypoxic caspase-1-/- mice showed lower pressure and reduced muscularization in pulmonary arteries, as well as reduced right ventricular remodeling compared with WT. Smooth muscle cell (SMC) proliferation was reduced in caspase-1-deficient pulmonary arteries and in WT arteries treated with a caspase-1 inhibitor. Impaired inflammation was shown in hypoxic caspase-1-/- mice by abolished pulmonary influx of immune cells and lower levels of IL-18, IL-1ß, and IL-6, which were also reduced in the medium surrounding caspase-1 abrogated pulmonary arteries. By adding IL-18 or IL-1ß to caspase-1-deficient pulmonary arteries, SMC proliferation was retained. Furthermore, inhibition of both IL-6 and phosphorylated STAT3 reduced proliferation of SMC in vitro, indicating IL-18, IL-6, and STAT3 as downstream mediators of caspase-1-induced SMC proliferation in pulmonary arteries. Caspase-1 induces SMC proliferation in pulmonary arteries through the caspase-1/IL-18/IL-6/STAT3 pathway, leading to pulmonary hypertension in mice exposed to hypoxia. We propose that caspase-1 inhibition is a potential target for treatment of pulmonary hypertension.

NLRP3 Inflammasome: A Novel Player in Metabolically Induced Inflammation-Potential Influence on the Myocardium.

Metabolic and immune systems are among the most fundamental requirements for survival. Many metabolic and immune response pathways or nutrient- and pathogen-sensing systems are evolutionarily conserved throughout species. As a result, the immune response and metabolic regulation are highly integrated and the proper function of each is dependent on the other. This interaction between metabolic disturbances and the immune system has been most extensively studied in disorders related to obesity such as insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease. Metabolically induced inflammation seems also to play a role in the development and progression of atherosclerosis including its complications such as myocardial infarction (MI) and post-MI remodeling. There are several lines of evidence suggesting that NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a sensor of metabolic stress linking metabolic disturbances to inflammation. Here, we will discuss the role of the NLRP3 inflammasome in the pathogenesis of obesity and diabetes, 2 important risk factors for atherosclerosis and MI. We will also discuss the role of NLRP3 inflammasome in the interaction between metabolic disturbances and myocardial inflammation during MI and during metabolically induced myocardial remodeling.

NLRP3 inflammasome mediates oxidative stress-induced pancreatic islet dysfunction.

Inflammasomes are multiprotein inflammatory platforms that induce caspase-1 activation and subsequently interleukin (IL)-1ß and IL-18 processing. The NLRP3 inflammasome is activated by different forms of oxidative stress, and, based on the central role of IL-1ß in the destruction of pancreatic islets, it could be related to the development of diabetes. We therefore investigated responses in wild-type C57Bl/6 (WT) mice, NLRP3-/- mice, and mice deficient in apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) after exposing islets to short-term hypoxia or alloxan-induced islet damage. NLRP3-deficient islets compared with WT islets had preserved function ex vivo and were protected against hypoxia-induced cell death. Furthermore, NLRP3 and ASC-deficient mice were protected against oxidative stress-induced diabetes caused by repetitive low-dose alloxan administration, and this was associated with reduced ß-cell death and reduced macrophage infiltration. This suggests that the beneficial effect of NLRP3 inflammasome deficiency on oxidative stress-mediated ß-cell damage could involve reduced macrophage infiltration and activation. To support the role of macrophage activation in alloxan-induced diabetes, we injected WT mice with liposomal clodronate, which causes macrophage depletion before induction of a diabetic phenotype by alloxan treatment, resulting in improved glucose homeostasis in WT mice. We show here that the NLRP3 inflammasome acts as a mediator of hypoxia and oxidative stress in insulin-producing cells, suggesting that inhibition of the NLRP3 inflammasome could have beneficial effects on ß-cell preservation.

Levels and prognostic impact of circulating markers of inflammation, endothelial activation and extracellular matrix remodelling in patients with lung cancer and chronic obstructive pulmonary disease.

BACKGROUND: The development of both chronic obstructive pulmonary disease (COPD) and lung cancer (LC) is influenced by smoking related chronic pulmonary inflammation caused by an excessive innate immune response to smoke exposure. In addition, the smoking induced formation of covalent bonds between the carcinogens and DNA and the accumulation of permanent somatic mutations in critical genes are important in the carcinogenic processes, and can also induce inflammatory responses. How chronic inflammation is mirrored by serum markers in COPD and LC and if these markers reflect prognosis in patients with LC is, however, largely unknown. METHODS: Serum levels of 18 markers reflecting inflammation, endothelial activation and extracellular matrix remodelling were analysed in 207 patients with non-small lung carcinoma (NSCLC) before surgery and 42 COPD patients. 56% of the LC patients also suffered from COPD. The serum samples were analysed by enzyme immunoassays. RESULTS: Serum levels of OPG, PTX3, AXL, ALCAM, sCD163, CD147, CatS and DLL1 were significantly higher in patients with COPD as compared to patients with LC. High sTNFR1 levels were associated with improved progression free survival (PFS) and overall survival (OS) in LC patients with (PFS hazard ratio (HR) 0.49, OS HR 0.33) and without COPD (OS HR 0.30). High levels of OPG were associated with improved PFS (HR 0.17) and OS (HR 0.14) for LC with COPD. CRP was significantly associated with overall survival regardless of COPD status. CONCLUSION: Several markers reflecting inflammation, endothelial activation and extracellular matrix remodelling are elevated in serum from patients with COPD compared to LC patients. Presence of COPD might influence the levels of circulating biomarkers. Some of these markers are also associated with prognosis.

Leukocyte Overexpression of Intracellular NAMPT Attenuates Atherosclerosis by Regulating PPARγ-Dependent Monocyte Differentiation and Function.

OBJECTIVE: Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) mediates inflammatory and potentially proatherogenic effects, whereas the role of intracellular NAMPT (iNAMPT), the rate limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD)+ generation, in atherogenesis is largely unknown. Here we investigated the effects of iNAMPT overexpression in leukocytes on inflammation and atherosclerosis. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice with hematopoietic overexpression of human iNAMPT (iNAMPThi), on a western type diet, showed attenuated plaque burden with features of lesion stabilization. This anti-atherogenic effect was caused by improved resistance of macrophages to apoptosis by attenuated chemokine (C-C motif) receptor 2-dependent monocyte chemotaxis and by skewing macrophage polarization toward an anti-inflammatory M2 phenotype. The iNAMPThi phenotype was almost fully reversed by treatment with the NAMPT inhibitor FK866, indicating that iNAMPT catalytic activity is instrumental in the atheroprotection. Importantly, iNAMPT overexpression did not induce any increase in eNAMPT, and eNAMPT had no effect on chemokine (C-C motif) receptor 2 expression and promoted an inflammatory M1 phenotype in macrophages. The iNAMPT-mediated effects at least partly involved sirtuin 1-dependent molecular crosstalk of NAMPT and peroxisome proliferator-activated receptor γ. Finally, iNAMPT and peroxisome proliferator-activated receptor γ showed a strong correlation in human atherosclerotic, but not healthy arteries, hinting to a relevance of iNAMPT/peroxisome proliferator-activated receptor γ pathway also in human carotid atherosclerosis. CONCLUSIONS: This study highlights the functional dichotomy of intracellular versus extracellular NAMPT, and unveils a critical role for the iNAMPT-peroxisome proliferator-activated receptor γ axis in atherosclerosis.

Palmitate promotes inflammatory responses and cellular senescence in cardiac fibroblasts.

Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3-/- and ASC-/-mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1ß, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated ß-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.

Toll-Like Receptor 9 Promotes Survival in SERCA2a KO Heart Failure Mice.

Aim. Inflammation is important in heart failure (HF). The role of the immune receptor toll-like receptor 9 (TLR9) in HF is not understood and not investigated in diastolic HF. We investigated the role of TLR9 in a murine diastolic HF model caused by cardiomyocyte SERCA2a excision. Methods and Results. We crossed SERCA2a KO and TLR9 KO mice to generate four mouse lines. Tamoxifen-induced cardiomyocyte SERCA2a gene excision was carried out in mice, causing diastolic HF. After 7.6 weeks, cardiac functions and dimensions were analyzed by echocardiography and heart tissues were processed. HF mice depleted of TLR9 demonstrated reduced survival compared to SERC2a KO mice, with a median life expectancy of 58 days compared to 63 days. Both HF groups displayed increased left atrium size, lung weight, fetal gene expressions, monocyte/macrophage infiltration, and fibrosis. However, there were no significant differences between the groups. Conclusion. In mice with SERCA2a KO-induced diastolic HF, the absence of TLR9 reduced median life expectancy. The cause remains elusive, as all investigated HF parameters were unaltered. Still, these findings support a salutary role of TLR9 in some subsets of HF conditions and underline the importance for future studies on the mechanisms of TLR9 in diastolic HF.

Low Circulating Levels of Mitochondrial and High Levels of Nuclear DNA Predict Mortality in Chronic Heart Failure.

BACKGROUND: Mitochondrial DNA (mtDNA) and possibly nuclear DNA (nDNA) are released as danger-associated molecular patterns during cardiac stress, and may activate several innate immune receptors. The purpose of this study was to investigate the regulation of these danger-associated molecular patterns during human heart failure (HF). METHODS AND RESULTS: Plasma levels of mtDNA and nDNA from HF patients (n = 84) were analyzed by reverse transcriptase-polymerase chain reaction and compared with controls (n = 72). Increased levels of mtDNA were found in New York Heart Association (NYHA) I-II and NYHA III-IV. There was evidence of increased nDNA in NYHA III-IV compared with controls and NYHA I-II. Kaplan-Meier analysis revealed higher mortality in patients with high nDNA levels, whereas high levels of mtDNA were associated with survival. CONCLUSIONS: Plasma levels of mtDNA and nDNA are elevated in human HF associated with increased and decreased mortality, respectively. This study may suggest a rationale for exploring interventions within inflammatory signaling pathways activated by nucleic acids as novel targets in treatment of HF.