Interleukin-17/interleukin-17 receptor axis elicits intestinal neutrophil migration, restrains gut dysbiosis and lipopolysaccharide translocation in high-fat diet-induced metabolic syndrome model.

Sound evidence supports a role for interleukin-17 (IL-17) -producing γδ T cells and IL-17-producing helper T (Th17) cells in intestinal homeostasis, especially in intestinal barrier integrity. In the present study, we aimed to evaluate the role of IL-17 cytokine in the regulation of intestinal immunity and obesity-induced metabolic syndrome (MetS) in an experimental murine model. C57BL/6 wild-type (WT) mice and mice lacking the IL-17 cytokine receptor (IL-17RA-/- ) were fed either a control diet (CD) or a high-fat diet (HFD) for 9 weeks. Our data demonstrate that IL-17RA-/- mice are protected against obesity, but develop hyperglycemia, hyperinsulinemia and insulin resistance. In parallel, HFD-fed IL-17RA-/- mice display intense inflammation in the ileum compared with WT mice on the HFD. IL-17RA-/- mice fed the HFD exhibit impaired neutrophil migration to the intestinal mucosa and reduced gene expression of the CXCL-1 chemokine and CXCR-2 receptor in the ileum. Interestingly, the populations of neutrophils (CD11b+  Ly6G+ ) and anti-inflammatory macrophages (CD11b+  CX3CR1+ ) are increased in the mesenteric lymph nodes of these mice. IL-17RA-/- mice on the HFD also display increased commensal bacterial translocation into the bloodstream and elevated lipopolysaccharide (LPS) levels in the visceral adipose tissue (VAT). Metagenomic analysis of bacterial 16S gene revealed increased Proteobacteria and Bacteroidetes phyla, the main representatives of Gram-negative bacteria, and reduced Akkermansia muciniphila in the fecal samples of IL-17RA-/- mice fed the HFD. Together, these data indicate that the IL-17/IL-17R axis drives intestinal neutrophil migration, limits gut dysbiosis and attenuates LPS translocation to VAT, resulting in protection to MetS.

Interleukin-23 promotes intestinal T helper type17 immunity and ameliorates obesity-associated metabolic syndrome in a murine high-fat diet model.

We addressed the role of interleukin-23 (IL-23) in driving the intestinal T helper type 17 (Th17) response during obesity and metabolic syndrome progression induced by a high-fat diet (HFD). Diet-induced obese and lean mice received HFD or control diet (CTD), respectively, for 20 weeks. The nutritional, metabolic and immune parameters were examined at weeks 9 and 20. Gene and protein IL-23p19 and IL-23 receptor expression was increased in the ileum of obese wild-type mice (WT) fed the HFD for 9 weeks. Mice lacking IL-23 and fed the HFD exhibited greater weight gain, higher fat accumulation, adipocyte hypertrophy and hepatic steatosis. Notably, these mice had more glucose intolerance, insulin resistance and associated metabolic alterations, such as hyperinsulinaemia and hyperlipidaemia. IL-23 deficiency also significantly reduced protein levels of IL-17, CCL20 and neutrophil elastase in the ileum and reduced Th17 cell expansion in the mesenteric lymph nodes of the HFD mice. Of importance, IL-23-deficient mice exhibited increased gut permeability and blood bacterial translocation compared with WT mice fed HFD. Finally, metagenomics analysis of gut microbiota revealed a dramatic outgrowth of Bacteroidetes over Firmicutes phylum with the prevalence of Bacteroides genera in the faeces of IL-23-deficient mice after HFD. In summary, IL-23 appears to maintain the Th17 response and neutrophil migration into the intestinal mucosa, minimizing the gut dysbiosis and protecting against obesity and metabolic disease development in mice.

The ability of baroreflex activation to improve blood pressure and resistance vessel function in spontaneously hypertensive rats is dependent on stimulation parameters.

Baroreflex activation by electric stimulation of the carotid sinus (CS) effectively lowers blood pressure. However, the degree to which differences between stimulation protocols impinge on cardiovascular outcomes has not been defined. To address this, we examined the effects of short- and long-duration (SD and LD) CS stimulation on hemodynamic and vascular function in spontaneously hypertensive rats (SHRs). We fit animals with miniature electrical stimulators coupled to electrodes positioned around the left CS nerve that delivered intermittent 5/25 s ON/OFF (SD) or 20/20 s ON/OFF (LD) square pulses (1 ms, 3 V, 30 Hz) continuously applied for 48 h in conscious animals. A sham-operated control group was also studied. We measured mean arterial pressure (MAP), systolic blood pressure variability (SBPV), heart rate (HR), and heart rate variability (HRV) for 60 min before stimulation, 24 h into the protocol, and 60 min after stimulation had stopped. SD stimulation reversibly lowered MAP and HR during stimulation. LD stimulation evoked a decrease in MAP that was sustained even after stimulation was stopped. Neither SD nor LD had any effect on SBPV or HRV when recorded after stimulation, indicating no adaptation in autonomic activity. Both the contractile response to phenylephrine and the relaxation response to acetylcholine were increased in mesenteric resistance vessels isolated from LD-stimulated rats only. In conclusion, the ability of baroreflex activation to modulate hemodynamics and induce lasting vascular adaptation is critically dependent on the electrical parameters and duration of CS stimulation.

Author Correction: The inflammasome NLRP3 plays a dual role on mouse corpora cavernosa relaxation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

NOD2 Deficiency Promotes Intestinal CD4+ T Lymphocyte Imbalance, Metainflammation, and Aggravates Type 2 Diabetes in Murine Model.

Type 2 diabetes (T2D) is a metabolic disease characterized by increased inflammation, NOD-like receptors (NLRs) activation and gut dysbiosis. Our research group has recently reported that intestinal Th17 response limits gut dysbiosis and LPS translocation to visceral adipose tissue (VAT), protecting against metabolic syndrome. However, whether NOD2 receptor contributes intestinal Th17 immunity, modulates dysbiosis-driven metabolic tissue inflammation, and obesity-induced T2D remain poorly understood. In this context, we observed that mice lacking NOD2 fed a high-fat diet (HFD) display severe obesity, exhibit greater adiposity, and more hepatic steatosis compared to HFD-fed wild-type (WT) mice. In addition, they develop increased hyperglycemia, worsening of glucose intolerance, and insulin resistance. Notably, the deficiency of NOD2 causes a deviation from M2 macrophage and regulatory T cells (Treg) to M1 macrophage and mast cells into VAT compared to WT mice fed HFD. An imbalance was also observed in Th17/Th1 cell populations, with reduced IL-17 and IL-22 gene expression in the mesenteric lymph nodes (MLNs) and ileum, respectively, of NOD2-deficient mice fed HFD. 16S rRNA sequencing indicates lower richness, alpha diversity, and a depletion of Allobaculum, Lactobacillus, and enrichment with Bacteroides genera in these mice compared to HFD-fed WT mice. These alterations were associated with disrupted tight-junctions expression, augmented serum LPS, and bacterial translocation into VAT. Overall, NOD2 activation is required for a protective Th17 over Th1 immunity in the gut, which seems to decrease gram-negative bacteria outgrowth in gut microbiota, attenuating the endotoxemia, metainflammation, and protecting against obesity-induced T2D.

Author Correction: Baroreceptor denervation reduces inflammatory status but worsens cardiovascular collapse during systemic inflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Baroreceptor denervation reduces inflammatory status but worsens cardiovascular collapse during systemic inflammation.

Beyond the regulation of cardiovascular function, baroreceptor afferents play polymodal roles in health and disease. Sepsis is a life-threatening condition characterized by systemic inflammation (SI) and hemodynamic dysfunction. We hypothesized that baroreceptor denervation worsens lipopolysaccharide (LPS) induced-hemodynamic collapse and SI in conscious rats. We combined: (a) hemodynamic and thermoregulatory recordings after LPS administration at a septic-like non-lethal dose (b) analysis of the cardiovascular complexity, (c) evaluation of vascular function in mesenteric resistance vessels, and (d) measurements of inflammatory cytokines (plasma and spleen). LPS-induced drop in blood pressure was higher in sino-aortic denervated (SAD) rats. LPS-induced hemodynamic collapse was associated with SAD-dependent autonomic disbalance. LPS-induced vascular dysfunction was not affected by SAD. Surprisingly, SAD blunted LPS-induced surges of plasma and spleen cytokines. These data indicate that baroreceptor afferents are key to alleviate LPS-induced hemodynamic collapse, affecting the autonomic control of cardiovascular function, without affecting resistance blood vessels. Moreover, baroreflex modulation of the LPS-induced SI and hemodynamic collapse are not dependent of each other given that baroreceptor denervation worsened hypotension and reduced SI.

Electrical stimulation of the carotid sinus lowers arterial pressure and improves heart rate variability in L-NAME hypertensive conscious rats.

We evaluated the effects of long-term (48 h) electrical stimulation of the carotid sinus (CS) in hypertensive rats. L-NAME-treated (10 days) Wistar rats were implanted with a catheter in the femoral artery and a miniaturized electrical stimulator attached to electrodes positioned around the left CS, encompassing the CS nerve. One day after implantation, arterial pressure (AP) was directly recorded in conscious animals for 60 min. Square pulses (1 ms, 3 V, 30 Hz) were applied intermittently (20/20 s ON/OFF) to the CS for 48 h. After the end of stimulation, AP was recorded again. Nonstimulated rats (control group) and rats without electrodes around the CS (sham-operated) were also studied. Next, the animals were decapitated, and segments of mesenteric resistance arteries were removed to study vascular function. After the stimulation period, AP was 16 ± 5 mmHg lower in the stimulated group, whereas sham-operated and control rats showed similar AP between the first and second recording periods. Heart rate variability (HRV) evaluated using time and frequency domain tools and a nonlinear approach (symbolic analysis) suggested that hypertensive rats with electrodes around the CS, stimulated or not, exhibited a shift in cardiac sympathovagal balance towards parasympathetic tone. The relaxation response to acetylcholine in endothelium-intact mesenteric arteries was enhanced in rats that underwent CS stimulation for 48 h. In conclusion, long-term CS stimulation is effective in reducing AP levels, improving HRV and increasing mesenteric vascular relaxation in L-NAME hypertensive rats. Moreover, only the presence of electrodes around the CS is effective in eliciting changes in HRV similar to those observed in stimulated rats.

Growth hormone attenuates myocardial fibrosis in rats with chronic pressure overload-induced left ventricular hypertrophy.

1. The role of growth hormone (GH) in cardiac remodelling and function in chronic and persistent pressure overload-induced left ventricular hypertrophy has not been defined. The aim of the present study was to assess short-term GH treatment on left ventricular function and remodelling in rats with chronic pressure overload-induced hypertrophy. 2. Twenty-six weeks after induction of ascending aortic stenosis (AAS), rats were treated with daily subcutaneous injections of recombinant human GH (1 mg/kg per day; AAS-GH group) or saline (AAS-P group) for 14 days. Sham-operated animals served as controls. Left ventricular function was assessed by echocardiography before and after GH treatment. Myocardial fibrosis was evaluated by histological analysis. 3. Before GH treatment, AAS rats presented similar left ventricular function and structure. Treatment of rats with GH after the AAS procedure did not change bodyweight or heart weight, both of which were higher in the AAS groups than in the controls. After GH treatment, posterior wall shortening velocity (PWSV) was lower in the AAS-P group than in the control group. However, in the AAS-GH group, PWSV was between that in the control and AAS-P groups and did not differ significantly from either group. Fractional collagen (% of total area) was significantly higher in the AAS-P and AAS-GH groups compared with control (10.34 +/- 1.29, 4.44 +/- 1.37 and 1.88 +/- 0.88%, respectively; P < 0.05) and was higher still in the AAS-P group compared with the AAS-GH group. 4. The present study has shown that short-term administration of GH to rats with chronic pressure overload-induced left ventricular hypertrophy induces cardioprotection by attenuating myocardial fibrosis.

The inflammasome NLRP3 plays a dual role on mouse corpora cavernosa relaxation.

NLRP3 plays a role in vascular diseases. Corpora cavernosa (CC) is an extension of the vasculature. We hypothesize that NLRP3 plays a deleterious role in CC relaxation. Male C57BL/6 (WT) and NLRP3 deficient (NLRP3-/-) mice were used. Intracavernosal pressure (ICP/MAP) measurement was performed. Functional responses were obtained from CC strips of WT and NLRP3-/- mice before and after MCC950 (NLRP3 inhibitor) or LPS + ATP (NLRP3 stimulation). NLRP3, caspase-1, IL-1ß, eNOS, nNOS, guanylyl cyclase-ß1 (GCß1) and PKG1 protein expressions were determined. ICP/MAP and sodium nitroprusside (SNP)-induced relaxation in CC were decreased in NLRP3-/- mice. Caspase-1, IL-1ß and eNOS activity were increased, but PKG1 was reduced in CC of NLRP3-/-. MCC950 decreased non-adrenergic non-cholinergic (NANC), acetylcholine (ACh), and SNP-induced relaxation in WT mice. MCC950 did not alter NLRP3, caspase-1 and IL-1ß, but reduced GCß1 expression. Although LPS + ATP decreased ACh- and SNP-, it increased NANC-induced relaxation in CC from WT, but not from NLRP3-/- mice. LPS + ATP increased NLRP3, caspase-1 and interleukin-1ß (IL-1ß). Conversely, it reduced eNOS activity and GCß1 expression. NLRP3 plays a dual role in CC relaxation, with its inhibition leading to impairment of nitric oxide-mediated relaxation, while its activation by LPS + ATP causes decreased CC sensitivity to NO and endothelium-dependent relaxation.

Mitochondrial DNA Promotes NLRP3 Inflammasome Activation and Contributes to Endothelial Dysfunction and Inflammation in Type 1 Diabetes.

Background: NLRP3 inflammasome activation in response to several signals, including mitochondrial DNA (mDNA), regulates inflammatory responses by caspase-1 activation and interleukin-1ß (IL-1ß) release. Circulating mDNA is linked to micro and macrovascular complications in diabetes. However, a role for mDNA in endothelial dysfunction is not clear. We tested the hypothesis that mDNA contributes to diabetes-associated endothelial dysfunction and vascular inflammation via NLRP3 activation. Methods: Vascular reactivity, reactive oxygen species (ROS) generation, calcium (Ca2+) influx and caspase-1 and IL-1ß activation were determined in mesenteric resistance arteries from normoglicemic and streptozotocin-induced diabetic C57BL/6 and NLRP3 knockout (Nlrp3-/- ) mice. Endothelial cells and mesenteric arteries were stimulated with mDNA from control (cmDNA) and diabetic (dmDNA) mice. Results: Diabetes reduced endothelium-dependent vasodilation and increased vascular ROS generation and caspase-1 and IL-1ß activation in C57BL/6, but not in Nlrp3-/- mice. Diabetes increased pancreatic cytosolic mDNA. dmDNA decreased endothelium-dependent vasodilation. In endothelial cells, dmDNA activated NLRP3 via mitochondrial ROS and Ca2+ influx. Patients with type 1 diabetes exhibited increased circulating mDNA as well as caspase-1 and IL-1ß activation. Conclusion: dmDNA activates endothelial NLRP3 inflammasome by mechanisms that involve Ca2+ influx and mitochondrial ROS generation. NLRP3 deficiency prevents diabetes-associated vascular inflammatory damage and endothelial dysfunction. Our study highlights the importance of NLRP3 inflammasome in diabetes-associated vascular dysfunction, which is key to diabetic complications.

Nrf2 as a Potential Mediator of Cardiovascular Risk in Metabolic Diseases.

Free radicals act as secondary messengers, modulating a number of important biological processes, including gene expression, ion mobilization in transport systems, protein interactions and enzymatic functions, cell growth, cell cycle, redox homeostasis, among others. In the cardiovascular system, the physiological generation of free radicals ensures the integrity and function of cardiomyocytes, endothelial cells, and adjacent smooth muscle cells. In physiological conditions, there is a balance between free radicals generation and the activity of enzymatic and non-enzymatic antioxidant systems. Redox imbalance, caused by increased free radical's production and/or reduced antioxidant defense, plays an important role in the development of cardiovascular diseases, contributing to cardiac hypertrophy and heart failure, endothelial dysfunction, hypertrophy and hypercontractility of vascular smooth muscle. Excessive production of oxidizing agents in detriment of antioxidant defenses in the cardiovascular system has been described in obesity, diabetes mellitus, hypertension, and atherosclerosis. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), a major regulator of antioxidant and cellular protective genes, is primarily activated in response to oxidative stress. Under physiological conditions, Nrf2 is constitutively expressed in the cytoplasm of cells and is usually associated with Keap-1, a repressor protein. This association maintains low levels of free Nrf2. Stressors, such as free radicals, favor the translocation of Nrf2 to the cell nucleus. The accumulation of nuclear Nrf2 allows the binding of this protein to the antioxidant response element of genes that code antioxidant proteins. Although little information on the role of Nrf2 in the cardiovascular system is available, growing evidence indicates that decreased Nrf2 activity contributes to oxidative stress, favoring the pathophysiology of cardiovascular disorders found in obesity, diabetes mellitus, and atherosclerosis. The present mini-review will provide a comprehensive overview of the role of Nrf2 as a contributing factor to cardiovascular risk in metabolic diseases.

Bonus Effects of Antidiabetic Drugs: Possible Beneficial Effects on Endothelial Dysfunction, Vascular Inflammation and Atherosclerosis.

Diabetes currently affects more than 400 million people worldwide. This metabolic disorder causes various micro- and macrovascular complications that accelerate atherosclerosis and yet trigger other cardiovascular diseases. The characteristic frame of hyperglycaemia, hyperinsulinaemia and hyperlipidaemia in diabetes increases several inflammatory mediators leading to endothelial dysfunction and pro-atherosclerotic processes. This MiniReview summarizes evidence that antidiabetic drugs have effects beyond lowering glycaemic levels. In experimental studies, antidiabetic drugs reduce the vascular production and release of pro-inflammatory cytokines, the recruitment, infiltration and activation of immune cells and pro-inflammatory mediators, thus decreasing vascular inflammatory responses; they also re-establish vascular redox homeostasis by reducing oxidative stress and balancing the release of vasoconstrictor and vasodilator factors, hence contributing to the improvement of endothelial function. These effects are associated with a reduction in vascular remodelling due to decreased matrix metalloproteinases expression/activity, reduced inflammatory processes and vascular wall fibrosis. In clinical studies, antidiabetic drugs also reduce the production and release of pro-inflammatory, pro-atherosclerotic and pro-oxidative mediators and improve flow-mediated dilatation, indicating beneficial effects on endothelial function. These bonus effects of antidiabetic drugs may delay and/or reduce the installation and development of the atherosclerotic disease, decrease cardiovascular risk and possibly impact mortality risk, life expectancy and quality.

Reactive oxygen species derived from NAD(P)H oxidase play a role on ethanol-induced hypertension and endothelial dysfunction in rat resistance arteries.

Chronic ethanol consumption is a risk factor for cardiovascular diseases. We studied whether NAD(P)H oxidase-derived reactive oxygen species (ROS) play a role in ethanol-induced hypertension, vascular dysfunction, and protein expression in resistance arteries. Male Wistar rats were treated with ethanol (20 % v/v) for 6 weeks. Ethanol treatment increased blood pressure and decreased acetylcholine-induced relaxation in the rat mesenteric arterial bed (MAB). These responses were attenuated by apocynin (30 mg/kg/day; p.o. gavage). Ethanol consumption increased superoxide anion (O2-) generation and decreased nitrate/nitrite (NO x ) concentration in the rat MAB and apocynin prevented these responses. Conversely, ethanol did not affect the concentration of hydrogen peroxide (H2O2) and reduced glutathione (GSH) or the activity of superoxide dismutase (SOD) and catalase (CAT) in the rat MAB. Ethanol increased interleukin (IL)-10 levels in the rat MAB but did not affect the levels of tumor necrosis factor (TNF)-α, IL-6, or IL-1ß. Ethanol increased the expression of Nox2 and the phosphorylation of SAPK/JNK, but reduced eNOS expression in the rat MAB. Apocynin prevented these responses. However, ethanol treatment did not affect the expression of Nox1, Nox4, p38MAPK, ERK1/2, or SAPK/JNK in the rat MAB. Ethanol increased plasma levels of TBARS, TNF-α, IL-6, IL-1ß, and IL-10, whereas it decreased NO x levels. The major finding of our study is that NAD(P)H oxidase-derived ROS play a role on ethanol-induced hypertension and endothelial dysfunction in resistance arteries. Moreover, ethanol consumption affects the expression and phosphorylation of proteins that regulate vascular function and NAD(P)H oxidase-derived ROS play a role in such responses.

Matrix Metalloproteinase-2 Activity is Associated with Divergent Regulation of Calponin-1 in Conductance and Resistance Arteries in Hypertension-induced Early Vascular Dysfunction and Remodelling.

Matrix metalloproteinase (MMP)-2 participates in hypertension-induced maladaptive vascular remodelling by degrading extra- and intracellular proteins. The consequent extracellular matrix rearrangement and phenotype switch of vascular smooth muscle cells (VSMCs) lead to increased cellular migration and proliferation. As calponin-1 degradation by MMP-2 may lead to VSMC proliferation during hypertension, the hypothesis of this study is that increased MMP-2 activity contributes to early hypertension-induced maladaptive remodelling in conductance and resistance arteries via regulation of calponin-1. The main objective was to analyse whether MMP-2 exerts similar effects on the structure and function of the resistance and conductance arteries during early hypertension. Two-kidney, one-clip (2K-1C) hypertensive male rats and corresponding controls were treated with doxycycline (30 mg/kg/day) or water until reaching one week of hypertension. Systolic blood pressure was increased in 2K-1C rats, and doxycycline did not reduce it. Aortas and mesenteric arteries were analysed. MMP-2 activity and expression were increased in both arteries, and doxycycline reduced it. Significant hypertrophic remodelling and VSMC proliferation were observed in aortas but not in mesenteric arteries of 2K-1C rats. The contractility of mesenteric arteries to phenylephrine was increased in 2K-1C rats, and doxycycline prevented this alteration. The potency of phenylephrine to contract aortas of 2K-1C rats was increased, and doxycycline decreased it. Whereas calponin-1 expression was increased in 2K-1C mesenteric arteries, calponin-1 was reduced in aortas. Doxycycline treatment reverted changes in calponin-1 expression. MMP-2 contributes to hypertrophic remodelling in aortas by decreasing calponin-1 levels, which may result in VSMC proliferation. On the other hand, MMP-2-dependent increased calponin-1 in mesenteric arteries may contribute to vascular hypercontractility in 2K-1C rats. Divergent regulation of calponin-1 by MMP-2 may be an important mechanism that leads to maladaptive vascular effects in hypertension.

Chronic treatment with fluoxetine modulates vascular adrenergic responses by inhibition of pre- and post-synaptic mechanisms.

Fluoxetine, a serotonin reuptake inhibitor (SSRI), has other effects in addition to blocking serotonin reuptake, including changes in the vasomotor tone. Whereas many studies focused on the acute effects of fluoxetine in the vasculature, its chronic effects are still limited. In the present study, we tested the hypothesis that chronic fluoxetine treatment modulates adrenergic vascular responses by interfering with post- and pre-synaptic mechanisms. Wistar rats were treated with vehicle (water) or chronic fluoxetine (10mg/kg/day) for 21 days. Blood pressure (BP) and heart rate were measured. Vascular reactivity was evaluated in perfused mesenteric arterial beds (MAB) and in mesenteric resistance arteries. Protein expression by western blot analysis or immunohistochemistry, ß-arrestin recruitment by BRET and calcium influx by FLIPR assay. Fluoxetine treatment decreased phenylephrine (PE)-induced, but not electrical-field stimulation (EFS)-induced vasoconstriction. Fluoxetine-treated rats exhibited increased KCl-induced vasoconstriction, which was abolished by prazosin. Desipramine, an inhibitor of norepinephrine (NA) reuptake, increased EFS-induced vasoconstrictor response in vehicle-treated, but not in fluoxetine-treated rats. Chronic treatment did not alter vascular expression of α1 adrenoceptor, phosphorylation of PKCα or ERK 1/2 and RhoA. On the other hand, vascular contractions to calcium (Ca2+) as well as Ca2+ influx in mesenteric arteries were increased, while intracellular Ca2+ storage was decreased by the chronic treatment with fluoxetine. In vitro, fluoxetine decreased vascular contractions to PE, EFS and Ca2+, but did not change ß-arrestin activity. In conclusion, chronic treatment with fluoxetine decreases sympathetic-mediated vascular responses by mechanisms that involve inhibition of NA release/reuptake and decreased Ca2+ stores.

Mitochondrial DNA Activates the NLRP3 Inflammasome and Predisposes to Type 1 Diabetes in Murine Model.

Although a correlation between polymorphisms of NOD-like receptor family-pyrin domain containing 3 (NLRP3) and predisposition to type 1 diabetes (T1D) has been identified, the potential function and activation of the NLRP3 inflammasome in T1D have not been clarified. The present study shows that non-obese diabetic mice exhibited increased NLRP3, and pro-IL-1ß gene expression in pancreatic lymph nodes (PLNs). Similar increases in gene expression of NLRP3, apoptosis associated speck like protein (ASC) and pro-IL-1ß were induced by multiple low doses of streptozotocin (STZ) in C57BL/6 mice. In addition, diabetic C57BL/6 mice also exhibited increased IL-1ß protein expression in the pancreatic tissue at day 7, which remained elevated until day 15. Diabetic mice also showed increased positive caspase-1 macrophages in the PLNs, which were decreased in NLRP3-/- mice, but not in ASC-/- mice, after STZ treatment. NLRP3- and IL-1R-deficient mice, but not ASC-deficient mice, showed reduced incidence of diabetes, less insulitis, lower hyperglycemia, and normal insulin levels compared to wild-type (WT) diabetic mice. Notably, these mice also displayed a decrease in IL-17-producing CD4 and CD8 T cells (Th17 and Tc17) and IFN-γ-producing CD4 and CD8 T cells (Th1 and Tc1) in the PLNs. Following STZ treatment to induce T1D, NLRP3-deficient mice also exhibited an increase in myeloid-derived suppressor cell and mast cell numbers in the PLNs along with a significant increase in IL-6, IL-10, and IL-4 expression in the pancreatic tissue. Interestingly, diabetic mice revealed increased circulating expression of genes related to mitochondrial DNA, such as cytochrome b and cytochrome c, but not NADH dehydrogenase subunit 6 (NADH). Mitochondrial DNA (mDNA) from diabetic mice, but not from non-diabetic mice, induced significant IL-1ß production and caspase-1 activation by WT macrophages, which was reduced in NLRP3-/- macrophages. Finally, mDNA administration in vivo increased Th17/Tc17/Th1/Tc1 cells in the PLNs and precipitated T1D onset, which was abolished in NLRP3-/- mice. Overall, our results demonstrate that mDNA-mediated NLRP3 activation triggers caspase-1-dependent IL-1ß production and contributes to pathogenic cellular responses during the development of STZ-induced T1D.

Chronic fluoxetine treatment increases NO bioavailability and calcium-sensitive potassium channels activation in rat mesenteric resistance arteries.

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), has effects beyond its antidepressant properties, altering, e.g., mechanisms involved in blood pressure and vasomotor tone control. Although many studies have addressed the acute impact of fluoxetine on the cardiovascular system, there is a paucity of information on the chronic vascular effects of this SSRI. We tested the hypothesis that chronic fluoxetine treatment enhances the vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling and activation of potassium (K+) channels. Wistar rats were divided into two groups: (I) vehicle (water for 21 days) or (II) chronic fluoxetine (10 mg/kg/day in the drinking water for 21 days). Fluoxetine treatment increased endothelium-dependent and independent vasorelaxation (analyzed by mesenteric resistance arteries reactivity) as well as constitutive NO synthase (NOS) activity, phosphorylation of eNOS at Serine1177 and NO production, determined by western blot and fluorescence. On the other hand, fluoxetine treatment did not alter vascular expression of neuronal and inducible NOS or guanylyl cyclase (GC). Arteries from fluoxetine-treated rats exhibited increased relaxation to pinacidil. Increased acetylcholine vasorelaxation was abolished by a calcium-activated K+ channel (KCa) blocker, but not by an inhibitor of KATP channels. On the other hand, vascular responses to Bay 41-2272 and 8-bromo-cGMP were similar between the groups. In conclusion, chronic fluoxetine treatment increases endothelium-dependent and independent relaxation of mesenteric resistance arteries by mechanisms that involve increased eNOS activity, NO generation, and KCa channels activation. These effects may contribute to the cardiovascular effects associated with chronic fluoxetine treatment.

Identification of a Passiflora alata Curtis dimeric peptide showing identity with 2S albumins.

Antifungal proteins and peptides, essential compounds for plant defense, have been isolated from several tissues of various plants. These proteins could be used as a natural alternative to control phytopathogenic fungi. In this report a heterodimeric antifungal protein named Pa-AFP1, showing higher identity with the 2S albumin family, was purified by using 70-100% ammonium sulfate saturation and further purification steps such as anionic exchange Q-Sepharose chromatography associated with HPLC reversed-phase C4 chromatography. Analysis by Tricine-SDS-PAGE revealed two peptidic molecular masses of approximately 4500 Da and 7000 Da, in the presence of ß-mercaptoethanol, while by removing the reducing agent a single protein with molecular mass of about 11,500 Da was obtained. Moreover, dimer mass was confirmed by MALDI-TOF analyses (11,569.76 Da). The antifungal protein, named Pa-AFP1, efficiently inhibited the growth of filamentous fungi Colletotrichum gloeosporioides, and was added to a short list of 2S albumins with antimicrobial properties. Otherwise, this same peptide showed no activity toward bacteria and yeasts. In summary, this compound could be used in the future to develop biotechnological products for the control of phytopathogenic fungi.