P2X7 receptor activation increases expression of caveolin-1 and formation of macrophage lipid rafts, thereby boosting CD39 activity.

Macrophages are tissue-resident immune cells that are crucial for the initiation and maintenance of immune responses. Purinergic signaling modulates macrophage activity and impacts cellular plasticity. The ATP-activated purinergic receptor P2X7 (also known as P2RX7) has pro-inflammatory properties, which contribute to macrophage activation. P2X7 receptor signaling is, in turn, modulated by ectonucleotidases, such as CD39 (also known as ENTPD1), expressed in caveolae and lipid rafts. Here, we examined P2X7 receptor activity and determined impacts on ectonucleotidase localization and function in macrophages primed with lipopolysaccharide (LPS). First, we verified that ATP boosts CD39 activity and caveolin-1 protein expression in LPS-primed macrophages. Drugs that disrupt cholesterol-enriched domains - such as nystatin and methyl-ß-cyclodextrin - decreased CD39 enzymatic activity in all circumstances. We noted that CD39 colocalized with lipid raft markers (flotillin-2 and caveolin-1) in macrophages that had been primed with LPS followed by treatment with ATP. P2X7 receptor inhibition blocked these ATP-mediated increases in caveolin-1 expression and inhibited the colocalization with CD39. Further, we found that STAT3 activation is significantly attenuated caveolin-1-deficient macrophages treated with LPS or LPS+BzATP. Taken together, our data suggest that P2X7 receptor triggers the initiation of lipid raft-dependent mechanisms that upregulates CD39 activity and could contribute to limit macrophage responses restoring homeostasis.

Injury-Induced Shedding of Extracellular Vesicles Depletes Endothelial Cells of Cav-1 (Caveolin-1) and Enables TGF-ß (Transforming Growth Factor-ß)-Dependent Pulmonary Arterial Hypertension.

Objective- To determine whether pulmonary arterial hypertension is associated with endothelial cell (EC)-Cav-1 (caveolin-1) depletion, EC-derived extracellular vesicle cross talk with macrophages, and proliferation of Cav-1 depleted ECs via TGF-ß (transforming growth factor-ß) signaling. Approach and Results- Pulmonary vascular disease was induced in Sprague-Dawley rats by exposure to a single injection of VEGFRII (vascular endothelial growth factor receptor II) antagonist SU5416 (Su) followed by hypoxia (Hx) plus normoxia (4 weeks each-HxSu model) and in WT (wild type; Tie2.Cre-; Cav1 lox/lox) and EC- Cav1-/- (Tie2.Cre+; Cav1 fl/fl) mice (Hx: 4 weeks). We observed reduced lung Cav-1 expression in the HxSu rat model in association with increased Cav-1+ extracellular vesicle shedding into the circulation. Whereas WT mice exposed to hypoxia exhibited increased right ventricular systolic pressure and pulmonary microvascular thickening compared with the group maintained in normoxia, the remodeling was further increased in EC- Cav1-/- mice indicating EC Cav-1 expression protects against hypoxia-induced pulmonary hypertension. Depletion of EC Cav-1 was associated with reduced BMPRII (bone morphogenetic protein receptor II) expression, increased macrophage-dependent TGF-ß production, and activation of pSMAD2/3 signaling in the lung. In vitro, in the absence of Cav-1, eNOS (endothelial NO synthase) dysfunction was implicated in the mechanism of EC phenotype switching. Finally, reduced expression of EC Cav-1 in lung histological sections from human pulmonary arterial hypertension donors was associated with increased plasma concentration of Cav-1, extracellular vesicles, and TGF-ß, indicating Cav-1 may be a plasma biomarker of vascular injury and key determinant of TGF-ß-induced pulmonary vascular remodeling. Conclusions- EC Cav-1 depletion occurs, in part, via Cav-1+ extracellular vesicle shedding into the circulation, which contributes to increased TGF-ß signaling, EC proliferation, vascular remodeling, and pulmonary arterial hypertension.

Sevoflurane Promotes Bactericidal Properties of Macrophages through Enhanced Inducible Nitric Oxide Synthase Expression in Male Mice.

BACKGROUND: Sevoflurane with its antiinflammatory properties has shown to decrease mortality in animal models of sepsis. However, the underlying mechanism of its beneficial effect in this inflammatory scenario remains poorly understood. Macrophages play an important role in the early stage of sepsis as they are tasked with eliminating invading microbes and also attracting other immune cells by the release of proinflammatory cytokines such as interleukin-1ß, interleukin-6, and tumor necrosis factor-α. Thus, the authors hypothesized that sevoflurane mitigates the proinflammatory response of macrophages, while maintaining their bactericidal properties. METHODS: Murine bone marrow-derived macrophages were stimulated in vitro with lipopolysaccharide in the presence and absence of 2% sevoflurane. Expression of cytokines and inducible NO synthase as well as uptake of fluorescently labeled Escherichia coli (E. coli) were measured. The in vivo endotoxemia model consisted of an intraperitoneal lipopolysaccharide injection after anesthesia with either ketamine and xylazine or 4% sevoflurane. Male mice (n = 6 per group) were observed for a total of 20 h. During the last 30 min fluorescently labeled E. coli were intraperitoneally injected. Peritoneal cells were extracted by peritoneal lavage and inducible NO synthase expression as well as E. coli uptake by peritoneal macrophages was determined using flow cytometry. RESULTS: In vitro, sevoflurane enhanced lipopolysaccharide-induced inducible NO synthase expression after 8 h by 466% and increased macrophage uptake of fluorescently labeled E. coli by 70% compared with vehicle-treated controls. Inhibiting inducible NO synthase expression pharmacologically abolished this increase in bacteria uptake. In vivo, inducible NO synthase expression was increased by 669% and phagocytosis of E. coli by 49% compared with the control group. CONCLUSIONS: Sevoflurane enhances phagocytosis of bacteria by lipopolysaccharide-challenged macrophages in vitro and in vivo via an inducible NO synthase-dependent mechanism. Thus, sevoflurane potentiates bactericidal and antiinflammatory host-defense mechanisms in endotoxemia.

Caveolin and Endothelial NO Signaling.

Pulmonary vascular diseases are associated with several factors including infection, cigarette smoking, abuse of dietary suppressants and drugs, prolonged exposure to high altitude, and other causes which in part induce significant oxidative stress resulting in endothelial cell injury, apoptosis, hyperproliferation, and vaso-occlusive disease. Maintenance of normal endothelial cell function is a critical role of endothelial nitric oxide synthase (eNOS) activity and physiologic nitric oxide (NO) signaling in the vascular wall. eNOS expression and activity is regulated by the membrane-associated scaffolding protein caveolin-1 (Cav-1), the main protein constituent of caveolae. This chapter summarizes the literature and highlights unanswered questions related to how inflammation-associated oxidative stress affects Cav-1 expression and regulatory functions, and how dysregulated eNOS enzymatic activity promotes endothelial dysfunction. Focus is given to how the conversion of eNOS from a NO-producing enzyme to a transient oxidant-generating system is associated twith Cav-1 depletion, endothelial cell injury, and pulmonary vascular diseases. Importantly, the vascular defects observed in absence of Cav-1 that give rise to injured or hyperproliferative endothelial cells and promote remodeled vasculature can be rescued by "re-coupling," inhibiting, or genetically deleting eNOS, supporting the notion that strict control of Cav-1 expression and eNOS activity and signaling is critical for maintaining pulmonary vascular homeostasis.

Inflammation-induced caveolin-1 and BMPRII depletion promotes endothelial dysfunction and TGF-ß-driven pulmonary vascular remodeling.

Endothelial cell (EC) activation and vascular injury are hallmark features of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Caveolin-1 (Cav-1) is highly expressed in pulmonary microvascular ECs and plays a key role in maintaining vascular homeostasis. The aim of this study was to determine if the lung inflammatory response to Escherichia coli lipopolysaccharide (LPS) promotes priming of ECs via Cav-1 depletion and if this contributes to the onset of pulmonary vascular remodeling. To test the hypothesis that depletion of Cav-1 primes ECs to respond to profibrotic signals, C57BL6 wild-type (WT) mice (Tie2.Cre-;Cav1fl/fl ) were exposed to nebulized LPS (10 mg; 1 h daily for 4 days) and compared with EC-specific Cav1-/- (Tie2.Cre+;Cav1fl/fl ). After 96 h of LPS exposure, total lung Cav-1 and bone morphogenetic protein receptor type II (BMPRII) expression were reduced in WT mice. Moreover, plasma albumin leakage, infiltration of immune cells, and levels of IL-6/IL-6R and transforming growth factor-ß (TGF-ß) were elevated in both LPS-treated WT and EC-Cav1-/- mice. Finally, EC-Cav1-/- mice exhibited a modest increase in microvascular thickness basally and even more so on exposure to LPS (96 h). EC-Cav1-/- mice and LPS-treated WT mice exhibited reduced BMPRII expression and endothelial nitric oxide synthase uncoupling, which along with increased TGF-ß promoted TGFßRI-dependent SMAD-2/3 phosphorylation. Finally, human lung sections from patients with ARDS displayed reduced EC Cav-1 expression, elevated TGF-ß levels, and severe pulmonary vascular remodeling. Thus EC Cav-1 depletion, oxidative stress-mediated reduction in BMPRII expression, and enhanced TGF-ß-driven SMAD-2/3 signaling promote pulmonary vascular remodeling in inflamed lungs.

CD39 limits P2X7 receptor inflammatory signaling and attenuates sepsis-induced liver injury.

BACKGROUND & AIMS: The severity of sepsis can be linked to excessive inflammatory responses resulting in hepatic injury. P2X7 receptor activation by extracellular ATP (eATP) exacerbates inflammation by augmenting cytokine production; while CD39 (ENTPD1) scavenges eATP to generate adenosine, thereby limiting P2X7 activation and resulting in A2A receptor stimulation. We aim to determine how the functional interaction of P2X7 receptor and CD39 control the macrophage response, and consequently impact on sepsis and liver injury. METHODS: Sepsis was induced by cecal ligation and puncture in C57BL/6 wild-type (WT) and CD39-/- mice. Several in vitro assays were performed using peritoneal or bone marrow derived macrophages to determine CD39 ectonucleotidase activity and its role in sepsis-induced liver injury. RESULTS: CD39 expression in macrophages limits ATP-P2X7 receptor pro-inflammatory signaling. P2X7 receptor paradoxically boosts CD39 activity. Inhibition and/or deletion of P2X7 receptor in LPS-primed macrophages attenuates cytokine production and inflammatory signaling as well as preventing ATP-induced increases in CD39 activity. Septic CD39-/- mice exhibit higher levels of inflammatory cytokines and show more pronounced liver injury than WT mice. Pharmacological P2X7 blockade largely prevents tissue damage, cell apoptosis, cytokine production, and the activation of inflammatory signaling pathways in the liver from septic WT, while only attenuating these outcomes in CD39-/- mice. Furthermore, the combination of P2X7 blockade with adenosine A2A receptor stimulation completely inhibits cytokine production, the activation of inflammatory signaling pathways, and protects septic CD39-/- mice against liver injury. CONCLUSIONS: CD39 attenuates sepsis-associated liver injury by scavenging eATP and ultimately generating adenosine. We propose boosting of CD39 would suppress P2X7 responses and trigger adenosinergic signaling to limit systemic inflammation and restore liver homeostasis during the acute phase of sepsis. Lay summary: CD39 expression in macrophages limits P2X7-mediated pro-inflammatory responses, scavenging extracellular ATP and ultimately generating adenosine. CD39 genetic deletion exacerbates sepsis-induced experimental liver injury. Combinations of a P2X7 antagonist and adenosine A2A receptor agonist are hepatoprotective during the acute phase of abdominal sepsis.

Targeting chalcone binding sites in living Leishmania using a reversible fluorogenic benzochalcone probe.

Chalcones (1,3-diphenyl-2-propen-1-ones) either natural or synthetic have a plethora of biological properties including antileishmanial activities, but their development as drugs is hampered by their largely unknown mechanisms of action. We demonstrate herein that our previously described benzochalcone fluorogenic probe (HAB) could be imaged by fluorescence microscopy in live Leishmania amazonensis promastigotes where it targeted the parasite acidocalcisomes, lysosomes and the mitochondrion. As in the live zebrafish model, HAB formed yellow-emitting fluorescent complexes when associated with biological targets in Leishmania. Further, we used HAB as a reversible probe to study the binding of a portfolio of diverse chalcones and analogues in live promastigotes, using a combination of competitive flow cytometry analysis and cell microscopy. This pharmacological evaluation suggested that the binding of HAB in promastigotes was representative of chalcone pharmacology in Leishmania, with certain exogenous chalcones exhibiting competitive inhibition (ca. 20-30%) towards HAB whereas non-chalconic inhibitors showed weak capacity (ca. 3-5%) to block the probe intracellular binding. However, this methodology was restricted by the strong toxicity of several competing chalcones at high concentration, in conjunction with the limited sensitivity of the HAB fluorophore. This advocates for further optimization of this undirect target detection strategy using pharmacophore-derived reversible fluorescent probes.

Ultraspecific live imaging of the dynamics of zebrafish neutrophil granules by a histopermeable fluorogenic benzochalcone probe.

Neutrophil granules (NGs) are key components of the innate immune response and mark the development of neutrophilic granulocytes in mammals. However, there has been no specific fluorescent vital stain up to now to monitor their dynamics within a whole live organism. We rationally designed a benzochalcone fluorescent probe (HAB) featuring high tissue permeability and optimal photophysics such as elevated quantum yield, pronounced solvatochromism and target-induced fluorogenesis. Phenotypic screening identified HAB as the first cell- and organelle-specific small-molecule fluorescent tracer of NGs in live zebrafish larvae, with no labeling of other cell types or organelles. HAB staining was independent of the state of neutrophil activation, labeling NGs of both resting and phagocytically active neutrophils with equal specificity. By high-resolution live imaging, we documented the dynamics of HAB-stained NGs during phagocytosis. Upon zymosan injection, labeled NGs were rapidly recruited to the forming phagosomes. Despite being a reversible ligand, HAB could not be displaced by high concentrations of pharmacologically relevant competing chalcones, indicating that this specific labeling was the result of the HAB's precise physicochemical signature rather than a general feature of chalcones. However, one of the competitors was discovered as a promising interstitial fluorescent tracer illuminating zebrafish histology, similarly to BODIPY-ceramide. As a yellow-emitting histopermeable vital stain, HAB functionally and spectrally complements most genetically incorporated fluorescent tags commonly used in live zebrafish biology, holding promise for the study of neutrophil-dependent responses relevant to human physiopathology such as developmental defects, inflammation and infection. Furthermore, HAB intensely labeled isolated live human neutrophils at the level of granulated subcellular structures consistent with human NGs, suggesting that the labeling of NGs by HAB is not restricted to the zebrafish model but also relevant to mammalian systems.

Neutralization of Apis mellifera bee venom activities by suramin.

In this work we evaluated the ability of suramin, a polysulfonated naphthylurea derivative, to antagonize the cytotoxic and enzymatic effects of the crude venom of Apis mellifera. Suramin was efficient to decrease the lethality in a dose-dependent way. The hemoconcentration caused by lethal dose injection of bee venom was abolished by suramin (30 µg/g). The edematogenic activity of the venom (0.3 µg/g) was antagonized by suramin (10 µg/g) in all treatment protocols. The changes in the vascular permeability caused by A. mellifera (1 µg/g) venom were inhibited by suramin (30 µg/g) in the pre- and posttreatment as well as when the venom was preincubated with suramin. In addition, suramin also inhibited cultured endothelial cell lesion, as well as in vitro myotoxicity, evaluated in mouse extensor digitorum longus muscle, which was inhibited by suramin (10 and 25 µM), decreasing the rate of CK release, showing that suramin protected the sarcolemma against damage induced by components of bee venom (2.5 µg/mL). Moreover, suramin inhibited the in vivo myotoxicity induced by i.m. injection of A. mellifera venom in mice (0.5 µg/g). The analysis of the area under the plasma CK vs. time curve showed that preincubation, pre- and posttreatment with suramin (30 µg/g) inhibited bee venom myotoxic activity in mice by about 89%, 45% and 40%, respectively. Suramin markedly inhibited the PLA2 activity in a concentration-dependent way (1-30 µM). Being suramin a polyanion molecule, the effects observed may be due to the interaction of its charges with the polycation components present in A. mellifera bee venom.

Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF). Nitric oxide (NO) donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS) increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially explained by a reduced eNOS expression. In addition, our data show that the disease primes endothelial cells in vivo, which keep the acquired phenotype in culture.