P-selectin mobility undergoes a sol-gel transition as it diffuses from exocytosis sites into the cell membrane.

In response to vascular damage, P-selectin molecules are secreted onto the surface of cells that line our blood vessels. They then serve as mechanical anchors to capture leucocytes from the blood stream. Here, we track individual P-selectin molecules released at the surface of live endothelial cells following stimulated secretion. We find P-selectin initially shows fast, unrestricted diffusion but within a few minutes, movement becomes increasingly restricted and ~50% of the molecules become completely immobile; a process similar to a sol-gel transition. We find removal of the extracellular C-type lectin domain (ΔCTLD) and/or intracellular cytoplasmic tail domain (ΔCT) has additive effects on diffusive motion while disruption of the adapter complex, AP2, or removal of cell-surface heparan sulphate restores mobility of full-length P-selectin close to that of ΔCT and ΔCTLD respectively. We have found P-selectin spreads rapidly from sites of exocytosis and evenly decorates the cell surface, but then becomes less mobile and better-suited to its mechanical anchoring function.

Phagocytosis depends on TRPV2-mediated calcium influx and requires TRPV2 in lipids rafts: alteration in macrophages from patients with cystic fibrosis.

Whereas many phagocytosis steps involve ionic fluxes, the underlying ion channels remain poorly defined. As reported in mice, the calcium conducting TRPV2 channel impacts the phagocytic process. Macrophage phagocytosis is critical for defense against pathogens. In cystic fibrosis (CF), macrophages have lost their capacity to act as suppressor cells and thus play a significant role in the initiating stages leading to chronic inflammation/infection. In a previous study, we demonstrated that impaired function of CF macrophages is due to a deficient phagocytosis. The aim of the present study was to investigate TRPV2 role in the phagocytosis capacity of healthy primary human macrophage by studying its activity, its membrane localization and its recruitment in lipid rafts. In primary human macrophages, we showed that P. aeruginosa recruits TRPV2 channels at the cell surface and induced a calcium influx required for bacterial phagocytosis. We presently demonstrate that to be functional and play a role in phagocytosis, TRPV2 might require a preferential localization in lipid rafts. Furthermore, CF macrophage displays a perturbed calcium homeostasis due to a defect in TRPV2. In this context, deregulated TRPV2-signaling in CF macrophages could explain their defective phagocytosis capacity that contribute to the maintenance of chronic infection.

Soluble CD14 acts as a DAMP in human macrophages: origin and involvement in inflammatory cytokine/chemokine production.

The innate immune system is able to detect bacterial LPS through the pattern recognition receptor CD14, which delivers LPS to various TLR signaling complexes that subsequently induce intracellular proinflammatory signaling cascades. In a previous study, we showed the overproduction of the soluble form of CD14 (sCD14) by macrophages from patients with cystic fibrosis (CF). CF is an autosomal recessive disorder that is caused by mutations in the gene that encodes the CFTR protein and is characterized by persistent inflammation. Macrophages play a significant role in the initial stages of this disease due to their inability to act as suppressor cells, leading to chronic inflammation in CF. In this work, we investigated the origin of sCD14 by human macrophages and studied the effect of sCD14 on the production of inflammatory cytokine/chemokine. Our data indicate that sCD14 stimulate proinflammatory cytokine/chemokine production in a manner that is independent of LPS but dependent on the TLR-4/CD14 membrane complex, NF-κB, and the inflammasome. Therefore, sCD14, overproduced by CF macrophages, originates primarily from the endocytosis/exocytosis process and should be considered to be a danger-associated molecular pattern. This elucidation of the origin and inflammation-induced mechanisms associated with sCD14 contributes to our understanding of maintained tissue inflammation.-Lévêque, M., Simonin-Le Jeune, K., Jouneau, S., Moulis, S., Desrues, B., Belleguic, C., Brinchault, G., Le Trionnaire, S., Gangneux, J.-P., Dimanche-Boitrel, M.-T., Martin-Chouly, C. Soluble CD14 acts as a DAMP in human macrophages: origin and involvement in inflammatory cytokine/chemokine production.

The impact of impaired macrophage functions in cystic fibrosis disease progression.

The underlying cause of morbidity in cystic fibrosis (CF) is the decline in lung function, which results in part from chronic inflammation. Inflammation and infection occur early in infancy in CF and the role of innate immune defense in CF has been highlighted in the last years. Once thought simply to be consumers of bacteria, macrophages have emerged as highly sensitive immune cells that are located at the balance point between inflammation and resolution of this inflammation in CF pathophysiology. In order to assess the potential role of macrophage in CF, we review the evidence that: (1) CF macrophage has a dysregulated inflammatory phenotype; (2) CF macrophage presents altered phagocytosis capacity and bacterial killing; and (3) lipid disorders in CF macrophage affect its function. These alterations of macrophage weaken innate defense of CF patients and may be involved in CF disease progression and lung damage.

The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro.

The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30-300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H2S donors (30-300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications.

AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro.

The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted H2S donor, AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], on bioenergetics, viability, and mitochondrial DNA integrity in bEnd.3 murine microvascular endothelial cells in vitro, under normal conditions, and during oxidative stress. Intracellular H2S was assessed by the fluorescent dye 7-azido-4-methylcoumarin. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer was used. Cell viability was estimated by the combination of the MTT and LDH methods. Oxidative protein modifications were measured by the Oxyblot method. Reactive oxygen species production was monitored by the MitoSOX method. Mitochondrial and nuclear DNA integrity were assayed by the Long Amplicon PCR method. Oxidative stress was induced by addition of glucose oxidase. Addition of AP39 (30-300 nM) to bEnd.3 cells increased intracellular H2S levels, with a preferential response in the mitochondrial regions. AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted of a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30-100 nM) and an inhibitory effect at the higher concentration of 300 nM. Under oxidative stress conditions induced by glucose oxidase, an increase in oxidative protein modification and an enhancement in MitoSOX oxidation was noted, coupled with an inhibition of cellular bioenergetic function and a reduction in cell viability. AP39 pretreatment attenuated these responses. Glucose oxidase induced a preferential damage to the mitochondrial DNA; AP39 (100 nM) pretreatment protected against it. In conclusion, the current paper documents antioxidant and cytoprotective effects of AP39 under oxidative stress conditions, including a protection against oxidative mitochondrial DNA damage.

Emerging role of hydrogen sulfide in health and disease: critical appraisal of biomarkers and pharmacological tools.

H2S (hydrogen sulfide) is a well known and pungent gas recently discovered to be synthesized enzymatically in mammalian and human tissues. In a relatively short period of time, H2S has attracted substantial interest as an endogenous gaseous mediator and potential target for pharmacological manipulation. Studies in animals and humans have shown H2S to be involved in diverse physiological and pathophysiological processes, such as learning and memory, neurodegeneration, regulation of inflammation and blood pressure, and metabolism. However, research is limited by the lack of specific analytical and pharmacological tools which has led to considerable controversy in the literature. Commonly used inhibitors of endogenous H2S synthesis have been well known for decades to interact with other metabolic pathways or even generate NO (nitric oxide). Similarly, commonly used H2S donors release H2S far too quickly to be physiologically relevant, but may have therapeutic applications. In the present review, we discuss the enzymatic synthesis of H2S and its emerging importance as a mediator in physiology and pathology. We also critically discuss the suitability of proposed 'biomarkers' of H2S synthesis and metabolism, and highlight the complexities of the currently used pharmacological H2S 'donor' molecules and 'specific' H2S synthesis inhibitors in their application to studying the role of H2S in human disease.