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Significance: Despite their intrinsic cytotoxic properties, mounting evidence indicates that reactive oxygen species (ROS) physiologically produced by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) of epithelial cells (NOX1, dual oxidase [DUOX]2) and phagocytes (NOX2) are critical for innate immune response and homeostasis of the intestinal mucosa. However, dysregulated ROS production could be a driving factor in inflammatory bowel diseases (IBDs). Recent Advances: In addition to NOX2, recent studies have demonstrated that NOX1- and DUOX2-derived ROS can regulate intestinal innate immune defense and homeostasis by impacting many processes, including bacterial virulence, expression of bacteriostatic proteins, epithelial renewal and restitution, and microbiota composition. Moreover, the antibacterial role of DUOX2 is a function conserved in evolution as it has been described in invertebrates, and lower and higher vertebrates. In humans, variants of the NOX2, NOX1, and DUOX2 genes, which are associated with impaired ROS production, have been identified in very early onset IBD, but overexpression of NOX/DUOX, especially DUOX2, has also been described in IBD, suggesting that loss-of-function or excessive activity of the ROS-generating enzymes could contribute to disease progression. Critical Issues: Therapeutic perspectives aiming at targeting NOX/DUOX in IBD should take into account the two sides of NOX/DUOX-derived ROS in intestinal inflammation. Hence, NOX/DUOX inhibitors or ROS inducers should be considered as a function of the disease context. Future Directions: A thorough understanding of the physiological and pathological regulation of NOX/DUOX in the gastrointestinal tract is an absolute pre-requisite for the development of therapeutic strategies that can modulate ROS levels in space and time.

Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Although neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we report that vascular permeability–enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of neutrophil rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the bloodstream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labeling method we provide direct evidence for the systemic dissemination of rTEM neutrophils, and showed them to exhibit an activated phenotype and be capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrate that increased microvascular leakage reverses the localization of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to reenter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.

Hutchinson–Gilford progeria syndrome (HGPS, progeria) is a rare genetic disease characterized by premature aging and death in childhood for which there were no approved drugs for its treatment until last November, when lonafarnib obtained long-sought FDA approval. However, the benefits of lonafarnib in patients are limited, highlighting the need for new therapeutic strategies. Here, we validate the enzyme isoprenylcysteine carboxylmethyltransferase (ICMT) as a new therapeutic target for progeria with the development of a new series of potent inhibitors of this enzyme that exhibit an excellent antiprogeroid profile. Among them, compound UCM-13207 significantly improved the main hallmarks of progeria. Specifically, treatment of fibroblasts from progeroid mice with UCM-13207 delocalized progerin from the nuclear membrane, diminished its total protein levels, resulting in decreased DNA damage, and increased cellular viability. Importantly, these effects were also observed in patient-derived cells. Using the LmnaG609G/G609G progeroid mouse model, UCM-13207 showed an excellent in vivo efficacy by increasing body weight, enhancing grip strength, extending lifespan by 20%, and decreasing tissue senescence in multiple organs. Furthermore, UCM-13207 treatment led to an improvement of key cardiovascular hallmarks such as reduced progerin levels in aortic and endocardial tissue and increased number of vascular smooth muscle cells (VSMCs). The beneficial effects go well beyond the effects induced by other therapeutic strategies previously reported in the field, thus supporting the use of UCM-13207 as a new treatment for progeria., Isoprenylcysteine carboxylmethyltransferase (ICMT) inhibitor induces progerin delocalization from the nuclear rim and decreases its levels, significantly improving the main hallmarks of progeria.

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function.

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47phox , p67phox , p40phox and Rac2) with the transmembrane proteins (p22phox and gp91phox , which form the cytochrome b558 ). gp91phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47phox and p40phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91phox , p22phox , p47phox , p67phox and p40phox , in the activation of this enzyme.

Background & Aims Myeloperoxidase exocytosis and production of hydrogen peroxide via the neutrophil superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contribute to efficient elimination of bacteria. Cirrhosis impairs immune functions and increases susceptibility to bacterial infection. We recently showed that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a severe impairment of formylpeptide receptor (fPR)-mediated intracellular signaling and superoxide production. Here, we performed ex vivo studies with these patients' neutrophils to further investigate myeloperoxidase release, bactericidal capacity and signaling events following fPR stimulation by the formylpeptide formyl-met-leu-phe (fMLP). Methods Myeloperoxidase release was studied by measuring extracellular myeloperoxidase activity. Activation of signaling effectors was studied by Western blot and their respective contribution to myeloperoxidase release studied using pharmacological antagonists. Results fMLP-induced myeloperoxidase release was strongly impaired in patients' neutrophils whereas the intracellular myeloperoxidase stock was unaltered. The fMLP-induced phosphorylation of major signaling effectors, AKT, ERK1/2 and p38-MAP-Kinases, was also strongly deficient despite a similar expression of signaling effectors or fPR. However, based on effector inhibition in healthy neutrophils, AKT and p38-MAPK but not ERK1/2 upregulated fMLP-induced myeloperoxidase exocytosis. Interestingly, patients' neutrophils exhibited a defective bactericidal capacity that was reversed ex vivo by the TLR7/8 agonist CL097, through potentiation of the fMLP-induced AKT/p38-MAPK signaling axis and myeloperoxidase release. Conclusions We provide first evidence that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a deficient AKT/p38-MAPK signaling, myeloperoxidase release and bactericidal activity, which can be reversed via TLR7/8 activation. These defects, together with the previously described severe deficient superoxide production, may increase cirrhotic patients' susceptibility to bacterial infections.

Cirrhosis is commonly accompanied by impaired defense functions of polymorphonuclear leucocytes (PMNs), increased patient susceptibility to infections, and hepatocellular carcinoma (HCC). PMN antimicrobial activity is dependent on a massive production of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) 2 (NADPH oxidase 2; NOX2), termed respiratory burst (RB). Rapamycin, an antagonist of mammalian target of rapamycin (mTOR), may be used in the treatment of HCC and in transplanted patients. However, the effect of mTOR inhibition on the PMN RB of patients with cirrhosis remains unexplored and was studied here using the bacterial peptide, formyl-Met-Leu-Phe (fMLP), as an RB inducer. fMLP-induced RB of PMN from patients with decompensated alcoholic cirrhosis was strongly impaired (30%-35% of control) as a result of intracellular signaling alterations. Blocking mTOR activation (phospho-S2448-mTOR) with rapamycin further aggravated the RB defect. Rapamycin also inhibited the RB of healthy PMNs, which was associated with impaired phosphorylation of the NOX2 component, p47phox (phox: phagocyte oxidase), on its mitogen-activated protein kinase (MAPK) site (S345) as well as a preferential inhibition of p38-MAPK relative to p44/42-MAPK. However, rapamycin did not alter the fMLP-induced membrane association of p47phox and p38-MAPK in patients' PMNs, but did prevent their phosphorylation at the membranes. The mTOR contribution to fMLP-induced RB, phosphorylation of p47phox and p38-MAPK was further confirmed by mTOR knockdown in HL-60 cells. Finally, rapamycin impaired PMN bactericidal activity, but not bacterial uptake. Conclusion: mTOR significantly up-regulates the PMN RB of patients with cirrhosis by p38-MAPK activation. Consequently, mTOR inhibition by rapamycin dramatically aggravates their PMN RB defect, which may increase patients' susceptibility to infection. Thus, concerns should be raised about the use of rapamycin in immuno-depressed patients. (HEPATOLOGY 2013)