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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.

Here we investigated the role of murine mast cell protease 4 (MCPT4), the functional counterpart of human mast cell chymase, in an experimental model of renal ischemia reperfusion injury, a major cause of acute kidney injury. MCPT4-deficient mice had worsened kidney function compared to wildtype mice. MCPT4 absence exacerbated pathologic neutrophil infiltration in the kidney and increased kidney myeloperoxidase expression, cell death and necrosis. In kidneys with ischemia reperfusion injury, when compared to wildtype mice, MCPT4-deficient mice showed increased surface expression of adhesion molecules necessary for leukocyte extravasation including neutrophil CD162 and endothelial cell CD54. In vitro, human chymase mediated the cleavage of neutrophil expressed CD162 and also CD54, P- and E-Selectin expressed on human glomerular endothelial cells. MCPT4 also dampened systemic neutrophil activation after renal ischemia reperfusion injury as neutrophils expressed more CD11b integrin and produced more reactive oxygen species in MCPT4-deficient mice. Accordingly, after renal injury, neutrophil migration to an inflammatory site distal from the kidney was increased in MCPT4-deficient versus wildtype mice. Thus, contrary to the described overall aggravating role of mast cells, one granule-released mediator, the MCPT4 chymase, exhibits a potent anti-inflammatory function in renal ischemia reperfusion injury by controlling neutrophil extravasation and activation thereby limiting associated damage.

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.

Summary Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging., Graphical abstract, Highlights • Aged mice show high levels of neutrophil reverse transendothelial migration (rTEM) • Mast cells (MC) and MC-derived CXCL1 drive neutrophil rTEM in inflamed aged tissues • Intensified endothelial ACKR1-CXCL1 axis promotes neutrophil CXCR2 internalization • Aged lungs program rTEM neutrophils toward an activated and noxious phenotype, Aging is a critical risk factor for inflammatory disorders. Barkaway, Rolas et al. show that inflamed aged tissues present a high frequency of neutrophil reverse transendothelial migration (rTEM) back into the circulation in a mast cell-dependent manner. rTEM neutrophils are retained in aged lungs and programmed toward an activated phenotype, capable of inducing tissue damage.

Leukocyte trafficking is a key component of steady-state tissue homing and in mounting an inflammatory response. Two recent publications in Immunity by He et al. (2018) and Adrover et al. (2019) report on the diurnal regulation of these responses and the associated pathophysiological implications.

Summary The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation., Graphical abstract, Highlights • Inflamed venular ECs exhibit an autophagic response that localizes to EC contacts • EC ATG5 deficiency promotes excessive and faster neutrophil TEM • Ablation of EC autophagy increases cell surface expression of adhesion molecules • Non-canonical autophagy operates in inflamed ECs and controls neutrophil migration, Transendothelial cell migration (TEM) is a vital step in neutrophil infiltration of tissues, but the molecular basis of its cessation is unclear. Reglero-Real et al. show that in inflamed tissues, endothelial cell autophagy remodels junctional architecture and acts as a negative regulator of neutrophil TEM.

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.

The Instituto Universitario de Oncología del Principado de Asturias is supported by Fundación Bancaria Caja de Ahorros de Asturias. J.M.P.F. is supported by Ministerio de Economía y Competitividad (MINECO/FEDER: No. SAF2015-64157-R) and Gobierno del Principado de Asturias. C.L.-O. is supported by grants from the European Research Council (ERC-2016-ADG, DeAge), Ministerio de Economía y Competitividad (MINECO/FEDER: Nos. SAF2014-52413-R and SAF2017-87655-R), Instituto de Salud Carlos III (RTICC) and Progeria Research Foundation (No. PRF2016-66). O.S.-F. is recipient of an FPU fellowship. A.R.F. is recipient of a Ramón y Cajal fellowship. The generation of progerin antibody was funded by the Wellcome Trust (No. 098291/Z/12/Z to S.N.).

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.