VAMPs sensitive to tetanus toxin are required for cortical granule exocytosis in mouse oocytes.

Fusion of cortical granules with oocyte plasma membrane is one of the most significant secretory events to prevent polyspermy during oocyte activation. Cortical granule exocytosis (CGE) is distinct from most other exocytosis because cortical granules are not renewed after secretion. However, it is thought to be mediated by SNARE complex, which mediates membrane fusion in other exocytoses. SNAREs proteins are divided into Q (glutamine)- and R (arginine)-SNAREs. Q-SNAREs include Syntaxins and SNAP25 family, and R-SNAREs include VAMPs family. In mouse oocytes, Syntaxin4 and SNAP23 have been involved in CGE; nevertheless, it is unknown if VAMP is required. Here, we demonstrated by RT-PCR and immunoblotting that VAMP1 and VAMP3 are expressed in mouse oocyte, and they localized in the cortical region of this cell. Using a functional assay to quantify CGE, we showed that tetanus toxin -which specifically cleavages VAMP1, VAMP2 or VAMP3- inhibited CGE suggesting that at least one VAMP was necessary. Function blocking assays demonstrated that only the microinjection of anti-VAMP1 or anti-VAMP3 antibodies abolished CGE in activated oocytes. These findings demonstrate that R-SNAREs sensitive to tetanus toxin, VAMP1 and VAMP3 -but not VAMP2-, are required for CGE and demonstrate that CGE is mediated by the SNARE complex.

Synaptotagmin 1 regulates cortical granule exocytosis during mouse oocyte activation.

Synaptotagmin 1 (Syt1) is an abundant and important presynaptic vesicle protein that binds Ca2+ for the regulation of synaptic vesicle exocytosis. Our previous study reported its localization and function on spindle assembly in mouse oocyte meiotic maturation. The present study was designed to investigate the function of Syt1 during mouse oocyte activation and subsequent cortical granule exocytosis (CGE) using confocal microscopy, morpholinol-based knockdown and time-lapse live cell imaging. By employing live cell imaging, we first studied the dynamic process of CGE and calculated the time interval between [Ca2+]i rise and CGE after oocyte activation. We further showed that Syt1 was co-localized to cortical granules (CGs) at the oocyte cortex. After oocyte activation with SrCl2, the Syt1 distribution pattern was altered significantly, similar to the changes seen for the CGs. Knockdown of Syt1 inhibited [Ca2+]i oscillations, disrupted the F-actin distribution pattern and delayed the time of cortical reaction. In summary, as a synaptic vesicle protein and calcium sensor for exocytosis, Syt1 acts as an essential regulator in mouse oocyte activation events including the generation of Ca2+ signals and CGE.

Filifactor alocis modulates human neutrophil antimicrobial functional responses.

Filifactor alocis is a newly appreciated pathogen in periodontal diseases. Neutrophils are the predominant innate immune cell in the gingival crevice. In this study, we examined modulation of human neutrophil antimicrobial functions by F. alocis. Both non-opsonised and serum-opsonised F. alocis were engulfed by neutrophils but were not efficiently eliminated. Challenge of neutrophils with either non-opsonised or serum-opsonised F. alocis induced a minimal intracellular as well as extracellular respiratory burst response compared to opsonised Staphylococcus aureus and fMLF, respectively. However, pretreatment or simultaneous challenge of neutrophils with F. alocis did not affect the subsequent oxidative response to a particulate stimulus, suggesting that the inability to trigger the respiratory response was only localised to F. alocis phagosomes. In addition, although neutrophils engulfed live or heat-killed F. alocis with the same efficiency, heat-killed F. alocis elicited a higher intracellular respiratory burst response compared to viable organisms, along with decreased surface expression of CD35, a marker of secretory vesicles. F. alocis phagosomes remained immature by delayed and reduced recruitment of specific and azurophil granules, respectively. These results suggest that F. alocis withstands neutrophil antimicrobial responses by preventing intracellular ROS production, along with specific and azurophil granule recruitment to the bacterial phagosome.

Natural killer cell activity and dysfunction in Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) encompasses disorders with abnormal function of lysosomes and lysosome-related organelles, and some patients who develop immunodeficiency. The basic mechanisms contributing to immune dysfunction in HPS are ill-defined. We analysed natural killer (NK) cells from patients diagnosed with HPS-1, HPS-2, HPS-4, and an unreported HPS subtype. NK cells from an HPS-2 and an unreported HPS subtype share a similar cellular phenotype with defective granule release and cytotoxicity, but differ in cytokine exocytosis. Defining NK cell activity in several types of HPS provides insights into cellular defects of the disorder and understanding of mechanisms contributing to HPS pathogenesis.

Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs.

Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs.

Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells.

Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2A activity. Loss-of-function mutations in MID1 lead to the X-linked Opitz G/BBB syndrome characterized by defective midline development during embryogenesis. Here, we show that MID1 is strongly upregulated in murine cytotoxic lymphocytes (CTLs), and that it controls TCR signaling, centrosome trafficking, and exocytosis of lytic granules. In accordance, we find that the killing capacity of MID1(-/-) CTLs is impaired. Transfection of MID1 into MID1(-/-) CTLs completely rescued lytic granule exocytosis, and vice versa, knockdown of MID1 inhibited exocytosis of lytic granules in WT CTLs, cementing a central role for MID1 in the regulation of granule exocytosis. Thus, MID1 orchestrates multiple events in CTL responses, adding a novel level of regulation to CTL activation and cytotoxicity.

Pharmacologic inhibition of dipeptidyl peptidase 1 (cathepsin C) does not block in vitro granzyme-mediated target cell killing by CD8 T or NK cells.

Recently developed small-molecule inhibitors of the lysosomal protease dipeptidyl peptidase 1 (DPP1), also known as cathepsin C (CatC), can suppress suppurative inflammation in vivo by blocking the processing of zymogenic (pro-) forms of neutrophil serine proteases (NSPs), including neutrophil elastase, proteinase 3, and cathepsin G. DPP1 also plays an important role in activating granzyme serine proteases that are expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Therefore, it is critical to determine whether DPP1 inhibition can also cause off-target suppression of CTL/NK-cell-mediated killing of virus-infected or malignant cells. Herein, we demonstrate that the processing of human granzymes A and B, transitioning from zymogen to active proteases, is not solely dependent on DPP1. Thus, the killing of target cells by primary human CD8+ T cells, NK cells, and gene-engineered anti-CD19 CAR T cells was not blocked in vitro even after prior exposure to high concentrations of the reversible DPP1 inhibitor brensocatib. Consistent with this observation, the turnover of model granzyme A/B peptide substrates in the human CTL/NK cell lysates was not significantly reduced by brensocatib. In contrast, preincubation with brensocatib almost entirely abolished (>90%) both the cytotoxic activity of mouse CD8+ T cells and granzyme substrate turnover. Overall, our finding that the effects of DPP1 inhibition on human cytotoxic lymphocytes are attenuated in comparison to those of mice indicates that granzyme processing/activation pathways differ between mice and humans. Moreover, the in vitro data suggest that human subjects treated with reversible DPP1 inhibitors, such as brensocatib, are unlikely to experience any appreciable deficits in CTL/NK-cell-mediated immunities.

Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion.

Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and small molecules in secretory granules require ion homeostasis to maintain their steady states, and thus need ion conductances in granule membranes. But granular ion channels are still elusive. Here we show that granule exocytosis in neuroendocrine cells delivers to cell surface dominant anion channels, to which chromogranin B (CHGB) is critical. Biochemical fractionation shows that native CHGB distributes nearly equally in soluble and membrane-bound forms, and both reconstitute highly selective anion channels in membrane. Confocal imaging resolves granular membrane components including proton pumps and CHGB in puncta on the cell surface after stimulated exocytosis. High pressure freezing immuno-EM reveals a major fraction of CHGB at granule membranes in rat pancreatic ß-cells. A cryo-EM structure of bCHGB dimer of a nominal 3.5 Å resolution delineates a central pore with end openings, physically sufficient for membrane-spanning and large single channel conductance. Together our data support that CHGB-containing (CHGB+) channels are characteristic of regulated secretion, and function in granule ion homeostasis near the plasma membrane or possibly in other intracellular processes.

All About (NK Cell-Mediated) Death in Two Acts and an Unexpected Encore: Initiation, Execution and Activation of Adaptive Immunity.

NK cells are key mediators of immune cell-mediated cytotoxicity toward infected and transformed cells, being one of the main executors of cell death in the immune system. NK cells recognize target cells through an array of inhibitory and activating receptors for endogenous or exogenous pathogen-derived ligands, which together with adhesion molecules form a structure known as immunological synapse that regulates NK cell effector functions. The main and best characterized mechanisms involved in NK cell-mediated cytotoxicity are the granule exocytosis pathway (perforin/granzymes) and the expression of death ligands. These pathways are recognized as activators of different cell death programmes on the target cells leading to their destruction. However, most studies analyzing these pathways have used pure recombinant or native proteins instead of intact NK cells and, thus, extrapolation of the results to NK cell-mediated cell death might be difficult. Specially, since the activation of granule exocytosis and/or death ligands during NK cell-mediated elimination of target cells might be influenced by the stimulus received from target cells and other microenvironment components, which might affect the cell death pathways activated on target cells. Here we will review and discuss the available experimental evidence on how NK cells kill target cells, with a special focus on the different cell death modalities that have been found to be activated during NK cell-mediated cytotoxicity; including apoptosis and more inflammatory pathways like necroptosis and pyroptosis. In light of this new evidence, we will develop the new concept of cell death induced by NK cells as a new regulatory mechanism linking innate immune response with the activation of tumour adaptive T cell responses, which might be the initiating stimulus that trigger the cancer-immunity cycle. The use of the different cell death pathways and the modulation of the tumour cell molecular machinery regulating them might affect not only tumour cell elimination by NK cells but, in addition, the generation of T cell responses against the tumour that would contribute to efficient tumour elimination and generate cancer immune memory preventing potential recurrences.

The Hunger Games: Aggregatibacter actinomycetemcomitans Exploits Human Neutrophils As an Epinephrine Source for Survival.

Aggregatibacter actinomycetemcomitans is a gram-negative facultative anaerobe and an opportunistic oral pathogen, strongly associated with periodontitis and other inflammatory diseases. Periodontitis is a chronic inflammation of the periodontium resulting from the inflammatory response of the host towards the dysbiotic microbial community present at the gingival crevice. Previously, our group identified catecholamines and iron as the signals that activate the QseBC two-component system in A. actinomycetemcomitans, necessary for the organism to acquire iron as a nutrient to survive in the anaerobic environment. However, the source of catecholamines has not been identified. It has been reported that mouse neutrophils can release catecholamines. In periodontitis, large infiltration of neutrophils is found at the subgingival pocket; hence, we wanted to test the hypothesis that A. actinomycetemcomitans exploits human neutrophils as a source for catecholamines. In the present study, we showed that human neutrophils synthesize, store, and release epinephrine, one of the three main types of catecholamines. Human neutrophil challenge with A. actinomycetemcomitans induced exocytosis of neutrophil granule subtypes: secretory vesicles, specific granules, gelatinase granules, and azurophilic granules. In addition, by selectively inhibiting granule exocytosis, we present the first evidence that epinephrine is stored in azurophilic granules. Using QseC mutants, we showed that the periplasmic domain of the QseC sensor kinase is required for the interaction between A. actinomycetemcomitans and epinephrine. Finally, epinephrine-containing supernatants collected from human neutrophils promoted A. actinomycetemcomitans growth and induced the expression of the qseBC operon under anaerobic conditions. Based on our findings, we propose that A. actinomycetemcomitans promotes azurophilic granule exocytosis by neutrophils as an epinephrine source to promote bacterial survival.

Ocean acidification increases polyspermy of a broadcast spawning bivalve species by hampering membrane depolarization and cortical granule exocytosis.

Ensuring that oocytes are fertilized by a single sperm during broadcast spawning is crucial for the fertilization success of many marine invertebrates. Although the adverse impacts of ocean acidification (OA) on various marine species have been revealed in recent years, its impact on polyspermy and the underlying mechanisms involved remain largely unknown. Therefore, in the present study, the effect of OA on polyspermy risk was assessed in a broadcast spawning bivalve, Tegillarca granosa. In addition, the impacts of OA on the two polyspermy blocking processes, the fast block (membrane depolarization) and the permanent block (cortical reaction), were investigated. The results show that the exposure of oocytes to two future OA scenarios (pH 7.8 and pH 7.4) leads to significant increases in polyspermy risk, about 1.70 and 2.38 times higher than the control, respectively. The maximum change in the membrane potential during oocyte membrane depolarization markedly decreased to 15.79 % (pH 7.8) and 34.06 % (pH 7.4) of the control value. Moreover, the duration of oocyte membrane depolarization was significantly reduced to approximately 63.38 % (pH 7.8) and 21.91 % (pH 7.4) of the control. In addition, cortical granule exocytosis, as well as microfilament migration, were significantly arrested by OA treatment. Exposure to future OA scenarios also led to significant reductions in the ATP and Ca2+ content of the oocytes, which may explain the hampered polyspermy blocking. Overall, the present study suggests that OA may significantly increase polyspermy risk in T. granosa by inhibiting membrane depolarization and arresting cortical granule exocytosis.

Case Report: Characterizing the Role of the STXBP2-R190C Monoallelic Mutation Found in a Patient With Hemophagocytic Syndrome and Langerhans Cell Histiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disorder. HLH can be considered as a threshold disease depending on the trigger and the residual NK-cell cytotoxicity. In this study, we analyzed the molecular and functional impact of a novel monoallelic mutation found in a patient with two episodes of HLH. A 9-month-old child was diagnosed at 2 months of age with cutaneous Langerhans cell histiocytosis (LCH). After successful treatment, the patient developed an HLH episode. At 16 month of age, the patient went through an HSCT losing the engraftment 5 months later concomitant with an HLH relapse. The genetic study revealed a monoallelic mutation in the STXBP2 gene (.pArg190Cys). We transfected COS7 cells to analyze the STXBP2-R190C expression and to test the interaction with STX11. We used the RBL-2H3 cell line expressing STXBP2-WT-EGFP or R190C-EGFP for degranulation assays. Mutation STXBP2-R190C did not affect protein expression or interaction with syntaxin-11. However, we have demonstrated that STXBP2-R190C mutation diminishes degranulation in the RBL-2H3 cell line compared with the RBL-2H3 cell line transfected with STXBP2-WT or nontransfected. These results suggest that STXBP2-R190C mutation acts as a modifier of the degranulation process producing a decrease in degranulation. Therefore, under homeostatic conditions, the presence of one copy of STXBP2-R190 could generate sufficient degranulation capacity. However, it is likely that early in life when adaptive immune system functions are not sufficiently developed, an infection may not be resolved with this genetic background, leading to a hyperinflammation syndrome and eventually develop HLH. This analysis highlights the need for functional testing of new mutations to validate their role in genetic susceptibility and to establish the best possible treatment for these patients.

Human Neutrophil Granule Exocytosis in Response to Mycobacterium smegmatis.

Mycobacterium smegmatis rarely causes disease in the immunocompetent, but reported cases of soft tissue infection describe abscess formation requiring surgical debridement for resolution. Neutrophils are the first innate immune cells to accumulate at sites of bacterial infection, where reactive oxygen species and proteolytic enzymes are used to kill microbial invaders. As these phagocytic cells play central roles in protection from most bacteria, we assessed human neutrophil phagocytosis and granule exocytosis in response to serum opsonized or non-opsonized M. smegmatis mc2. Although phagocytosis was enhanced by serum opsonization, M. smegmatis did not induce exocytosis of secretory vesicles or azurophilic granules at any time point tested, with or without serum opsonization. At early time points, opsonized M. smegmatis induced significant gelatinase granule exocytosis compared to non-opsonized bacteria. Differences in granule release between opsonized and non-opsonized M. smegmatis decreased in magnitude over the time course examined, with bacteria also evoking specific granule exocytosis by six hours after addition to cultured primary single-donor human neutrophils. Supernatants from neutrophils challenged with opsonized M. smegmatis were able to digest gelatin, suggesting that complement and gelatinase granule exocytosis can contribute to neutrophil-mediated tissue damage seen in these rare soft tissue infections.

Protocol for Determining the Effect of Neuroendocrine Hormones on Murine ILC Function.

Neuroendocrine hormones are recognized as important mediators of inflammation that participate in the regulation of the magnitude and length of the immune response. It was demonstrated that endogenous glucocorticoids control the function of innate lymphoid cells (ILCs), and this regulatory mechanism is both cell type- and tissue-specific and is required for host protection during infections. We describe here how to analyze in vitro the effects of corticosterone on murine ILCs, using flow cytometry. The protocols described allow for the identification of the specific combination of stimuli with which glucocorticoids cooperate to regulate the function of ILCs. These methods are instrumental to understanding the molecular mechanisms downstream of glucocorticoid receptor activation and can explain the tissue specificity of ILC response to glucocorticoids.

STXBP2-R190C Variant in a Patient With Neonatal Hemophagocytic Lymphohistiocytosis (HLH) and G6PD Deficiency Reveals a Critical Role of STXBP2 Domain 2 on Granule Exocytosis.

Neonatal hemophagocytic lymphohistiocytosis (HLH) is a medical emergency that can be associated with significant morbidity and mortality. Often these patients present with familial HLH (f-HLH), which is caused by gene mutations interfering with the cytolytic pathway of cytotoxic T-lymphocytes (CTLs) and natural killer cells. Here we describe a male newborn who met the HLH diagnostic criteria, presented with profound cholestasis, and carried a maternally inherited heterozygous mutation in syntaxin-binding protein-2 [STXBP2, c.568C>T (p.Arg190Cys)] in addition to a severe pathogenic variant in glucose 6-phosphate dehydrogenase [G6PD, hemizygous c.1153T>C (Cys385Arg)]. Although mutations in STXBP2 gene are associated with f-HLH type 5, the clinical and biological relevance of the p.Arg190Cys mutation identified in this patient was uncertain. To assess its role in disease pathogenesis, we performed functional assays and biochemical and microscopic studies. We found that p.Arg190Cys mutation did not alter the expression or subcellular localization of STXBP2 or STX11, neither impaired the STXBP2/STX11 interaction. In contrast, forced expression of the mutated protein into normal CTLs strongly inhibited degranulation and reduced the cytolytic activity outcompeting the effect of endogenous wild-type STXBP2. Interestingly, arginine 190 is located in a structurally conserved region of STXBP2 where other f-HLH-5 mutations have been identified. Collectively, data strongly suggest that STXBP2-R190C is a deleterious variant that may act in a dominant-negative manner by probably stabilizing non-productive interactions between STXBP2/STX11 complex and other still unknown factors such as the membrane surface or Munc13-4 protein and thus impairing the release of cytolytic granules. In addition to the contribution of STXBP2-R190C to f-HLH, the accompanied G6PD mutation may have compounded the clinical symptoms; however, the extent by which G6PD deficiency has contributed to HLH in our patient remains unclear.

Human Neutrophil Isolation and Degranulation Responses to Yersinia pestis Infection.

Neutrophils are the primary immune cell recruited to the site of bacterial infection, where they can rapidly deploy vesicles filled with various pro-inflammatory and anti-microbial proteins. This degranulation process, combined with oxidative and nitrosative mechanisms, is a major part of the initial host response to kill microorganisms. Neutrophils are one of the main cell types that interact with Yersinia pestis during infection, which is often lethal in the absence of prompt antibiotic treatment. Intradermal inoculation of Y. pestis results in bubonic plague, and inhalation of aerosolized droplets containing Y. pestis results in pneumonic plague. Although neutrophils are recruited to the site of inoculation during both bubonic and pneumonic plague, the neutrophils fail to clear Y. pestis, and, during pneumonic plague, contribute to the development of severe pneumonia. Subverting neutrophil responses is critical to the development of fulminant disease, yet the mechanisms by which Y. pestis impairs neutrophils are poorly understood. Cell culture models are important tools for studying Y. pestis interactions with immune cells. We describe a cell culture model for the infection of human neutrophils with Y. pestis. Neutrophils are isolated from human peripheral blood at high purity and subsequently infected with Y. pestis. We specifically focus on the application of this in vitro infection assay to the analysis of neutrophil degranulation responses.

LPA5 Is an Inhibitory Receptor That Suppresses CD8 T-Cell Cytotoxic Function via Disruption of Early TCR Signaling.

Persistent T cell antigen receptor (TCR) signaling by CD8 T cells is a feature of cancer and chronic infections and results in the sustained expression of, and signaling by, inhibitory receptors, which ultimately impair cytotoxic activity via poorly characterized mechanisms. We have previously determined that the LPA5 GPCR expressed by CD8 T cells, upon engaging the lysophosphatidic acid (LPA) bioactive serum lipid, functions as an inhibitory receptor able to negatively regulate TCR signaling. Notably, the levels of LPA and autotaxin (ATX), the phospholipase D enzyme that produces LPA, are often increased in chronic inflammatory disorders such as chronic infections, autoimmune diseases, obesity, and cancer. In this report, we demonstrate that LPA engagement selectively by LPA5 on human and mouse CD8 T cells leads to the inhibition of several early TCR signaling events including intracellular calcium mobilization and ERK activation. We further show that, as a consequence of LPA5 suppression of TCR signaling, the exocytosis of perforin-containing granules is significantly impaired and reflected by repressed in vitro and in vivo CD8 T cell cytolytic activity. Thus, these data not only document LPA5 as a novel inhibitory receptor but also determine the molecular and biochemical mechanisms by which a naturally occurring serum lipid that is elevated under settings of chronic inflammation signals to suppress CD8 T cell killing activity in both human and murine cells. As diverse tumors have repeatedly been shown to aberrantly produce LPA that acts in an autocrine manner to promote tumorigenesis, our findings further implicate LPA in activating a novel inhibitory receptor whose signaling may be therapeutically silenced to promote CD8 T cell immunity.

Review of Defective NADPH Oxidase Activity and Myeloperoxidase Release in Neutrophils From Patients With Cirrhosis.

Patients with decompensated cirrhosis are highly susceptible to develop bacterial infections and these can trigger multiorgan failure associated with high in-hospital mortality. Neutrophils from patients with decompensated cirrhosis exhibit marked alterations that may explain the susceptibility of these patients to develop bacterial infections. These neutrophil alterations include marked defects in intracellular signaling pathways involving serine/threonine kinases such as protein kinase B (AKT), p38-mitogen-activated protein kinase (MAPK), and the MAP kinases1/2; activation of the NADPH oxidase complex; myeloperoxidase (MPO) release; and bactericidal activity of neutrophils stimulated by the bacterial peptide formyl-Methionine-Leucine-Phenylalanine (fMLF). Impaired activity of the NADPH oxidase 2 (NOX2) complex is also related to reduced levels of expression of its major components through post-transcriptional mechanisms. In addition, the catalytic NOX2 component gp91 phox is subject to degradation by elastase highly present in patients' plasma. A defect in the protein kinase B (AKT) and p38 MAPK-mediated signaling pathways may explain the decrease in phosphorylation of p47 phox (an important component of the NADPH oxidase complex) and MPO release, in response to neutrophil stimulation by fMLF. Most of these alterations are reversible ex vivo with TLR7/8 agonists (CL097, R848), raising the possibility that these agonists might be used in the future to restore neutrophil antibacterial functions in patients with cirrhosis.

Live imaging of cortical granule exocytosis reveals that in vitro matured mouse oocytes are not fully competent to secrete their content.

Oocyte in vitro maturation does not entirely support all the nuclear and cytoplasmic changes that occur physiologically, and it is poorly understood whether in vitro maturation affects the competence of cortical granules to secrete their content during cortical reaction. Here, we characterize cortical granule exocytosis (CGE) in live mouse oocytes activated by strontium chloride using the fluorescent lectin FITC-LCA. We compared the kinetic of CGE between ovulated (in vivo matured, IVO) and in vitro matured (IVM) mouse oocytes. Results show that: (1) IVM oocytes have a severely reduced response to strontium chloride; (2) the low response was confirmed by quantification of remnant cortical granules in permeabilized cells and by a novel method to quantify the exudate in non-permeabilized cells; (3) the kinetic of CGE in IVO oocytes was rapid and synchronous; (4) the kinetic of CGE in IVM oocytes was delayed and asynchronous; (5) cortical granules in IVM oocytes show an irregular limit in regards to the cortical granule free domain. We propose the analysis of CGE in live oocytes as a biological test to evaluate the competence of IVM mouse oocytes.This article has an associated First Person interview with the first author of the paper.

Hepatic stellate cell interferes with NK cell regulation of fibrogenesis via curcumin induced senescence of hepatic stellate cell.

Hepatic fibrosis, a common scarring response to various forms of chronic liver injury, is a precursor to cirrhosis and liver cancer. During liver fibrosis, hepatic stellate cells (HSCs) initially activate and proliferate, which are responsible for the secretion of extracellular matrix components. However, these cells eventually senesce and are cleared by natural killer (NK) cells. Our previous researches have shown that the natural product curcumin could promote the senescence of activated HSC. In this study, we investigated how NK cells target senescent HSC and assessed the effect of this process on liver fibrosis. We found that senescent HSC induced by curcumin are susceptible to NK cells killing, due to the increased expression of NK cell activating ligand major histocompatibility complex class I chain-related genes A (MICA) and UL16-binding proteins 2 (ULBP2), but not Poliovirus Receptor (PVR). Further studies displayed that the interaction between NK cells and senescent LX2 cells stimulated granule exocytosis. Moreover, the inhibition of granule exocytosis weakened the cytotoxicity of NK cells and promoted the accumulation of senescent LX2 cells. Therefore, these aggregated data indicated that NK cells mediated clearance of senescent LX2 cells and granule exocytosis could play a protective role in the improvement of liver fibrosis.