Ketamine reduces inducible superoxide generation in human neutrophils in vitro by modulating the p38 mitogen-activated protein kinase (MAPK)-mediated pathway

Many cellular stresses and inflammatory stimuli can activate p38 mitogen-activated protein kinase (MAPK), a serine/threonine kinase in the MAPK family. The different stimuli act via different receptors or signalling pathways to induce phosphorylation of the cytosolic protein p47^phox, one subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Formyl–methionyl–leucyl–phenylalanine (fMLP) has been shown to induce the p38 MAPK phosphorylation during the respiratory burst in human neutrophils. Here, we show that treatment with S(+)-ketamine or R(-)-ketamine at different concentrations (50, 100, 200, 400 µM) reduced fMLP-induced superoxide anion generation and p47^phox phosphorylation in neutrophils in a concentration-dependent manner (y = −0·093x + 93·35 for S(+)-ketamine and y = −0·0982x + 95·603 for R(-)-ketamine, respectively). While treatment with 50 µM ketamine inhibited fMLP-induced superoxide generation by 10%, treatment with 400 µM S(+)-ketamine and R(-)-ketamine reduced fMLP-induced superoxide generation to 60·5 ± 8·3% and 60·0 ± 8·5%, respectively, compared with that in neutrophils treated with fMLP alone. Furthermore, treatment with ketamine down-regulated both fMLP-induced p47^phox and isoproterenol-induced p38 MAPK phosphorylation and superoxide production. Interestingly, treatment with SB203580, the p38 MAPK inhibitor, also mitigated fMLP-induced superoxide anion generation and p38 MAPK and p47^phox phosphorylation as well as apoptosis in a concentration-dependent fashion in neutrophils. Therefore, ketamine racemes inhibited fMLP-induced superoxide anion generation and p47^phox phosphorylation by modulating fMLP-mediated p38 MAPK activation in neutrophils.     

Endocytosis is required for exocytosis and priming of respiratory burst activity in human neutrophils

Objective and design

Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47^phox phosphorylation-dependent translocation of cytosolic NADPH oxidase components. Clathrin-mediated endocytosis was recently reported to be necessary for priming, but the mechanism linking endocytosis to priming was not identified. The present study examined the hypothesis that endocytosis regulates neutrophil priming by controlling granule exocytosis.

Materials and methods

Clathrin-mediated endocytosis by isolated human neutrophils was inhibited by chlorpromazine, monodansylcadaverine, and sucrose. Exocytosis of granule subsets was measured as release of granule components by ELISA or chemiluminescence. ROS generation was measured as extracellular release of superoxide as reduction of ferrocytochrome c. p38 MAPK activation and p47^phox phosphorylation were measured by immunoblot analysis. Statistical analysis was performed using a one-way ANOVA with the Tukey–Kramer multiple-comparison test.

Results

Inhibition of endocytosis prevented priming of superoxide release by TNFα and inhibited TNFα stimulation and priming of exocytosis of all four granule subsets. Inhibition of endocytosis did not reduce TNFα-stimulated p38 MAPK activation or p47^phox phosphorylation. Inhibition of NADPH oxidase activity blocked TNFα stimulation of secretory vesicle and gelatinase granule exocytosis.

Conclusions

Endocytosis is linked to priming of respiratory burst activity through ROS-mediated control of granule exocytosis.

     

Emerging role of oxidative stress in metabolic syndrome and cardiovascular diseases: important role of Rac/NADPH oxidase

'Oxidative stress' is a term defining states of elevated reactive oxygen species (ROS) levels. Normally, ROS control several physiological processes, such as host defence, biosynthesis of hormones, fertilization and cellular signalling. However, oxidative stress has been involved in different pathologies, including metabolic syndrome and numerous cardiovascular diseases. A major source of ROS involved in both metabolic syndrome and cardiovascular pathophysiology is the NADPH oxidase (NOX) family of enzymes. NOX is a multi‐component enzyme complex that consists of membrane‐bound cytochrome b‐558, which is a heterodimer of gp91phox and p22phox, cytosolic regulatory subunits p47phox and p67phox, and the small GTP‐binding protein Rac1. Rac1 plays many important biological functions in cells, but perhaps the most unique function of Rac1 is its ability to bind and activate the NOX complex. Furthermore, Rac1 has been reported to be a key regulator of oxidative stress through its co‐regulatory effects on both nitric oxide (NO) synthase and NOX. Therefore, the main goal of this review is to give a brief outline about the important role of the Rac1–NOX axis in the pathophysiology of both metabolic syndrome and cardiovascular disease. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Adiponectin and its globular fragment differentially modulate the oxidative burst of primary human phagocytes

Adiponectin (Acrp30) belongs to the family of C1q/tumor necrosis factor α (TNFα)-related proteins. Acrp30 circulates as multimers of high, middle, and low molecular weight. In this study, we detected Acrp30 and its globular fragment (gAcrp30) in synovial fluid from rheumatoid arthritis patients. Intriguingly, the LMW form was more abundant in synovial fluid than in serum from both rheumatoid arthritis patients and healthy subjects. We also investigated the effects of Acrp30 and gAcrp30 on reactive oxygen species (ROS) production via the phagocytic NADPH oxidase. Acrp30 inhibited fMLF-induced ROS production by human phagocytes, whereas gAcrp30 enhanced it. gAcrp30's effect is additive with TNFα, whereas Acrp30 inhibited TNFα-induced priming. gAcrp30 enhanced NOX-2 expression at the plasma membrane, with a concomitant increase in p47(phox) phosphorylation. Selective inhibitors of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1 (ERK1)/2 abrogated p47(phox) phosphorylation by gAcrp30. In contrast, p47(phox) phosphorylation was inhibited by Acrp30 in association with increased AMP-activated protein kinase (AMPK) phosphorylation in phagocytes. These results suggest that human phagocyte ROS production is regulated by different mechanisms selective for Acrp30 versus gAcrp30. An imbalance between gAcrp30 and higher molecular weight isoforms of Acrp30 might contribute to chronic inflammation by regulating NADPH oxidase.     

Prolactin-induced activation of phagocyte NADPH oxidase in the teleost fish gilthead seabream involves the phosphorylation of p47phox by protein kinase C

The pituitary hormone prolactin (PRL) is a multifunctional polypeptide which act as a key component of the neuroendocrine-immune loop and as a local regulator of the macrophage response. The involvement of PRL in regulating monocyte/macrophage functions is suggested by the presence of PRL receptors in these cells. Recently, we reported that physiological concentrations of native PRL were able to induce the expression of the pro-inflammatory cytokines IL-1β and TNFα, and the production of reactive oxygen species (ROS) in head kidney leukocytes and macrophages from the teleost fish gilthead seabream (Sparus aurata L.). In this study, we show that the NADPH oxidase subunit p47phox becomes phosphorylated in leukocytes stimulated with PRL, an effect that is blocked when neutralizing polyclonal antibodies to PRL are added. Additionally, the pharmacological inhibition of either protein kinase C (PKC) with calphostin C or the Jak/Stat signaling pathway with AG490 impaired PKC activation, p47phox phosphorylation and ROS production in seabream leukocytes activated with PRL. Taken together, our results demonstrate for the first time the need for PKC in regulating the PRL-mediated phosphorylation of p47phox, the activation of NADPH oxidase and the production of ROS by macrophages in vertebrates.     

Dexamethasone increases ROS production and T cell suppressive capacity by anti-inflammatory macrophages

Macrophages have been demonstrated to suppress T cell responses by producing reactive oxygen species (ROS) leading to the subsequent induction of T regulatory cells in a ROS-dependent manner. Macrophages may therefore be instrumental in downregulating T cell responses in situations of exacerbated immune responses. Here we investigated the effect of immunosuppressive drugs on ROS production by macrophage subsets and the subsequent effects on T cell activation. Macrophage types 1 and 2 were differentiated with GM-CSF or M-CSF, in presence or absence of dexamethasone, cyclosporine A, FK506, rapamycin, or mycophenolic acid. The ROS producing capacity of fully differentiated Mph was highest in anti-inflammatory Mph2 and not affected by exposure to immunosuppressive drugs. However, presence of rapamycin during Mph2 differentiation decreased the ROS production of these cells. In contrast, other immunosuppressive drugs, with dexamethasone being the most potent, increased the ROS producing capacity of Mph2. Intriguingly although the ROS producing ability of Mph1 was unaffected, dexamethasone strongly increased the ROS producing capabilities of dendritic cells. Both at the mRNA and protein level we found that dexamethasone enhanced the expression of NOX2 protein p47^phox. Functionally, dexamethasone further enhanced the capacity of Mph2 to suppress T cell mediated IFN-γ and IL-4 production. In vivo, only in rats with normal ROS production (congenic DA.Ncf1^E3/E3) it was observed that dexamethasone injection resulted in long-lasting upregulation of ROS production by macrophages and induced higher levels of Treg in a ROS-dependent manner. In conclusion, we show that the anti-inflammatory drug dexamethasone increases the ROS producing capacity of macrophages.     

Three common polymorphisms in the CYBA gene form a haplotype associated with decreased ROS generation

NOX enzymes are reactive oxygen species (ROS)‐generating NADPH oxidases. Several members of the NOX family depend on the p22^phox subunit, encoded by the CYBA gene. CYBA is highly polymorphic, and has been widely studied as a potential risk factor for various diseases, with conflicting results. In the present study, we used Epstein‐Barr (EBV)‐transformed B‐lymphocytes from 50 healthy unrelated individuals to analyze their CYBA mRNA sequence and NOX2‐dependent ROS generation. Seven single‐nucleotide polymorphisms (SNPs) were identified (five previously described, two novel). The combination of these SNPs yielded 11 distinct haplotypes, which could be grouped into seven haplogroups (A–G). Haplogroup C (c.214T>C, c.521T>C, and c.^*24G>A) showed a significantly lower ROS generation, as compared to the most frequent haplogroup, A. CYBA variants from the seven haplogroups were transduced into p22^phox‐deficient B‐lymphocytes. The haplogroup C variant showed significantly lower ROS production. c.214T>C and c.521T>C lead to nonsynonymous codon changes, while c.^*24G>A lies within the 3′UTR. Using a luciferase/3′UTR construct, we showed that the ^*24A allele led to decreased reporter gene activity. These results help to unravel the complex nature of how genetic variations in CYBA influence NOX2 activity, and indicate that haplotypes, rather than individual SNPs, define the effect on ROS generation. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

NOX1-derived ROS drive the expression of Lipocalin-2 in colonic epithelial cells in inflammatory conditions

Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract, associated with altered patterns of cytokine synthesis, excessive reactive oxygen species (ROS) production, and high levels of the innate immune protein, lipocalin-2 (LCN-2), in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1, which consists of the transmembrane proteins, NOX1 and p22^PHOX, and the cytosolic proteins, NOXO1, NOXA1, and Rac1. Here, we investigated whether NOX1 activation and ROS production induced by key inflammatory cytokines in IBD causally affects LCN-2 production in colonic epithelial cells. We found that the combination of TNFα and IL-17 induced a dramatic upregulation of NOXO1 expression that was dependent on the activation of p38MAPK and JNK1/2, and resulted into an increase of NOX1 activity and ROS production. NOX1-derived ROS drive the expression of LCN-2 by controlling the expression of IκBζ, a master inducer of LCN-2. Furthermore, LCN-2 production and colon damage were decreased in NOX1-deficient mice during TNBS-induced colitis. Finally, analyses of biopsies from patients with Crohn's disease showed increased JNK1/2 activation, and NOXO1 and LCN-2 expression. Therefore, NOX1 might play a key role in mucosal immunity and inflammation by controlling LCN-2 expression.     

Interleukin-18 Primes the Oxidative Burst of Neutrophils in Response to Formyl-Peptides: Role of Cytochrome b558 Translocation and N-Formyl Peptide Receptor Endocytosis

Using flow cytometry, we observed that interleukin-18 (IL-18) primed human neutrophils (PMNs) in whole blood to produce superoxide anion (O₂°⁻) in response to N-formyl peptide (fMLP) stimulation, whereas IL-18 alone had no significant effect. In contrast to tumor necrosis factor alpha (TNF-α), which is a cytokine known to strongly prime O₂°⁻ production, IL-18 did not induce either p47^phox phosphorylation or its translocation from the cytosol to the plasma membrane. However, IL-18 increased PMN degranulation, as shown by increased levels of cytochrome b558 and CD11b expression at the PMN surface. Moreover, addition of IL-18 to whole blood for 45 min reduced the ability of PMNs to bind to fMLP, suggesting endocytosis of fMLP receptors, as visualized by confocal microscopy. 2,3-Butanedione 2-monoxime, which inhibits endosomal recycling of plasma membrane components back to the cell surface, concomitantly accentuated the diminution of fMLP binding at the PMN surface and increased IL-18 priming of O₂°⁻ production by PMNs in response to fMLP. This suggests that fMLP receptor endocytosis could account, at least in part, for the priming of O₂°⁻ production. In addition, genistein, a tyrosine kinase inhibitor, and SB203580, a p38 mitogen-activated protein kinase (p38MAPK) inhibitor, completely reversed the decreased level of fMLP binding and increased the level of CD11b expression after IL-18 treatment. Flow cytometric analysis of intact PMNs in whole blood showed that IL-18 increased p38MAPK phosphorylation and tyrosine phosphorylation. In particular, IL-18 induced phosphorylation of focal adhesion kinase (p125^FAK), which has been implicated in cytoskeleton reorganization. Taken together, our findings suggest several mechanisms that are likely to regulate cytokine-induced priming of the oxidative burst in PMNs in their blood environment.     

Src kinase participates in LPS-induced activation of NADPH oxidase

The production of superoxide from NADPH oxidase by macrophages in response to endotoxin (LPS) is an important innate immune response, yet it is not clear how LPS signals the activation of NADPH oxidase. The hypothesis is that LPS-induced src kinase and PI3 kinase (PI3K) facilitates the activation of p47^phox, the regulatory subunit of NADPH oxidase. In mouse macrophage RAW264.7 cells, inhibition of src tyrosine family kinases inhibited LPS-induced activation of NADPH oxidase, phosphorylation of p47^phox, activation of PI3K and phosphorylation of the TLR4. Moreover, inhibition of LPS-induced increases in intracellular calcium blunted src kinase activation, PI3K association with TLR4, as well as PI3 kinase activation. These data suggest that both src kinase and PI3 kinase are involved in LPS-induced NADPH oxidase activation. Importantly, these data suggest that LPS-induced src kinase activation is critical for PI3 kinase activation as well as TLR4 phosphorylation and is dependent upon LPS-induced increase in intracellular calcium. These signaling events fill critical gaps in our understanding of LPS-induced free radical production as well as may potentially responsible for the mechanism of innate immune tolerance or desensitization caused by steroids or ethanol.     

Assembly of NADPH Oxidase in Human Neutrophils Is Modulated by the Opacity-Associated Protein Expression State of Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc survives exposure to neutrophils despite these cells'' antimicrobial products, such as reactive oxygen species (ROS). ROS production in neutrophils is initiated by NADPH oxidase, which converts oxygen into superoxide. The subunits of NADPH oxidase are spatially separated between granules (gp91^phox/p22^phox) and the cytoplasm (p47^phox, p67^phox, and p40^phox). Activation of neutrophils promotes the coassembly of NADPH oxidase subunits at phagosome and/or plasma membranes. While Gc-expressing opacity-associated (Opa) proteins can induce neutrophils to produce ROS, Opa-negative (Opa⁻) Gc does not stimulate neutrophil ROS production. Using constitutively Opa⁻ and OpaD-positive (OpaD⁺) Gc bacteria in strain FA1090, we now show that the difference in ROS production levels in primary human neutrophils between these backgrounds can be attributed to differential assembly of NADPH oxidase. Neutrophils infected with Opa⁻ Gc showed limited translocation of NADPH oxidase cytoplasmic subunits to cellular membranes, including the bacterial phagosome. In contrast, these subunits rapidly translocated to neutrophil membranes following infection with OpaD⁺ Gc. gp91^phox and p22^phox were recruited to Gc phagosomes regardless of bacterial Opa expression. These results suggest that Opa⁻ Gc interferes with the recruitment of neutrophil NADPH oxidase cytoplasmic subunits to membranes, in particular, the p47^phox “organizing” subunit, to prevent assembly of the holoenzyme, resulting in an absence of the oxidative burst.     

Anti-inflammatory properties of the type 1 and type 2 vasoactive intestinal peptide receptors: role in lethal endotoxic shock

Vasoactive intestinal peptide (VIP) is a neuropeptide that can modulate several immune aspects. Previous reports showed that VIP attenuates the deleterious consequences of septic shock by inhibiting the production of pro-inflammatory agents and stimulating the production of anti-inflammatory cytokines in activated macrophages. In this study, by using selective VIP agonists, we investigated the differential involvement of the VIP receptors in the anti-inflammatory role of VIP. Both the type 1 VIP receptor (VPAC1) agonist, [K(15), R(16), L(27)] VIP 1-7-GRF 8-27, and the type 2 VIP receptor (VPAC2) agonist, Ro25-1553, protected mice from lethal endotoxemia by inhibiting the macrophage-derived pro-inflammatory mediators IL-6, TNF-alpha, IL-12 and NO, and by stimulating the production of the anti-inflammatory cytokine IL-10. In addition, both VIP and VPAC1 agonist, but not the VPAC2 agonist, reduced in vitro and in vivo the expression of the co-stimulatory B7. 1/B7.2 molecules, and the subsequent stimulatory activity for T helper cells in stimulated macrophages. The higher effectiveness of the VPAC1 agonist compared with the VPAC2 agonist suggests that VPAC1 is the major mediator of the anti-inflammatory action of VIP. Since VIP and the two agonists appear to affect multiple cytokines and inflammatory factors, they might provide a more efficient therapeutical alternative to the use of specific cytokine antibodies or antagonists.     

Angiotensin II-Induced Early and Late Inflammatory Responses Through NOXs and MAPK Pathways

Angiotensin II (Ang II) dysregulation has been determined as cause or an effect of many diseases. The relationship between Ang II and reactive oxygen species (ROS), which are generated by enzymes in the nicotinamide adenine dinucleotide phosphate oxidase (NOX) family, has been the focus of many researchers for years. Inflammation in response to the activities of various NOXs with differing time-dependent characteristics was reported. It is still unclear how these factors interplay over the course of the inflammatory response and how signal transduction through mitogen-activated protein kinase (MAPK) pathways. Our study collected data on the effects of Ang II on human umbilical vascular endothelial cells (HUVECs) over a comprehensive time period. Our results demonstrated that NOXs had two time-dependent reactions in response to Ang II stimulation via MAPK pathways. First, ROS was produced only during the early inflammatory phase. NOX4 promoted more rapid generation of H₂O₂ via the JNK pathway than generation of O₂·^? via ERK1/2 and p38 pathways. During both the early and late phases of the inflammatory response, NOX4 activity was transduced through the JNK pathway, whereas NOX1 and NOX2 signals were transmitted via the ERK1/2 and p38 pathways. Signal transduction via ROS generation was more likely during the early phase of the inflammatory response, and increased cytokine levels were more likely induced by the late phase of the inflammatory response.     

BCR‐induced superoxide negatively regulates B‐cell proliferation and T‐cell‐independent type 2 Ab responses

Superoxide and its derivatives have been implicated as secondary messenger molecules that influence signaling cascades in non‐phagocytes. B lymphocytes produce superoxide after BCR ligation. We found that these ROS regulate B‐cell signaling and entry into the cell cycle. B cells from mice deficient in the gp91^phox subunit of the NADPH oxidase complex are unable to generate ROS after BCR ligation. However, after BCR stimulation, more gp91^phox KO B cells enter the G1 stage of the cell cycle and proliferate than WT B cells. BCR ligation leads to a more rapid decrease in p27^Kip1 levels in gp91^phox KO B cells. Gp91^phox KO mice display enhanced T‐cell‐independent type 2, but normal T‐dependent Ab responses. ROS‐dependent regulation of BCR‐induced proliferation may help modulate the size of the humoral response to T‐cell‐independent type 2 Ag immunization.     

VIP differentially activates beta2 integrins, CR1, and matrix metalloproteinase-9 in human monocytes through cAMP/PKA, EPAC, and PI-3K signaling pathways via VIP receptor type 1 and FPRL1

The neuropeptide vasoactive intestinal peptide (VIP) regulates the exocytosis of secretory granules in a wide variety of cells of neuronal and non-neuronal origin. In human monocytes, we show that the proinflammatory effects of VIP are associated with stimulation of exocytosis of secretory vesicles as well as tertiary (gelatinase) granules with, respectively, up-regulation of the membrane expression of the beta2 integrin CD11b, the complement receptor 1 (CD35), and the matrix metalloproteinase-9 (MMP-9). Using the low-affinity formyl peptide receptor-like 1 (FPRL1) antagonist Trp-Arg-Trp-Trp-Trp-Trp (WRW4) and the exchange protein directly activated by cAMP (EPAC)-specific compound 8CPT-2Me-cAMP and measuring the expression of Rap1 GTPase-activating protein as an indicator of EPAC activation, we found that the proinflammatory effect of VIP is mediated via the specific G protein-coupled receptor VIP/pituitary adenylate cyclase-activating protein (VPAC1) receptor as well as via FPRL1: VIP/VPAC1 interaction is associated with a cAMP increase and activation of a cAMP/p38 MAPK pathway, which regulates MMP-9, CD35, and CD11b exocytosis, and a cAMP/EPAC/PI-3K/ERK pathway, which regulates CD11b expression; VIP/FPRL1 interaction results in cAMP-independent PI-3K/ERK activation with downstream integrin up-regulation. In FPRL1-transfected Chinese hamster ovary-K1 cells lacking VPAC1, VIP exposure also resulted in PI-3K/ERK activation. Thus, the proinflammatory effects of VIP lie behind different receptor interactions and multiple signaling pathways, including cAMP/protein kinase A, cAMP/EPAC-dependent pathways, as well as a cAMP-independent pathway, which differentially regulates p38 and ERK MAPK and exocytosis of secretory vesicles and granules.     

Inhibition of NLRP3 Inflammasome Prevents LPS-Induced Inflammatory Hyperalgesia in Mice: Contribution of NF-κB,Caspase-1/11, ASC,NOX, and NOS Isoforms

The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3), an intracellular signaling molecule that senses many environmental- and pathogen/host-derived factors, has been implicated in the pathogenesis of several diseases associated with inflammation. It has been suggested that NLRP3 inflammasome inhibitors may have a therapeutic potential in the treatment of NLRP3-related inflammatory diseases. The aim of this study was to determine whether inhibition of NLRP3 inflammasome prevents inflammatory hyperalgesia induced by lipopolysaccharide (LPS) in mice as well as changes in expression/activity of nuclear factor κB (NF-κB), caspase-1/11, nicotinamide adenine dinucleotide phosphate oxidase (NOX), and endothelial/neuronal/inducible nitric oxide synthase (eNOS/nNOS/iNOS) that may regulate NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/pro-caspase-1 inflammasome formation and activity by using a selective NLRP3 inflammasome inhibitor, MCC950. Male mice received saline (10 ml/kg; i.p.), LPS (10 mg/kg; i.p.), and/or MCC950 (3 mg/kg; i.p.). Reaction time to thermal stimuli within 1 min was evaluated after 6 h. The mice were killed and the brains, hearts, and lungs were collected for measurement of NF-κB, caspase-1, caspase-11, NLRP3, ASC, NOX subunits (gp91^phox; NOX2), and p47^phox; NOXO2), nitrotyrosine, eNOS, nNOS, iNOS, and β-actin protein expression, NOS activity, and interleukin (IL)-1β levels. LPS-induced hyperalgesia was associated with a decrease in eNOS, nNOS, and iNOS protein expression and activity as well as an increase in expression of NF-κB p65, caspase-1 p20, caspase-11 p20, NLRP3, ASC, gp91^phox, p47^phox, and nitrotyrosine proteins in addition to elevated IL-1β levels. The LPS-induced changes were prevented by MCC950. The results suggest that inhibition of NLRP3/ASC/pro-caspase-1 inflammasome formation and activity prevents inflammatory hyperalgesia induced by LPS in mice as well as changes in NF-κB, caspase-11, NOX2, NOXO2, and eNOS/nNOS/iNOS expression/activity.     

NADPH oxidases and MMP-9/TIMP-1 balance in human glomeruli: putative links to diabetic nephropathy

Study aim Glomerular basement membrane thickening, the hallmark of diabetic nephropathy, is thought to be related to an enhanced oxidative stress and reduced matrix proteolysis. Our study concerned the mRNA and protein expression of NADPH oxidase (NOX) components, MMP‐2, MMP‐9 and TIMP‐1 in freshly isolated human glomeruli as well as enzymatic activities and their modulation by glucose, H₂O₂ and angiotensin‐2. Material and methods NOX, cytosolic and membrane‐bound associated proteins and mRNA were analysed by RT‐PCR and Western blotting after glomerular extraction. Oxidase activity was identified by cytochrome c reduction and chemiluminescence. Gelatinases and inhibitors were semiquantitatively assessed by RT‐PCR, gelatin zymography and ELISA in a model of glomerular conditioned survival. Results NOX‐2, NOX‐4 and membrane‐bound and cytosolic factors could be observed in freshly extracted glomeruli (RNA + protein). p40^phox, p67^phox and p47^phox molecular weights were increased compared to their phagocytic counterparts advocating for specific glomerular analogues, and a slight specific oxidase activity was retrieved in isolated glomeruli. Also, mRNA coding for MMP‐2, ‐9 and TIMP‐1, ‐2 were detected. High glucose concentrations (25 mm) reduced TIMP‐1 release in glomerular survival media and MMP‐2 activity in glomerular extracts. On the opposite, angiotensin‐2 significantly induced MMP‐2 and ‐9 activities in the survival media as well as H₂O₂ in glomerular extracts, while addition of 25 mm glucose blunted these findings. Conclusion Glomerular matrix remodelling, the backbone of renal fibrosis in diabetic patients, could be induced by H₂O₂ from specific glomerular NADPH oxidases under the influence of extra‐cellular glucose and angiotensin‐2 and could participate in the control of MMP activities.     

Defects in the vasoactive intestinal peptide (VIP)/VPAC system during early stages of the placental–maternal leucocyte interaction impair the maternal tolerogenic response

Successful embryo implantation occurs followed by a local inflammatory/T helper type 1 (Th1) response, subsequently redirected towards a tolerogenic predominant profile. The lack of control of this initial local inflammatory response may be an underlying cause of early pregnancy complications as recurrent spontaneous abortions (RSA). Considering that vasoactive intestinal peptide (VIP) mediates anti-inflammatory and tolerogenic effects in several conditions we hypothesized that VIP might contribute to tolerance towards trophoblast antigens during the early interaction of maternal leucocytes and trophoblast cells. In this study we investigated VIP/VPAC system activity and expression on maternal peripheral blood mononuclear cells (PBMCs) after interaction with immortalized trophoblast cells (Swan-71 cell line) as an in-vitro model of feto–maternal interaction, and we analysed whether it modulates maternal regulatory T cell (T_reg)/Th1 responses. We also investigated the contribution of the endogenous VIP/VPAC system to RSA pathogenesis. VIP decreased T-bet expression significantly, reduced monocyte chemotactic protein-1 (MCP-1) and nitrite production in co-cultures of PBMCs from fertile women with trophoblast cells; while it increased the frequency of CD4⁺CD25⁺ forkhead box protein 3 (Foxp3)⁺ cells, transforming growth factor (TGF)-β expression and interleukin (IL)-10 secretion. These effects were prevented by VIP-specific antagonist. Interestingly, PBMCs from RSA patients displayed significantly higher T-bet expression, lower T_reg frequency and lower frequency of VIP-producer CD4 lymphocytes after the interaction with trophoblast cells. Moreover, the patients displayed a significantly lower frequency of endometrial CD4⁺VIP⁺ cells in comparison with fertile women. VIP showed a Th1-limiting and T_reg-promoting response in vitro that would favour early pregnancy outcome. Because RSA patients displayed defects in the VIP/VPAC system, this neuropeptide could be a promising candidate for diagnostic biomarker or surrogate biomarker for recurrent spontaneous abortions.     

Colitis susceptibility in mice with reactive oxygen species deficiency is mediated by mucus barrier and immune defense defects

Reactive oxygen species (ROS) generated by NADPH oxidases (NOX/DUOX) provide antimicrobial defense, redox signaling, and gut barrier maintenance. Inactivating NOX variants are associated with comorbid intestinal inflammation in chronic granulomatous disease (CGD; NOX2) and pediatric inflammatory bowel disease (IBD; NOX1); however Nox-deficient mice do not reflect human disease susceptibility. Here we assessed if a hypomorphic patient-relevant CGD mutation will increase the risk for intestinal inflammation in mice. Cyba (p22^phox) mutant mice generated low intestinal ROS, while maintaining Nox4 function. The Cyba variant caused profound mucus layer disruption with bacterial penetration into crypts, dysbiosis, and a compromised innate immune response to invading microbes, leading to mortality. Approaches used in treatment-resistant CGD or pediatric IBD such as bone marrow transplantation or oral antibiotic treatment ameliorated or prevented disease in mice. The Cyba mutant mouse phenotype implicates loss of both mucus barrier and efficient innate immune defense in the pathogenesis of intestinal inflammation due to ROS deficiency, supporting a combined-hit model where a single disease variant compromises different cellular functions in interdependent compartments.