scholarly journals The Inhibitory Receptor CLEC12A Regulates PI3K-Akt Signaling to Inhibit Neutrophil Activation and Cytokine Release

2021 ◽  
Vol 12 ◽  
Author(s):  
Guillaume Paré ◽  
Julien Vitry ◽  
Michael L. Merchant ◽  
Myriam Vaillancourt ◽  
Andréa Murru ◽  
...  
Keyword(s):  
Uric Acid ◽  
Akt Signaling ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Cross Linking ◽  
Dependent Manner ◽  
Membrane Domains ◽  
Lectin Receptor ◽  
Molecular Events ◽  
Inhibitory Signaling

The myeloid inhibitory C-type lectin receptor CLEC12A limits neutrophil activation, pro-inflammatory pathways and disease in mouse models of inflammatory arthritis by a molecular mechanism that remains poorly understood. We addressed how CLEC12A-mediated inhibitory signaling counteracts activating signaling by cross-linking CLEC12A in human neutrophils. CLEC12A cross-linking induced its translocation to flotillin-rich membrane domains where its ITIM was phosphorylated in a Src-dependent manner. Phosphoproteomic analysis identified candidate signaling molecules regulated by CLEC12A that include MAPKs, phosphoinositol kinases and members of the JAK-STAT pathway. Stimulating neutrophils with uric acid crystals, the etiological agent of gout, drove the hyperphosphorylation of p38 and Akt. Ultimately, one of the pathways through which CLEC12A regulates uric acid crystal-stimulated release of IL-8 by neutrophils is through a p38/PI3K-Akt signaling pathway. In summary this work defines early molecular events that underpin CLEC12A signaling in human neutrophils to modulate cytokine synthesis. Targeting this pathway could be useful therapeutically to dampen inflammation.

scholarly journals Recruitment of the cross-linked opsonic receptor CD32A (FcγRIIA) to high-density detergent-resistant membrane domains in human neutrophils

2004 ◽  
Vol 381 (3) ◽  
pp. 919-928 ◽  
Author(s):  
Emmanuelle ROLLET-LABELLE ◽  
Sébastien MAROIS ◽  
Kathy BARBEAU ◽  
Stephen E. MALAWISTA ◽  
Paul H. NACCACHE
Keyword(s):  
Tyrosine Phosphorylation ◽  
Plasma Membranes ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
High Density ◽  
Human Neutrophils ◽  
Cross Linking ◽  
Src Kinases ◽  
Membrane Domains ◽  
Detergent Resistant Membranes

We have previously shown that CD32A (or FcγRIIA), one of the main opsonin receptors, was rapidly insolubilized and degraded in intact neutrophils after its cross-linking. In view of these experimental difficulties, the early signalling steps in response to CD32A activation were studied in purified plasma membranes of neutrophils. After CD32A cross-linking in these fractions, the tyrosine phosphorylation of two major substrates, the receptor itself and the tyrosine kinase Syk, was observed. Phosphorylation of these two proteins was observed only in the presence of orthovanadate, indicating the presence, in the membranes, of one or more tyrosine phosphatases that maintain CD32A dephosphorylation. The tyrosine phosphorylation of these two proteins was inhibited by the Src kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). The ligation of CD32A led to its recruitment to a previously uncharacterized subset of high-density flotillin-1-positive DRMs (detergent-resistant membranes). The changes in the solubility properties of CD32A were observed in the absence of added ATP; therefore, they were probably not secondary to the tyrosine phosphorylation of the receptor, rather they preceded it. Src kinases as well as Syk were constitutively present in DRMs of high and low density and no evident changes in their distribution were detected after cross-linking of CD32A. Pretreatment of plasma membranes with methyl-β-cyclodextrin did not inhibit the recruitment of CD32A to DRMs, although it led to the loss of the Src kinase Lyn from these fractions. In addition, methyl-β-cyclodextrin inhibited the tyrosine phosphorylation of CD32A and Syk induced by cross-linking of CD32A. This membrane model allowed us to observe a movement of CD32A from detergent-soluble regions of the membranes to DRMs, where it joined Src kinases and Syk and became tyrosine-phosphorylated.

scholarly journals Cold-Inducible RNA-Binding Protein (CIRP) Potentiates Uric Acid-Induced IL-1β Production

2021 ◽  
Author(s):  
Yuya Fujita ◽  
Toru Yago ◽  
Haruki Matsumoto ◽  
Tomoyuki Asano ◽  
Naoki Matsuoka ◽  
...  
Keyword(s):  
Uric Acid ◽  
Nlrp3 Inflammasome ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inflammasome Activation ◽  
Dependent Manner ◽  
Caspase 1

Abstract Background Gout is an autoinflammatory disease driven by interleukin-1 (IL-1) induction in response to uric acid crystals. IL-1β production is dependent on inflammasome activation, which requires a priming signal, followed by an activating signal. The cold-inducible RNA-binding protein (CIRP) has been recently identified as a damage-associated molecular pattern (DAMP). In this study, we evaluated the roles of CIRP in monosodium urate (MSU)-mediated IL-1β secretion using human neutrophils. Methods Human neutrophils were stimulated by MSU in the presence or absence of CIRP priming to determine NLRP3 inflammasome activation and subsequent caspase-1 activation and IL-1β production. Cellular supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) to determine the presence of IL-1β or caspase-1 (p20). The cellular supernatants and lysates were also analyzed by immunoblotting using anti-cleaved IL-1β or anti-cleaved caspase-1 antibodies. Additionally, pro-IL-1β and NLRP3 transcript levels were analyzed by real-time reverse transcription-PCR (RT-PCR). Results Neither CIRP nor MSU stimulation alone induced sufficient IL-1β secretion from neutrophils. However, MSU stimulation induced IL-1β secretion from CIRP-primed neutrophils in a dose-dependent manner. This MSU-induced IL-1β secretion from CIRP-primed neutrophils was accompanied by the induction of cleaved IL-1β (p17). Furthermore, cleaved caspase-1 was induced in the cellular lysates of CIRP/MSU-treated neutrophils. Additionally, CIRP stimulation induced the expression of pro-IL-1β mRNA and protein in neutrophils. Conclusions Our data indicate that CIRP, an endogenous stress molecule, triggers uric acid-induced mature IL-1β induction as a priming stimulus for NLRP3 inflammasome in human neutrophils. We propose that CIRP acts as an important proinflammatory stimulant that primes and activates inflammasome and pro-IL-1β processing in response to uric acid in innate immune cells.

Uric Acid Induces NADPH Oxidase–Independent Neutrophil Extracellular Trap Formation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2270-2270
Author(s):  
Yasuyuki Arai ◽  
Yoko Nishinaka ◽  
Toshiyuki Arai ◽  
Kiyomi Mizugishi ◽  
Souichi Adachi ◽  
...  
Keyword(s):  
Uric Acid ◽  
Cardiovascular Diseases ◽  
Nadph Oxidase ◽  
Western Blotting ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Direct Effects ◽  
High Concentration ◽  
Independent Manner ◽  
Dose Dependent

Abstract Introduction Neutrophils play a critical role in the innate immune response against various kinds of microbes. In addition to phagocytosis, neutrophil extracellular traps (NETs) have been identified as a novel killing mechanism, capturing microbes with extracellular structures consisting of DNA fibers and antimicrobial granule proteins. On the other hand, aberrant NET formation has been associated with the development of autoimmune and cardiovascular diseases. Various kinds of stimuli, including phorbol myristate acetate (PMA), are known to induce NETs by stimulating the production of reactive oxygen species (ROS), especially singlet oxygen (1O2), through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) in neutrophils. Uric acid (UA), a product of purine metabolism, is a scavenger of 1O2 and regulates oxidative stress in humans. UA is believed to suppress Nox-dependent NET formation. By contrast, monosodium urate crystals, an endogenous danger signal, stimulate NET formation in patients with gout, suggesting that a high concentration of UA itself causes NET formation. At present, there are no data explaining the dual action of UA. In this study, we examined the direct effects of UA on NET formation. Materials and Methods Human neutrophils were isolated from the peripheral blood of healthy volunteers and two patients with chronic granulomatous disease (CGD), who provided written informed consent. Detection of 1O2 from the stimulated neutrophils was carried out by chemiluminescence assay using trans-1-(2'-methoxyvinyl)pyrene (MVP) as a 1O2-specific probe. NET formation was analyzed by scanning electron microscope (SEM) or confocal laser microscopy after staining with Sytox Green (dsDNA), and quantified by measuring the Sytox-positive area per cell. Involvement of the NF-κB pathway in NET formation was examined by Western blotting using anti-phospho-NF-κB p65 antibody. Data were compared with Student's t-test, and p values < 0.05 were considered significant. Results First, we examined the effects of UA on 1O2 production and NET formation by PMA-stimulated neutrophils. The addition of UA (0.05 – 5.0 mg/dL) significantly suppressed 1O2 production by PMA-stimulated neutrophils in a dose-dependent manner. At lower concentrations of UA (≤ 0.5 mg/dL), PMA-induced NET formation was suppressed, as predicted, while a high concentration of UA (5.0 mg/dL) failed to suppress PMA-induced NET formation. Next, we explored the direct effects of UA on 1O2 production and NET formation. UA treatment alone did not produce any detectable levels of 1O2 or other types of ROS. However, unexpectedly, UA-stimulated neutrophils formed NETs in a dose-dependent manner (1.0 – 8.0 mg/dL of UA). Nox inhibitors (diphenyleneiodonium [DPI] or apocynin) and a 1O2 scavenger (α-phenyl-N-tert-butyl nitrone [PBN]) did not suppress UA-induced NET formation. Moreover, neutrophils in CGD patients produced NETs after treatment with UA, although they had a defect in ROS formation and failed to make NETs after PMA stimulation. These data demonstrate that UA induces NET formation in a Nox-independent manner. Finally, we explored the signaling pathway in UA-stimulated neutrophils for inducing NET formation. Treatment with NF-κB inhibitor significantly suppressed UA-stimulated NET formation. In addition, using Western blotting, we detected phospho-NF-κB p65 in UA-activated neutrophils. These results indicate that NF-κB activation is indispensable for UA-induced NET formation. Conclusion UA suppressed PMA-induced NET formation at low concentrations (≤ 0.5 mg/dL), working as a 1O2 scavenger. By contrast, high concentrations of UA (≥ 5.0 mg/dL) induced NET formation in a Nox- or ROS-independent manner through NF-κB activation. To our knowledge, this is the first study to report on UA-induced NETs. Our findings are clinically significant in that a high level of serum UA may promote the chronic formation of NETs in circulating neutrophils, thereby contributing to vascular damage. Our data may explain one of the missing links between hyperuricemia and the risk of cardiovascular diseases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cold-inducible RNA-binding protein (CIRP) potentiates uric acid-induced IL-1β production

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Yuya Fujita ◽  
Toru Yago ◽  
Haruki Matsumoto ◽  
Tomoyuki Asano ◽  
Naoki Matsuoka ◽  
...  
Keyword(s):  
Uric Acid ◽  
Nlrp3 Inflammasome ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inflammasome Activation ◽  
Dependent Manner ◽  
Caspase 1

Abstract Background Gout is an autoinflammatory disease driven by interleukin-1 (IL-1) induction in response to uric acid crystals. IL-1β production is dependent on inflammasome activation, which requires a priming signal, followed by an activating signal. The cold-inducible RNA-binding protein (CIRP) has been recently identified as a damage-associated molecular pattern (DAMP). In this study, we evaluated the roles of CIRP in monosodium urate (MSU)-mediated IL-1β secretion using human neutrophils. Methods Human neutrophils were stimulated by MSU in the presence or absence of CIRP priming to determine NLRP3 inflammasome activation and subsequent caspase-1 activation and IL-1β production. Cellular supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) to determine the presence of IL-1β or caspase-1 (p20). The cellular supernatants and lysates were also analyzed by immunoblotting using anti-cleaved IL-1β or anti-cleaved caspase-1 antibodies. Results Neither CIRP nor MSU stimulation alone induced sufficient IL-1β secretion from neutrophils. However, MSU stimulation induced IL-1β secretion from CIRP-primed neutrophils in a dose-dependent manner. This MSU-induced IL-1β secretion from CIRP-primed neutrophils was accompanied by the induction of cleaved IL-1β (p17), which was inhibited by the pretreatment of MCC950, a specific inhibitor for NLRP3. Furthermore, cleaved caspase-1 was induced in the cellular lysates of CIRP/MSU-treated neutrophils. Additionally, CIRP stimulation induced the protein expression of pro-IL-1β in neutrophils. Conclusions Our data indicate that CIRP, an endogenous stress molecule, triggers uric acid-induced mature IL-1β induction as a priming stimulus for NLRP3 inflammasome in human neutrophils. We propose that CIRP acts as an important proinflammatory stimulant that primes and activates inflammasome and pro-IL-1β processing in response to uric acid in innate immune cells.

Ceramide-Enriched Membrane Domains Contribute to Targeted and Nontargeted Effects of Radiation Through Modulation of PI3K/AKT Signaling in HNSCC Cells

2020 ◽  
Author(s):  
Riad Ladjohounlou ◽  
Safa Louati ◽  
Alexandra Lauret ◽  
Arnaud Gauthier ◽  
Dominique Ardail ◽  
...  
Keyword(s):  
Akt Signaling ◽  
Membrane Domains ◽  
Effects Of Radiation

scholarly journals Control of microtubule dynamics using an optogenetic microtubule plus end–F-actin cross-linker

2017 ◽  
Vol 217 (2) ◽  
pp. 779-793 ◽  
Author(s):  
Rebecca C. Adikes ◽  
Ryan A. Hallett ◽  
Brian F. Saway ◽  
Brian Kuhlman ◽  
Kevin C. Slep
Keyword(s):  
Blue Light ◽  
Actin Binding ◽  
Cross Linking ◽  
Dependent Manner ◽  
Exclusion Zone ◽  
Actin Networks ◽  
Drosophila Melanogaster S2 Cells ◽  
Actin Binding Domain ◽  
Specific Factors ◽  
Insight Into

We developed a novel optogenetic tool, SxIP–improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein–dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes.

scholarly journals Tissue-specific distribution of cross-linked somatostatin receptor proteins in the rat

1992 ◽  
Vol 282 (2) ◽  
pp. 339-344 ◽  
Author(s):  
C B Srikant ◽  
K K Murthy ◽  
Y C Patel
Keyword(s):  
Exocrine Pancreas ◽  
Receptor Protein ◽  
Cross Linking ◽  
Molecular Heterogeneity ◽  
Dependent Manner ◽  
Tissue Specific ◽  
Receptor Proteins ◽  
Specific Distribution ◽  
A Minor ◽  
The Brain

Pharmacological studies have suggested that the somatostatin (SS) receptor is heterogeneous and exhibits SS-14-and SS-28-selective subtypes. Whether such subtypes arise from molecular heterogeneity of the receptor protein has not been definitively established. Previous reports characterizing the molecular properties of the SS receptor by the cross-linking approach have yielded divergent size estimates ranging from 27 kDa to 200 kDa. In order to resolve this discrepancy, as well as to determine whether SS-14 and SS-28 interact with specific receptor proteins, we have cross-linked radioiodinated derivatives of [125I-Tyr11]SS-14 (T*-SS-14) and [Leu8,D-Trp22,125I-Tyr25]SS-28 (LTT*-SS-28) to membrane SS receptors in rat brain, pituitary, exocrine pancreas and adrenal cortex using a number of chemical and photoaffinity cross-linking agents. The labelled cross-linked receptor proteins were analysed by SDS/PAGE under reducing conditions followed by autoradiography. Our findings indicate that the pattern of specifically labelled cross-linked SS receptor proteins is sensitive to the concentration of chemical cross-linking agents such as disuccinimidyl suberate and dithiobis-(succinimidyl propionate). Labelled high-molecular-mass complexes of cross-linked receptor-ligand proteins were observed only when high concentrations of these cross-linkers were employed. Using optimized low concentrations of cross-linkers, however, two major labelled bands of 58 +/- 3 kDa and 27 +/- 2 kDa were detected. These two bands were identified as specifically labelled SS receptor proteins subsequent to cross-linking with a number of photoaffinity cross-linking agents as well. We demonstrate here that the 58 kDa protein is the major SS receptor protein in the rat pituitary, adrenal and exocrine pancreas, whereas the 27 kDa moiety represents the principal form in the brain. Additionally, the presence of a minor specifically labelled band of 32 kDa was detected uniquely in the brain, and a minor labelled protein of 42 kDa was observed in the pancreas. The labelling pattern obtained with LTT*-SS-28 was identical to that observed with T*-SS-14. Labelling of the 27 kDa band by either ligand was inhibited by SS-14 and SS-28 in a dose-dependent manner. Densitometric quantification showed that SS-14 exhibited greater than 2-fold greater potency than SS-28 for inhibiting the labelling of the 27 kDa species. These findings emphasize the need for careful interpretation of cross-linking data obtained for SS receptors, and provide evidence for molecular heterogeneity and for a tissue-specific distribution of the two principal SS receptor proteins.

scholarly journals Differential role of SIRT1/MAPK pathway during cerebral ischemia in rats and humans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sireesh Kumar Teertam ◽  
Phanithi Prakash Babu
Keyword(s):  
Cerebral Ischemia ◽  
Caspase 3 ◽  
Mapk Pathway ◽  
Protective Role ◽  
Akt Signaling ◽  
Sirtuin 1 ◽  
Neurological Dysfunction ◽  
Dependent Manner ◽  
Erk Mapk

AbstractCerebral ischemia (CI) is a severe cause of neurological dysfunction and mortality. Sirtuin-1 (Silent information regulator family protein 1, SIRT1), an oxidized nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, plays an important role in protection against several neurodegenerative disorders. The present study aims to investigate the protective role of SIRT1 after CI in experimental young and aged rats and humans. Also, the study examines the possible regulatory mechanisms of neuronal death in CI settings. Immunoblotting and immunohistochemistry were used to evaluate changes in the expression of SIRT1, JNK/ERK/MAPK/AKT signaling, and pro-apoptotic caspase-3 in experimental rats and CI patients. The study findings demonstrated that, in aged experimental rats, SIRT1 activation positively influenced JNK and ERK phosphorylation and modulated neuronal survival in AKT-dependent manner. Further, the protection conferred by SIRT1 was effectively reversed by JNK inhibition and increased pro-apoptotic caspase-3 expression. In young experimental rats, SIRT1 activation decreased the phosphorylation of stress-induced JNK, ERK, caspase-3, and increased the phosphorylation of AKT after CI. Inhibition of SIRT1 reversed the protective effect of resveratrol. More importantly, in human patients, SIRT1 expression, phosphorylation of JNK/ERK/MAPK/AKT signaling and caspase-3 were up-regulated. In conclusion, SIRT1 could possibly be involved in the modulation of JNK/ERK/MAPK/AKT signaling pathway in experimental rats and humans after CI.

scholarly journals Garcinia Multiflora Inhibits FPR1-Mediated Neutrophil Activation and Protects Against Acute Lung Injury

2018 ◽  
Vol 51 (6) ◽  
pp. 2776-2793 ◽  
Author(s):  
Yung-Fong Tsai ◽  
Shun-Chin Yang ◽  
Wen-Yi Chang ◽  
Jih-Jung Chen ◽  
Chun-Yu Chen ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Superoxide Anion ◽  
Calcium Influx ◽  
Neutrophil Activation ◽  
Lung Damage ◽  
Hek293 Cells ◽  
Human Neutrophils ◽  
Therapeutic Effects

Background/Aims: Formyl peptide receptors (FPRs) recognize different endogenous and exogenous molecular stimuli and mediate neutrophil activation. Dysregulation of excessive neutrophil activation and the resulting immune responses can induce acute lung injury (ALI) in the host. Accordingly, one promising approach to the treatment of neutrophil-dominated inflammatory diseases involves therapeutic FPR1 inhibition. Methods: We extracted a potent FPR1 antagonist from Garcinia multiflora Champ. (GMC). The inhibitory effects of GMC on superoxide anion release and elastase degranulation from activated human neutrophils were determined with spectrophotometric analysis. Reactive oxygen species (ROS) production and the FPR1 binding ability of neutrophils were assayed by flow cytometry. Signaling transduction mediated by GMC in response to chemoattractants was assessed with a calcium influx assay and western blotting. A lipopolysaccharide (LPS)-induced ALI mouse model was used to determine the therapeutic effects of GMC in vivo. Results: GMC significantly reduced superoxide anion release, the reactive oxidants derived therefrom, and elastase degranulation mediated through selective, competitive FPR1 blocking in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF)-stimulated human neutrophils. In cell-free systems, GMC was unable to scavenge superoxide anions or suppress elastase activity. GMC produced a right shift in fMLF-activated concentration-response curves and was confirmed to be a competitive FPR1 antagonist. GMC binds to FPR1 not only in neutrophils, but also FPR1 in neutrophil-like THP-1 and hFPR1-transfected HEK293 cells. Furthermore, the mobilization of calcium and phosphorylation of mitogen-activated protein kinases and Akt, which are involved in FPR1-mediated downstream signaling, was competitively blocked by GMC. In an in vivo study, GMC significantly reduced pulmonary edema, neutrophil infiltration, and alveolar damage in LPS-induced ALI mice. Conclusion: Our findings demonstrate that GMC is a natural competitive FPR1 inhibitor, which makes it a possible anti-inflammatory treatment option for patients critically inflicted with FPR1-mediated neutrophilic lung damage.

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