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During recovery from septic shock, circulating mitochondrial N-formyl peptides predispose to secondary infection by occupying formyl peptide receptor 1 on the neutrophil (polymorphonuclear leukocyte) membrane, suppressing cytosolic calcium ([Ca2+]i)-dependent responses to secondarily encountered bacteria. However, no study has yet investigated therapeutic clearance of circulating mitochondrial N-formyl peptides in clinical settings. Thus, we studied how to remove mitochondrial N-formyl peptides from septic-shock plasma and whether such removal could preserve cell-surface formyl peptide receptor 1 and restore sepsis-induced polymorphonuclear leukocyte dysfunction by normalizing [Ca2+]i flux. In in vitro model systems, mitochondrial N-formyl peptide removal rescued polymorphonuclear leukocyte formyl peptide receptor 1-mediated [Ca2+]i flux and chemotaxis that had been suppressed by prior mitochondrial N-formyl peptide exposure. However, polymorphonuclear leukocyte functional recovery occurred in a stepwise fashion over 30 to 90 min. Intracellular Ca2+-calmodulin appears to contribute to this delay. In ex vivo model, systems using blood samples obtained from patients with septic shock, antimitochondrial N-formyl peptide antibodies alone failed to eliminate mitochondrial N-formyl peptides from septic-shock plasma or inhibit mitochondrial N-formyl peptide activity. We therefore created a beads-based antimitochondrial N-formyl peptide antibody cocktail by combining protein A/sepharose with antibodies specific for the most potent human mitochondrial N-formyl peptide chemoattractants. The beads-based antimitochondrial N-formyl peptide antibody cocktail treatment successfully removed those active mitochondrial N-formyl peptides from septic-shock plasma. Furthermore, the beads-based antimitochondrial N-formyl peptide antibody cocktail treatment significantly restored chemotactic and bactericidal dysfunction of polymorphonuclear leukocytes obtained from patients with septic shock who developed secondary infections. By clearing circulating mitochondrial N-formyl peptides, the immobilized antimitochondrial N-formyl peptide antibody therapy prevented mitochondrial N-formyl peptide interactions with surface formyl peptide receptor 1, thereby restoring [Ca2+]i-dependent polymorphonuclear leukocyte antimicrobial function in clinical septic-shock environments. This approach may help prevent the development of secondary, nosocomial infections in patients recovering from septic shock., Competing Interests: Conflict of interest statement. W.Y.K., Y.S.J., G.J.S., S.H.K., A.L., J.Y.K., H.Y.K., H.P., T.K., and K.S.K. are inventors on a patent application submitted by Seoul National University that covers “Blood perfusion device to reduce secondary infection in hospital”. The authors have declared no additional conflict of interest exists., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

Introduction: G-protein coupled receptors (GPCRs) expressed on neutrophils regulate their mobilization from the bone marrow into the blood, their half-live in the circulation, and their pro- and anti-inflammatory activities during inflammation. Chronic kidney disease (CKD) is associated with systemic inflammatory responses, and neutrophilia is a hallmark of CKD onset and progression. Nonetheless, the role of neutrophils in CKD is currently unclear., Methods: Blood and renal tissue were collected from non-dialysis CKD (grade 3 - 5) patients to evaluate GPCR neutrophil expressions and functions in CKD development., Results: CKD patients presented a higher blood neutrophil-to-lymphocyte ratio (NLR), which was inversely correlated with the glomerular filtration rate (eGFR). A higher frequency of neutrophils expressing the senescent GPCR receptor (CXCR4) and activation markers (CD18 + CD11b + CD62L + ) was detected in CKD patients. Moreover, CKD neutrophils expressed higher amounts of GPCR formyl peptide receptors (FPR) 1 and 2, known as neutrophil pro- and anti-inflammatory receptors, respectively. Cytoskeletal organization, migration, and production of reactive oxygen species (ROS) by CKD neutrophils were impaired in response to the FPR1 agonist (fMLP), despite the higher expression of FPR1. In addition, CKD neutrophils presented enhanced intracellular, but reduced membrane expression of the protein Annexin A1 (AnxA1), and an impaired ability to secrete it into the extracellular compartment. Secreted and phosphorylated AnxA1 is a recognized ligand of FPR2, pivotal in anti-inflammatory and efferocytosis effects. CKD renal tissue presented a low number of neutrophils, which were AnxA1 + ., Conclusion: Together, these data highlight that CKD neutrophils overexpress GPCRs, which may contribute to an unbalanced aging process in the circulation, migration into inflamed tissues, and efferocytosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Scharf, Sandri, Rizzetto, Xavier, Grosso, Correia-Silva, Farsky, Gil and Farsky.)

Gradual loss of neuronal structure and function due to impaired blood-brain barrier (BBB) and neuroinflammation are important factors in multiple sclerosis (MS) progression. Our previous studies demonstrated that the C16 peptide and angiopoietin 1 (Ang-1) compound (C + A) could modulate inflammation and vascular protection in many models of MS. In this study, nanotechnology and a novel nanovector of the leukocyte chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) were used to examine the effects of C + A on MS. The acute experimental autoimmune encephalomyelitis (EAE) model of MS was established in Lewis rats. The C + A compounds were conjugated to control nano-carriers and fMLP-nano-carriers and administered to animals by intravenous injection. The neuropathological changes in the brain cortex and spinal cord were examined using multiple approaches. The stimulation of vascular injection sites was examined using rabbits. The results showed that all C + A compounds (C + A alone, nano-carrier C + A, and fMLP-nano-carrier C + A) reduced neuronal inflammation, axonal demyelination, gliosis, neuronal apoptosis, vascular leakage, and BBB impairment induced by EAE. In addition, the C + A compounds had minimal side effects on liver and kidney functions. Furthermore, the fMLP-nano-carrier C + A compound had better effects compared to C + A alone and the nano-carrier C + A. This study indicated that the fMLP-nano-carrier C + A could attenuate inflammation-related pathological changes in EAE and may be a potential therapeutic strategy for the treatment of MS and EAE.

Status epilepticus (SE) is described as continuous and self-sustaining seizures, which triggers hippocampal neurodegeneration, inflammation, and gliosis. N-formyl peptide receptor (FPR) has been associated with inflammatory process. N-formyl-methionyl-leucyl-phenylalanine (fMLP) peptide plays an anti-inflammatory role, mediated by the activation of G-protein-coupled FPR. Here, we evaluated the influence of fMLP peptides on the behavior of limbic seizures, memory consolidation, and hippocampal neurodegeneration process. Male Wistar rats (Rattus norvegicus) received microinjections of pilocarpine in hippocampus (H-PILO, 1.2 mg/μL, 1 μL) followed by fMLP (1 mg/mL, 1 μL) or vehicle (VEH, saline 0.9%, 1 μL). During the 90 min of SE, epileptic seizures were analyzed according to the Racine's Scale. After 24 h of SE, memory impairment was assessed by the inhibitory avoidance test and the neurodegeneration process was evaluated in hippocampal areas. There was no change in latency and number of wet dog shake (WDS) after administration of fMLP. However, our results showed that the intrahippocampal infusion of fMLP reduced the severity of seizures, as well as the number of limbic seizures. In addition, fMLP infusion protected memory dysfunction followed by SE. Finally, the intrahippocampal administration of fMLP attenuated the process of neurodegeneration in both hippocampi. Taken together, our data suggest a new insight into the functional role of fMLP peptides, with important implications for their potential use as a therapeutic agent for the treatment of brain disorders, such as epilepsy. Schematic drawing on the neuroprotective and anticonvulsant role of fMLP during status epilepticus. Initially, a cannula was implanted in hippocampus and pilocarpine/saline was administered into the hippocampus followed by fMLP/saline (A-C). fMLP reduced seizure severity and neuronal death in the hippocampus, as well as protecting against memory deficit (D)., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase. Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin 1A, a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes and relate this to intracellular coronin 1A localization. We used bone marrow neutrophils from wild-type and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase), secondary (lactoferrin), and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of coronin 1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in myeloperoxidase, MMP-2, and MMP-9 but not lactoferrin release, along with diminished filopodia formation, CD63 polarization, and colocalization of coronin 1A with CD63 in immune complex-stimulated Rac2-/- bone marrow neutrophils. Rac2 and coronin 1A were found associated with granules in cytochalasin B/f-Met-Leu-Phe-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on coronin 1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Six previously undescribed components, bis(4-glycosyloxybenzyl) 2-isobutyltartrate derivatives (pholidotoside A-E) and phenolic glycoside (pholidotosin A), together with twenty known compounds were isolated from the pseudobulbs of Pholidota chinensis. Their structures and absolute configuration were elucidated and established through various spectroscopic and chemical methods. The anti-inflammatory potential of selected compounds was examined using a human neutrophil cell model activated by N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB). Among these, dihydrophenanthrenes exhibited potent inhibitory effect on both superoxide anion generation and elastase release assays with IC 50 values ranging from 0.41 ± 0.05 to 7.14 ± 0.30 μM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms. However, excessively activated PMNs can also cause damage to host tissues under inflammatory conditions. Here we developed simple assays to determine the activation state of PMNs in human whole blood that contains soluble mediators known to influence PMN functions. Because mouse models are widely used to study the role of PMNs in infectious and inflammatory diseases, we adapted these assays for the rapid and reliable assessment of PMN functions in murine blood samples. Freshly collected whole blood samples were stimulated with agonists of the formyl peptide receptors (FPR) of PMNs and changes in reactive oxygen species (ROS) production and the expression of CD11b, CD62L (L-selectin), CD66b, and CD63 on the cell surface were analyzed with flow cytometry. We optimized these assays to minimize inadvertent interferences such as cell stress generated during sample handling and the loss of plasma mediators that regulate PMN functions. Human PMNs readily responded to the FPR agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP). The most sensitive responses of human PMNs to fMLP were CD11b, CD62L, and CD66b expression with half maximal effective concentrations (EC 50 ) of 5, 8, and 6 nM fMLP, respectively. CD63 expression and ROS production required markedly higher fMLP concentrations with EC 50 values of 19 and 50 nM fMLP, respectively. Mouse PMNs did not respond well to fMLP and required significantly higher concentrations of the FPR agonist WKYMVm (W-peptide) to achieve equivalent cell activation. The most sensitive response of mouse PMNs was ROS production with an EC 50 of 38 nM W-peptide. Because mice do not express CD66b, we only assessed the expression of CD62L, CD11b, and CD63 with EC 50 values of 54, 119, and 355 nM W-peptide, respectively. Validation of our optimized assays showed that they sensitively detect the responses of human PMNs to priming with endotoxin in vitro as well as the corresponding responses of murine PMNs to bacterial infection in a sepsis model. We conclude that these optimized assays could be useful tools for the monitoring of patients with infections, sepsis, and other inflammatory conditions as well as for the design and interpretation of preclinical studies of these diseases in mouse models., (Copyright © 2022. Published by Elsevier B.V.)

The bacteria-derived formyl peptide fMet-Leu-Phe (fMLF) is a potent chemoattractant of phagocytes that induces chemotaxis at subnanomolar concentrations. At higher concentrations, fMLF inhibits chemotaxis while stimulating degranulation and superoxide production, allowing phagocytes to kill invading bacteria. How an agonist activates distinct cellular functions at different concentrations remains unclear. Using a bioluminescence resonance energy transfer-based FPR1 biosensor, we found that fMLF at subnanomolar and micromolar concentrations induced distinct conformational changes in FPR1, a Gi-coupled chemoattractant receptor that activates various phagocyte functions. Neutrophil-like HL-60 cells exposed to subnanomolar concentrations of fMLF polarized rapidly and migrated along a chemoattractant concentration gradient. These cells also developed an intracellular Ca 2+ concentration gradient. In comparison, high nanomolar and micromolar concentrations of fMLF triggered the PLC-β/diacyl glycerol/inositol trisphosphate pathway downstream of the heterotrimeric Gi proteins, leading to Ca 2+ mobilization from intracellular stores and Ca 2+ influx from extracellular milieu. A robust and uniform rise in cytoplasmic Ca 2+ level was required for degranulation and superoxide production but disrupted cytoplasmic Ca 2+ concentration gradient and inhibited chemotaxis. In addition, elevated ERK1/2 phosphorylation and β-arrestin2 membrane translocation were associated with diminished chemotaxis in the presence of fMLF above 1 nM. These findings suggest a mechanism for FPR1 agonist concentration-dependent signaling that leads to a switch from migration to bactericidal activities in phagocytes.

The over-activated neutrophils through G-protein-coupled receptors (GPCRs) caused inflammation or tissue damage. Therefore, GPCRs or their downstream molecules are major targets for inhibiting uncontrolled neutrophil activation. Our studies investigate the action and underlying mechanism of larixol, a diterpene extract from the root of euphorbia formosana, on fMLP-induced neutrophil respiratory burst, chemotaxis, and granular release. The immunoprecipitation assay was performed to investigate whether larixol inhibits fMLP-induced respiratory burst by interfering with the interaction of fMLP receptor Gi-protein βγ subunits with its downstream molecules. Briefly, larixol inhibited fMLP (0.1 μM)-induced superoxide anion production (IC 50 :1.98 ± 0.14 μM), the release of cathepsin G (IC 50 :2.76 ± 0.15 μM) and chemotaxis in a concentration-dependent manner; however, larixol did not inhibit these functions induced by PMA (100 nM). Larixol inhibited fMLP-induced Src kinase phosphorylation. Therefore, larixol attenuated the downstream signaling of Src kinases, ERK1/2, p38, and AKT phosphorylation. Moreover, larixol inhibited fMLP-induced intracellular calcium mobilization, PKC phosphorylation, and p47 phox translocation from the cytosol to the plasma membrane. Larixol inhibited the interaction of the βγ subunits of Gi-protein of fMLP receptor with Src kinase or with PLCβ by the immunoprecipitation and duolink assay. Furthermore, larixol did not antagonize the formyl peptide receptors. Larixol did not increase cyclic nucleotide levels in neutrophils. These results suggest that larixol modulated fMLP-induced neutrophils superoxide anion production, chemotaxis, and granular releases by interrupting the interaction of the βγ subunits of Gi-protein with downstream signaling of the fMLP receptor., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Three-dimensional tracking of cells is one of the most powerful methods to investigate multicellular phenomena, such as ontogenesis, tumor formation or wound healing. However, 3D tracking in a biological environment usually requires fluorescent labeling of the cells and elaborate equipment, such as automated light sheet or confocal microscopy. Here we present a simple method for 3D tracking large numbers of unlabeled cells in a collagen matrix. Using a small lensless imaging setup, consisting of an LED and a photo sensor only, we were able to simultaneously track ~3000 human neutrophil granulocytes in a collagen droplet within an unusually large field of view (>50 mm2) at a time resolution of 4 seconds and a spatial resolution of ~1.5 μm in xy- and ~30 μm in z-direction. The setup, which is small enough to fit into any conventional incubator, was used to investigate chemotaxis towards interleukin-8 (IL-8 or CXCL8) and N-formylmethionyl-leucyl-phenylalanine (fMLP). The influence of varying stiffness and pore size of the embedding collagen matrix could also be quantified. Furthermore, we demonstrate our setup to be capable of telling apart healthy neutrophils from those where a condition of inflammation was (I) induced by exposure to lipopolysaccharide (LPS) and (II) caused by a pre-existing asthma condition. Over the course of our experiments we have tracked more than 420.000 cells. The large cell numbers increase statistical relevance to not only quantify cellular behavior in research, but to make it suitable for future diagnostic applications, too., Competing Interests: The authors have declared that no competing interests exist.

Chemotaxis is the directed movement of neutrophils towards an infected site. This physiological process can be reproduced using a modified Boyden chamber, such as the Transwell® support. Different techniques can be used to count neutrophils after migration to the lower chamber of the holder. The present study supports the use of an optimized Transwell® assay coupled with a flow cytometry-based method (Sysmex XN-9000) to detect chemotaxis abnormalities. A reference interval of neutrophil's chemotaxis was determined as part of this work. A first step involves the extraction of neutrophils from whole blood. The migration of neutrophils from the upper to the lower support chamber is subsequently directed by a chemoattractant gradient using N-formyl-l-Methionyl-l-Leucyl-l-Phenylalanine (fMLP). Neutrophils collected in the lower chamber are finally counted by flow cytometry. The original protocol was optimized through the comparison of different parameters. The use of Polymorphprep®, in the extraction of neutrophils, showed an improvement of the neutrophils yield of 1.65 times (57.5% of recovery) compared to the extraction using the Ficoll-Hypaque® gradient. A solution containing 5% of Bovin Serum Albumin (BSA) was used to suspend the extracted neutrophils, stabilize their viability and preserve their integrity. The mechanical agitation of the Transwell® permeable supports during migration did not show an increase in neutrophil yield. A migration time of 1 h 30 was identified as the best time for collecting the largest number of neutrophils after migration. Finally, we demonstrated that scraping the bottom of the well after migration improved neutrophil collection from the lower chamber by 1.9-fold compared to a non-scraping method. In conclusion, our results support the use of Polymorphprep® and a 5% BSA solution in the suspension, without agitation of the medium. An incubation time of 1 h 30 was identified as optimal for neutrophil migration through the chamber. Scraping the bottom after neutrophil migration improved neutrophil collection yield. Normal adult values were obtained with directed migration equal to 32.4% ±13.41% on 15 men and 18 women., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Exaggerated neutrophil activation and formation of neutrophil extracellular traps (NETs) are reported in systemic sclerosis (SSc) but its involvement in SSc pathogenesis is not clear. In the present study we assessed markers of neutrophil activation and NET formation in SSc patients in relation to markers of inflammation and disease phenotype. Factors promoting neutrophil activation in SSc remain largely unknown. Among the neutrophil activating factors, mitochondrial-derived N-formyl methionine (fMet) has been reported in several autoinflammatory conditions. The aim of the current study is to assess whether SSc patients have elevated levels of fMet and the role of fMet in neutrophil-mediated inflammation on SSc pathogenesis. Markers of neutrophil activation (calprotectin, NETs) and levels of fMet were analyzed in plasma from two SSc cohorts (n=80 and n=20, respectively) using ELISA. Neutrophil activation assays were performed in presence or absence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporin H. Elevated levels of calprotectin and NETs were observed in SSc patients as compared to healthy controls (p<0.0001) associating with SSc clinical disease characteristics. Further, SSc patients had elevated levels of circulating fMet as compared to healthy controls (p<0.0001). Consistent with a role for fMet-mediated neutrophil activation, fMet levels correlated with levels of calprotectin and NETs (r=0.34, p=0.002; r=0.29, p<0.01 respectively). Additionally, plasma samples from SSc patients with high levels of fMet induced de novo neutrophil activation through FPR1-dependent mechanisms. Our data for the first time implicates an important role for the mitochondrial component fMet in promoting neutrophil-mediated inflammation in SSc., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kuley, Stultz, Duvvuri, Wang, Fritzler, Hesselstrand, Nelson and Lood.)

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.

Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that in human neutrophils, calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI ( capri kd ) exhibit chemoattractant-induced, nonadaptive Ras activation; significantly increased phosphorylation of AKT, GSK-3α/3β, and cofilin; and excessive actin polymerization. capri kd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants, as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher-concentration range of chemoattractant gradients., Competing Interests: The authors declare no competing interest.

Neutrophils fight with invading pathogens through various mechanisms including degranulation, phagocytosis, and the release of neutrophil extracellular traps (NETs). This study aimed to determine the impact of a synthetic formyl-peptide (FMLP) on human neutrophils in vitro, and to determine the role of mitoxantrone (MTX), a pharmacological blocker of mitochondrial Ca^(2+) Uniporter (MCU), on FMLP-induced alterations. Isolated neutrophils and a whole-blood preparation of neutrophils were pre-treated with MTX and then stimulated with FMLP. Field's-stained smears and brightfield microscopy were employed for morphological characterization and quantification of neutrophils. The release of cell-free DNA (cfDNA) was also measured for determining neutrophil damage. Our data demonstrated degenerative changes in neutrophils and a greater cfDNA release upon stimulation with FMLP which was negatively associated with the presence of resting platelets in whole blood preparation. Interestingly, MTX pre-treatment significantly reduced FMLP-triggered neutrophil damage and cfDNA release. Metformin, a known inhibitor of NETs formation, also decreased the FMLP-induced changes in neutrophils. In addition to confirming the degenerative potential of FMLP, this study reveals a novel contribution of MCU in regulating FMLP-induced morphological alterations in human neutrophils.

Two new isosarcophine derivatives, cherbonolides M ( 1 ) and N ( 2 ), were further isolated from a Formosan soft coral Sarcophyton cherbonnieri . The planar structure and relative configuration of both compounds were established by the detailed analysis of the IR, MS, and 1D and 2D NMR data. Further, the absolute configuration of both compounds was determined by the comparison of CD spectra with that of isosarcophine ( 3 ). Notably, cherbonolide N ( 2 ) possesses the unique cembranoidal scaffold of tetrahydrooxepane with the 12,17-ether linkage fusing with a γ -lactone. In addition, the assay for cytotoxicity of both new compounds revealed that they showed to be noncytotoxic toward the proliferation of A549, DLD-1, and HuCCT-1 cell lines. Moreover, the anti-inflammatory activities of both metabolites were carried out by measuring the N -formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced generation of superoxide anion and elastase release in the primary human neutrophils. Cherbonolide N ( 2 ) was found to reduce the generation of superoxide anion (20.6 ± 6.8%) and the elastase release (30.1 ± 3.3%) in the fMLF/CB-induced human neutrophils at a concentration of 30 μM.

Owing to the excellent antibacterial and antiviral activity, silver nanoparticles have a widespread use in the food and pharmaceutical industries. With the increase in the production and use of the related products, the potential hazard of silver nanoparticles has aroused public attention. The main purpose of this study is to explore the toxicity of silver nanoparticles and induction of lung inflammation in vitro and in vivo. Here, we validated that small amounts of silver ions dissolved from silver nanoparticles caused the depolarization of plasma membrane, resulting in an overload of intracellular sodium and calcium, and eventually led to the cell necrosis. The blockers of calcium or sodium channels inversed the toxicity of silver ions. Then, we instilled silver nanoparticles or silver nitrate (50 μg per mouse) into the lungs of mice, and this induced pulmonary injury and mitochondrial content release, led to the recruitment of neutrophils to the lung tissue via p38 MAPK pathway. Altogether, these data show that released silver ions from nanoparticles induced cell necrosis through Na + and Ca 2+ influx and triggered pulmonary inflammation through elevating mitochondrial-related contents released from these necrotic cells.

Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications-a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.

A new dimeric quaternary protoberberine alkaloid, bispalmatrubine ( 1 ), and thirteen known compounds ( 2 - 14 ) were purified from the tubers of Tinospora dentata . Their structures were determined by spectroscopic and spectrometric analytical methods. Among the isolates, eight compounds were examined for their in vitro anti-inflammatory potential and several tested alkaloids displayed moderate inhibitory effects of N -formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release.

Qin Pi ( Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8 E )-4''- O -methylligstroside ( 1 ), (8 E )-4''- O -methyldemethylligstroside ( 2 ), and 3'',4''-di- O -methyl-demethyloleuropein ( 3 ), have been isolated from the stem bark of Fraxinus chinensis , together with 23 known compounds ( 4 - 26 ). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8 E )-4''- O -methylligstroside ( 1 ), (8 E )-4''- O -methyldemethylligstroside ( 2 ), 3'',4''-di- O -methyldemethyloleuropein ( 3 ), oleuropein ( 6 ), aesculetin ( 9 ), isoscopoletin ( 11 ), aesculetin dimethyl ester ( 12 ), fraxetin ( 14 ), tyrosol ( 21 ), 4-hydroxyphenethyl acetate ( 22 ), and (+)-pinoresinol ( 24 ) exhibited inhibition (IC 50 ≤ 7.65 μg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1 , 9 , 11 , 14 , 21 , and 22 inhibited fMLP/CB-induced elastase release with IC 50 ≤ 3.23 μg/mL. In addition, compounds 2 , 9 , 11 , 14 , and 21 showed potent inhibition with IC 50 values ≤ 27.11 μM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines , tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1 , 9 , and 14 . Compounds 1 , 9 , and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1 , 9 , and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1 , 9 , and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

Following nerve injury, disintegrated axonal mitochondria distal to the injury site release mitochondrial formylated peptides and DNA that can induce activation and inflammatory profiling of Schwann cells via formyl peptide receptor 2 (Fpr2) and toll-like receptor 9 (TLR9), respectively. We studied RT4 schwannoma cells to investigate the regulation of Fpr2 and TLR9 after stimulation with fMLF as a prototypical formylated peptide. RT4 cells were treated with fMLF at various concentrations and times with and without pretreatment with inhibitors (chloroquine for activated TLR9, PBP10 for Fpr2). Western blots of Fpr2, TLR9, p-p38, p-NFκB, and IL-6 were compared in relation to inflammatory profiling of RT4 cells and chemokine receptors (CCR2, CXCR4) as potential co-receptors of Fpr2. fMLF stimulation upregulated Fpr2 in RT4 cells at low concentrations (10 nM and 100 nM) but higher concentrations were required (10 µM and 50 µM) when the cells were pretreated with an activated TLR9 inhibitor. Moreover, the higher concentrations of fMLF could modulate TLR9 and inflammatory markers. Upregulation of Fpr2 triggered by 10 nM and 100 nM fMLF coincided with higher levels of chemokine receptors (CCR2, CXCR4) and PKCβ. Treating RT4 cells with fMLF, as an in vitro model of Schwann cells, uncovered Schwann cells' complex responses to molecular patterns of release from injured axonal mitochondria.

The formyl peptide receptor 2 (ALX/FPR2), a G-protein-coupled receptor (GPCR), plays an important role in host defense and inflammation. This receptor can be driven as pro- or anti-inflammatory depending on its agonist, such as N -formyl-Met-Leu-Phe-Lys (fMLFK) and resolvin D1 (RvD1) or its aspirin-triggered 17 (R)-epimer, AT-RvD1, respectively. However, the activation mechanism of ALX/FPR2 by pro- and anti-inflammatory agonists remains unclear. In this work, on the basis of molecular dynamics simulations, we evaluated a model of the ALX/FPR2 receptor activation process using two agonists, fMLFK and AT-RvD1, with opposite effects. The simulations by both fMLFK and AT-RvD1 induced the ALX/FPR2 activation through a set of receptor-core residues, in particular, R205, Q258, and W254. In addition, the activation was dependent on the disruption of electrostatic interactions in the cytoplasmic region of the receptor. We also found that in the AT-RvD1 simulations, the position of the H8 helix was similar to that of the same helix in other class-A GPCRs coupled to arrestin. Thus our results shed light on the mechanism of activation of the ALX/FPR2 receptor by pro-inflammatory and pro-resolution agonists.

Phosphatidylinositol-specific phospholipase C (PI-PLC) and cytosolic phospholipase A 2 (cPLA 2 ) regulate both eosinophil degranulation and leukotriene (LT) synthesis via PI-PLC-mediated calcium influx and cPLA 2 activation. Phosphatidylcholine-specific phospholipase C (PC-PLC) likely plays a key role in cellular signaling, including the eosinophilic allergic inflammatory response. This study examined the role of PC-PLC in eosinophil LT synthesis and degranulation using tricyclodecan-9-yl-xanthogenate (D609), a PC-specific PLC inhibitor. D609 inhibited N-formyl-met-leu-phe + cytochalasin B (fMLP/B)-induced arachidonic acid (AA) release and leukotriene C 4 (LTC 4 ) secretion. However, at concentrations that blocked both AA release and LTC 4 secretion, D609 had no significant inhibitory effect on stimulated cPLA 2 activity. D609 also partially blocked fMLP/B-induced calcium influx, indicating that inhibition of AA release and LTC 4 secretion by D609 is due to inhibition of calcium-mediated cPLA 2 translocation to intracellular membranes, not inhibition of cPLA 2 activity. In addition, D609 inhibited fMLP/B-stimulated eosinophil peroxidase release, indicating that PC-PLC regulates fMLP/B-induced eosinophil degranulation by increasing the intracellular calcium concentration ([Ca 2+ ] i ). Overall, our results showed that PC-PLC is critical for fMLP/B-stimulated eosinophil LT synthesis and degranulation. In addition, degranulation requires calcium influx, while PC-PLC regulates LTC 4 synthesis through calcium-mediated cPLA 2 activation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aims: Takotsubo syndrome (TTS) episodes are primarily initiated by 'pulse' release of catecholamines inducing neutrophil infiltration and myocardial inflammation in susceptible individuals (largely ageing women). Evidence of myocardial inflammation and associated energetic impairment persists for ≥ 3 months post-acute TTS episodes, suggesting the existence of additional 'perpetuating' mechanisms. The effects of B-type natriuretic peptide (BNP) in suppressing superoxide (O 2 - ) release from neutrophils are transiently impaired in acute heart failure. We also evaluated the extent and duration of BNP-induced suppression of O 2 - release post-TTS., Methods and Results: TTS patients were studied acutely (n = 34) and 3 months thereafter (n = 13) and compared with control subjects (n = 25). O 2 - generation from neutrophils, triggered by N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate, and its suppression by BNP, were measured in vitro. Determinants of variability in BNP effect were sought via univariate and multivariate analyses. Relative to control subjects, in TTS patients, BNP suppression of both phorbol myristate acetate and N-formyl-methionyl-leucyl-phenylalanine-induced O 2 - release was impaired acutely (P < 0.05 for both); this did not improve over the 3-month recovery period, despite treatment with conventional anti-failure medication in 85% of patients. No significant correlates of BNP effect (other than TTS) were identified., Conclusions: (1) While TTS is associated with marked and prolonged release of BNP, there is virtually total loss of the ability of BNP to suppress neutrophil O 2 - release and its impact on tissue inflammation. (2) BNP responses do not recover for at least 3 months post-attacks, suggesting that this might contribute to perpetuation of myocardial inflammation in TTS patients., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

The importance of the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in neutrophil function has been intensely studied. However, the role of the intracellular Na + concentration ([Na + ] i ) which is closely linked to the intracellular Ca 2+ regulation has been largely overlooked. The [Na + ] i is regulated by Na + transport proteins such as the Na + /Ca 2+ -exchanger (NCX1), Na + /K + -ATPase, and Na + -permeable, transient receptor potential melastatin 2 (TRPM2) channel. Stimulating with either N-formylmethionine-leucyl-phenylalanine (fMLF) or complement protein C5a causes distinct changes of the [Na + ] i . fMLF induces a sustained increase of [Na + ] i , surprisingly, reaching higher values in TRPM2 -/- neutrophils. This outcome is unexpected and remains unexplained. In both genotypes, C5a elicits only a transient rise of the [Na + ] i . The difference in [Na + ] i measured at t = 10 min after stimulation is inversely related to neutrophil chemotaxis. Neutrophil chemotaxis is more efficient in C5a than in an fMLF gradient. Moreover, lowering the extracellular Na + concentration from 140 to 72 mM improves chemotaxis of WT but not of TRPM2 -/- neutrophils. Increasing the [Na + ] i by inhibiting the Na + /K + -ATPase results in disrupted chemotaxis. This is most likely due to the impact of the altered Na + homeostasis and presumably NCX1 function whose expression was shown by means of qPCR and which critically relies on proper extra- to intracellular Na + concentration gradients. Increasing the [Na + ] i by a few mmol/l may suffice to switch its transport mode from forward (Ca 2+ -efflux) to reverse (Ca 2+ -influx) mode. The role of NCX1 in neutrophil chemotaxis is corroborated by its blocker, which also causes a complete inhibition of chemotaxis., (Copyright © 2020 Najder, Rugi, Lebel, Schröder, Oster, Schimmelpfennig, Sargin, Pethő, Bulk and Schwab.)

Human neutrophils have a vital role in host defense and inflammatory responses in innate immune systems. Growing evidence shows that the overproduction of reactive oxygen species and granular proteolytic enzymes from activated neutrophils is linked to the pathogenesis of acute inflammatory diseases. However, adequate therapeutic targets are still lacking to regulate neutrophil functions. Herein, we report that MVBR-28, synthesized from the Mannich bases of heterocyclic chalcone, has anti-neutrophilic inflammatory effects through regulation of intracellular pH. MVBR-28 modulates neutrophil functions by attenuating respiratory burst, degranulation, and migration. Conversely, MVBR-28 has no antioxidant effects and fails to alter elastase activity in cell-free systems. The anti-inflammatory effects of MVBR-28 are not seen through cAMP pathways. Significantly, MVBR-28 potently inhibits extracellular Ca 2+ influx in N-formyl-methionyl-leucyl-phenylalanine (fMLF)- and thapsigargin-activated human neutrophils. Notably, MVBR-28 attenuates fMLF-induced intracellular alkalization in a K + -dependent manner, which is upstream of Ca 2+ pathways. Collectively, these findings provide new insight into Mannich bases of heterocyclic chalcone regarding the regulation of neutrophil functions and the potential for the development of MVBR-28 as a lead compound for treating neutrophilic inflammatory diseases., (© 2020 Federation of American Societies for Experimental Biology.)

High-level reactive oxygen species (ROS) production in neutrophils is tightly regulated, as it can damage host cells. Neutrophils also undergo low-level ROS production when stimulated by cytokines or chemoattractants, but its biologic significance remains largely unknown. Voltage-gated proton channels (Hv1/VSOP) activity reportedly supports ROS production in neutrophils; however, we show here that Hv1/VSOP balances ROS production to suppress neutrophil directional migration in the presence of low concentrations of N-formyl-Met-Leu-Phe (fMLF). Neutrophils derived from Hvcn1 gene knockout mice produced more ROS than neutrophils from wild-type mice in the stimulation with fMLF at concentration of 1 µM and nonstimulus condition. They also exhibited stronger chemotactic responses to low concentrations of fMLF than did wild-type neutrophils. Receptor sensitivity to fMLF and evoked Ca 2+ responses did not differ between Hv1/VSOP-deficient and wild-type neutrophils. Activation of ERK, but not p38, was enhanced and prolonged during the increased ROS production seen after fMLF stimulation in Hv1/VSOP-deficient neutrophils. Inhibiting ROS production suppressed the enhanced ERK activation in Hv1/VSOP-deficient neutrophils and their directional migration. These results indicate that Hv1/VSOP balances ROS production to reduce ERK signaling and suppress excessive neutrophil migration in response to fMLF. Our findings thus reveal a novel role for ROS in the directional migration of neutrophils., (©2020 Society for Leukocyte Biology.)

Neauvia hydrogel (N-Gel) is a hyaluronic acid-based dermal filler, cross-linked with polyethylene glycol. This filler contains sodium hyaluronate at different concentrations, poly(ethylene glycol) diglycidyl ether cross-linked, glycine, and l-prolyne. Assessing any effects of N-Gel on immunity and inflammation is of crucial importance. The aim of the study was to characterize the ability of N-Gel to modulate human polymorphonuclear leukocyte (PMN) functions, including migration, oxidative metabolism, and production of proinflammatory mediators. N-Gel was tested on isolated human PMN. Spontaneous and N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated migration were examined using the Boyden Chamber technique, whereas the oxidative metabolism was assessed through spectrofluorometric measurement of reactive oxygen species (ROS) production under resting conditions and after stimulation with fMLP. Tumor necrosis factor (TNF)-α and interleukin (IL)-8 mRNA levels were measured by real-time PCR after stimulation with fMLP or Escherichia coli lipopolysaccharide. This study showed that N-Gel reduced fMLP-induced migration and ROS production without affecting these functions in resting cells. In addition, incubation of PMN with N-Gel effectively reduced both TNF-α and IL-8 mRNA levels. N-Gel modulates critical functions of human PMN such as migration and oxidative metabolism, indicating its potential as an anti-inflammatory agent., (© 2020 Wiley Periodicals LLC.)

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.

Background: Host-directed therapies are a comparatively new and promising method for the treatment of tuberculosis. A variety of host pathways, vaccines and drugs have the potential to provide novel adjunctive therapies for the treatment of tuberculosis. In this connection, we have earlier reported the immunotherapeutic potential of N-formylated N-terminal peptide of glutamine synthetase of Mycobacterim tuberculosis H37Rv (Mir SA and Sharma S, 2014). Now in the present study, we investigated the immunotherapeutic effect of N-terminally formylated internal-peptide 'f- MLLLPD' of mycobacterial glutamine synthetase (Rv2220) in mouse model of tuberculosis., Methods: The N-terminally formylated peptide, f-MLLLPD was tested for its potential to generate Reactive Oxygen Species (ROS) in murine neutrophils. Further, its therapeutic effect alone or in combination with anti-tubercular drugs was evaluated in mouse model of tuberculosis., Results: The f-MLLLPD peptide treatment alone and in combination with ATDs reduced the bacterial load (indicated as colony forming units) in lungs of infected mice by 0.58 (p<0.01) and 2.92 (p<0.001) log10 units respectively and in their spleens by 0.46 (p<0.05) and 2.46 (p<0.001) log10 units respectively. In addition, the observed histopathological results correlated well with the CFU data., Conclusion: The results of the current study show that f-MLLLPD peptide confers an additional therapeutic efficacy to the anti-tuberculosis drugs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Here we report the effects of exogenous and endogenous galectin-1 (Gal-1) in modulating the functional responses of human and murine neutrophils at different stages of activation, i.e. naive, primed, and activated. Exposure to Gal-1 did not induce ROS production in either naive or N-formyl-methionyl-leucyl-phenylalanine-primed (fMLP; 10 -9 M) neutrophils. However, Gal-1 elicited a concentration-dependent ROS production in neutrophils activated with fMLP at concentrations ranging from 10 -8 M to 10 -6 M. Additional fMLP (10 -7 M) stimulation of fMLP-activated neutrophils increased ROS production, whose intensity was inversely related to the fMLP concentration used in the first activation step (10 -8 M to 10 -6 M), and was not influenced by the presence of Gal-1. Naive neutrophils treated with Gal-1 and then exposed to fMLP (10 -6 M) or phorbol-12-myristate-13-acetate (10 -7 M) produced less ROS, as compared to naive neutrophils not treated with Gal-1. Interestingly, these in vitro Gal-1 effects were associated with Gal-1 carbohydrate-binding activity and the ability to decrease FPR-1 (formyl peptide receptor 1) expression in naive human neutrophils. Conversely, positive ROS modulation by Gal-1 in activated neutrophils was not associated with FPR-1 expression but it was related to its carbohydrate recognition. In vitro, fMLP stimulation of Gal-1 -/- mouse neutrophils produced more ROS than fMLP stimulation of Gal-1 +/+ neutrophils and this effect may be associated with increased FPR-1 expression. Exogenous Gal-1 induced ROS production in Gal-1 -/- mouse neutrophils more effectively than in Gal-1 +/+ mouse neutrophils. Compared to Gal-1 +/+ mice, Gal-1 -/- mice exhibited lower bacterial load in the peritoneal fluid and peripheral blood, thus indicating a greater bactericidal activity in vivo. These findings demonstrate that endogenous Gal-1 restricts ROS generation that correlates with bacterial killing capacity in inflammatory neutrophils. Thus, endogenous and exogenous Gal-1 may either positively or negatively modulate the effector functions of neutrophils according to the cell activation stage., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments., Competing Interests: The authors have declared that no competing interests exist.

Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen-killing responses. In neutrophils G protein-coupled receptor (GPCR)-driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), are highly dependent on PI3K-γ, a Ras-GTP, and Gβγ coincidence detector. In unprimed cells, the major GPCR-triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR-PIP 3 signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM-CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP 3 accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation., (©2019 Society for Leukocyte Biology.)

Neutrophil chemotactic defects have been reported previously in patients with hyper-IgE syndrome. Bi-allelic mutations in dedicator of cytokinesis 8 (DOCK8) gene usually cause an autosomal recessive hyper-IgE syndrome phenotype. Data are lacking about expression of DOCK8 protein in neutrophils or the possible role of DOCK8 in neutrophil function. We sought to determine if DOCK8 protein is expressed in neutrophils and if DOCK8 plays a role in neutrophil function. The expression of DOCK8 protein was assessed in neutrophils from healthy volunteers with and without activators. Neutrophil chemotaxis, phagocytosis and superoxide generation were studied in neutrophils from DOCK8-deficient patients compared to neutrophils from healthy controls before and after stimulation with activators: phorbol 12-myristate 13-acetate (PMA) or N-Formylmethionyl-leucyl-phenylalanine (fMLP). DOCK8 protein is expressed in resting neutrophils from healthy controls, with a significant increase in DOCK8 expression after stimulation. Neutrophil functions were assessed in 6 DOCK8-deficient patients. All patients had the same non-sense mutation (c.C5134A, p.S1711X). Normal chemotaxis was recorded in 4/6 patients while a mild to moderate chemotaxis defect was recorded in 2/6. Superoxide generation was mainly normal in neutrophils from all six patients and phagocytosis was normal in five patients tested. We conclude that DOCK8 protein is expressed in resting human neutrophils and DOCK8 expression is increased after stimulation with either PMA or fMLP. Most patients with a disease-causing mutation in DOCK8 have normal neutrophil functions, while a minority showed a mild to moderate chemotactic defect.

Microtubules (MTs) are critically involved in the transport of material within cells, but their roles in chemotactic leukocyte motility and effector functions are still obscure. Resting neutrophils contain few MTs assembled in an MT organizing center (MTOC) behind their multilobular nuclei. Using a probe of real-time tubulin polymerization, SiR-tubulin, we found that neutrophils elongated their MTs within minutes in response to signals from the two prototypic chemotactic peptides, CXCL1 and fMLP. Taxol, a beta-tubulin binding and MT stabilizing drug, was found to abolish this CXCL1- and fMLP-stimulated MT polymerization. Nevertheless, taxol treatment as well as disruption of existing and de novo generated MTs did not impair neutrophil protrusion and squeezing through IL-1β-stimulated endothelial monolayers mediated by endothelial deposited CXCL1 and neutrophil CXCR2. Notably, CXCL1-dependent neutrophil TEM was not associated with neutrophil MT polymerization. Chemokinetic neutrophil motility on immobilized CXCL1 was also not associated with MT polymerization, and taxol treatment did not interfere with this motility. Nevertheless, and consistent with its ability to suppress MT polymerization induced by soluble CXCL1 and fMLP, taxol treatment inhibited neutrophil chemotaxis toward both chemotactic peptides. Taxol treatment also suppressed CXCL1- and fMLP-triggered elastase-dependent neutrophil invasion through collagen I barriers. Collectively, our results highlight de novo chemoattractant-triggered MT polymerization as key for neutrophil chemotaxis and elastase-dependent invasion but not for chemotactic neutrophil crossing of inflamed endothelial barriers., (©2019 Society for Leukocyte Biology.)

Several studies showed that hydroxyethyl starch (HES), a synthetic colloid used in volume replacement therapies, interferes with leukocyte-endothelium interactions. Although still unclear, the mechanism seems to involve the inhibition of neutrophils' integrin. With the aim to provide direct evidence of the binding of HES to neutrophils and to investigate the influence of HES on neutrophil chemotaxis, we isolated and treated the cells with different concentrations of fluorescein-conjugated HES (HES-FITC), with or without different stimuli (N-Formylmethionine-leucyl-phenylalanine, fMLP, or IL-8). HES internalization was evaluated by trypan blue quenching and ammonium chloride treatment. Chemotaxis was evaluated by under-agarose assay after pretreatment of the cells with HES or a balanced saline solution. The integrin interacting with HES was identified by using specific blocking antibodies. Our results showed that HES-FITC binds to the plasma membrane of neutrophils without being internalized. Additionally, the cell-associated fluorescence increased after stimulation of neutrophils with fMLP ( p < 0.01) but not IL-8. HES treatment impaired the chemotaxis only towards fMLP, event mainly ascribed to the inhibition of CD-11b (Mac-1 integrin) activity. Therefore, the observed effect mediated by HES should be taken into account during volume replacement therapies. Thus, HES treatment could be advantageous in clinical conditions where a low activation/recruitment of neutrophils may be beneficial, but may be harmful when unimpaired immune functions are mandatory.

A dogma of innate immunity is that neutrophils use G-protein-coupled receptors (GPCRs) for chemoattractant to chase bacteria through chemotaxis and then use phagocytic receptors coupled with tyrosine kinases to destroy opsonized bacteria via phagocytosis. Our current work showed that G-protein-coupled formyl peptide receptors (FPRs) directly mediate neutrophil phagocytosis. Mouse neutrophils lacking formyl peptide receptors (Fpr1/2 -/- ) are defective in the phagocytosis of Escherichia coli and the chemoattractant N-formyl-Met-Leu-Phe (fMLP)-coated beads. fMLP immobilized onto the surface of a bead interacts with FPRs, which trigger a Ca 2+ response and induce actin polymerization to form a phagocytic cup for engulfment of the bead. This chemoattractant GPCR/Gi signaling works independently of phagocytic receptor/tyrosine kinase signaling to promote phagocytosis. Thus, in addition to phagocytic receptor-mediated phagocytosis, neutrophils also utilize the chemoattractant GPCR/Gi signaling to mediate phagocytosis to fight against invading bacteria.

The ability to detect and image secreted peroxynitrite (ONOO - ) along the extracellular surface of a single cell is biologically significant, as ONOO - generally exerts its function for host defense and signal transductions at the plasma membrane. However, as a result of the short lifetime and fast diffusion rate of small ONOO - , precise determination of the ONOO - level at the cell surface remains a challenging task. In this paper, the use of a membrane-anchored streptavidin-biotin-controlled binding probe (CBP), ONOO-CBP, to determine quantitatively the ONOO - level at the cell surface and to investigate the effect of different stimulants on the production of ONOO - along the plasma membrane of macrophages is reported. Our results revealed that the combination of NO synthase (iNOS) and NADPH oxidase (NOX) activators was highly effective in inducing ONOO - secretion, achieving more than a 25-fold increase in ONOO - relative to untreated cells. After 1 h of phorbol-12-myristate-13-acetate (PMA) stimulation, the amount of ONOO - secreted by RAW264.7 macrophages was similar to the condition treated with 25 μm 3-morpholinosydnonimine hydrochloride (SIN-1), which was estimated to release about 20 μm of ONOO - into Dulbecco's modified Eagle's medium (DMEM) in 1 h. This novel approach should open up new opportunities to image various reactive oxygen and nitrogen species secreted at the plasma membrane that cannot be simply achieved by conventional analytical methods., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Neutrophils are recruited to infection sites and kill bacteria by phagocytosis and reactive oxygen species (ROS) production. It has been reported that vacuoles are present in neutrophils that produce ROS and are present in large numbers in blood smears of patients with bacterial infections. The leukocyte differentiation function on the Sysmex automated hematology analyzer classifies leukocytes by flow cytometry. Particularly, side-scattered light is known to reflect the quantity of organelles. This study investigated the possibility of detecting vacuoles or invagination of cell membrane in neutrophils producing ROS using a hematology analyzer. Whole blood and polymorphonuclear (PMN) cell fractions were activated with phorbol myristate acetate (PMA) or formylmethionylleucylphenylalanine (fMLP) and analyzed using the Sysmex XE-2100 automated hematology analyzer. PMN fractions were morphologically analyzed with a confocal laser scanning microscope (CLSM), electron microscope (EM), and general-purpose conventional flow cytometer. In the white blood cell differentiation scattergram obtained in this analysis, a new cluster separate from the original neutrophil cluster appeared in the eosinophil area in an area of higher side-scattering (SSC) intensity. Flow cytometry analysis of the PMN fractions revealed that the cells in this new cluster were CD16b- and APF-positive, indicating that the cells were activated neutrophils that produced ROS. CLSM and EM findings revealed that ROS production occurred in the cytoplasm and that the activated neutrophils contained some vacuole-like structures of vacuoles or invagination of cell membrane. Vacuole-like Sstructures were found within the cytoplasm of neutrophils producing ROS. These neutrophils were detected as an independent cluster in the eosinophil area with higher SSC intensity than that shown by neutrophils in the traditional cluster on the white blood cell differentiation scattergram, likely because the vacuole-like structures increased the SSC intensity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neutrophils reach the sites of inflammation and infection in a timely manner by navigating efficiently through mechanically complex interstitial spaces, following the guidance of chemical gradients. However, our understanding of how neutrophils that follow chemical cues overcome mechanical obstacles in their path is restricted by the limitations of current experimental systems. Observations in vivo provide limited insights due to the complexity of the tissue environment. Here, we developed microfluidic devices to study the effect of progressive mechanical confinement on the migration patterns of human neutrophils toward chemical attractants. Using these devices, we identified four migration patterns: arrest, oscillation, retrotaxis, and persistent migration. The proportion of these migration patterns is different in patients receiving immunosuppressant treatments after kidney transplant, patients in critical care, and neonatal patients with infections and is distinct from that in healthy donors. The occurrence of these migration patterns is independent of the nuclear lobe number of the neutrophils and depends on the integrity of their cytoskeletal components. Our study highlights the important role of mechanical cues in moving neutrophils and suggests the mechanical constriction-induced migration patterns as potential markers for infection and inflammation., (©2018 Society for Leukocyte Biology.)

Background: Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches., Objectives: We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the in vitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function., Methods: We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function., Results: Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved in vitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients., Conclusion: Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved in vitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Trauma causes inflammation by releasing mitochondria that act as Danger-Associated Molecular Patterns (DAMPs). Trauma also increases susceptibility to infection. Human mitochondria contain 13 N-formyl peptides (mtFPs). We studied whether mtFPs released into plasma by clinical injury induce neutrophil (PMN) inflammatory responses, whether their potency reflects their similarity to bacterial FPs and how their presence at clinically relevant concentration affects PMN function., Methods: N-terminal sequences of the 13 mtFPs were synthesized. Changes in human PMN cytosolic Ca concentration ([Ca]i) and chemotactic responses to mtFPs were studied. Sequence similarity of mtFPs to the canonical bacterial peptide f-Met-Leu-Phe (fMLF/fMLP) was studied using the BLOcks SUbstitution Matrix 62 (BLOSUM 62) system. The presence of mtFPs in plasma of trauma patients was assayed by Enzyme-linked immunosorbent assay (ELISA). The effects of the most potent mtFP (ND6) on PMN signaling and function were then studied at ambient clinical concentrations by serial exposure of native PMN to ND6, chemokines and leukotrienes., Results: Five mtFPs (ND6, ND3, ND4, ND5, and Cox 1) induced [Ca]i flux and chemotaxis in descending order of potency. Evolutionary similarity to fMLF predicted [Ca]i flux and chemotactic potency linearly (R = 0.97, R = 0.95). Chemoattractant potency was also linearly related to [Ca]i flux induction (R = 0.92). Active mtFPs appear to circulate in significant amounts immediately after trauma and persist through the first week. The most active mtFP, ND6, suppresses responses to physiologic alveolar chemoattractants (CXCL-1, leukotriene B4) as well as to fMLF where CXCL-1 and leukotriene B4 do not suppress N-formyl peptide receptor (FPR)-1 responses to mtFPs. Prior FPR-1 inhibition rescues PMN from heterologous suppression of CXCR-1 and BLT-1 by mtFPs., Conclusion: The data suggest mtFPs released by injured tissue may attract PMN to trauma sites while suppressing PMN responses to other chemoattractants. Inhibition of mtFP-FPR1 interactions might increase PMN recruitment to lung bacterial inoculation after trauma. These findings suggest new paradigms for preventing infections after trauma., Level of Evidence: Therapeutic, Level IV.

N-Formyl-Met-Leu-Phe (fMLP), a mimic of N-formyl oligopeptides that are released from bacteria, is a potent leukocyte chemotactic factor. It induces intracellular calcium ([Ca]i) transient that is important for various neutrophil biological functions, e.g., adhesion, ROS, and cytokine productions. Toll-like receptors (TLRs), an essential part of host innate immunity, regulate neutrophil activities, but their role in [Ca]i signaling is less clear. In the present study, we examined the effect of several TLR ligands, including Pam3Cys4 (TLR1/2), lipopolysaccharide (LPS, TLR4), and lipoteichoic acid (LTA, TLR2/6), on calcium signaling and on the fMLP-induced [Ca]i transients in mouse neutrophils loaded with Fura-2/AM. We found that unlike fMLP, the three TLR ligands tested did not elicit any detectable Ca flux. However, Pam3Cys4, but not LPS or LTA, markedly synergized the fMLP-induced [Ca]i transients, and had no effect on the host component keratinocyte-derived cytokine (KC)- or C5a-induced calcium flux. The effect of Pam3Cys4 on the fMLP-induced [Ca]i transients is by enhancing extracellular Ca influx, not intracellular Ca release. Surprisingly, deletion of TLR2 or MyD88 in neutrophils had no impact on the Pam3Cys4's effect, suggesting a TLR2-MyD88-independent mechanism. Finally, using the pan PKC activator and inhibitor, we demonstrated that PKC negatively regulated fMLP-induced [Ca]i transients and that inhibition of PKC did not prohibit Pam3Cys4's synergistic effect on the fMLP-induced calcium influx. In conclusion, the present study identified a novel synergistic effect of Pam3Cys4 on fMLP-induced [Ca]i transients, a process important for many neutrophil biological functions.

Neutrophils are important mediators of the innate immune defense and of the host response to a physical trauma. Because aberrant infiltration of injured sites by neutrophils was shown to cause adverse effects after trauma, we investigated how neutrophil infiltration could be modulated at the cellular level. Our data indicate that protein kinase D (PKD) is a vital regulator of neutrophil transmigration. PKD phosphorylates the Cofilin-phosphatase Slingshot-2L (SSH-2L). SSH-2L in turn dynamically regulates Cofilin activity and actin polymerization in response to a chemotactic stimulus for neutrophils, for example, fMLP. Here, we show that inhibition of PKD by two specific small molecule inhibitors results in broad, unrestricted activation of Cofilin and strongly increases the F-actin content of neutrophils even under basal conditions. This phenotype correlates with a significantly impaired neutrophil deformability as determined by optical stretcher analysis. Consequently, inhibition of PKD impaired chemotaxis as shown by reduced extravasation of neutrophils. Consequently, we demonstrate that transendothelial passage of both, neutrophil-like NB4 cells and primary PMNs recovered from a hemorrhagic shock trauma model was significantly reduced. Thus, inhibition of PKD may represent a promising modulator of the neutrophil response to trauma., (©2018 Society for Leukocyte Biology.)

Five new cembranoid-related diterpenoids, namely, flexibilisins D and E ( 1 and 2 ), secoflexibilisolides A and B ( 3 and 4 ), and flexibilisolide H ( 5 ), along with nine known compounds ( 6 ⁻ 14 ), were isolated from the soft coral Sinularia flexibilis. Their structures were established by extensive spectral analysis. Compound 3 possesses an unusual skeleton that could be biogenetically derived from cembranoids. The cytotoxicity and anti-inflammatory activities of the isolates were investigated, and the results showed that dehydrosinulariolide ( 7 ) and 11- epi -sinulariolide acetate ( 8 ) exhibited cytotoxicity toward a limited panel of cancer cell lines and 14-deoxycrassin ( 9 ) displayed anti-inflammatory activity by inhibition of superoxide anion generation and elastase release in N -formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophils.

Six new cembranoids, cherbonolides A-E ( 1 ⁻ 5 ) and bischerbolide peroxide ( 6 ), along with one known cembranoid, isosarcophine ( 7 ), were isolated from the Formosan soft coral Sarcophyton cherbonnieri . The structures of these compounds were elucidated by detailed spectroscopic analysis and chemical methods. Compound 6 was discovered to be the first example of a molecular skeleton formed from two cembranoids connected by a peroxide group. Compounds 1 ⁻ 7 were shown to have the ability of inhibiting the production of superoxide anions and elastase release in N -formyl-methionyl-leucyl-phenyl-alanine/cytochalasin B (fMLF/CB)-induced human neutrophils.

Previous studies have demonstrated that lipid rafts and β‑adducin serve an important role in leukocyte rolling. In the present study the migratory ability and behavior of neutrophils was demonstrated to rely on the integrity of the lipid raft structure. β‑adducin was demonstrated to have a critical role in neutrophil migration. Knockdown of β‑adducin attenuated the migratory ability of dHL‑60 cells and the distribution of β‑adducin in lipid raft structures was changed by N‑formylmethionyl‑leucyl‑phenyl‑alanine treatment. Furthermore, the findings demonstrated that the tyrosine phosphorylation of β‑adducin was required for its relocation. The results of the present study suggested that the lipid raft‑associated protein β‑adducin may be a novel control point for the excessive infiltration of neutrophils during inflammation.

Objective and Design: The objective of this study is to uncover the signal transduction pathways of N-formyl methionyl-leucyl-phenylalanine (fMLP) in monocyte., Materials or Subjects: Freshly isolated human peripheral blood monocytes (PBMC) were used for in vitro assessment of signal transduction pathways activated by fMLP., Treatment: Time-course and dose-response experiments were used to evaluate the effect of fMLP along with the specific inhibitors/stimulators on the activation of downstream signaling kinases., Methods: Freshly isolated human PBMC were stimulated with fMLP for the desired time. Western blot and siRNA analysis were used to evaluate the activated intracellular signaling kinases, and flow analysis was performed to assess the levels of CD11b. Furthermore, luminescence spectrometry was performed to measure the levels of released hydrogen peroxide in the media., Results: fMLP strongly stimulated the activation of AKT and ERK1/2 through a RhoA-GTPase-dependent manner and also induced H 2 O 2 release by monocytes. Furthermore, fMLP mediated its effects through restricted activation of formylpeptide receptor-like 1 (FPRL1/FPR2), but independently of either EGFR transactivation or intracellular calcium release. In addition, NAC reversed fMLP- and H 2 O 2 -induced activation of Akt and RhoA-GTPase., Conclusion: Collectively, these data suggested that fMLP-activated ERK1/2 and Akt pathways through specific activation of the FPRL1/ROS/RoA-GTPase pathway.

This study investigates the effect and the underlying mechanism of 2',3-dihydroxy-5-methoxybiphenyl (RIR-2), a lignan extracted from the roots of Rhaphiolepis indica (L.) Lindl. ex Ker var. tashiroi Hayata ex Matsum. & Hayata (Rosaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst and cathepsin G in human neutrophils. Signaling pathways regulated by RIR-2 which modulated fMLP-induced respiratory burst were evaluated by an interaction between β subunit of G-protein (Gβ) with downstream signaling induced by fMLP and by immunoblotting analysis of the downstream targets of Gβ-protein. RIR-2 inhibited fMLP-induced superoxide anion production (IC 50 :2.57 ± 0.22 μM), cathepsin G release (IC 50 :18.72 ± 3.76 μM) and migration in a concentration dependent manner. RIR-2 specifically suppresses fMLP-induced Src family kinases phosphorylation by inhibiting the interaction between Gβ-protein with Src kinases without inhibiting Src kinases activities, therefore, RIR-2 attenuated the downstream targets of Src kinase, such as phosphorylation of Raf/ERK, AKT, P38, PLCγ2, PKC and translocation Tec, p47 ph ° x and P40 ph ° x from the cytosol to the inner leaflet of the plasma membrane. Furthermore, RIR-2 attenuated fMLP-induced intracellular calcium mobilization by inhibiting the interaction between Gβ-protein with PLCβ2. RIR-2 was not a competitive or allosteric antagonist of fMLP. On the contrary, phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of Src, AKT, P38, PKC and membrane localization of p47 ph ° x and P40 ph ° x remained unaffected. RIR-2 specifically modulates fMLP-mediated neutrophil superoxide anion production and cathepsin G release by inhibiting the interaction between Gβ-protein with downstream signaling which subsequently interferes with the activation of intracellular calcium, PLCγ2, AKT, p38, PKC, ERK, p47 ph ° x and p40 phox ., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the vasculature, NADPH oxidase is the main contributor of reactive oxygen species (ROS) which play a key role in endothelial signalling and functions. We demonstrate that ECV304 cells express p47 phox , p67 phox , and p22 phox subunits of NADPH oxidase, as well as formyl peptide receptors 1 and 3 (FPR1/3), which are members of the GPCR family. By RT-PCR, we also detected Flt-1 and Flk-1/KDR in these cells. Stimulation of FPR1 by N-fMLP induces p47 phox phosphorylation, which is the crucial event for NADPH oxidase-dependent superoxide production. Transphosphorylation of RTKs by GPCRs is a biological mechanism through which the information exchange is amplified throughout the cell. ROS act as signalling intermediates in the transactivation mechanism. We show that N-fMLP stimulation induces the phosphorylation of cytosolic Y951, Y996, and Y1175 residues of VEGFR2, which constitute the anchoring sites for signalling molecules. These, in turn, activate PI3K/Akt and PLC- γ 1/PKC intracellular pathways. FPR1-induced ROS production plays a critical role in this cross-talk mechanism. In fact, inhibition of FPR1 and/or NADPH oxidase functions prevents VEGFR2 transactivation and the triggering of the downstream signalling cascades. N-fMLP stimulation also ameliorates cellular migration and capillary-like network formation ability of ECV304 cells.