Concise synthesis of deuterium-labelled proanthocyanidin metabolite 5-(3',4'-dihydroxyphenyl)-γ-valerolactone as an analytical tool.

Deuterated proanthocyanidin metabolite 5-(3',4'-dihydroxyphenyl)-γ-valerolactone has been successfully produced. This metabolite is responsible for several proanthocyanidin protective effects in the field of cancer chemoprevention, skin wrinkle-prevention, and antimicrobials. The synthetic approach applied employs a short reaction sequence and allows the incorporation of four deuterium atoms on non-exchangeable sites, making it an attractive strategy to produce a stable isotopically labeled internal standard for quantitative mass spectrometry isotope dilution-based methods, as demonstrated by developing an LC-MS/MS method to quantify DHPV in urine samples. Overall, this efficient synthesis provides a valuable analytical tool for the study of the metabolic conversion of proanthocyanidins thus helping to investigate the biological effect and establishing the active dose of the key catabolite 5-(3',4'-dihydroxyphenyl)-γ-valerolactone.

A Novel Chromatographic Method to Assess the Binding Ability towards Dicarbonyls.

Human exposure to dicarbonyls occurs via ingestion (e.g., food), inhalation (e.g., electronic cigarettes) and dysregulation of endogenous metabolic pathways (e.g., glycolysis). Dicarbonyls are electrophiles able to induce carbonylation of endogenous substrate. They have been associated with the onset and progression of several human diseases. Several studies have advocated the use of dicarbonyl binders as food preservatives or as drugs aimed at mitigating carbonylation. This study presents the setup of an easy and cheap assay for the screening of selective and potent dicarbonyl binders. The method is based on the incubation of the candidate molecules with a molecular probe. The activity is then determined by measuring the residual concentration of the molecular probe over time by liquid chromatography (LC). However, the naturally occurring dicarbonyls (e.g., glyoxal, methylglyoxal) are not appealing as probes since they are hard to separate and detect using the most popular LC variants. Benzylglyoxal (BGO) was therefore synthesized and tested, proving to be a convenient probe that allows a direct quantification of residual dicarbonyls by reversed phase LC without derivatization. The method was qualified by assessing the binding ability of some molecules known as binders of natural occurring dicarbonyls, obtaining results consistent with literature.

Chemoenzymatic approach towards the synthesis of the antitumor and antileishmanial marine metabolite (+)-Harzialactone A via the stereoselective, biocatalyzed reduction of a prochiral ketone.

As a rich source of biological active compounds, marine natural products have been increasingly screened as candidates for developing new drugs. Among the several marine products and metabolites, (+)-Harzialactone A has drawn considerable attention for its antitumor and antileishmanial activity. In this work a chemoenzymatic approach has been implemented for the preparation of the marine metabolite (+)-Harzialactone A. The synthesis involved a stereoselective, biocatalyzed reduction of the prochiral ketone 4-oxo-5-phenylpentanoic acid or the corresponding esters, all generated by chemical reactions. A collection of different promiscuous oxidoreductases (both wild-type and engineered) and diverse microorganism strains were investigated to mediate the bioconversions. After co-solvent and co-substrate investigation in order to enhance the bioreduction performance, T. molischiana in presence of NADES (choline hydrochloride-glucose) and ADH442 were identified as the most promising biocatalysts, allowing the obtainment of the (S)-enantiomer with excellent ee (97% to > 99% respectively) and good to excellent conversion (88% to 80% respectively). The successful attempt in this study provides a new chemoenzymatic approach for the synthesis of (+)-Harzialactone A.

Obesity and Type 2 Diabetes: Adiposopathy as a Triggering Factor and Therapeutic Options.

Obesity and type 2 diabetes (T2DM) are major public health concerns associated with serious morbidity and increased mortality. Both obesity and T2DM are strongly associated with adiposopathy, a term that describes the pathophysiological changes of the adipose tissue. In this review, we have highlighted adipose tissue dysfunction as a major factor in the etiology of these conditions since it promotes chronic inflammation, dysregulated glucose homeostasis, and impaired adipogenesis, leading to the accumulation of ectopic fat and insulin resistance. This dysfunctional state can be effectively ameliorated by the loss of at least 15% of body weight, that is correlated with better glycemic control, decreased likelihood of cardiometabolic disease, and an improvement in overall quality of life. Weight loss can be achieved through lifestyle modifications (healthy diet, regular physical activity) and pharmacotherapy. In this review, we summarized different effective management strategies to address weight loss, such as bariatric surgery and several classes of drugs, namely metformin, GLP-1 receptor agonists, amylin analogs, and SGLT2 inhibitors. These drugs act by targeting various mechanisms involved in the pathophysiology of obesity and T2DM, and they have been shown to induce significant weight loss and improve glycemic control in obese individuals with T2DM.

CRISPR/Cas9 screen in human iPSC-derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity.

INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.

Discovery of a Potent and Highly Selective Dipeptidyl Peptidase IV and Carbonic Anhydrase Inhibitor as "Antidiabesity" Agents Based on Repurposing and Morphing of WB-4101.

The management of patients with type 2 diabetes mellitus (T2DM) is shifting from cardio-centric to weight-centric or, even better, adipose-centric treatments. Considering the downsides of multidrug therapies and the relevance of dipeptidyl peptidase IV (DPP IV) and carbonic anhydrases (CAs II and V) in T2DM and in the weight loss, we report a new class of multitarget ligands targeting the mentioned enzymes. We started from the known α1-AR inhibitor WB-4101, which was progressively modified through a tailored morphing strategy to optimize the potency of DPP IV and CAs while losing the adrenergic activity. The obtained compound 12 shows a satisfactory DPP IV inhibition with a good selectivity CA profile (DPP IV IC50: 0.0490 µM; CA II Ki 0.2615 µM; CA VA Ki 0.0941 µM; CA VB Ki 0.0428 µM). Furthermore, its DPP IV inhibitory activity in Caco-2 and its acceptable pre-ADME/Tox profile indicate it as a lead compound in this novel class of multitarget ligands.

Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells.

The regulatory role of protein tyrosine kinases in ß1- and ß2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.

Efficient long-range conduction in cable bacteria through nickel protein wires.

Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures.

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition.

Neuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.

Analytical Profile and Antioxidant and Anti-Inflammatory Activities of the Enriched Polyphenol Fractions Isolated from Bergamot Fruit and Leave.

The aim of the study is to compare the qualitative and semi-quantitative profile of the polyphenol fraction purified from the leaf (BLPF) and fruit (BFPF) of bergamot (Citrus bergamia), and to evaluate their antioxidant and anti-inflammatory activity. The analytical qualitative profile was carried out by LC-ESI/MS using three different approaches: targeted (searching analytes already reported in bergamot extract), semi-targeted (a selective search of 3-hydroxy-3-methylglutarate [HMG] derivatives involved in the cholesterol reducing activity of BPF) and untargeted. A total number of 108 compounds were identified by using the three approaches, 100 of which are present in both the extracts thus demonstrating a good qualitative overlapping of polyphenols between the two extracts. The antioxidant activity was higher for BLPF in respect to BFPF but when normalized in respect to the polyphenol content they were almost overlapping. Both the extracts were found to dose dependently inhibit cell inflammation stimulated with IL-1α. In conclusion, the comparison of the qualitative and quantitative profile of polyphenols as well as of the antioxidant and anti-inflammatory activity of bergamot leaf and fruit well indicates that leaf is a valid source of bergamot polyphenol extraction and an even richer source of polyphenol in respect to the fruit.

Profiling Vaccinium macrocarpon components and metabolites in human urine and the urine ex-vivo effect on Candida albicans adhesion and biofilm-formation.

The aim of this work was to profile, by using an HPLC-MS/MS method, cranberry compounds and metabolites found in human urine after ingestion of a highly standardized cranberry extract (Anthocran®). Two different strategies were adopted for the data analysis: a targeted and an untargeted approach. These strategies allowed the identification of 42 analytes including cranberry components, known metabolites and metabolites hitherto unreported in the literature, including six valerolactones/valeric acid derivatives whose presence in urine after cranberry consumption has never been described before. Absolute concentrations of 26 over 42 metabolites were obtained by using pure available standards. Urine collected at different time points after the last dosage of Anthocran® were tested on the reference strain C. albicans SC5314, a biofilm-forming strain. Fractions collected after 12 h were found to significantly reduce the adhesion and biofilm formation compared to the control (p < 0.05). A similar effect was then obtained by using Anthocran™ Phytosome™, the lecithin formulation containing 1/3 of standardized cranberry extract and formulated to enhance the absorption of the cranberry components. The urinary profile of cranberry components and metabolites in the urine fractions collected at 1 h, 6 h and 12 h after the last capsule intake were then reproduced by using the pure standards at the concentration ranges found in the urine fraction, and tested on C. albicans. Only the mixture mimicking the urinary fraction collected at 12 h and containing as main components, quercetin and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone was found effective thus confirming the ex-vivo results.

Differentiation but not ALS mutations in FUS rewires motor neuron metabolism.

Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.

Development of a direct ESI-MS method for measuring the tannin precipitation effect of proline-rich peptides and in silico studies on the proline role in tannin-protein interactions.

Tannins are a heterogeneous class of polyphenols that are present in several plants and foods. Their ability to interact and precipitate proline-rich proteins leads to different effects such as astringency or antidiarrheal activity. Thus, evaluation of the tannin content in plant extracts plays a key role in understanding their potential use as pharmaceuticals and nutraceuticals. Several methods have been proposed to study tannin-protein interactions but few of them are focused on quantification. The purpose of the present work is to set up a suitable and time efficient method able to quantify the extent of tannin protein precipitation. Bradykinin, chosen as a model, was incubated with increasing concentrations of 1,2,3,4,6-penta-O-galloyl-ß-d-glucose and tannic acid selected as reference of tannic compounds. Bradykinin not precipitated was determined by a mass spectrometer TSQ Quantum Ultra Triple Quadrupole (direct infusion analysis). The results were expressed as PC50, which is the concentration able to precipitate 50% of the protein. The type of tannin-protein interaction was evaluated also after precipitate solubilisation. The involvement of proline residues in tannin-protein interactions was confirmed by repeating the experiment using a synthesized peptide (RR-9) characterized by the same bradykinin sequence, but having proline residues replaced by glycine residues: no interaction occurred between the peptide and the tannins. Moreover, modelling studies on PGG-BK and PGG-RR-9 were performed to deeply investigate the involvement of prolines: a balance of hydrophobic and H-bond contacts stabilizes the PGG-BK cluster and the proline residues exert a crucial role thus allowing the PGG molecules to elicit a sticking effect.

Direct HPLC separation of carnosine enantiomers with two chiral stationary phases based on penicillamine and teicoplanin derivatives.

Carnosine is present in high concentrations in specific human tissues such as the skeletal muscle, and among its biological functions, the remarkable scavenging activity toward reactive carbonyl species is noteworthy. Although the two enantiomers show almost identical scavenging reactivity toward reactive carbonyl species, only d-carnosine is poorly adsorbed at the gastrointestinal level and is stable in human plasma. Direct methods for the enantioselective analysis of carnosine are still missing even though they could find more effective applications in the analysis of complex matrices. In the present study, the use of two different chiral stationary phases is presented. A chiral ligand-exchange chromatography stationary phase based on N,S-dioctyl-d-penicillamine resulted in the direct enantioseparation of carnosine. Indeed, running the analysis at 25°C and 1.0 mL/min with a 1.5 mM copper(II) sulfate concentration allowed us to obtain separation and resolution factors of 3.37 and 12.34, respectively. However, the use of a copper(II)-containing eluent renders it hardly compatible with mass spectrometry detectors. With the teicoplanin-based stationary phase, a mass spectrometry compatible method was successfully developed. Indeed, a water/methanol 60:40 v/v pH 3.1 eluent flowed at 1.0 mL/min and with a 25°C column temperature produced separation and resolution factors of 2.60 and 4.16, respectively.

HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)-mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs.Amyotrophic lateral sclerosis (ALS) leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition.

Diadenosine tetraphosphate (Ap4A) - an E. coli alarmone or a damage metabolite?

Under stress, metabolism is changing: specific up- or down-regulation of proteins and metabolites occurs as well as side effects. Distinguishing specific stress-signaling metabolites (alarmones) from side products (damage metabolites) is not trivial. One example is diadenosine tetraphosphate (Ap4A) - a side product of aminoacyl-tRNA synthetases found in all domains of life. The earliest observations suggested that Ap4A serves as an alarmone for heat stress in Escherichia coli. However, despite 50 years of research, the signaling mechanisms associated with Ap4A remain unknown. We defined a set of criteria for distinguishing alarmones from damage metabolites to systematically classify Ap4A. In a nutshell, no indications for a signaling cascade that is triggered by Ap4A were found; rather, we found that Ap4A is efficiently removed in a constitutive, nonregulated manner. Several fold perturbations in Ap4A concentrations have no effect, yet accumulation at very high levels is toxic due to disturbance of zinc homeostasis, and also because Ap4A's structural overlap with ATP can result in spurious binding and inactivation of ATP-binding proteins. Overall, Ap4A met all criteria for a damage metabolite. While we do not exclude any role in signaling, our results indicate that the damage metabolite option should be considered as the null hypothesis when examining Ap4A and other metabolites whose levels change upon stress.

A selective alpha1D-adrenoreceptor antagonist inhibits human prostate cancer cell proliferation and motility "in vitro".

The progression of prostate cancer (PC) to a metastatic hormone refractory disease is the major contributor to the overall cancer mortality in men, mainly because the conventional therapies are generally ineffective at this stage. Thus, other therapeutic options are needed as alternatives or in addition to the classic approaches to prevent or delay tumor progression. Catecholamines participate to the control of prostate cell functions by the activation of alpha1-adrenoreceptors (alpha1-AR) and increased sympathetic activity has been linked to PC development and evolution. Molecular and pharmacological studies identified three alpha1-AR subtypes (A, B and D), which differ in tissue distribution, cell signaling, pharmacology and physiological role. Within the prostate, alpha1A-ARs mainly control stromal cell functions, while alpha1B- and alpha1D- subtypes seem to modulate glandular epithelial cell growth. The possible direct contribution of alpha1D-ARs in tumor biology is supported by their overexpression in PC. The studies here presented investigate the "in vitro" antitumor action of A175, a selective alpha1D-AR antagonist we have recently obtained by modifying the potent, but not subtype-selective alpha1-AR antagonist (S)-WB4101, in the hormone-refractory PC3 and DU145 PC cell lines. The results indicate that A175 has an alpha1D-AR-mediated significant and dose-dependent antiproliferative action that possibly involves the induction of G0/G1 cell cycle arrest, but not apoptosis. In addition, A175 reduces cell migration and adhesiveness to culture plates. In conclusion, our work clarified some cellular aspects promoted by alpha1D-AR activity modulation and supports a further pharmacological approach in the cure of hormone-refractory PC, by targeting specifically this AR subtype.

Chemistry and Pharmacology of a Series of Unichiral Analogues of 2-(2-Pyrrolidinyl)-1,4-benzodioxane, Prolinol Phenyl Ether, and Prolinol 3-Pyridyl Ether Designed as α4ß2-Nicotinic Acetylcholine Receptor Agonists.

Some unichiral analogues of 2R,2'S-2-(1'-methyl-2'-pyrrolidinyl)-7-hydroxy-1,4-benzodioxane, a potent and selective α4ß2-nAChR partial agonist, were designed by opening dioxane and replacing hydroxyl carbon with nitrogen. The resulting 3-pyridyl and m-hydroxyphenyl ethers have high α4ß2 affinity and good subtype selectivity, which get lost if OH is removed from phenyl or the position of pyridine nitrogen is changed. High α4ß2 affinity and selectivity are also attained by meta hydroxylating the 3-pyridyl and the phenyl ethers of (S)-N-methylprolinol and the phenyl ether of (S)-2-azetidinemethanol, known α4ß2 agonists, although the interaction mode of the aryloxymethylene substructure cannot be assimilated to that of benzodioxane. Indeed, the α4ß2 and α3ß4 functional tests well differentiate behaviors that the binding tests homologize: both the 3-hydroxyphenyl and the 5-hydroxy-3-pyridyl ether of N-methylprolinol are α4ß2 full agonists, but only the latter is highly α4ß2/α3ß4 selective, while potent and selective partial α4ß2 agonism characterizes the hydroxybenzodioxane derivative and its two opened semirigid analogues.

Src family kinases and Syk are required for neutrophil extracellular trap formation in response to ß-glucan particles.

We report that particles of ß-glucan, one of the surface components of yeasts, are powerful inducers of neutrophil extracellular trap (NET) formation in human neutrophils. ß-Glucan triggered a prolonged phosphorylation of Src family kinases and Syk that were suppressed by the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d] pyrimidine (PP2) and a novel Syk inhibitor, PRT-060318, respectively. PP2 and PRT-060318 also inhibited ß-glucan-induced NET formation and reactive oxygen species (ROS) generation, suggesting that both responses are triggered by a Src/Syk-regulated signaling pathway. Given that the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) markedly inhibited NET formation, our findings suggest that ROS are required for the full-blown formation of NETs in response to ß-glucan particles. Contrary to ß-glucan, ROS generation triggered by phorbol myristate acetate (PMA) was unaffected by PP2 and PRT-060318, but these compounds, as well as DPI, suppressed Src/Syk phosphorylation triggered by PMA. Whereas PP2 had no effect on PMA-induced NET formation, PRT-060318 had a significant, albeit partial, inhibitory effect, thus suggesting that ROS induce NET formation in part via activation of Syk. These findings were substantiated by the evidence that neutrophils from mice with the conditional deletion of Syk were defective in formation of NETs in response to ß-glucan.

Class I phosphoinositide-3-kinases and SRC kinases play a nonredundant role in regulation of adhesion-independent and -dependent neutrophil reactive oxygen species generation.

Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very rapid and transient, and the second one is delayed and lasts up to 30-40 min. We examined the role of phosphoinositide 3-kinases (PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine neutrophils deficient of PI3Kγ or Hck, Fgr, and Lyn. Our studies show that PI3Kγ is indispensable for the early, fMLF-induced ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3Kγ-deficient neutrophils. In contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the late one. Selective inhibitors of PI3Kα and PI3Kδ markedly reduced both the early and late response to fMLF and TNF in human neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3Kγ via Ras in the early phase of the response and by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for Vav phosphorylation.