Neutrophil Superoxide Production Research Articles

Semaglutide outperforms insulin in restoring neutrophil function against implant-related infection in diabetic and obese mice: experimental research.

Bone and joint infections (BJI) are a significant complication after arthroplasty and fracture fixation, particularly challenging in patients with type 2 diabetes mellitus (T2DM) and obesity. Semaglutide, a glucagon-like peptide-1 receptor agonist (GLP-1RA), have shown efficacy in managing T2DM and obesity. However, its impact on BJI risk and neutrophil function remains unclear. To investigate whether preoperative semaglutide treatment (1) reduces the risk of BJI in diabetic and obese mice undergoing intra-articular implants, and (2) outperforms insulin in restoring neutrophil function to mitigate implant-related infection. A C57BL/6 mouse model of T2DM/obesity was induced using a high-fat diet (HFD) for 12 weeks. Mice received preoperative insulin or semaglutide therapy for 1-28 days. BJI risk was assessed using an intraarticular-implant model challenged with S. aureus or E. coli. Neutrophil function was evaluated through bactericidal activity, superoxide production, and migration ability. Semaglutide treatment led to a significant and sustained reduction in body weight and improved glucose tolerance in HFD mice. Both insulin and semaglutide therapies significantly reduced BJI risk, with semaglutide showing a more pronounced effect over time. Semaglutide therapy also enhanced neutrophil bactericidal activity, superoxide production, and migration ability compared to insulin therapy. Preoperative semaglutide treatment effectively reduces BJI risk and improves neutrophil function in diabetic and obese mouse models. These findings suggest that semaglutide may be a promising pharmacological intervention to mitigate infection risk in orthopedic patients with T2DM or obesity.

Neutrophil response to Porphyromonas gingivalis is modulated by low-level laser application.

Neutrophil response is critical in inflammatory regulation and immune response to bacterial infections. During periodontal disease, pathogenic bacteria lead to exaggerated neutrophil responses. We hypothesized that low-level laser application (LLLT), therapeutic strategy for dampening inflammatory processes, will regulate neutrophil activity in response to periodontopathogens. The impact of LLLT on neutrophil responses was measured by light delivered at wavelength of 850 nm. The direct effect of LLLT on P. gingivalis A7436 was determined by flow cytometry using LIVE/DEADTM Cell Vitality kit. The phagocytosis of P. gingivalis A7436 by human neutrophils was measured using flow cytometry. Superoxide generation was measured by cytochrome-C-reduction in the presence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 mM). Cytokine release by neutrophils was measured by multiplex immunoassay. The phagocytosis of P. gingivalis by primary human neutrophils was significantly reduced in response to LLLT (p < 0.05). While LLLT led to increased superoxide production in neutrophils that were not challenged by P. gingivalis, it dampened the increased superoxide and IL-6 release by the neutrophils in response to P. gingivalis. LLLT did not directly affect the viability of P. gingivalis. These results suggested that LLLT can provide therapeutic strategy in periodontal disease, regulating the neutrophil response to P. gingivalis.

CHRONIC GRANULOMATOUS DISEASE PRESENTING AS COW'S MILK ALLERGY AND EOSINOPHILIC ESOPHAGITIS

<h3>Introduction</h3> An infant with failure to thrive was diagnosed with cow's milk allergy (CMA) and eosinophilic esophagitis (EOE). Further work up by multiple specialists considered inborn error of metabolism, cystic fibrosis, infection, or malignancy. He was diagnosed with chronic granulomatous disease (CGD) after 3 weeks in the hospital. <h3>Case</h3> A 2 month-old male with history of formula intolerance was admitted for poor weight gain, vomiting, and bloody stools in the setting of social challenges. The infant was pale-appearing with baseline tachypnea however chest x-ray was unremarkable. Initial immune workup was limited to immunoglobulin levels which were normal. Given his elevated fecal calprotectin, he was diagnosed with CMA. EGD revealed EOE and gastritis. He had persistent leukocytosis to 59,000/mm^3 with 32% eosinophils, anemia, thrombocytosis, and elevated inflammatory markers. This prompted suggestion of bone marrow biopsy to evaluate for leukemia. Prior to this, dihydrorhodamine (DHR) assay confirmed absence of superoxide production in neutrophils. Genetic testing revealed hemizygous pathogenic variant c.469C>T in the CYBB gene associated with X-linked CGD. Mother's DHR was normal suggesting de novo mutation. Chest CT after diagnosis showed right lower lobe inflammatory chronic granulomatous process which was confirmed with lung biopsy. Bacterial and fungal prophylaxis were initiated and curative treatment with hematopoietic is currently being pursued. This patient's genetic variant has been described in a case series to cause very early onset GI manifestations, hypereosinophilia, and hyperleukocytosis with no infections. Persistent failure to thrive and enteropathy with leukocytosis warrants an extensive workup as CGD and other primary immunodeficiencies may present with inflammatory findings.

Hypoparathyroidism-Retardation-Dysmorphism Syndrome due to a Variant in the Tubulin-Specific Chaperone E Gene as a Cause of Combined Immune Deficiency.

BackgroundHypoparathyroidism-retardation-dysmorphism (HRD) syndrome is a disease composed of hypoparathyroidism, growth retardation, severe developmental delay, and typical dysmorphic features caused by the tubulin-specific chaperone E gene variant. Many patients succumb in infancy to HRD due to overwhelming infections mainly caused by Pneumococcus spp. Knowledge related to the immune system in these patients is scarce.PurposeTo define the immune phenotype of a cohort of HRD patients including their cellular, humoral, and neutrophil functions.MethodsThe study included HRD patients followed at Soroka University Medical Center. Clinical and immunological data were obtained, including immunoglobulin concentrations, specific antibody titers, lymphocyte subpopulations, lymphocyte proliferation, and neutrophil functions.ResultsNine patients (5 females and 4 males) were enrolled, aged 6 months to 15 years. All received amoxicillin prophylaxis as part of a routine established previously. Three patients had bacteremia with Klebsiella, Shigella spp., and Candida. Three patients had confirmed coronavirus disease 19 (COVID-19), and two of them died from this infection. All patients had normal blood counts. Patients showed high total IgA and IgE levels, low anti-pneumococcal antibodies in spite of a routine vaccination schedule, and reduced frequency of naive B cells with increased frequency of CD21lowCD27- B cells. All patients had abnormal T-cell population distributions, including reduced terminally differentiated effector memory CD8, inverted CD4/CD8 ratios, and impaired phytohemagglutinin (PHA)-induced lymphocyte proliferation. Neutrophil superoxide production and chemotaxis were normal in all patients tested.ConclusionHRD is a combined immunodeficiency disease with syndromic features, manifesting in severe invasive bacterial and viral infections.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10875-022-01380-9.

Increased Dietary Manganese Impairs Neutrophil Extracellular Trap Formation Rendering Neutrophils Ineffective at Combating Staphylococcus aureus.

Dietary metals can modify the risk to infection. Previously, we demonstrated that heightened dietary manganese (Mn) during systemic Staphylococcus aureus infection increases S. aureus virulence. However, immune cells also operate in these same environments and the effect of dietary Mn on neutrophil function in vivo has not been assessed. This study reveals that increased concentrations of Mn impairs mitochondrial respiration and superoxide production in neutrophils responding to S. aureus. As a result, high Mn accelerates primary degranulation, while impairing suicidal neutrophil extracellular trap (NET) formation, which decreases bactericidal activity. In vivo, elevated dietary Mn accumulated extracellularly in the heart, indicating that excess Mn may be more bioavailable in the heart. Coinciding with this phenotype, neutrophil function in the heart was most impacted by a high Mn diet, as neutrophils produced lower levels of mitochondrial superoxide and underwent less suicidal NET formation. Consistent with an ineffective neutrophil response when mice are on a high Mn diet, S. aureus burdens were increased in the heart and mice were more susceptible to systemic infection. Therefore, elevated dietary Mn not only affects S. aureus but also renders neutrophils less capable of restricting staphylococcal infection.

Astragalus mongholicus Bunge Water Extract Exhibits Anti-inflammatory Effects in Human Neutrophils and Alleviates Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice.

Neutrophils are the primary immune cells in innate immunity, which are related to various inflammatory diseases. Astragalus mongholicus Bunge is a Chinese medicinal herb used to treat various oxidative stress-related inflammatory diseases. However, there are limited studies that elucidate the effects of Astragalus mongholicus Bunge in human neutrophils. In this study, we used isolated human neutrophils activated by various stimulants to investigate the anti-inflammatory effects of Astragalus mongholicus Bunge water extract (AWE). Cell-free assays were used to examine free radicals scavenging capabilities on superoxide anion, reactive oxygen species (ROS), and nitrogen-centered radicals. Imiquimod (IMQ) induced psoriasis-like skin inflammation mouse model was used for investigating anti-psoriatic effects. We found that AWE inhibited superoxide anion production, ROS generation, and elastase release in human neutrophils, which exhibiting a direct anti-neutrophil effect. Moreover, AWE exerted a ROS scavenging ability in the 2,2’-Azobis (2-amidinopropane) dihydrochloride assay, but not superoxide anion in the xanthine/xanthine oxidase assay, suggesting that AWE exhibited anti-oxidation and anti-inflammatory capabilities by both scavenging ROS and by directly inhibiting neutrophil activation. AWE also reduced CD11b expression and adhesion to endothelial cells in activated human neutrophils. Meanwhile, in mice with psoriasis-like skin inflammation, administration of topical AWE reduced both the affected area and the severity index score. It inhibited neutrophil infiltration, myeloperoxidase release, ROS-induced damage, and skin proliferation. In summary, AWE exhibited direct anti-inflammatory effects by inhibiting neutrophil activation and anti-psoriatic effects in mice with IMQ-induced psoriasis-like skin inflammation. Therefore, AWE could potentially be a pharmaceutical Chinese herbal medicine to inhibit neutrophilic inflammation for anti-psoriasis.

An ACE inhibitor reduces bactericidal activity of human neutrophils in vitro and impairs mouse neutrophil activity in vivo.

Angiotensin-converting enzyme inhibitors (ACEIs) are used by millions of patients to treat hypertension, diabetic kidney disease, and heart failure. However, these patients are often at increased risk of infection. To evaluate the impact of ACEIs on immune responses to infection, we compared the effect of an ACEI versus an angiotensin receptor blocker (ARB) on neutrophil antibacterial activity. ACEI exposure reduced the ability of murine neutrophils to kill methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Klebsiella pneumoniae in vitro. In vivo, ACEI-treated mice infected with MRSA had increased bacteremia and tissue bacteria counts compared to mice treated with an ARB or with no drug. Similarly, ACEIs, but not ARBs, increased the incidence of MRSA-induced infective endocarditis in mice with aortic valve injury. Neutrophils from ACE knockout (KO) mice or mice treated with an ACEI produced less leukotriene B4 (LTB4) upon stimulation with MRSA or lipopolysaccharide, whereas neutrophils overexpressing ACE produced more LTB4 compared to wild-type neutrophils. As a result of reduced LTB4 production, ACE KO neutrophils showed decreased survival signaling and increased apoptosis. In contrast, neutrophils overexpressing ACE had an enhanced survival phenotype. Last, in a cohort of human volunteers receiving the ACEI ramipril for 1 week, ACEI administration reduced neutrophil superoxide and reactive oxygen species production and neutrophils isolated from volunteers during ramipril treatment had reduced bactericidal activity. Together, these data demonstrate that ACEI treatment, but not ARB treatment, can reduce the bacterial killing ability of neutrophils.

Characterization of Blood Immune Cells in Patients With Decompensated Cirrhosis Including ACLF.

Background and AimsPatients with cirrhosis and acute-on-chronic liver failure (ACLF) have immunosuppression, indicated by an increase in circulating immune-deficient monocytes. The aim of this study was to investigate simultaneously the major blood-immune cell subsets in these patients.Material and MethodsBlood taken from 67 patients with decompensated cirrhosis (including 35 critically ill with ACLF in the intensive care unit), and 12 healthy subjects, was assigned to either measurements of clinical blood counts and microarray (genomewide) analysis of RNA expression in whole-blood; microarray (genomewide) analysis of RNA expression in blood neutrophils; or assessment of neutrophil antimicrobial functions.ResultsSeveral features were found in patients with ACLF and not in those without ACLF. Indeed, clinical blood count measurements showed that patients with ACLF were characterized by leukocytosis, neutrophilia, and lymphopenia. Using the CIBERSORT method to deconvolute the whole-blood RNA-expression data, revealed that the hallmark of ACLF was the association of neutrophilia with increased proportions of macrophages M0-like monocytes and decreased proportions of memory lymphocytes (of B-cell, CD4 T-cell lineages), CD8 T cells and natural killer cells. Microarray analysis of neutrophil RNA expression revealed that neutrophils from patients with ACLF had a unique phenotype including induction of glycolysis and granule genes, and downregulation of cell-migration and cell-cycle genes. Moreover, neutrophils from these patients had defective production of the antimicrobial superoxide anion.ConclusionsGenomic analysis revealed that, among patients with decompensated cirrhosis, those with ACLF were characterized by dysregulation of blood immune cells, including increases in neutrophils (that had a unique phenotype) and macrophages M0-like monocytes, and depletion of several lymphocyte subsets (including memory lymphocytes). All these lymphocyte alterations, along with defective neutrophil superoxide anion production, may contribute to immunosuppression in ACLF, suggesting targets for future therapies.

QOL-36. USE OF CANNABINOIDS IN THE PEDIATRIC CENTRAL NERVOUS SYSTEM TUMOR POPULATION

BACKGROUNDCannabinoids, including cannabidiol (CBD) and tetrahydrocannabinol (THC), are a class of compounds found in marijuana. Numerous studies in adults have examined cannabinoid use in management of cancer-related symptoms such as nausea, anorexia, and pain. Less is known about the use in the pediatric oncology population.METHODSA prospective observational study has been ongoing since 2016 at Children’s Hospital Colorado to evaluate cannabinoids’ impact using PedsQL™ modules on quality of life of pediatric patients with central nervous system (CNS) tumors who are 2–18 years old. Laboratory assessments of T-cell activity and pharmacokinetics of CBD, THC and associated metabolites are in process. Diaries with exploratory information on cannabinoid use patterns are being collected.RESULTSThirty-three patients (14:19; male:female) have been enrolled with a median age of 6.4 years (range, 2.9–17.7 years). The most common tumor type in enrolled patients is embryonal tumors (13/33; 39%). Nine (27%) patients have low-grade glial/glioneuronal tumors, and eight (24%) had high-grade/diffuse midline gliomas. The remaining patients had ependymoma or craniopharyngioma. The median time on cannabinoids is 9 months. Most (n=20) patients have used oral products with CBD and THC. One patient continues on cannabinoid therapy in follow up. Preliminary immune function analyses identified impaired neutrophil superoxide anion production and chemotaxis in patients taking cannabinoids at early time points on therapy.CONCLUSIONSFamilies of children with various CNS tumors are pursuing cannabinoid therapy for both antitumor and supportive care purposes. Analysis of the impact of cannabinoids on patients’ quality of life is ongoing.

The effect of pamidronate delivery in bisphosphonate-na\xefve patients on neutrophil chemotaxis and oxidative burst

The pathogenesis of medication-related osteonecrosis of the jaw (MRONJ), a morbid condition associated with bisphosphonate administration, has not been fully elucidated. Recent research utilizing a murine model has revealed that the neutrophil becomes dysfunctional following exposure to bisphosphonates. Accordingly, the impairment of neutrophil function could play an important role in the pathogenesis of MRONJ via an infectious mechanism mediated by the suppression of the innate immune system. Currently, the existing human data are insufficient to substantiate this theory. To investigate, we isolated neutrophils from blood and oral rinse samples from bisphosphonate-naïve patients who were recently diagnosed with multiple myeloma both prior to and one month following their initial infusion of pamidronate, an intravenous bisphosphonate agent. Stimulated blood and oral neutrophil superoxide production and chemotactic capabilities were found to be impaired relative to baseline values. These results suggest that impaired neutrophil function may partially contribute to the aetiology underlying the pathophysiological processes linked to the development of MRONJ. Further, as the functional status of circulating neutrophils was reflected in the oral cavity where sampling can be accomplished in a non-invasive fashion, it is conceivable that neutrophil function could serve as a potential biomarker for MRONJ prognostication.

Data showing effects of a PI3K-δ inhibitor on neutrophil superoxide production during FPR2 activation and reactivation

Reactive oxygen species (ROS) generated by the NADPH oxidase are crucial for antimicrobial host defense and also play a role in the regulation of inflammatory processes. Signals generated by formyl peptide receptor 2 (FPR2) activate the neutrophil ROS generating NADPH oxidase; such signals are mediated when the receptors bind an activating agonist, as well as when agonist desensitized FPR2 are reactivated by the receptor for platelet-activating factor (PAF). We present data on the effects of Idelalisib, a specific inhibitor for the PI3Kδ isoform, on ROS production during FPR2 activation and reactivation by PAF, respectively. Neutrophils were isolated from peripheral blood of healthy adults obtained from the blood bank at Sahlgrenska University Hospital and ROS release was measured using isoluminol-amplified chemiluminescence.

Caffeinated instant coffee prevents an increase in exercise-mediated superoxide anion production in rat peritoneal neutrophils

The present study analyzed the in vivo effects of drinking caffeinated and decaffeinated instant coffee (8% w/v) by adult male Wistar rats submitted to high-intensity exercises. The parameters used in the evaluation were the determination of the activities of NADPH oxidase, myeloperoxidase and other antioxidant enzymes present in neutrophils of rats. It was observed that exercise-induced superoxide anion production depends on the NADPH oxidase activity (estimated by the cytochrome C reduction test) in peritoneal neutrophils (p < 0.05). The intake of caffeinated and decaffeinated instant coffee beverages and of a caffeine solution to 1.67% did not induced changes in myeloperoxidase, superoxide dismutase and glutathione peroxidase activities in the peritoneal neutrophils of rats after exercise (p < 0.05). From the obtained results, it was concluded that moderate intake of caffeinated instant coffee (equivalent to a daily human consumption of 4 50-mL cups of coffee) may have beneficial effects on health, contributing to a reduction in superoxide anion generation triggered in neutrophils after high-intensity exercise.

Substance P receptor blocker, aprepitant, inhibited cutaneous and other neurogenic inflammation side effects of the EGFR1-TKI, erlotinib.

Cutaneous changes like rash and hair loss, as well as other neurogenic inflammation side effects, occur frequently during anticancer treatment with the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), erlotinib. These adverse events may be so severe that they impair the patient's compliance with the treatment or even cause its discontinuation. In the current preclinical study, rats (9.2weeks) were treated with erlotinib (10mg/kg/day) ± aprepitant (2mg/kg/day) for 12weeks. Visual changes in the development of facial skin lesions/hair loss and SP-receptor expression (immunohistochemically) in facial skin tissue were assessed; also changes in plasma magnesium, 8-isoprostane, substance P (SP), neutrophil superoxide production, and cardiac function (echocardiography) were measured. Erlotinib lowered plasma magnesium 14%, elevated SP 65%, caused 3.7-fold higher basal superoxide production, 2.5-fold higher 8-isoprostane levels, 11.6% lower cardiac systolic, and 10.9% lower diastolic function. Facial dermatological changes (alopecia, skin reddening, scabbing, nose crusting) occurred by 4weeks (± + to ++) in erlotinib-treated rats, and progressively worsened (±++ to +++) by week 12. Facial skin SP-receptor upregulation (78% higher) occurred in epidermal and hair follicle cells. All adverse effects were substantially and significantly mitigated by aprepitant, including a 62% lowering of skin SP-receptors (p < 0.05). Elevated SP levels mediated the side effects of erlotinib treatment, but aprepitant's significant prevention of the systemic and cutaneous adverse events indicates a novel potential therapy against the side effects of this anticancer treatment.

Serum amyloid A3 confers protection against acute lung injury in Pseudomonas aeruginosa-infected mice.

Pseudomonas aeruginosa is a gram-negative bacterium associated with serious illnesses, including ventilator-associated pneumonia and various sepsis syndromes in humans. Understanding the host immune mechanisms against P. aeruginosa is, therefore, of clinical importance. The present study identified serum amyloid A3 (SAA3) as being highly inducible in mouse bronchial epithelium following P. aeruginosa infection. Genetic deletion of Saa3 rendered mice more susceptible to P. aeruginosa infection with decreased neutrophil superoxide anion production, and ex vivo treatment of mouse neutrophils with recombinant SAA3 restored the ability of neutrophils to produce superoxide anions. The SAA3-deficient mice showed exacerbated inflammatory responses, which was characterized by pronounced neutrophil infiltration, elevated expression of TNF-α, KC/CXCL1, and MIP-2/CXCL2 in bronchoalveolar lavage fluid (BALF), and increased lung microvascular permeability compared with their wild-type littermates. BALF neutrophils from Saa3 knockout mice exhibited reduced superoxide anion production compared with neutrophils from wild-type mice. Adoptive transfer of SAA3-treated neutrophils to Saa3 knockout mice ameliorated P. aeruginosa-induced acute lung injury. These findings demonstrate that SAA3 not only serves as a biomarker for infection and inflammation, but also plays a protective role against P. aeruginosa infection-induced lung injury in part through augmentation of neutrophil bactericidal functions.

Eugenol prevents fMLF-induced superoxide anion production in human neutrophils by inhibiting ERK1/2 signaling pathway and p47phox phosphorylation

Eugenol is a polyphenol extracted from Syzygium aromaticum essential oil. It is known to have anti-inflammatory and chemoprotective properties as well as a potent anti-oxidant activity due the presence of its phenolic group. In this study, we examined the effects of eugenol on neutrophil superoxide production, a key process involved in innate immunity and inflammation. Superoxide anion generationin human neutrophils was measured by cytochrome c reduction assay. Western blotting was used to analyze the phosphorylation of, p47phox, MAPKinases (p38 and ERK1/2), MEK1/2 and Raf, key proteins involved in the activation of NADPH oxidase. Pretreatment of neutrophils by increasing concentrations (2.5 µg/mL–20 µg/mL) of eugenol for 30 min, inhibited significantly (p < 0.001) superoxide anion generation induced by the chemotactic peptide formyl-Met-Leu-Phe (fMLF) with an IC50 of 5 µg/mL. Phorbolmyristate acetate (PMA)-stimulated O2− production was affected only at the highest eugenol concentration (20 µg/mL). Results showed that eugenol decreased the phosphorylation of p47phox onSer-345 and Ser-328, the translocation of p47phox to the membranesand the phosphorylation of Raf, MEK1/2 and ERK1/2 proteins. Taken together, our results suggest that eugenol inhibits the generation of superoxide anion by neutrophils via the inhibition of Raf/MEK/ERK1/2/p47phox-phosphorylation pathway.

The Prolyl Isomerase Pin1 Controls Lipopolysaccharide-Induced Priming of NADPH Oxidase in Human Neutrophils.

Production of superoxide anion and other reactive oxygen species (ROS) by neutrophils has a vital role in host defense against microbes. However, over-production can induce cell injury participating to inflammation. Superoxide anion is produced by the phagocyte NADPH oxidase/NOX2, a multicomponent enzyme system consisting of six proteins: two trans-membrane proteins (gp91phox and p22phox) and four soluble cytosolic proteins (p40phox, p67phox, p47phox, and the small G-proteins, Rac1/2). Phosphorylation of p47phox on several serines regulates NADPH oxidase activation. LPS released by gram negative bacteria can enhance or prime neutrophil superoxide production in combination with other agonists such as the bacterial peptide formyl-Met-Leu-Phe (fMLP). Since the pathways involved in LPS-induced priming are not completely understood, we investigated the role of the prolyl cis/trans isomerase Pin1 in this process. Two different Pin1 inhibitors, PiB, and Juglone are able to block LPS-induced priming of ROS production by human neutrophils in a concentration dependent manner. PiB and Juglone did not inhibit LPS-induced CD11b translocation neither CD62L shedding. LPS induced an increase of Pin1 activity in neutrophils similar to TNFα and fMLP. Since the phosphorylation of p47phox on Ser345 is critical for NADPH oxidase up-regulation, we investigated the effect of LPS on this process. Results show that LPS induced the phosphorylation of p47phox mainly on serine 345 and induced the activation of p38MAPKinase and ERK1/2. These results suggest that the prolyl cis/trans isomerase Pin1 may control LPS-induced priming of superoxide production in human neutrophils. Pharmacological targeting of Pin1 could be a valuable approach in sepsis.

Estradiol inhibits fMLP-induced neutrophil migration and superoxide production by upregulating MKP-2 and dephosphorylating ERK

Estrogen has been reported to inhibit neutrophil infiltration related inflammation and suppress neutrophils migration in vitro, but the underlying mechanism is not fully understood. By using HL-60 differentiated neutrophil-like cells (dHL-60) and human neutrophils, we examined the effect of 17-β estradiol (E2) on cell migration and superoxide production in response to chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) and explored the mechanisms involved. We found that fMLP significantly induced dHL-60 cell and neutrophil migration and superoxide production, which was inhibited by ERK inhibitor PD98059. E2 significantly inhibited fMLP-induced dHL-60 cell and neutrophil migration and superoxide production at both physiological and pharmacological concentrations. Mechanistic studies showed that pretreatment of these cells with E2 rapidly elevated the protein level of mitogen-activated protein kinase phosphatase 2 (MKP-2) and inhibited fMLP-induced ERK phosphorylation. Pretreatment of these cells with estrogen receptor (ER) antagonist ICI 182780 reversed the inhibition of fMP-induced cell migration and superoxide production, and the induction of MKP-2 expression and the suppression of fMP-induced ERK phosphorylation by E2. However, pretreatment of cells with G-protein coupled ER antagonist G15 had no such effect. Collectively, these results demonstrate that fMLP stimulates neutrophil chemotaxis and superoxide production through activating ERK, and indicate that ER-mediated upregulation of MKP-2 may dephosphorylate ERK and contribute to the inhibitory effect of E2 on neutrophil activation by fMLP. Our study reveals new mechanisms involved in the anti-inflammatory activity of estrogen.

Lysophosphatidic Acid Enhances In Vivo Infiltration and Activation of Guinea Pig Eosinophils and Neutrophils via a Rho/Rho-Associated Protein Kinase-Mediated Pathway

Lysophosphatidic acid (LPA) has been shown to be a chemoattractant in in vitro studies. The present study was carried out to determine whether LPA enhances infiltration of inflammatory cells in in vivo studies with guinea pigs. LPA (1 – 10 μg/ml), when by guinea pigs for 5 min, substantially increased the numbers of eosinophils and neutrophils in the bronchoalveolar lavege fluid (BALF), which was recovered at over 4 h after the inhalation of LPA. Infiltration in BALF was significantly inhibited by inhalation of Y-27632, an inhibitor of Rho-associated protein kinase (ROCK). LPA also increased superoxide production of eosinophils and neutrophils. In contrast, Y-27632 inhibited superoxide production. These findings suggest that LPA may contribute to infiltration and activation of inflammatory cells in bronchial asthma; furthermore, the Rho/ROCK-mediated pathway may be involved.

Dominant activating RAC2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects

AbstractRas-related C3 botulinum toxin substrate 2 (RAC2), through interactions with reduced NAD phosphate oxidase component p67phox, activates neutrophil superoxide production, whereas interactions with p21-activated kinase are necessary for fMLF-induced actin remodeling. We identified 3 patients with de novo RAC2[E62K] mutations resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Neutrophils from RAC2[E62K] patients exhibited excessive superoxide production, impaired fMLF-directed chemotaxis, and abnormal macropinocytosis. Cell lines transfected with RAC2[E62K] displayed characteristics of active guanosine triphosphate (GTP)–bound RAC2 including enhanced superoxide production and increased membrane ruffling. Biochemical studies demonstrated that RAC2[E62K] retains intrinsic GTP hydrolysis; however, GTPase-activating protein failed to accelerate hydrolysis resulting in prolonged active GTP-bound RAC2. Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients. This gain-of-function mutation highlights a specific, nonredundant role for RAC2 in hematopoietic cells that discriminates RAC2 from the related, ubiquitous RAC1.

Phospholipid transfer protein and alpha-1 antitrypsin regulate Hck kinase activity during neutrophil degranulation

Excessive neutrophil degranulation is a common feature of many inflammatory disorders, including alpha-1 antitrypsin (AAT) deficiency. Our group has demonstrated that phospholipid transfer protein (PLTP) prevents neutrophil degranulation but serine proteases, which AAT inhibits, cleave PLTP in diseased airways. We propose to identify if airway PLTP activity can be restored by AAT augmentation therapy and how PLTP subdues degranulation of neutrophils in AAT deficient subjects. Airway PLTP activity was lower in AAT deficient patients but elevated in the airways of patients on augmentation therapy. Functional AAT protein (from PiMM homozygotes) prevented PLTP cleavage unlike its mutated ZZ variant (PiZZ). PLTP lowered leukotriene B4 induced degranulation of primary, secondary and tertiary granules from neutrophils from both groups (n = 14/group). Neutrophils isolated from Pltp knockout mice have enhance neutrophil degranulation. Both AAT and PLTP reduced neutrophil degranulation and superoxide production, possibly though their inhibition of the Src tyrosine kinase, Hck. Src kinase inhibitors saracatinib and dasatinib reduced neutrophil degranulation and superoxide production. Therefore, AAT protects PLTP from proteolytic cleavage and both AAT and PLTP mediate degranulation, possibly via Hck tyrosine kinase inhibition. Deficiency of AAT could contribute to reduced lung PLTP activity and elevated neutrophil signaling associated with lung disease.