Human Peripheral Blood Neutrophils Research Articles

Evasion of Human Neutrophil-Mediated Host Defense during Toxoplasma gondii Infection.

ABSTRACTNeutrophils are a major player in host immunity to infection; however, the mechanisms by which human neutrophils respond to the intracellular protozoan parasite Toxoplasma gondii are still poorly understood. In the current study, we found that, whereas primary human monocytes produced interleukin-1beta (IL-1β) in response to T. gondii infection, human neutrophils from the same blood donors did not. Moreover, T. gondii inhibited lipopolysaccharide (LPS)-induced IL-1β synthesis in human peripheral blood neutrophils. IL-1β suppression required active parasite invasion, since heat-killed or mycalolide B-treated parasites did not inhibit IL-1β release. By investigating the mechanisms involved in this process, we found that T. gondii infection of neutrophils treated with LPS resulted in reduced transcript levels of IL-1β and NLRP3 and reduced protein levels of pro-IL-1β, mature IL-1β, and the inflammasome sensor NLRP3. In T. gondii-infected neutrophils stimulated with LPS, the levels of MyD88, TRAF6, IKKα, IKKβ, and phosphorylated IKKα/β were not affected. However, LPS-induced IκBα degradation and p65 phosphorylation were reduced in T. gondii-infected neutrophils, and degradation of IκBα was reversed by treatment with the proteasome inhibitor MG-132. Finally, we observed that T. gondii inhibited the cleavage and activity of caspase-1 in human neutrophils. These results indicate that T. gondii suppression of IL-1β involves a two-pronged strategy whereby T. gondii inhibits both NF-κB signaling and activation of the NLRP3 inflammasome. These findings represent a novel mechanism of T. gondii evasion of human neutrophil-mediated host defense by targeting the production of IL-1β.

Impairment of Several Immune Functions and Redox State in Blood Cells of Alzheimer's Disease Patients. Relevant Role of Neutrophils in Oxidative Stress.

Since aging is considered the most risk factor for sporadic Alzheimer’s Disease (AD), the age-related impairment of the immune system (immunosenescence), based on a chronic oxidative-inflammatory stress situation, could play a key role in the development and progression of AD. Although AD is accompanied by systemic disturbance, reflecting the damage in the brain, the changes in immune response and redox-state in different types of blood cells in AD patients have been scarcely studied. The aim was to analyze the variations in several immune functions and oxidative-inflammatory stress and damage parameters in both isolated peripheral neutrophils and mononuclear blood cells, as well as in whole blood cells, from patients diagnosed with mild (mAD) and severe AD, and of age-matched controls (elderly healthy subjects) as well as of adult controls. The cognitive decline of all subjects was determined by Mini-Mental State Examination (MMSE) test (mAD stage was established at 20 ≤ MMSE ≤ 23 score; AD stage at <18 MMSE; elderly subjects >27 MMSE). The results showed an impairment of the immune functions of human peripheral blood neutrophils and mononuclear cells of mAD and AD patients in relation to healthy elderly subjects, who showed the typical immunosenescence in comparison with the adult individuals. However, several alterations were only observed in severe AD patients (lower chemotaxis, lipopolysaccharide lymphoproliferation, and interleukin (IL)-10 release; higher basal proliferation, tumor necrosis factor (TNF)-α release, and IL-10/TNF-α ratio), others only in mAD subjects (higher adherence), meanwhile others appeared in both mAD and AD patients (lower phytohemaglutinin lymphoproliferation and higher IL-6 release). This impairment of immune functions could be mediated by: (1) the higher oxidative stress and damage also observed in blood cells from mAD and AD patients and in isolated neutrophils [lower glutathione (GSH) levels, high oxidized glutathione (GSSG)/GSH ratio, and GSSG and malondialdehyde contents], and (2) the higher release of basal pro-inflammatory cytokines (IL-6 and TNF-α) found in AD patients. Because the immune system parameters studied are markers of health and rate of aging, our results supported an accelerated immunosenescence in AD patients. We suggest the assessment of oxidative stress and function parameters in peripheral blood cells as well as in isolated neutrophils and mononuclear cells, respectively, as possible markers of AD progression.

Celastrol, a pentacyclic triterpene, attenuates fMLP-induced elastase release in human peripheral blood neutrophils

Celastrol, a pentacyclic triterpene, attenuates fMLP-induced elastase release in human peripheral blood neutrophils

Human canonical CD157/Bst1 is an alternatively spliced isoform masking a previously unidentified primate-specific exon included in a novel transcript

CD157/Bst1 is a dual-function receptor and β-NAD+-metabolizing ectoenzyme of the ADP-ribosyl cyclase family. Expressed in human peripheral blood neutrophils and monocytes, CD157 interacts with extracellular matrix components and regulates leukocyte diapedesis via integrin-mediated signalling in inflammation. CD157 also regulates cell migration and is a marker of adverse prognosis in epithelial ovarian cancer and pleural mesothelioma. One form of CD157 is known to date: the canonical sequence of 318 aa from a 9-exon transcript encoded by BST1 on human chromosome 4. Here we describe a second BST1 transcript, consisting of 10 exons, in human neutrophils. This transcript includes an unreported exon, exon 1b, located between exons 1 and 2 of BST1. Inclusion of exon 1b in frame yields CD157-002, a novel proteoform of 333 aa: exclusion of exon 1b by alternative splicing generates canonical CD157, the dominant proteoform in neutrophils and other tissues analysed here. In comparative functional analyses, both proteoforms were indistinguishable in cell surface localization, specific mAb binding, and behaviour in cell adhesion and migration. However, NAD glycohydrolase activity was detected in canonical CD157 alone. Comparative phylogenetics indicate that exon 1b is a genomic innovation acquired during primate evolution, pointing to the importance of alternative splicing for CD157 function.

Ehrlichia chaffeensis and Its Invasin EtpE Block Reactive Oxygen Species Generation by Macrophages in a DNase X-Dependent Manner.

ABSTRACTThe obligatory intracellular pathogen Ehrlichia chaffeensis lacks most genes that confer resistance to oxidative stress but can block reactive oxygen species (ROS) generation by host monocytes-macrophages. Bacterial and host molecules responsible for this inhibition have not been identified. To infect host cells, Ehrlichia uses the C terminus of its surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C), which directly binds the mammalian cell surface receptor glycosylphosphatidylinositol-anchored protein DNase X. We investigated whether EtpE-C binding to DNase X blocks ROS production by mouse bone marrow-derived macrophages (BMDMs). On the basis of a luminol-dependent chemiluminescence assay, E. chaffeensis inhibited phorbol myristate acetate (PMA)-induced ROS generation by BMDMs from wild-type, but not DNase X−/−, mice. EtpE-C is critical for inhibition, as recombinant EtpE-C (rEtpE-C)-coated latex beads, but not recombinant N-terminal EtpE-coated or uncoated beads, inhibited PMA-induced ROS generation by BMDMs from wild-type mice. DNase X is required for this inhibition, as none of these beads inhibited PMA-induced ROS generation by BMDMs from DNase X−/− mice. Previous studies showed that E. chaffeensis does not block ROS generation in neutrophils, a cell type that is a potent ROS generator but is not infected by E. chaffeensis. Human and mouse peripheral blood neutrophils did not express DNase X. Our findings point to a unique survival mechanism of ROS-sensitive obligate intramonocytic bacteria that involves invasin EtpE binding to DNase X on the host cell surface. This is the first report of bacterial invasin having such a subversive activity on ROS generation.

Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.

There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.

Abstract 2701: Large-scale ex vivo generation of human neutrophils from cord blood CD34+ cells

Abstract Ex vivo expansion of hematopoietic stem cell and subsequent differentiation into mature neutrophils remains a challenge. Here, we have developed a three-stage culture system to produce efficiently functional neutrophils derived from cord blood CD34+ cells. A procedure of ex vivo expansion and differentiation in a large-scale was developed in a modified IMDM basal medium supplemented with transferrin, insulin, fetal bovine serum, and some other nutrients with selected cytokine combination that contained stem cell factor (SCF), Flt-3 ligand (FL), granulocyte-colony stimulating factor(G-CSF), granulocyte macrophage colony-stimulating factor(GM-CSF) and thrombopoietin (TPO) in stage I (days 0~6); SCF, FL, G-CSF, interleukin 3 (IL-3), and GM-CSF in stage II (days 6~9); SCF, FL, and G-CSF in stage III (days 9~18), respectively. Enriched CD34+ cells were firstly cultured and expanded in 25-T flasks. After 6 day-culture, the cells were transferred to a 2-L bottle with 500 ml of medium in the bottle-turning device system. During the differentiation process, neutrophil marker CD66b was evaluated by flow cytometry. Ex vivo generated neutrophils or medium only were incubated with E.coli overnight for its bacteria killing assay, and then the E.coli colony-forming units were counted separately. Matured neutrophils or vehicle were transplanted into NOD/SCID mice intravenously for chemotactic activity in vivo. The cells that accumulated in the pouch with chemoattractant were collected and subjected to flow cytometric analysis for human CD66b antigen. The ex vivo generated neutrophils/progenitors were then injected into sub-lethal irradiated NOD/SCID mice to monitor the viability and maturation in vivo. After the three-stage culture, proliferation fold of total cell reached 30013 ± 286.5 with 60.2% ± 2.4% for CD66b+ neutrophils. The calculated yield of matured neutrophils from each CD34+ cell was ranged from 1.8 × 104 to 1.87 × 104 for 18-day culture. There was no E.coli colony formed after incubation with neutrophils in bacteria killing assay, indicating that the generated neutrophils was functional. For in vivo chemotaxis assay, the neutrophils collected from 18-day culture were injected into mice and detected at 1.08% ±0.16% for human CD66b+ cells and no any CD66b+ cell observed for negative control group. In addition, the CD66b+ cells were extended for 4 days from a 15-day cultured neutrophil group in mouse peripheral blood (PB), while only for 2 days from a freshly isolated human PB neutrophils post injection, indicating that the ex vivo generated neutrophils/progenitors could further matured in vivo. Taken together, we have established a pilot-scale culture system to produce functional human neutrophils ex vivo. Considering that one neutrophil transfusion unit (100ml) contains 2×1010 cells, the CD34+ cells from one CB unit (80 ml) would generate 1.6×1011 neutrophils, which are equivalent to 8 unit doses of neutrophils in the clinical application. Citation Format: Zhenwang Jie, Yu Zhang, Chen Wang, Bin Shen, Xin Guan, Zhihua Ren, Xinxin Ding, Wei Dai, Yongping Jiang. Large-scale ex vivo generation of human neutrophils from cord blood CD34+ cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2701. doi:10.1158/1538-7445.AM2017-2701

P 018 - Free radical pathway of 2-hexadecenal formation in cells and its biological role

In our earlier studies it has been established that the action of gamma-, UV-irradiation and HOCl on aqueous deaerated sphingolipids dispersions causes destruction of studied biomolecules with the formation of 2-hexadecenal. HOCl has powerful cytotoxic properties because it is a strong oxidizer and a source of reactive chlorine species. We investigated the effect of HOCl on human erythrocytes, HEK293 and astroglial cells. For the sensitive quantitative analysis of 2-hexadecenal in cells we used extraction procedure and applied the method based on HPLC with fluorescence detector. We found that the HOCl-treatment of cells at concentrations from 10 µM to 1 mM provokes 2-hexadecenal formation. It has been found that 2-hexadecenal at micromolar concentrations regulates reactive oxygen species generation in human peripheral blood neutrophils stimulated by adhesion and fMLP, through reallocation of myeloperoxidase, phospholipase A2, cyclooxygenase and 5-lipoxygenase contributions to this process. 2-Hexadecenal modifies the functions of astroglial cells in culture by changing their morphological characteristics. This is associated with the redistribution of F-actin and the subsequent cytoskeleton reorganization. It results in cells’ mitotic and proliferative activity reduction through the initiation of apoptosis involving JNK and p38 mitogen-activated protein kinase pathways.

Anti-inflammatory Effects of Quercetin and Vitexin on Activated Human Peripheral Blood Neutrophils: - The effects of quercetin and vitexin on human neutrophils.

ObjectivesPolymorphonuclear neutrophils (PMNs) constitute the first line of defense against invading microbial pathogens. Early events in inflammation involve the recruitment of neutrophils to the site of injury or damage where changes in intracellular calcium can cause the activation of pro-inflammatory mediators from neutrophils including superoxide generation, degranulation and release of myeloperoxidase (MPO), productions of interleukin (IL)-8 and tumor necrosis factor α (TNF-α), and adhesion to the vascular endothelium. To address the anti-inflammatory role of flavonoids, in the present study, we investigated the effects of the flavonoids quercetin and vitexin on the stimulus-induced nitric oxide (NO), TNF-α, and MPO productions in human neutrophils.MethodsHuman peripheral blood neutrophils were isolated, and their viabilities were determined by using the Trypan Blue exclusion test. The polymorphonuclear leukocyte (PMNL) preparations contained more than 98% neutrophils as determined by morphological examination with Giemsa staining. The viabilities of cultured neutrophils with various concentrations of quercetin and vitexin (1 – 100 μM) were studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without quercetin and vitexin (25 μM) for 45 min, and stimulated with phorbol 12-myristate 13-acetate (PMA) (10−7 M). NO production was carried out through nitrite determination by using the Griess method. Also, the TNF-α and the MPO productions were measured using enzyme-linked immunosorbent assay (ELISA) kits and MPO assay kits.ResultsNeutrophil viability was not affected up to a concentration of 100 μM of quercetin or vitexin. Both quercetin and vitexin significantly inhibited TNF-α, NO, and MPO productions in human neutrophils (P < 0.001).ConclusionThe present study showed that both quercetin and vitexin had significant anti-inflammatory effects. Thus, treatment with either quercetin or vitexin may be considered as a therapeutic strategy for treating patients with neutrophil-mediated inflammatory diseases.

Antioxidant Capacity, Phenolic Constituents and Toxicity of Hot Water Extract from Red Maple Buds.

The present study reports, for the first time, the results of the antioxidant capacity and the phenolic composition of a hot water extract from red maple buds (RMB), as well as its safety. In this regard and comparatively to antioxidant standards, this extract exhibits a significant antiradical capacity when tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH· ) and anion superoxide trapping assays. High-resolution mass spectrometric and nuclear magnetic resonance analyses permitted to determine for the first time, in red maple species, cyanidin-3-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-arabinoside, and quercetin. Also, the quantification of individual phenolics by high-performance liquid chromatography method revealed that ginnalin A at 117.0 mg/g is the major compound of RMB hot water extract. Finally, using flow cytometry evaluation, the extract of RMB was determined to have no toxicity neither to cause significant modification of apoptosis process, up to concentration of 100 μg/ml, on human peripheral blood neutrophils. These results allow anticipating various fields of application of RMB water extract.

Treatment with Rutin - A Therapeutic Strategy for Neutrophil-Mediated Inflammatory and Autoimmune Diseases: - Anti-inflammatory Effects of Rutin on Neutrophils.

Objectives:Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO) and myeloperoxidase (MPO). These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor (TNF)-α productions and MPO activity in human neutrophils were investigated.Methods:Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without rutin (25 μM) for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA). Then, the TNF-α, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA), Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and neutrophils were treated with various concentrations of rutin (1 - 100 μM), after which MTT was appended and incubated at 37ºC for 4 hour.Results:Rutin at concentrations up to 100 μM did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and TNF-α productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001). Also, MPO activity was significantly reduced by rutin (P < 0.001).Conclusion:In this in vitro study, rutin had an anti-inflammatory effect due to its inhibiting NO and TNF-α productions, as well as MPO activity, in activated human neutrophils. Treatment with rutin may be considered as a therapeutic strategy for neutrophil-mediated inflammatory/ autoimmune diseases.

Recombinant human thrombomodulin inhibits neutrophil extracellular trap formation in vitro.

The aim of this study was to investigate the effects of recombinant human-soluble thrombomodulin (rTM) on lipopolysaccharide (LPS)-induced, platelet-dependent neutrophil extracellular trap (NET) formation (NETosis). Human peripheral blood neutrophils and platelets were co-incubated with or without LPS (0.2 μg/ml) in the presence and absence of rTM (2 μg/ml). NETosis was confirmed by immunostaining and confocal microscopy. In the absence of platelets, LPS did not induce NETosis in the neutrophils. NETosis, however, was induced by LPS when neutrophils were co-cultured with platelets (64 % of neutrophils). Notably, rTM was able to fully inhibit NETosis in neutrophils cultured with platelets and in the presence of LPS. rTM did not induce NETosis in this co-culture system (p < 0.01 versus LPS in the absence of rTM). These results show that rTM can suppress LPS-induced platelet-dependent NETosis in vitro.

CFTR targeting during activation of human neutrophils.

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, plays critical roles in phagocytic host defense. However, how activated neutrophils regulate CFTR channel distribution subcellularly is not well defined. To investigate, we tested multiple Abs against different CFTR domains, to examine CFTR expression in human peripheral blood neutrophils by flow cytometry. The data confirmed that resting neutrophils had pronounced CFTR expression. Activation of neutrophils with soluble or particulate agonists did not significantly increase CFTR expression level, but induced CFTR redistribution to cell surface. Such CFTR mobilization correlated with cell-surface recruitment of formyl-peptide receptor during secretory vesicle exocytosis. Intriguingly, neutrophils from patients with ΔF508-CF, despite expression of the mutant CFTR, showed little cell-surface mobilization upon stimulation. Although normal neutrophils effectively targeted CFTR to their phagosomes, ΔF508-CF neutrophils had impairment in that process, resulting in deficient hypochlorous acid production. Taken together, activated neutrophils regulate CFTR distribution by targeting this chloride channel to the subcellular sites of activation, and ΔF508-CF neutrophils fail to achieve such targeting, thus undermining their host defense function.

Porphyromonas gingivalis-induced production of reactive oxygen species, tumor necrosis factor-α, interleukin-6, CXCL8 and CCL2 by neutrophils from localized aggressive periodontitis and healthy donors: modulating actions of red blood cells and resolvin E1.

Porphyromonas gingivalis is regarded as a significant contributor in the pathogenesis of periodontitis and certain systemic diseases, including atherosclerosis. P.gingivalis occasionally translocates from periodontal pockets into the circulation, where it adheres to red blood cells (RBCs). This may protect the bacterium from contact with circulating phagocytes without affecting its viability. In this in vitro study, we investigated whether human peripheral blood neutrophils from 10 subjects with localized aggressive periodontitis (LAgP) and 10 healthy controls release the proinflammatory cytokines interleukin (IL)-6, tumor necrosis factor α (TNF-α), the chemokine (C-X-C motif) ligand 8 (CXCL8; also known as IL-8) and chemokine (C-C motif) ligand 2 (CCL2; also known as monocyte chemotactic protein-1) and intracellular reactive oxygen species (ROS) in response to challenge with P.gingivalis. In addition, the impact of RBC interaction with P.gingivalis was investigated. The actions of resolvin E1 (RvE1), a known regulator of P.gingivalis induced neutrophil responses, on the cytokine and ROS responses elicited by P.gingivalis in cultures of neutrophils were investigated. Upon stimulation with P.gingivalis, neutrophils from subjects with LAgP and healthy controls released similar quantities of IL-6, TNF-α, CXCL8, CCL2 and intracellular ROS. The presence of RBCs amplified the release of IL-6, TNF-α and CCL2 statistically significant in both groups, but reduced the generation of ROS in the group of healthy controls, and showed a similar tendency in the group of subjects with LAgP. RvE1 had no impact on the production of intracellular ROS, TNF-α, IL-6, CXCL8 and CCL2 by neutrophils from either group, but tended to reduce the generation of ROS in subjects with LAgP in the absence of RBCs. Our data support that binding to RBCs protects P.gingivalis from ROS and concomitantly enhances neutrophil release of proinflammatory cytokines providing a selective advantage for P.gingivalis growth.

IL-21 regulates the innate immune response to Staphylococcus aureus pulmonary infection.

Abstract Methicillin-resistant Staphylococcus aureus (MRSA) infection is a major complication of viral respiratory infections and immunodeficient states. Although the innate immune response mediated by neutrophils plays a major role in the clearance of MRSA, the cytokine regulation of this process is poorly understood. Here, we investigated the role of IL-21 in the host response to pulmonary MRSA infection. In experiments using a mouse model, Il21r-deficient mice more efficiently cleared pulmonary MRSA infection, suggesting a negative role for IL-21 in MRSA clearance. Interestingly, Il21r KO mice displayed an enhanced “interferon signature” response after pulmonary MRSA infection as compared to WT mice, and blocking the type I IFN response eliminated the ability of Il21r KO mice to more efficiently clear the infection. Despite the more efficient clearing of pulmonary MRSA in Il21r KO than in wild-type mice, when IL-21 was administered to wild-type mice, there was enhanced clearance of MRSA, and this clearance was accompanied by the induction of cytolytic proteases and other components of pulmonary defense. Correspondingly, human peripheral blood neutrophils responded directly to IL-21 with the induction of cytolytic proteases including granzymes, and they displayed enhanced IL-21-induced killing of MRSA that was granzyme-dependent. These data demonstrate that although the chronic absence of IL-21 signaling in Il21r KO leads to the dominant action of type I IFN responses that result in MRSA clearance, IL-21 directly induces a cytolytic program in both human and mouse neutrophils that results in MRSA killing. These data suggest that manipulation of IL-21 signaling may be a strategy for controlling bacterial infections.

JAK/STAT regulation of Aspergillus fumigatus corneal infections and IL-6/23-stimulated neutrophil, IL-17, elastase, and MMP9 activity.

IL-6 and IL-23 (IL-6/23) induce IL-17A (IL-17) production by a subpopulation of murine and human neutrophils, resulting in autocrine IL-17 activation, enhanced production of reactive oxygen species, and increased fungal killing. As IL-6 and IL-23 receptors trigger JAK1, -3/STAT3 and JAK2/STAT3 phosphorylation, respectively, we examined the role of this pathway in a murine model of fungal keratitis and also examined neutrophil elastase and gelatinase (matrix metalloproteinase 9) activity by IL-6/23-stimulated human neutrophils in vitro. We found that STAT3 phosphorylation of neutrophils in Aspergillus fumigatus-infected corne as was inhibited by the JAK/STAT inhibitor Ruxolitinib, resulting in impaired fungal killing and decreased matrix metalloproteinase 9 activity. In vitro, we showed that fungal killing by IL-6/23-stimulated human peripheral blood neutrophils was impaired by JAK/STAT inhibitors Ruxolitinib and Stattic, and by the retinoic acid receptor-related orphan receptor γt inhibitor SR1001. This was also associated with decreased reactive oxygen species, IL-17A production, and retinoic acid receptor-related orphan receptor γt translocation to the nucleus. We also demonstrate that IL-6/23-activated neutrophils exhibit increased elastase and gelatinase (matrix metalloproteinase 9) activity, which is inhibited by Ruxolitinib and Stattic but not by SR1001. Taken together, these observations indicate that the regulation of activity of IL-17-producing neutrophils by JAK/STAT inhibitors impairs reactive oxygen species production and fungal killing activity but also blocks elastase and gelatinase activity that can cause tissue damage.

Isolation and co-cultivation of human macrophages and neutrophils with Plasmodium falciparum-parasitized erythrocytes: An optimized system to study the phagocytic activity to malarial parasites

Macrophages and neutrophils are our front line of defense against invading pathogens. They are professional phagocytic cells that play a key role in the clearance of Plasmodium falciparum-parasitized erythrocytes from the host circulation. A stable in vitro culture system for these cells and parasitized erythrocytes would provide the means to study their immunological responses to infection, potentially revealing important clues for new therapeutic interventions for malaria. Here, we present an optimized protocol for cultivating human peripheral blood monocyte-derived macrophages and neutrophils with Plasmodium falciparum-parasitized erythrocytes.

The effect of continuous low-intensity laser irradiation of the red spectrum on the changes in the functional activity and speed of NADPH-oxidase response of human peripheral blood neutrophils

Registration of the changes in the functional metabolic status of neutrophils under the influence of low-intensity laser radiation (LILR) can be useful for the choice of the exposure parameters in the studies designed to evaluate immunotropic effects. Such investigations, in their turn, are very promising in clinical terms since their results can be used to optimize the laser therapy techniques. The objective of the present study was to evaluate the degranulation potential of neutrophils and the intensity of their NADPH-oxidase reaction in response to the exposure to laser radiation as exemplified by the release of lysosomal granules in vitro. The source of radiation was the LASMIK device operated in the continuous mode at the following parameters: wavelength 635 nm, power density 0.12 mW/cm2, exposure time 10, 30, 90, 120, 150 s in the case of the automatic timer control and 100 s in the case of manual shutdown. It was shown that the maximum lysosomal activity and release of lysosomal granules took place at a 90-10 s exposure and a wavelength of 635 nm with the appearance of a plateau within 120 and 150 s after the onset of irradiation. In the case of a shorter exposure of neutrophil granulocytes to LILR (10 and 30 s) no pronounced effect was observed. It means that low-intensity laser radiation of the red spectrum with a wavelength of 635 nm is a physical stimulus reinforcing exocytosis of lysosomal granules by neutrophils in vitro. The reliable changes of NADPH-oxidase activity of neutrophil granulocytes isolated from donor peripheral blood are recorded at an optimum exposure time of 90-100 s. It is concluded that the laser therapy techniques, at least those designed to regulate neutrophils should be applied with the optimum exposure time of 90-100 s.

Identification of FAM3D as a new endogenous chemotaxis agonist for the formyl peptide receptors.

The family with sequence similarity 3 (FAM3) gene family is a cytokine-like gene family with four members FAM3A, FAM3B, FAM3C and FAM3D. In this study, we found that FAM3D strongly chemoattracted human peripheral blood neutrophils and monocytes. To identify the FAM3D receptor, we used chemotaxis, receptor internalization, Ca(2+) flux and radioligand-binding assays in FAM3D-stimulated HEK293 cells that transiently expressed formyl peptide receptor (FPR)1 or FPR2 to show that FAM3D was a high affinity ligand of these receptors, both of which were highly expressed on the surface of neutrophils, and monocytes and macrophages. After being injected into the mouse peritoneal cavity, FAM3D chemoattracted CD11b+ Ly6G+ neutrophils in a short time. In response to FAM3D stimulation, phosphorylated ERK1/2 and phosphorylated p38 MAPK family proteins were upregulated in the mouse neutrophils, and this increase was inhibited upon treatment with an inhibitor of FPR1 or FPR2. FAM3D has been reported to be constitutively expressed in the gastrointestinal tract. We found that FAM3D expression increased significantly during colitis induced by dextran sulfate sodium. Taken together, we propose that FAM3D plays a role in gastrointestinal homeostasis and inflammation through its receptors FPR1 and FPR2.

Involvement of PI3Kγ in Superoxide Anion Production in Response to IL-8, RANTES, and fMLP in Human Peripheral Blood Neutrophils

Involvement of PI3Kγ in Superoxide Anion Production in Response to IL-8, RANTES, and fMLP in Human Peripheral Blood Neutrophils Neutrophils are essential components of the immune system and have a critical role in combating bacterial and fungal infections. A key weapon in the neutrophil armory is the “respiratory burst,” which is the generation of reactive oxygen species (ROS) by a multicomponent oxidase complex. It is well established that pre-exposure of human neutrophils to proinflammatory cytokines and chemokines markedly augments the production of reactive oxygen species (ROS) to subsequent stimuli. In inflammatory reactions there are complex interactions of protein mediators (cytokines) and mediators derived from lipids. An important event in inflammation is superoxide production, in relation to microbicidal activity as well as tissue damage. A better understanding of phenomena involved in the regulation of NADPH oxidase could help developing novel therapeutic agents for inflammatory diseases involving abnormal neutrophil superoxide. Therefore, stimulating superoxide production by human neutrophils was investigated for this reason and because it sheds a light on intracellular signals that activate this response. Pretreatment of human neutrophils with N-formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8), and regulated on activation, normal T cell expressed and secreted (RANTES) markedly augmented the amount of superoxide anion produced, which was inhibited completely (IL-8 and fMLP) or partially (RANTES) by a specific isoform of phosphoinositide 3-kinase (PI3K), the PI3KγII inhibitor.