Human Polymorphonuclear Neutrophils Research Articles

CXCR4 and CXCR7 Inhibition Ameliorates the Formation of Platelet-Neutrophil Complexes and Neutrophil Extracellular Traps through Adora2b Signaling.

Peritonitis and peritonitis-associated sepsis are characterized by an increased formation of platelet–neutrophil complexes (PNCs), which contribute to an excessive migration of polymorphonuclear neutrophils (PMN) into the inflamed tissue. An important neutrophilic mechanism to capture and kill invading pathogens is the formation of neutrophil extracellular traps (NETs). Formation of PNCs and NETs are essential to eliminate pathogens, but also lead to aggravated tissue damage. The chemokine receptors CXCR4 and CXCR7 on platelets and PMNs have been shown to play a pivotal role in inflammation. Thereby, CXCR4 and CXCR7 were linked with functional adenosine A2B receptor (Adora2b) signaling. We evaluated the effects of selective CXCR4 and CXCR7 inhibition on PNCs and NETs in zymosan- and fecal-induced sepsis. We determined the formation of PNCs in the blood and, in addition, their infiltration into various organs in wild-type and Adora2b−/− mice by flow cytometry and histological methods. Further, we evaluated NET formation in both mouse lines and the impact of Adora2b signaling on it. We hypothesized that the protective effects of CXCR4 and CXCR7 antagonism on PNC and NET formation are linked with Adora2b signaling. We observed an elevated CXCR4 and CXCR7 expression in circulating platelets and PMNs during acute inflammation. Specific CXCR4 and CXCR7 inhibition reduced PNC formation in the blood, respectively, in the peritoneal, lung, and liver tissue in wild-type mice, while no protective anti-inflammatory effects were observed in Adora2b−/− animals. In vitro, CXCR4 and CXCR7 antagonism dampened PNC and NET formation with human platelets and PMNs, confirming our in vivo data. In conclusion, our study reveals new protective aspects of the pharmacological modulation of CXCR4 and CXCR7 on PNC and NET formation during acute inflammation.

The Chlamydia psittaci Inclusion Membrane Protein 0556 Inhibits Human Neutrophils Apoptosis Through PI3K/AKT and NF-κB Signaling Pathways.

Inclusion membrane proteins (Incs) play an important role in the structure and stability of chlamydial inclusion and the interaction between Chlamydia spp. and their hosts. Following Chlamydia infection through the respiratory tract, human polymorphonuclear neutrophils (hPMN) not only act as the primary immune cells reaching the lungs, but also serve as reservoir for Chlamydia. We have previously identified a Chlamydia psittaci hypothetical protein, CPSIT_0556, as a medium expressed inclusion membrane protein. However, the role of inclusion membrane protein, CPSIT_0556 in regulating hPMN functions remains unknown. In the present study, we found that CPSIT_0556 could not only inhibit hPMN apoptosis through the PI3K/Akt and NF-κB signaling pathways by releasing IL-8, but also delays procaspase-3 processing and inhibits caspase-3 activity in hPMN. Up-regulating the expression of anti-apoptotic protein Mcl-1 and down-regulating the expression of pro-apoptotic protein Bax could also inhibit the translocalization of Bax in the cytoplasm into the mitochondria, as well as induce the transfer of p65 NF-κB from the cytoplasm to the nucleus. Overall, our findings demonstrate that CPSIT_0556 could inhibit hPMN apoptosis through PI3K/Akt and NF-κB pathways and provide new insights towards understanding a better understanding of the molecular pathogenesis and immune escape mechanisms of C. psittaci.

Determination of tigecycline in human lung epithelial cells and polymorphonuclear neutrophils by liquid chromatography/tandem mass spectrometry and its application in a cellular pharmacokinetics study.

In order to characterize the intracellular pharmacokinetic properties of tigecycline, we developed and fully validated a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantification of tigecycline in human lung epithelial (BEAS-2B) cells and polymorphonuclear neutrophils (PMNs). Tetracycline was used as an internal standard and chromatographic separation was achieved on a C18 Hypersil Gold aQ column using two mobile phases, a solution of water (containing 0.1% formic acid) and acetonitrile. The flow rate was 0.4mL/min for 5.0min. Tigecycline drug uptake was evaluated by incubating the BEAS-2B cells and the PMNs for up to 3h at tigecycline concentrations of 1mg/L. The assay was linear over the tested concentration range of 0.01-2mg/L for tigecycline in BEAS-2B cells and PMNs (r2 >0.99). The inter- and inter-day precisions (RSD, %) were <10.02% and the accuracies (%) were within the range of 85-115%. The uptake study showed that after incubation with tigecycline (1mg/L) for 3h at 37°C, the intracellular peak concentration of BEAS-2B cells was 14.44 ± 7.12 mg/L at 1 h, and 41.43 ± 25.66 mg/L in PMNs at 20 min. The mean intracellular concentrations fluctuated in the range of 0.8-14.44 mg/L in BEAS-2B cells and 10.14-41.43 mg/L in PMNs for 1mg/L tigecycline exposure. Validated LC/MS/MS is a simple, rapid, and sensitive method for determining the intracellular concentration of tigecycline, and tigecycline has good penetrations both in human BEAS-2B cells and PMNs. The method can be efficiently used for future studies of the intracellular pharmacokinetics of tigecycline.

The Modulation of NADPH Oxidase Activity in Human Neutrophils by Moroccan Strains of Leishmania major and Leishmania tropica Is Not Associated with p47phox Phosphorylation.

Polymorphonuclear neutrophils (PMNs) are the first phagocyte recruited and infected by Leishmania. They synthetize superoxide anions (O2−) under the control of the NADPH oxidase complex. In Morocco, Leishmania major and L. tropica are the main species responsible for cutaneous leishmaniasis (CL). The impact of these parasites on human PMN functions is still unclear. We evaluated the in vitro capacity of primary Moroccan strains of L. major and L. tropica to modulate PMN O2− production and p47phox phosphorylation status of the NADPH oxidase complex. PMNs were isolated from healthy blood donors, and their infection rate was measured by microscopy. O2− production was measured by superoxide dismutase–inhibitable reduction of cytochrome C. P47phox phosphorylation was analyzed by Western blot using specific antibodies against Ser328 and Ser345 sites. Whereas we did not observe any difference in PMN infectivity rate, our results indicated that only L. tropica promastigotes inhibited both fMLF- and PMA-mediated O2− production independently of p47phox phosphorylation. Leishmania soluble antigens (SLAs) from both species significantly inhibited O2− induced by fMLF or PMA. However, they only decreased PMA-induced p47phox phosphorylation. L. major and L. tropica modulated differently O2− production by human PMNs independently of p47phox phosphorylation. The inhibition of ROS production by L. tropica could be a mechanism of its survival within PMNs that might explain the reported chronic pathogenicity of L. tropica CL.

E-selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity.

Transendothelial migration (TEM) of neutrophils under blood flow is critical in the inflammatory cascade. However, the role of endothelial plasticity in this process is not fully understood. Therefore, we used an in vitro model to test the dynamics of human polymorphonuclear neutrophil (PMN) TEM across lipopolysaccharide-treated human umbilical vein endothelial cell (HUVEC) monolayers. Interestingly, shRNA-E-selectin knockdown in HUVECs destabilized endothelial junctional integrity by reducing actin branching and increasing stress fiber at cell-cell junctions. This process is accomplished by downregulating the activation of cortactin and Arp2/3, which in turn alters the adhesive function of VE-cadherin, enhancing PMN transmigration. Meanwhile, redundant P-selectins possess overlapping functions in E-selectin-mediated neutrophil adhesion, and transmigration. These results demonstrate, to our knowledge, for the first time, that E-selectins negatively regulate neutrophil transmigration through alterations in endothelial plasticity. Furthermore, it improves our understanding of the mechanisms underlying actin remodeling, and junctional integrity, in endothelial cells mediating leukocyte TEM.

Fractalkine Is Linked to the Necrosome Pathway in Acute Pulmonary Inflammation.

Acute pulmonary inflammation affects over 10% of intensive care unit (ICU) patients and is associated with high mortality. Fractalkine (CX3CL1) and its receptor, CX3CR1, have been shown to affect pulmonary inflammation, but previous studies have focused on macrophages. In a murine model of acute pulmonary inflammation, we identified inflammatory hallmarks in C57BL/6J and CX3CR1−/− mice. Pulmonary inflammation was significantly enhanced in the CX3CR1−/− animals compared to the C57BL/6J animals, as assessed by microvascular permeability, polymorphonuclear neutrophil (PMN) migration into lung tissue and alveolar space. The CX3CR1−/− mice showed increased levels of apoptotic PMNs in the lungs, and further investigations revealed an increased activation of necrosome-related receptor-interacting serine/threonine-protein kinases 1 (RIPK1), 3 (RIPK3), and mixed-lineage kinase domain-like pseudokinase (MLKL). Phosphorylated MLKL leads to membrane rupture and damage-associated molecular pattern (DAMP) release, which further enhance inflammation. The release of DAMPs was significantly higher in the CX3CR1−/− mice and led to the activation of various cascades, explaining the increased inflammation. RIPK3 and MLKL inhibition improved the inflammatory response in human PMNs in vitro and confirmed our in vivo findings. In conclusion, we linked CX3CL1 to the necrosome complex in pulmonary inflammation and demonstrated a pivotal role of the necrosome complex in human PMNs.

Inhibition of Arginase 1 Liberates Potent T Cell Immunostimulatory Activity of Human Neutrophil Granulocytes.

Myeloid cell arginase-mediated arginine depletion with consecutive inhibition of T cell functions is a key component of tumor immune escape. Both, granulocytic myeloid-derived suppressor cells (G-MDSC) and conventional mature human polymorphonuclear neutrophil granulocytes (PMN) express high levels of arginase 1 and can act as suppressor cells of adaptive anti-cancer immunity. Here we demonstrate that pharmacological inhibition of PMN-derived arginase 1 not only prevents the suppression of T cell functions but rather leads to a strong hyperactivation of T cells. Human PMN were incubated in cell culture medium in the absence or presence of an arginase inhibitor. T cells from healthy donors were then activated either polyclonally or in an antigen-specific manner in the supernatants of the PMN cultures at different PMN-T cell ratios. T cell proliferation was completely suppressed in these supernatants in the absence of an arginase inhibitor. Arginase inhibition led to a strong hyperinduction of T cell proliferation, which exceeded control activation conditions up to 25-fold. The hyperinduction was correlated with higher PMN-T cell ratios and was only apparent when PMN arginase activity was blocked sufficiently. The T cell stimulatory factor was liberated very early by PMN and was present in the < 3 kDa fraction of the PMN supernatants. Increased T cell production of specific proinflammatory cytokines by PMN supernatant in the presence of arginase inhibitor was apparent. Upon arginase inhibition, downregulation of important T cell membrane activation and costimulation proteins was completely prevented or de novo induction accelerated. Antigen-specific T cell cytotoxicity against tumor cells was enhanced by PMN supernatant itself and could be further increased by PMN arginase blockade. Finally, we analyzed anergic T cells from multiple myeloma patients and noticed a complete reversal of anergy and the induction of strong proliferation upon T cell activation in PMN supernatants by arginase inhibition. In summary, we discovered a potent PMN-mediated hyperactivation of human T cells, which is apparent only when PMN arginase-mediated arginine depletion is concurrently inhibited. Our findings are clearly relevant for the analysis and prevention of human tumor immune escape in conjunction with the application of arginase inhibitors already being developed clinically.

Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape.

Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.

Induction of Krüppel-Like Factor 4 Mediates Polymorphonuclear Neutrophil Activation in Streptococcus pneumoniae Infection.

The recruitment and activation of polymorphonuclear neutrophils (PMNs) are of central importance for the elimination of pathogens in bacterial infections. We investigated the Streptococcus pneumoniae-dependent induction of the transcription factor Krüppel-like factor (KLF) 4 in PMNs as a potential regulator of PMN activation. We found that KLF4 expression is induced in human blood-derived PMNs in a time- and dose-dependent manner by wild-type S. pneumoniae and capsule knockout mutants. Unencapsulated knockout mutants induced stronger KLF4 expression than encapsulated wild types. The presence of autolysin LytA-competent (thus viable) pneumococci and LytA-mediated bacterial autolysis were required for KLF4 induction in human and murine PMNs. LyzMcre-mediated knockdown of KLF4 in murine blood-derived PMNs revealed that KLF4 influences pneumococci killing and increases the release of the proinflammatory cytokines tumor necrosis factor α and keratinocyte chemoattractant and decreases the release of the anti-inflammatory cytokine interleukin-10. Thus, S. pneumoniae induces KLF4 expression in PMNs, which contributes to PMN activation in S. pneumoniae infection.

Manipulation of Autophagy and Apoptosis Facilitates Intracellular Survival of Staphylococcus aureus in Human Neutrophils.

Polymorphonuclear neutrophils (PMN) are critical for first line innate immune defence against Staphylococcus aureus. Mature circulating PMN maintain a short half-life ending in constitutive apoptotic cell death. This makes them unlikely candidates as a bacterial intracellular niche. However, there is significant evidence to suggest that S. aureus can survive intracellularly within PMN and this contributes to persistence and dissemination during infection. The precise mechanism by which S. aureus parasitizes these cells remains to be established. Herein we propose a novel mechanism by which S. aureus subverts both autophagy and apoptosis in PMN in order to maintain an intracellular survival niche during infection. Intracellular survival of S. aureus within primary human PMN was associated with an accumulation of the autophagic flux markers LC3-II and p62, while inhibition of the autophagy pathway led to a significant reduction in intracellular survival of bacteria. This intracellular survival of S. aureus was coupled with a delay in neutrophil apoptosis as well as increased expression of several anti-apoptotic factors. Importantly, blocking autophagy in infected PMN partially restored levels of apoptosis to that of uninfected PMN, suggesting a connection between the autophagic and apoptotic pathways during intracellular survival. These results provide a novel mechanism for S. aureus intracellular survival and suggest that S. aureus may be subverting crosstalk between the autophagic and apoptosis pathways in order to maintain an intracellular niche within human PMN.

Azithromycin potentiates avian IgY effect against Pseudomonas aeruginosa in a murine pulmonary infection model

Cystic fibrosis (CF) patients are at risk of acquiring chronic Pseudomonas aeruginosa lung infections. The biofilm mode of growth of P. aeruginosa induces tolerance to antibiotics and the host response; accordingly, treatment failure occurs. Supplemental azithromycin has proven beneficial in CF owing to potential immunomodulatory mechanisms. Clinical studies have demonstrated a reduction in exacerbations in CF patients by avian IgY anti-Pseudomonas immunotherapy. We hypothesise that azithromycin pre-treatment could potentiate the observed anti-Pseudomonas effect of IgY opsonisation in vivo. Evaluation of phagocytic cell capacity was performed using in vitro exposure of azithromycin pre-treated human polymorphonuclear neutrophils to IgY opsonised P. aeruginosa PAO3. A murine lung infection model using nasal planktonic P. aeruginosa inoculation and successive evaluation 24 h post-infection was used to determine lung bacteriology and subsequent pulmonary inflammation. Combined azithromycin treatment and IgY opsonisation significantly increased bacterial killing compared with the two single-treated groups and controls. In vivo, significantly increased bacterial pulmonary elimination was revealed by combining azithromycin and IgY. A reduction in the inflammatory markers mobiliser granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein 2 (MIP-2) and interleukin 1 beta (IL-1β) paralleled this effect. Combination of azithromycin and anti-Pseudomonas IgY potentiated the killing and pulmonary elimination of P. aeruginosa in vitro and in vivo. The augmented effect of combinatory treatment with azithromycin and IgY constitutes a potential clinical application for improving anti-Pseudomonas strategies.

Inorganic arsenic administration suppresses human neutrophil function in vitro.

Arsenic, a major environmental toxicant and pollutant, is a global public health concern. Among its many adverse effects, arsenic is immunotoxic, but its effects on human neutrophil functions are not yet well-defined. In this study, we aimed to evaluate the in vitro effects of acute low-dose NaAsO2 exposure on human polymorphonuclear neutrophils (PMNs) for 12 h on the following innate defense mechanisms: formation of neutrophil extracellular traps (NETs), production of reactive oxygen species (ROS), and phagocytosis. Phorbol myristate acetate (PMA) was added to induce NETs formation, which was quantified by measuring cell-free extracellular DNA (cf-DNA), myeloperoxidase-conjugated (MPO)-DNA and neutrophil elastase-conjugated (NE)-DNA, and confirmed by immunofluorescence labeling and imaging. Extracellular bactericidal activity by NETs was evaluated by co-culturing Escherichia coli and PMNs in the presence of a phagocytic inhibitor. Levels of NETs in the culture medium after PMA stimulation was significantly lower in PMNs pre-exposed to arsenic than those not exposed to arsenic. Immunofluorescence staining and extracellular bactericidal activity by NETs revealed similar results. Phagocytosis and ROS production by PMNs were also significantly reduced by arsenic pre-exposure. Together, our findings provide new insights in arsenic immunotoxicity and suggest how it increases susceptibility to infectious diseases in humans.

Immunomodulatory effects of colistin on host responses against carbapenem-resistant Klebsiella pneumoniae biofilms

Colistin (CST) is a last-resort therapeutic option for carbapenem-resistant Klebsiella pneumoniae (CR-Kp) infections in critically ill patients. The effect of subinhibitory CST concentrations (sub-MICs) on biofilm formation is organism-dependent. We investigated the interactions between CST and innate immune cells against CR-Kp biofilms (CR-KpBF) by studying the effect of biofilm sub-MICs of CST on (i) damage induced by human polymorphonuclear neutrophils (PMNs) on CR-KpBF and (ii) the immunomodulatory potential on human mononuclear cells (MNCs) exposed to CR-KpBF. The impact of CST on PMN-induced biofilm damage was assessed by XTT reduction assay. Signal transduction and gene expression profiles in response to CST sub-MICs of MNCs exposed to CR-KpBF were studied by RT-PCR and multiplex ELISA. Pre-exposure of CR-Kp to 0.06 mg/L CST led to subsequent increased PMN-mediated biofilm damage against CR-KpBF in the presence of CST biofilm sub-MICs: there was an additive effect at 2, 4, 8 and 16 mg/L. However, the overall biofilm damage was not >52%. MNCs responded to CR-KpBF through Toll-like receptor 2 (TLR2) by 2.5-fold upregulation and NLRP3 inflammasome activation. CR-KpBF stimulated increased production of interleukin 1-beta (IL-1β), tumour necrosis factor-alpha (TNFα), IL-8 and IL-6. In the combination treatment, 0.5 mg/L CST reduced IL-1β, TNFα and IL-8 levels, whereas at 2 mg/L and 8 mg/L it increased the anti-inflammatory cytokine IL-10 (P < 0.05). Biofilm sub-MICs of CST enhance PMN killing capacity and attenuate production of inflammatory cytokines by MNCs exposed to CR-KpBF, playing a potentially important immunotherapeutic role especially for patients with cytokine deregulation.

Gene Expression in Human Polymorphonuclear Neutrophils (PMNs) Stimulated by Bacillus Calmette-Guérin (BCG).

Neutrophils are the most abundant leukocytes in the blood. Moreover, neutrophils form the first line of host immune defense against bacterial and fungal invasion, and also play an important part in inflammatory and immune system responses. Intravesical bacillus Calmette-Guérin (BCG) has been shown to reduce and delay tumor progression to muscle-invasive disease after transurethral resection of bladder tumors (TRUBTs). Following intravesical BCG, neutrophils gather around tissues infected by BCG in the early stage of inflammatory and immune responses. In our previous study, we reported that BCG induced the formation of neutrophil extracellular traps (NETs), which play an important role in tumor treatment. Therefore, in the present study, we analyzed the gene expression profile of neutrophils stimulated by BCG through high-throughput arrays, which helped us determine the potential roles of neutrophils in BCG immunotherapy. The results showed that the expression of neutrophil genes led to changes in the early stage of BCG stimulation. The changed genes were involved in many functions of neutrophils such as mobility, proliferation, and secretion of cytokines, chemokines, and adhesion molecules. These changes in neutrophil biological functions may play an essential role in BCG induction of inflammatory and immune responses, and in anti-tumor processes.

Regulation of Neutrophil Function by Selective Targeting of Glycan Epitopes Expressed on the Integrin CD11b/CD18

Polymorphonuclear neutrophils (PMN) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMN and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease and chronic obstructive pulmonary disease. The glycoprotein CD11b/CD18 plays a well described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N‐linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMN. MS analyses revealed a surprising lack of sialylation on CD11b glycans. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis (GNA) lectin and biantennary galactosylated N‐glycans recognized by the Phaseolus Vulgaris erythroagglutinin (PHA‐E) lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN associated tissue damage in chronic inflammatory diseases.Support or Funding Information This work was supported by the Crohn’s and colitis foundation (SRA # 544596)

The Pore-forming Leukotoxins from &amp;lt;i&amp;gt;S. aureus&amp;lt;/i&amp;gt; Involve Ca&amp;lt;sup&amp;gt;2+&amp;lt;/sup&amp;gt; Release-Activated Ca&amp;lt;sup&amp;gt;2+&amp;lt;/sup&amp;gt; Channels and Other Types of Ca&amp;lt;sup&amp;gt;2+&amp;lt;/sup&amp;gt; Channels in Ca&amp;lt;sup&amp;gt;2+&amp;lt;/sup&amp;gt; Entry into Neutrophils

The pore-forming bi-component leukotoxins from <i>Staphylococcus aureus</i> induce two independent cellular events <i>1)</i> the formation of trans-membrane pores not permeable to divalent ions and <i>2)</i> the opening of pre-existing Ca²⁺ channels in human polymorphonuclear neutrophils (PMNs). The influx of Ca²⁺ and Mn²⁺ (Mn²⁺ was used as a Ca²⁺ surrogate) in Fura2-loaded human PMNs was determined by spectrofluorometry techniques. The present study showed that, in the presence of extracellular Ca²⁺, the staphylococcal HlgA/HlgB γ-hemolysin induced a rapid Ca²⁺ release from internal Ca²⁺ stores before the onset of a Mn²⁺ (Ca²⁺) influx. The sustained increase of Ca²⁺ and Mn²⁺ influx was partially inhibited by the ionic blockers of Ca²⁺ Release-Activated Ca²⁺ (CRAC) channels, La³⁺ and Ni²⁺. Furthermore, the incubation of human PMNs with either TMB8 or thapsigargin did inhibit significantly the Ca²⁺ release mediated by leukotoxins simultanously to a clear decrease of Ca²⁺ and Mn²⁺ influx. The internal Ca²⁺ release induced by γ-hemolysin was also inhibited by PMNs pretreatment with a pertussis toxin, NaF, caffeine, ryanodine, cinnarizine and flunarizine and consequently, the Mn²⁺ (Ca²⁺) influx was significantly reduced. Moreover, different Ca²⁺ signaling pathways blockers such as U73122, staurosporine, thyrphostin A9 and okadaic acid were tested on the leukotoxins activity. Taken together, this work provided evidence that, in the presence of extracellular Ca²⁺, bi-component staphylococcal leukotoxins provoked in human PMNs after a specific binding to their membrane receptors, a rapid depletion of internal Ca²⁺ stores mediating a CRAC channels activation. This Ca²⁺-dependent mechanism seems likely to be associated to the heterotrimeric G-proteins activation. Interestingly, in the absence of extracellular Ca²⁺, the staphylococcal leukotoxins tested induced the opening of an important divalent ions (Ca²⁺, Mn²⁺, Ni²⁺) pathway not sensitive to CRAC channels blockers. Consequently, we strongly suggested that other types of Ca²⁺ channels might be involved in bi-component leukotoxins activity, including Ca²⁺ channels dependent on the protein kinase C activation.

Bi-component Staphylococcal Leukotoxins Induce Chloride Ions Fluxes in Human Neutrophils: Opening of Ca&amp;lt;sup&amp;gt;2+&amp;lt;/sup&amp;gt;-activated Cl&amp;lt;sup&amp;gt;-&amp;lt;/sup&amp;gt; Channels

The bi-component leukotoxins; γ-hemolysin and Panton and Valentin Leukocidin (PVL) from Staphylococcus aureus induce two independent cellular events 1) the formation of trans-membrane pores not permeable to chloride (Cl-) ions and 2) the activation of at least, two modes of chloride fluxes (efflux/influx), including pre-existing Ca2+-activated Cl- channels (CaCC) in human polymorphonuclear neutrophils (PMNs). This was investigated by using spectrofluorometry techniques and the chloride-sensitive quencher fluorescent indicator, MQAE (N-(6-methoxyquinolyl) acetoacethyl ester). The ethidium bromide was used as an indicator for the trans-membrane pores formation by staphylococcal leukotoxins. In the absence of extracellular Ca2+, HlgA/HlgB, HlgC/HlgB and LukS-PV/LukF-PV leukotoxins from S. aureus induced a massive efflux of chloride (Cl-) ions. Interestingly, in the presence of extracellular Ca2+, the HlgA/HlgB γ-hemolysin provoked a biphasic response of Cl- movements (efflux/influx). Conversely to HlgA/HlgB and LukS-PV/LukF-PV, HlgC/HlgB leukotoxins did not induce any Cl- movement under this condition (e.g. in the presence of extracellular Ca2+). The potent Cl- channel inhibitor, DIDS, did inhibit significantly the Cl- fluxes caused by all pairs of staphylococcal leukotoxins tested in both conditions. In the present study, we found that the inhibitory effect of flufenamic acid, known as a Cl- channel inhibitor, was restricted only to the Ca2+-dependent Cl- influx triggered only by HlgA/HlgB and LukS-PV/LukF-PV leukotoxins. These findings might suggest that, Cl- fluxes in human neutrophils did involve at least, two different types of Cl- pathways, depending on the absence or presence of extracellular Ca2+. Both Cl- channels blockers, DIDS and flufenamic acid did not alter the pores formation by staphylococcal leukotoxins. Furthermore, under conditions when the membrane pores formation was blocked by divalent ions (Ca2+ and/or Zn2+), Cl- ions movements were still observed. Taken together, our results strongly provide an evidence that: i) trans-membrane pores formed by staphylococcal leukotoxins: HlgA/HlgB, HlgC/HlgB (γ-hemolysin) and LukS-PV/LukF-PV (PVL) do not drive Cl- ions fluxes ii) at least, two different types of Cl- ions pathways are activated, depending on the absence or presence of extracellular Ca2+, including Ca2+-activated Cl- channels (CaCC) and, iii) Ca2+-activated Cl- channels are mediated only by HlgA/HlgB and LukS-PV/LukF-PV leukotoxins.

Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18.

Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well-described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N-linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation-dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N-glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN-associated tissue damage in chronic inflammatory diseases.

43 kDa Glycoprotein (gp43) from Paracoccidioides brasiliensis Induced IL-17A and PGE2 Production by Human Polymorphonuclear Neutrophils: Involvement of TLR2 and TLR4.

The glycoprotein gp43 is the major antigenic/diagnostic component of Paracoccidioides brasiliensis, one of the etiologic agents of paracoccidioidomycosis (PCM). Gp43 has protective roles in mice, but due to adhesive properties, this glycoprotein has also been associated with immune evasion mechanisms. The present study evaluated gp43 interaction in vitro with Toll-like receptors 2 and 4 (TLR2 and TLR4) present in polymorphonuclear neutrophils (PMNs) from healthy human individuals and the consequent modulation of the immune response through the expression and release of cytokines and eicosanoids. PMNs were incubated in the absence or presence of monoclonal antibodies anti-TLR2 and anti-TLR4 (individually or in combination) before gp43 stimulation. Then, PMNs were analyzed for the expression of both surface receptors and the detection of intracytoplasmic IL-17A and IL-4 using flow cytometry, while the production of PGE2, LTB4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α was evaluated in the supernatants by enzyme-linked immunosorbent assay (ELISA). Our results showed that gp43 increased TLR2 and TLR4 expression by PMNs and induced PGE2 and IL-17A via TLR4 and TLR2, respectively. Thus, our data suggest that gp43 from P. brasiliensis might modulate host susceptibility to the fungal infection by affecting PGE2 and IL-17A production.

Standardized ethanol extract, essential oil and zerumbone of Zingiber zerumbet rhizome suppress phagocytic activity of human neutrophils

BackgroundZingiber zerumbet rhizome and its bioactive metabolites have previously been reported to exhibit innumerable pharmacological properties particularly anti-inflammatory activities. In the present study, the 80% ethanol extract, essential oil and zerumbone of Z. zerumbet rhizomes were explored for their in vitro immunosuppressive properties on chemotaxis, CD11b/CD18 expression, phagocytosis and chemiluminescence of isolated human polymorphonuclear neutrophils (PMNs).MethodsThe extract was analyzed quantitatively by performing a validated reversed phase high performance liquid chromatography (RP-HPLC). Zerumbone was isolated by chromatographic technique while the essential oil was acquired through hydro-distillation of the rhizomes and further analyzed by gas chromatography (GC) and GC-MS. Chemotaxis assay was assessed by using a 24-well cell migration assay kit, while CD18 integrin expression and phagocytic engulfment were measured using flow cytometry. The reactive oxygen species (ROS) production was evaluated by applying lucigenin- and luminol-enhanced chemiluminescence assays.ResultsZerumbone was found to be the most abundant compound in the extract (242.73 mg/g) and the oil (58.44%). Among the samples tested, the oil revealed the highest inhibition on cell migration with an IC50 value of 3.24 μg/mL. The extract, oil and zerumbone showed moderate inhibition of CD18 integrin expression in a dose-dependent trend. Z. zerumbet extract showed the highest inhibitory effect on phagocytic engulfment with percentage of phagocytizing cells of 55.43% for PMN. Zerumbone exhibited strong inhibitory activity on oxidative burst of zymosan- and PMA-stimulated neutrophils. Zerumbone remarkably inhibited extracellular ROS production in PMNs with an IC50 value of 17.36 μM which was comparable to that of aspirin.ConclusionThe strong inhibition on the phagocytosis of neutrophils by Z. zerumbet extract and its essential oil might be due the presence of its chemical components particularly zerumbone which was capable of impeding phagocytosis at different stages.