Human Polymorphonuclear Neutrophils Research Articles

C-Terminal Alpha-1 Antitrypsin Peptide: A New Sepsis Biomarker with Immunomodulatory Function.

Systemic inflammatory response syndrome (SIRS) is a life threatening condition and the leading cause of death in intensive care units. Although single aspects of pathophysiology have been described in detail, numerous unknown mediators contribute to the progression of this complex disease. The aim of this study was to elucidate the pathophysiological role of CAAP48, a C-terminal alpha-1 antitrypsin fragment, that we found to be elevated in septic patients and to apply this peptide as diagnostic marker for infectious and noninfectious etiologies of SIRS. Incubation of human polymorphonuclear neutrophils with synthetic CAAP48, the SNP-variant CAAP47, and several control peptides revealed intense neutrophil activation, induction of neutrophil chemotaxis, reduction of neutrophil viability, and release of cytokines. We determined the abundance of CAAP48 in patients with severe sepsis, severe SIRS of noninfectious origin, and viral infection. CAAP48 levels were 3-4-fold higher in patients with sepsis compared to SIRS of noninfectious origin and allowed discrimination of those patients with high sensitivity and specificity. Our results suggest that CAAP48 is a promising discriminatory sepsis biomarker with immunomodulatory functions, particularly on human neutrophils, supporting its important role in the host response and pathophysiology of sepsis.

Neutrophil Recruitment Is Regulated By Adamts-13 in a Murine Model of Invasive Aspergillosis

▪Introduction: During inflammation von Willebrand factor (VWF) multimers are secreted as an acute phase protein whereupon the size and the prothrombotic activity play an essential role. The size of VWF multimers is regulated by the specific proteolytic activity of ADAMTS-13 (a disintegrin and metalloprotease with ThromboSpondin type 1 repeats-13) which is diminished under several pathological conditions. Employing a murine model of invasive pulmonary aspergillosis (IPA) we aimed to determine the relevance of this regulatory pathway for innate inflammatory responses and polymorphonuclear neutrophil (PMN) recruitment which is crucial for fungal clearance and survival.Methods: IPA was induced by intratracheal application of Aspergillus fumigatus (A. fumigatus) conidia in wildtype (129/Sv/Pas) or Adamts13 deficient (Adamts13-/-) mice. 24 h after infection some mice were sacrificed for analysis of fungal load and histology. To assess fungal load as CFU lung homogenates were plated and cultured on Sabourough agar plates. Paraffin sections of the lungs were prepared and stained with mouse complement component C3d antibody for histological analysis. In addition, degranulation, oxidative burst activity and CD62L shedding were assessed in PMN of wildtype and Adamts13-/- mice. Chemotactic properties of A. fumigatus -activated and control serum from wildtype and knock-out mice was evaluated by migration of purified human PMN, isolated by dextran sedimentation and Histopaque® centrifugation, in a transwell assay (Corning® HTS Transwell®-96 well permeable support; 3µm).Results: Adamts13 deficiency in mice was accompanied by a worse outcome of IPA infections compared to wildtype controls. Adamts13-/- mice displayed more severe signs of disease and a lethal course was observed in about 24% of animals. Compared to Adamts13-/- mice all neutropenic mice developed lethal IPA after infection, but all wild-type mice survived the disease. Besides, examination of the lungs revealed a higher fungal burden along with increased signs of acute lung injury, complement deposition and levels of pro-inflammatory cytokines in Adamts13-/- mice. Furthermore a more pronounced perivascular leukocyte infiltration was observed in histological sections indicating a dysregulated inflammatory response in Adamts13-/- mice. Importantly, the activation of neutrophil effector functions in response to TLR agonist or in PMN-mediated fungal killing of conidia or hyphae revealed no general defect in vitro. Despite enhanced complement deposition in the lungs in Adamts13-/- mice, PMN migration towards complement-activated serum revealed unaltered chemotactic properties comparing A. fumigatus -activated serum of wildtype and knock-out mice.Conclusion: In acute inflammatory processes such as fungal pneumonia the proteolytic regulation of VWF by ADAMTS-13 is an important mechanism to control PMN recruitment and outcome of infections. DisclosuresNo relevant conflicts of interest to declare.

Silver nanoparticles of 70 nm and 20 nm affect differently the biology of human neutrophils

The influence of size of nanoparticles (NP), especially in regard to pulmonary toxicity, has been widely investigated. In general, NP with smaller diameters are more pro-inflammatory in vivo, at least in terms of neutrophil influx. Nevertheless, the influence of size of NP on polymorphonuclear neutrophil (PMN) cell biology is poorly documented. In the study here, it was decided to determine if AgNP with a diameter of 70 nm (AgNP70) will alter the biology of human PMN similarly to AgNP20 previously reported to induce apoptosis and inhibit de novo protein synthesis. The results here indicated that, in contrast to AgNP20, AgNP70 delayed PMN apoptosis. However, both AgNP20 and AgNP70 inhibited de novo protein synthesis. Both forms of AgNP did not significantly increase reactive oxygen species (ROS) production, but AgNP20 significantly increased the cell production of the CXCL8 chemokine (IL-8). In addition, AgNP20, but not AgNP70, induced the release of albumin and matrix metalloproteinase-9 (MMP-9/gelatinase B) into culture supernatants. Consistent with this latter observation, gelatinase activity was increased by AgNP20, as assessed by zymography. From these outcomes, it is concluded that two NP with different initial diameters can possess similar – as well as distinct – biological properties in modulating human PMN functions. These outcomes are testimony to the complexity of the modes of action of NP at the cellular level.

Human Periodontal Stem Cells Release Specialized Proresolving Mediators and Carry Immunomodulatory and Prohealing Properties Regulated by Lipoxins.

Unresolved inflammation and tissue destruction are underlying mechanisms of periodontitis, which is linked to dysregulated polymorphonuclear neutrophil (PMN) functions. Lipoxin A4 (LXA4) is a specialized proresolving lipid mediator (SPM) that dampens excessive inflammation, promotes resolution, and protects from leukocyte-mediated tissue damage. Human periodontal ligament stem cells (hPDLSCs) represent key players during tissue regeneration and may contribute to resolution of inflammation; thus, they may represent a promising tool in regenerative dentistry. In the present study, we investigated the actions of hPDLSCs on PMN apoptosis and antimicrobial functions, and determined the impact of LXA4 on hPDLSCs. hPDLSCs significantly reduced apoptosis and stimulated microbicidal activity of human PMNs, via both cell-cell interactions and paracrine mechanisms. Lipid mediator metabololipidomics analysis demonstrated that hPDLSCs biosynthesize SPMs, including resolvin D1, D2, D5, and D6; protectin D1; maresins; and LXB4; as well as prostaglandins D2, E2, and F2α. LXA4 significantly enhanced proliferation, migration, and wound healing capacity of hPDLSCs through the activation of its cognate receptor ALX/FPR2, expressed on hPDLSCs. Together, these results demonstrate that hPDLSCs modulate PMN functions, and provide the first evidence that stem cells generate SPM and that the LXA4-ALX/FPR2 axis regulates regenerative functions of hPDLSCs by a novel receptor-mediated mechanism. These findings uncovered unappreciated features of stem cells from the periodontal ligament, supporting the notion that these cells may act as master regulators of pathophysiological events through the release of mediators that promote the resolution of inflammation and bacterial killing. The study also demonstrated that it is possible to modulate important functions of periodontal stem cells using lipoxin A4, a potent endogenous stop signal of inflammation. Thus, this study revealed an unappreciated anti-inflammatory proregenerative circuit that may be exploited to combat periodontal pathologies using resident stem cells. Moreover, the data may represent a more general template to explain the immunomodulatory functions of stem cells.

Isolation and Functional Analysis of Human Neutrophils.

This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll-Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described.

Immunomodulatory effects of Santolina chamaecyparissus leaf extracts on human neutrophil functions

Context: Santolina chamaecyparissus L. (Asteraceae) is an aromatic plant wide spread in the Mediterranean region. It is used in folk medicine for its anti-inflammatory properties.Objective: The effects of S. chamaecyparissus aqueous extract (SCAE) and polyphenolic extract (SCPE) on human polymorphonuclear neutrophil (PMN) degranulation, chemotaxis, phagocytosis, and microbicidal capacity were examined in vitro.Materials and methods: Aqueous and polyphenolic extracts were prepared from S. chamaecyparissus leaves. The elastase release was used as a marker for measuring PMN degranulation, while chemotaxis was performed using a 48-microwell chemotaxis chamber. The phagocytosis and the microbicidal capacity were evaluated using fresh cultures of Candida albicans.Results: The treatment of neutrophils with different concentrations (10–200 µg/ml) of SCAE and SCPE caused a significant (p < 0.001) and dose-dependent inhibitory effect on elastase release in fMLP/Cytochalasin B (CB)-stimulated neutrophils. Indeed, 100 µg/ml of SCAE exerted an inhibitory effect of 51.97 ± 6.2%, whereas SCPE at the same concentration abolished completely PMN degranulation. Moreover, both extracts inhibited markedly (p < 0.01) fMLP-induced chemotactic migration. At 200 µg/ml, SCAE and SCPE exerted an inhibitory effect of 54.61 ± 7.3% and 57.71 ± 7.44%, respectively. In addition, a decline in both phagocytosis and microbicidal capacity against Candida albicans was observed when PMNs were exposed to 100 and 200 µg/ml of SCAE or SCPE.Conclusion: The exerted effects on neutrophil functions support the anti-inflammatory activity and show new mechanisms of action and effectiveness of S. chamaecyparissus leaf extracts. This plant may be considered as an interesting source of anti-inflammatory and immunomodulatory agents.

Inhibition of Neutrophils by Hypertonic Saline Involves Pannexin-1, CD39, CD73, and Other Ectonucleotidases

Hypertonic saline (HS) resuscitation has been studied as a possible strategy to reduce polymorphonuclear neutrophil (PMN) activation and tissue damage in trauma patients. Hypertonic saline blocks PMNs by adenosine triphosphate (ATP) release and stimulation of A2a adenosine receptors. Here, we studied the underlying mechanisms in search of possible reasons for the inconsistent results of recent clinical trials with HS resuscitation. Purified human PMNs or PMNs in whole blood were treated with HS to simulate hypertonicity levels found after HS resuscitation (40 mmol/L beyond isotonic levels). Adenosine triphosphate release was measured with a luciferase assay. Polymorphonuclear neutrophil activation was assessed by measuring oxidative burst. The pannexin-1 (panx1) inhibitor panx1 and the gap junction inhibitor carbenoxolone (CBX) blocked ATP release from PMNs in purified and whole blood preparations, indicating that HS releases ATP via panx1 and gap junction channels. Hypertonic saline blocked N-formyl-Met-Leu-Phe-induced PMN activation by 40% in purified PMN preparations and by 60% in whole blood. These inhibitory effects were abolished by panx1 but only partially reduced by CBX, which indicates that panx1 has a central role in the immunomodulatory effects of HS. Inhibition of the ectonucleotidases CD39 and CD73 abolished the suppressive effect of HS on purified PMN cultures but only partially reduced the effect of HS in whole blood. These findings suggest redundant mechanisms in whole blood that may strengthen the immunomodulatory effect of HS in vivo. We conclude that HS resuscitation exerts anti-inflammatory effects that involve panx1, CD39, CD73, and other ectonucleotidases, which produce the adenosine that blocks PMNs by stimulating their A2a receptors. Our findings shed new light on the immunomodulatory mechanisms of HS and suggest possible new strategies to improve the clinical efficacy of hypertonic resuscitation.

α-Defensins partially protect human neutrophils against Panton-Valentine leukocidin produced by Staphylococcus aureus.

α-Defensins produced by neutrophils are important effector molecules of the innate immune system. In addition to their microbicidal effects, α-defensins have the ability to neutralize bacterial toxins. Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus. Staphylococcus aureus that produce PVL are responsible for severe diseases, including necrotizing pneumonia. Polymorphonuclear neutrophils (PMNs) are the target cells of PVL action. The goal of this study was to elucidate the effect of a group of α-defensins known as the human neutrophil peptides (HNPs) on the interactions between LukS-PV and LukF-PV, which compose PVL, and human PMNs. We observed that HNPs bound to both subunits of PVL and significantly decreased PVL pore formation in PMNs, with a maximum inhibition of 27%. When various HNP molecules were tested individually under the same conditions, we observed that HNP3, but not HNP1 or 2, decreased pore formation. Similarly, HNP3 significantly decreased PVL-induced PMN lysis, with a maximum inhibition of 31%. Interestingly, HNPs did not affect LukS-PV LukF-PV oligomerization, LukS-PV LukF-PV binding to PMNs or calcium influx induced by PVL in PMNs. Our results suggest that HNP3 partially protects neutrophils against PVL by interfering with the conformational changes of PVL required to form a functional pore. Panton-Valentine leukocidin (PVL) is a pore-forming toxin produced by Staphylococcus aureus, responsible for neutrophil damage and key player of severe staphylococcal diseases. Antimicrobial peptides produced by neutrophils (HNP1-3) neutralize several other bacterial cytotoxins. We examined the impact of human neutrophil peptides (HNPs) on PVL cytotoxicity against human neutrophils and we found that HNPs bind to both LukS and LukF components of PVL, thereby inhibiting pore formation and neutrophil lysis. Our results suggest that HNP3 may impair PVL conformational changes required to form a functional pore and provide insight into the pathogenesis of PVL-related staphylococcal infection, with potential impact on the disease outcome.

Effect of Ambroxol and Beclomethasone on Lipopolysaccharide-Induced Nitrosative Stress in Bronchial Epithelial Cells

Background: Nitrosative stress is involved in different airway diseases. Lipopolysaccharide (LPS) induces neutrophil-related cytokine release and nitrosative stress in human bronchial epithelial (BEAS-2B) cells alone or with human polymorphonuclear neutrophils (PMNs). Ambroxol protects against oxidative stress, and beclomethasone dipropionate is an anti-inflammatory drug. Objectives: We evaluated the ability of ambroxol and/or beclomethasone dipropionate to inhibit LPS-induced expression/release of RANTES, IL-8, inducible NO synthase (iNOS), myeloperoxidase (MPO) and 3-nitrotyrosine (3-NT: nitrosative stress biomarker) in BEAS-2B ± PMNs stimulated with LPS (1 μg/ml). Methods: The effect of ambroxol and/or beclomethasone dipropionate on IL-8, RANTES and iNOS levels was assessed by Western blot analysis; IL-8, MPO and 3-NT levels were measured by ELISA. Cell viability was assessed by the trypan blue exclusion test. Results: In BEAS-2B alone, LPS (at 12 h) increased RANTES/iNOS expression and IL-8 levels (p < 0.001). Ambroxol suppressed LPS-induced RANTES expression and IL-8 release (p < 0.001), whilst inhibiting iNOS expression (p < 0.05). Beclomethasone dipropionate had no effect on RANTES but halved iNOS expression and IL-8 release. Coculture of BEAS-2B with PMNs stimulated IL-8, MPO and 3-NT production (p < 0.001), potentiated by LPS (p < 0.001). Ambroxol and beclomethasone dipropionate inhibited LPS-stimulated IL-8, MPO and 3-NT release (p < 0.05). Ambroxol/beclomethasone dipropionate combination potentiated the inhibition of IL-8 and 3-NT production in BEAS-2B with PMNs (p < 0.05 and p < 0.01, respectively). Ambroxol and/or beclomethasone dipropionate inhibited nitrosative stress and the release of neutrophilic inflammatory products in vitro. Conclusion: The additive effect of ambroxol and beclomethasone dipropionate on IL-8 and 3-NT inhibition suggests new therapeutic options in the treatment of neutrophil-related respiratory diseases such as chronic obstructive pulmonary disease and respiratory infections.

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide.

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.

Sjögren's Syndrome Antigen B Acts as an Endogenous Danger Molecule to Induce Interleukin-8 Gene Expression in Polymorphonuclear Neutrophils.

BackgroundSjögren’s syndrome antigen B is expressed in the nucleus and surface membrane of human polymorphonuclear neutrophils and is released after cell death. However, its biological role is not clear. This study is aimed to investigate the effect of Sjögren’s syndrome antigen B on human polymorphonuclear neutrophils.MethodsHuman recombinant Sjögren’s syndrome antigen B (rSSB) purified from E. coli was incubated with human polymorphonuclear neutrophils as well as retinoid acid-induced granulocytic differentiated HL-60 cells, HL-60 (RA). Interleukin (IL)-8 protein production and mRNA expressions were measured by enzyme-linked immunosorbent assay and quantitative-polymerase chain reaction, respectively. Uptake of fluorescein isothiocyanate (FITC)-rSSB was assessed by flow cytometry and fluorescence microscopy. Moreover, mitogen-activated protein kinase (MAPK) pathways and nuclear factor-kappaB activation were investigated.ResultsHuman rSSB stimulated IL-8 production from normal human neutrophils and HL-60 (RA) cells in a time- and dose-dependent manner. This IL-8-stimulated activity was blocked by chloroquine and NH4Cl, indicating that endosomal acidification is important for this effect. We found rSSB activated both MAPK pathway and nuclear factor-kappaB signaling to transcribe the IL-8 gene expression of cells. Furthermore, tumor necrosis factor-α exerted an additive effect and rSSB-anti-SSB immune complex exhibited a synergistic effect on rSSB-induced IL-8 production.ConclusionsSjögren’s syndrome antigen B might act as an endogenous danger molecule to enhance IL-8 gene expression in human polymorphonuclear neutrophils.

The impact of cationic solid lipid nanoparticles on human neutrophil activation and formation of neutrophil extracellular traps (NETs)

Cationic solid lipid nanoparticles (cSLNs) are extensively employed as the nanocarriers for drug/gene targeting to tumors and the brain. Investigation into the possible immune response of cSLNs is still lacking. The aim of this study was to evaluate the impact of cSLNs upon the activation of human polymorphonuclear neutrophil cells (PMNs). The cytotoxicity, pro-inflammatory mediators, Ca2+ mobilization, mitogen-activated protein kinases (MAPKs), and neutrophil extracellular traps (NETs) as the indicators of PMN stimulation were examined in this work. The cSLNs presented a diameter of 195nm with a zeta potential of 44mV. The cSLNs could interact with the cell membrane to produce a direct membrane lysis and the subsequent cytotoxicity according to lactate dehydrogenase (LDH) elevation. The interaction of cSLNs with the membrane also triggered a Ca2+ influx, followed by the induction of oxidative stress and degranulation. The cationic nanoparticles elevated the levels of superoxide anion and elastase by 24- and 9-fold, respectively. The PMN activation by cSLNs promoted the phosphorylation of p38 and Jun-N-terminal kinases (JNK) but not extracellular signal-regulated kinases (ERK). The imaging of scanning electron microscopy (SEM) and immunofluorescence demonstrated the production of NETs by cSLNs. This phenomenon was not significant for the neutral SLNs (nSLNs), although histones in NETs also increased after treatment of nSLNs. Our results suggest an important role of cSLNs in governing the activation of human neutrophils.

Anti-Pseudomonas aeruginosa IgY Antibodies Induce Specific Bacterial Aggregation and Internalization in Human Polymorphonuclear Neutrophils.

Polymorphonuclear neutrophils (PMNs) are essential cellular constituents in the innate host response, and their recruitment to the lungs and subsequent ubiquitous phagocytosis controls primary respiratory infection. Cystic fibrosis pulmonary disease is characterized by progressive pulmonary decline governed by a persistent, exaggerated inflammatory response dominated by PMNs. The principal contributor is chronic Pseudomonas aeruginosa biofilm infection, which attracts and activates PMNs and thereby is responsible for the continuing inflammation. Strategies to prevent initial airway colonization with P. aeruginosa by augmenting the phagocytic competence of PMNs may postpone the deteriorating chronic biofilm infection. Anti-P. aeruginosa IgY antibodies significantly increase the PMN-mediated respiratory burst and subsequent bacterial killing of P. aeruginosa in vitro. The mode of action is attributed to IgY-facilitated formation of immobilized bacteria in aggregates, as visualized by fluorescence microscopy and the induction of increased bacterial hydrophobicity. Thus, the present study demonstrates that avian egg yolk immunoglobulins (IgY) targeting P. aeruginosa modify bacterial fitness, which enhances bacterial killing by PMN-mediated phagocytosis and thereby may facilitate a rapid bacterial clearance in airways of people with cystic fibrosis.

Mitochondria Orchestrate Chemotaxis of Neutrophils by Fueling Their Autocrine Purinergic Signaling Systems

Polymorphonuclear neutrophils (PMNs) need chemotaxis to find invading bacteria. We have previously demonstrated that chemotaxis depends on ATP release and autocrine purinergic signaling via P2Y2, A2a, and A3 receptors (Chen 2006, Bao 2013). Here we investigate the role of mitochondria in the production of the ATP that fuels these purinergic signaling mechanisms. Blocking mitochondria with CCCP, KCN, or oligomycin impaired ATP release from human PMNs following stimulation with the chemotactic bacterial peptide fMLP. CCCP also significantly impaired chemotaxis, abolishing gradient sensing as well as reducing migration speed in a chemotactic gradient field generated with a micropipette that was loaded with 100 nM fMLP (Fig. 1A). Addition of the non-hydrolysable ATP analog ATPγS restored cell motility but was unable to restore gradient sensing. These findings indicate that localized purinergic signaling is required for proper PMN chemotaxis. We found two subsets of mitochondria in polarized PMNs (Fig. 1B). A small number of mitochondria with high membrane potential (Δψm) was found at the front (red) and the remaining mass of mitochondria with low Δψm stayed at the back (green) during chemotaxis. Our results suggest that mitochondria play an important role in regulating neutrophil chemotaxis by generating the ATP that fuels purinergic signaling. This study was supported by NIH grants R01 GM51477, R01 GM60475, R01 AI080582, and T32 GM103702.

Probing unexplored neutrophil GPCR signaling pathways to discover novel anti‐bacterial targets

Polymorphonuclear neutrophils (PMNs), the primary phagocytic leukocytes in the blood, play a central role in the innate immune response to endovascular pathogens such as Staphylococcus aureus (SA). However, excessive PMN activation, such as in septic shock or traumatic injury, can severely damage tissues, including in the cardiovascular system, and is associated with high mortality rates (30‐50%). Enhanced understanding of signaling, in particular by GPCRs that regulate PMN function, may reveal new therapeutic approaches. We thus used Taqman GPCR arrays to define the basal expression of GPCRs (which represented ~190 receptors) by human PMNs and after 3 hr incubation with methicillin‐resistant SA (MRSA). MRSA‐incubated PMNs had altered expression of numerous Gi‐, Gs‐ and Gq‐coupled GPCRs. Antagonists of one newly recognized GPCR, GPR30/GPER, inhibited the formation of DNA‐based neutrophil extracellular traps (NETs) but not phagocytosis or reactive oxygen species production by PMNs. The potential to selectively inhibit NETs, which have been implicated as important mediators of vascular inflammation, while not blocking other responses to pathogens, suggests that PMN‐expressed GPCRs, including newly recognized GPCRs, such as GPR30/GPER are novel targets for the therapy of endovascular pathogens.

Galanin modulates human and murine neutrophil activation in vitro.

Polymorphonuclear neutrophils are key players in innate immunity. The innate immune system needs to be tightly controlled to ensure proper activation but also no overactivation. Galanin has been shown to regulate inflammatory reactions, and therefore, we aimed to elucidate the expression of galanin and its three receptors (GAL1 -GAL3 ) in polymorphonuclear neutrophils and to evaluate whether galanin exerts direct or indirect effects on human and murine polymorphonuclear neutrophils. Human peripheral polymorphonuclear neutrophils were isolated from fresh blood of healthy donors, and murine polymorphonuclear neutrophils were isolated from bone marrow of C57BL/6N mice. Gene expression was evaluated by qRT-PCR. As a marker for polymorphonuclear neutrophil activation, CD11b integrin surface expression was measured by FACS analysis. Furthermore, a label-free technology measuring ligand-induced dynamic mass redistribution was used to evaluate the response of polymorphonuclear neutrophils to galanin. GAL2 receptor expression was found in both human and murine polymorphonuclear neutrophils, galanin and GAL3 receptor were exclusively expressed in murine bone marrow polymorphonuclear neutrophils, and GAL1 receptor was not detectable in polymorphonuclear neutrophils of either species. Galanin treatment was not able to induce CD11b integrin surface expression or dynamic mass redistribution in human polymorphonuclear neutrophils and murine bone marrow polymorphonuclear neutrophils. However, galanin treatment significantly enhanced the response of polymorphonuclear neutrophils of both species to interleukin-8. Galanin can be regarded as an immunomodulatory peptide as it can sensitize polymorphonuclear neutrophils towards pro-inflammatory cytokines in humans and mice.

IFNγ effect in the induction of the Neutrophil Extracellular Traps (NETs) in Chronic Granulomatous Disease patients

IFNγ effect in the induction of the Neutrophil Extracellular Traps (NETs) in Chronic Granulomatous Disease patients

Comparison between the Effects of Regular Cigarettes Smoke and Electronic Nicotine Delivery Devices Vapor on the Pathogenesis of Pseudomonas Aeruginosa = مقارنة بين تأثير دخان السجائر العادية و بخار أجهزة توصيل النيكوتين الإلكترونية على ضراوة ميكروب الزائفة الزنجارية ( سيدوموناس ايرجينوزا )

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative pathogen associated with pneumonia, bronchitis, and chronic colonization of the respiratory tract. It easily affected by the environmental inhalant pollutants that may change its virulence and alter the host innate immunity against it. In this study, we aimed to establish the effect of cigarette smoke and electronic nicotine delivery device vapor on the virulence of P. aeruginosa. Bacteria were grown in control media, different concentrations of cigarette smoke exposed media, and range of doses of nicotine vapor exposed media. The growth ability of bacteria, pH change, and the pigment production capability were monitored for each culture. Sensitivities of P. aeruginosa to the reactive oxygen species and to killing by purified human polymorphonuclear neutrophils were established as well as their ability to change their surface charge and their susceptibility to antimicrobial peptides. Smoke exposed P. aeruginosa showed inhibition in their growth in a dose-dependent manner and inhibition of the pyocyanin production whilst the nicotine vapor did not affect their growth kinetics but increased the pigment production dose dependently too. When treated with H 2 O 2 , smoke and vapor exposed bacteria had significantly higher survival rates than the control. Where both smoke and vapor exposed cells grew significantly in the presence of human neutrophils while control bacteria were killed, exposure to 75% cigarette smoke showed the higher survival rate. Moreover, cigarette smoke was found to increase P. aeruginosa resistance to the antimicrobial peptides. Consequently, cigarette smoke and electronic nicotine vapor were found to boost P. aeruginosa pathogenesis and virulence.

A Role for NFAT in Innate Immunity: Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation Under Cyclosporine Α Treatment

Background and Aims: Patients after allogeneic hematopoietic stem cell transplantation (HSCT) suffer from immunodeficiency, in part due to long-term immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA). Additionally, these patients have an increased risk for opportunistic fungal infections like invasive aspergillosis (IA). The nuclear factor of activated T cells (NFAT) is known as an important transcription factor in signaling-pathways downstream of calcineurin in the adaptive immune systems, e.g. in T cells, but also plays an important role in innate immune response as indicated by recent data in rodent models. These studies showed a relevant impact of NFAT-/calcineurin inhibition on the innate immune response by polymorphonuclear neutrophils (PMN) in a candida sepsis model. To clarify whether this is also relevant in humans, we investigated the role of NFAT signaling pathways in PMN activation and effector functions in healthy volunteer donors and patients under immunosuppressive treatment with CsA after HSCT.Methods: Firstly, we performed in vitro experiments using PMN from healthy donors analyzing their effector functions in absence or presence of CsA in titrated doses according to therapeutic levels. In detail, we examined phagocytosis, activation, generation of reactive oxygen species (ROS) and release of inflammatory mediators like IL-8 and TNF-α. After activation with lipopolysaccharide (LPS) and zymosan, phagocytosis and activation-induced shedding of CD62L were measured by flow cytometry using polychromatic microspheres and matching surface markers (CD11b, CD62L, CD66b). In addition, generation of ROS was analyzed by dichlorofluorescein assay (DCF), whereas activation-induced release of inflammatory mediators was measured by enzyme-linked immunosorbent assay (ELISA) and intracellular flow cytometry. In addition, blood samples of patients after HSCT under continuous CsA medication (n=6) and healthy volunteer donors (n=6) were analyzed ex vivo at two different time points after allogeneic HSCT (day 25-35 and 125-135) concerning their PMN effector functions in the same manner as described above.Results: Analysis of healthy donors PMN in vitro showed that CsA had no significant influence on expression of activation markers and shedding of CD62L. Moreover, no substantial influence of CsA on generation of ROS was detected compared to untreated controls (5245 RFU +/- 354 (CsA) vs. 5763 +/- 520 (control) after stimulation with LPS, mean +/- SEM). Furthermore, activation-induced synthesis of IL-8 was not reduced in presence of CsA (519pg/ml +/- 81 vs. 463 +/- 131 (control) after stimulation with LPS). In contrast, CsA rather enhanced phagocytosis after stimulation with LPS (83.5% +/- 1.7 vs. 71.0 +/- 1.5 (control)). Regarding the ex vivo analysis of HSCT patient and healthy donor blood samples, production of ROS was not affected under CsA therapy (35.1% +/- 9.4 vs. 31.1 +/- 6.6 (control) after stimulation with zymosan). Furthermore, CsA medication showed a stimulating effect on PMN phagocytosis which is in line with our in vitro data (58.4% +/- 7.1 vs. 44.3 +/- 2.3 (control) after stimulation with LPS). Interestingly, in several patients an increased production of IL-8 and TNF-α was detectable after stimulation with zymosan (IL-8: 9.1% +/- 3.6 vs. 1.5 +/- 0.3 (control); TNF-α: 16.6% +/- 6.2 vs. 1.0 +/- 0.4 (control)).Conclusions: In contrast to previous results by others in murine model systems, we found an increased phagocytic activity in vitro and ex vivo in human PMN upon NFAT/calcineurin inhibition, whereas other effector mechanisms were unaffected. In addition, HSCT patients under CsA treatment displayed enhanced inflammatory mediators production in PMN. It is currently unclear whether these findings are clinically relevant for the innate immune response after HSCT, e. g. in terms of anti-fungal immunity. Further studies are needed to address whether these enhanced PMN functions in the presence of CsA contribute to a dysregulated innate immune response in humans. Alternatively, our discrepant results may be due to differences in PMN functionality and CsA responsiveness in mice and man. However, while CsA strongly suppresses adaptive immune responses, i.e. T-cell responses in graft-versus-host disease (GvHD), our results so far suggest that CsA does not affect innate immune effector functions by humans PMN in a comparable way. DisclosuresRadsak:Celgene: Research Funding.

Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells.

Polymorphonuclear neutrophils (PMN) play a central role in inflammation and participate in its control, notably by modulating dendritic cell (DC) functions via soluble mediators or cell-cell contacts. Neutrophil extracellular traps (NETs) released by PMN could play a role in this context. To evaluate NET effects on DC maturation, we developed a model based on monocyte-derived DC (moDC) and calibrated NETs isolated from fresh human PMN. We found that isolated NETs alone had no discernable effect on moDC. In contrast, they downregulated LPS-induced moDC maturation, as shown by decreased surface expression of HLA-DR, CD80, CD83, and CD86, and by downregulated cytokine production (TNF-α, IL-6, IL-12, IL-23), with no increase in the expression of tolerogenic DC genes. Moreover, the presence of NETs during moDC maturation diminished the capacity of these moDC to induce T lymphocyte proliferation in both autologous and allogeneic conditions, and modulated CD4(+) T lymphocyte polarization by promoting the production of Th2 cytokines (IL-5 and IL-13) and reducing that of Th1 and Th17 cytokines (IFN-γ and IL-17). Interestingly, the expression and activities of the lymphoid chemokine receptors CCR7 and CXCR4 on moDC were not altered when moDC matured in the presence of NETs. Together, these findings reveal a new role for NETs in adaptive immune responses, modulating some moDC functions and thereby participating in the control of inflammation.