Modulation Of Neutrophil Function Research Articles

Neutrophil gelatinase-associated lipocalin and innate immune responses to bacterial infections.

Neutrophil gelatinase-associated lipocalin (NGAL), an essential component of the antimicrobial innate immune system, is present in neutrophils and multiple other tissues. It prevents iron acquisition by microorganisms by sequestering iron-loaded bacterial siderophores. NGAL also modulates neutrophil functions. Its production is inducible following Toll-like receptor 4 activation and release of pro-inflammatory cytokines. NGAL is employed clinically in the diagnosis of acute kidney injury and may be useful in general in the differential diagnosis of a bacterial-mediated infectious process. Elevated levels of NGAL have been detected in the blood of patients with bacterial urinary tract infection, community-acquired pneumonia, sepsis, as well as in the cerebrospinal fluid and peritoneal fluid of patients with bacterial meningitis and peritonitis. Some bacteria have developed resistance to NGAL-mediated iron sequestration by production of modified siderophores that are not recognized by NGAL.

Balancing Innate Immunity and Inflammatory State via Modulation of Neutrophil Function: A Novel Strategy to Fight Sepsis.

Sepsis and SIRS (systemic inflammatory response syndrome) belong to a severe disease complex characterized by infection and/or a whole-body inflammatory state. There is a growing body of evidence that neutrophils are actively involved in sepsis and are responsible for both release of cytokines and phagocytosis of pathogens. The neutrophil level is mainly regulated by G-CSF, a cytokine and drug, which is widely used in the septic patient with neutropenia. This review will briefly summarize the role of neutrophils and the therapeutic effect of G-CSF in sepsis. We further suggest that targeting neutrophil function to modulate the balance between innate immunity and inflammatory injury could be a worthwhile therapeutic strategy for sepsis.

Modulation Of Neutrophil Function By Moderate Aerobic Exercise In Eutrophic And Overweight

Modulation Of Neutrophil Function By Moderate Aerobic Exercise In Eutrophic And Overweight

Role of PARP‐1 in the modulation of neutrophil function: relevance for inflammatory bowel disease (902.5)

Aberrant immune response has been proposed as the underlying cause of Inflammatory Bowel Diseases. Disease progression and relapse are attributed to a hyperactive adaptive immune response to the bowel luminal contents accompanied by a disproportionate and persistent neutrophil (PMN) influx. Poly(ADP‐ribose) polymerases (PARPs), including its most abundant isoform PARP1, catalyzes the transfer of ADP‐ribose units from NAD+ to a number of target proteins. In vivo studies have demonstrated that genetic ablation or pharmacological inhibition of PARP1 ameliorates the experimental colitis pathophysiological changes. Considering the essential role of PARP1 in the regulation of inflammation, we propose to evaluate the effect of Parp1 deletion on neutrophil function in a model of Dextran Sulfate Sodium (DSS)‐induced colitis. In comparison to WT mice, Parp1‐/‐ were protected from the effects of DSS, with significantly lower weight loss, and reduced proinflammatory mediators, including metalloproteinases and chemoattractants. The regulation of neutrophil‐related gene in the colon was also studied by microarray. Similarly, Parp1‐/‐ neutrophils demonstrated an impaired chemotaxis in vitro and reduced motility in vivo in a model of aseptic peritonitis. In light of these current findings, we postulate that the pathogenic role of PARP1 may be mediated in part by the regulation of neutrophil function.Grant Funding Source: Supported by NIH Grant 1K01DK099268‐01

Neutrophil phenotype model for extracorporeal treatment of sepsis

Neutrophils play a central role in eliminating bacterial pathogens, but may also contribute to end-organ damage. Interleukin-8 (IL-8), a key modulator of neutrophil function, signals through neutrophil specific surface receptors CXCR-1 and CXCR-2. Expression of these surface receptors can be altered by perfusion through an extracorporeal device. Extracorporeal methods of immune modulation have shown promise for treatment of sepsis; however, achieving an appropriate response is a major challenge [1]. In this study a mechanistic mathematical model was used to evaluate and deploy an extracorporeal sepsis treatment that modulates CXCR-1/2 levels.

Comparison of innate immune activation after prolonged feeding of milk fermented with three species of Lactobacilli

The present investigation aimed at identifying the abilities of three different species of probiotic lactobacilli to modulate cellular immune responses in mouse neutrophils and macrophages in vivo over a study period of 60 days. Neutrophil respiratory burst enzymes (cytochrome c reductase and MPO) showed remarkable increased activity (P ≤ 0.01) after consumption of milks fermented by different species of probiotics over 30 and 60 days of feeding trials. Enzyme activities (β-galactosidase and β-glucuronidase) and nitric oxide production also increased considerably (P ≤ 0.01) in macrophages, both in peritoneal fluid and in enriched cell cultures. The effects of enhanced enzyme activities were corroborated by simultaneous increases in the phagocytic activities of neutrophils and macrophages. The increases in cellular functions were invariably maximal during the first 30 days of study and were maintained, but did not increase, over the next 30 days. Further, Lactobacillus helveticus-fed groups were most effective at modulating neutrophil functions whereas Lactobacillus paracasei-fed groups were more potent at enhancing macrophage functions. Together, our results indicate that probiotics have strain specific effects on stimulating cellular functions while not causing excessive stimulation of the immune system over longer feeding periods, thereby resulting in maximum and stable health benefits.

Fish oil is a modulator of neutrophil function in chronic laminitis

s / Journal of Equine Veterinary Science 33 (2013) 838–859 855 Magnesium, chromium, and cinnamon have also been recommended for the control insulin resistance in horses, but there is a paucity of scientific evidence to support their use. It is conceivable that some horses respond to specific supplements whereas others do not, and this may be explained by the multiple manifestations of insulin dysregulation. However, veterinarians should be cautious about horse owners substituting supplements for good management practices until further scientific evidence is presented. The practice of harvesting blood to lower iron concentrations in horses is not condoned. This recommendation arose from findings in other species linking iron overload with insulin resistance. In a study recently performed by the University of Kentucky, resveratrol lowered insulin concentrations in horses with insulin dysregulation. Results have only been presented as an abstract to date; the full article must be evaluated before recommendations for resveratrol supplementation are made. 017 Fish oil is a modulator of neutrophil function in chronic laminitis Samantha M. Steelman , David M. Hood , Yang Y. Fan , Robert S. Chapkin , and Bhanu P. Chowdhary 1 Department of Veterinary Integrative Biosciences, Texas A&M University, Hoof Diagnostic and Rehabilitation Clinic, Bryan, TX, 3 Program in Integrative Nutrition & Complex Diseases, Texas A&M University Take-home message: Dietary supplementation with marine-based omega-3 fatty acids might be beneficial in chronic laminitis. Introduction: Recent experiments in our lab have shown that horses with chronic laminitis exhibit neutrophilic infiltration of the laminar tissue and that neutrophils from these animals produce elevated levels of inflammatory cytokines, suggesting a role for these cells in chronic laminitis. Fish oil has been shown to reduce chronic, neutrophil-driven inflammation in a number of other species. We therefore conducted a pilot experiment to determine the ability of fish oil supplementation to attenuate inflammation in horses with chronic laminitis. Materials and Methods: Four horses with chronic laminitis were fed 80 g of powdered fish oil daily for 45 days. Blood was collected on days 0, 7, 14, and 21, and force plate analyses were performed on days 0 and 45. Data were analyzed using ANOVA for repeated measures. Results: Fish oil supplementation significantly decreased neutrophil chemotaxis in response to zymosan and increased neutrophil production of resolvin D1, an antiinflammatory lipid. No changes in lameness or mean stance load pattern were noticed. Discussion and Conclusions: Contrary to some published reports, we found ample evidence to indicate that fish oil supplementation can reduce inflammation in horses. The lack of effect on measurements of lameness is likely due to the short duration of the study. Dietary supplementation with marine-based omega-3 fatty acids might be beneficial in chronic laminitis. 018 Equine Insulin Resistance (EIR): private veterinary practice prevention and management learned in over 1,000 cases in the last 3 years Frank K. Reilly Equine Medical & Surgical Associates, West Chester, PA Take-homemessage: With care, it is possible to return the horse to some grass turnout in a safe, practical, and tested manner. Equine Insulin Resistance (EIR) is now the leading cause of laminitis worldwide. Owners, farriers, and veterinarians are seeing an increase in the number of cases and want information on what works and what does not in avoiding EIR, and practical ways to manage the problem. Recent advances in diagnostic testing have helped to identify EIR through blood tests, but widely varying test techniques make it difficult for the owner to understand what the numbers mean. Telling owners to feed lousy hay and keep the horse on a dirt lot is not being accepted as an answer by most owners with EIR horses. The ability to return to some grass turnout in a safe, practical, and tested manner, so that the horse can bewith other horses and not be segregated, is important information.

Equine Insulin Resistance (EIR): private veterinary practice prevention and management learned in over 1,000 cases in the last 3 years

s / Journal of Equine Veterinary Science 33 (2013) 838–859 855 Magnesium, chromium, and cinnamon have also been recommended for the control insulin resistance in horses, but there is a paucity of scientific evidence to support their use. It is conceivable that some horses respond to specific supplements whereas others do not, and this may be explained by the multiple manifestations of insulin dysregulation. However, veterinarians should be cautious about horse owners substituting supplements for good management practices until further scientific evidence is presented. The practice of harvesting blood to lower iron concentrations in horses is not condoned. This recommendation arose from findings in other species linking iron overload with insulin resistance. In a study recently performed by the University of Kentucky, resveratrol lowered insulin concentrations in horses with insulin dysregulation. Results have only been presented as an abstract to date; the full article must be evaluated before recommendations for resveratrol supplementation are made. 017 Fish oil is a modulator of neutrophil function in chronic laminitis Samantha M. Steelman , David M. Hood , Yang Y. Fan , Robert S. Chapkin , and Bhanu P. Chowdhary 1 Department of Veterinary Integrative Biosciences, Texas A&M University, Hoof Diagnostic and Rehabilitation Clinic, Bryan, TX, 3 Program in Integrative Nutrition & Complex Diseases, Texas A&M University Take-home message: Dietary supplementation with marine-based omega-3 fatty acids might be beneficial in chronic laminitis. Introduction: Recent experiments in our lab have shown that horses with chronic laminitis exhibit neutrophilic infiltration of the laminar tissue and that neutrophils from these animals produce elevated levels of inflammatory cytokines, suggesting a role for these cells in chronic laminitis. Fish oil has been shown to reduce chronic, neutrophil-driven inflammation in a number of other species. We therefore conducted a pilot experiment to determine the ability of fish oil supplementation to attenuate inflammation in horses with chronic laminitis. Materials and Methods: Four horses with chronic laminitis were fed 80 g of powdered fish oil daily for 45 days. Blood was collected on days 0, 7, 14, and 21, and force plate analyses were performed on days 0 and 45. Data were analyzed using ANOVA for repeated measures. Results: Fish oil supplementation significantly decreased neutrophil chemotaxis in response to zymosan and increased neutrophil production of resolvin D1, an antiinflammatory lipid. No changes in lameness or mean stance load pattern were noticed. Discussion and Conclusions: Contrary to some published reports, we found ample evidence to indicate that fish oil supplementation can reduce inflammation in horses. The lack of effect on measurements of lameness is likely due to the short duration of the study. Dietary supplementation with marine-based omega-3 fatty acids might be beneficial in chronic laminitis. 018 Equine Insulin Resistance (EIR): private veterinary practice prevention and management learned in over 1,000 cases in the last 3 years Frank K. Reilly Equine Medical & Surgical Associates, West Chester, PA Take-homemessage: With care, it is possible to return the horse to some grass turnout in a safe, practical, and tested manner. Equine Insulin Resistance (EIR) is now the leading cause of laminitis worldwide. Owners, farriers, and veterinarians are seeing an increase in the number of cases and want information on what works and what does not in avoiding EIR, and practical ways to manage the problem. Recent advances in diagnostic testing have helped to identify EIR through blood tests, but widely varying test techniques make it difficult for the owner to understand what the numbers mean. Telling owners to feed lousy hay and keep the horse on a dirt lot is not being accepted as an answer by most owners with EIR horses. The ability to return to some grass turnout in a safe, practical, and tested manner, so that the horse can bewith other horses and not be segregated, is important information.

The Neutrophil in Chronic Obstructive Pulmonary Disease. Too Little, Too Late or Too Much, Too Soon?

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and has few effective therapies. It is characterized by anomalous and persistent inflammation, both local and systemic. Neutrophilic inflammation predominates in the COPD airway wall and lumen, but, despite the presence of abundant innate immune cells, the progressive clinical course of the disease is punctuated by recurrent infection-driven exacerbations. An extensive body of evidence (from cell culture to murine models and finally to the susceptibility of human patients with α1-antitrypsin deficiency to develop COPD) implicates neutrophil elastase and other neutrophil-derived proteases as key mediators of the tissue damage and relentless decline in lung function that occurs in this condition. In addition to the well recognized role of cytokines in modulating neutrophil function and survival, it has recently become apparent that hypoxia can influence neutrophil function, with impaired killing of pathogenic bacteria, enhanced release of proteases, and delayed apoptosis. This destructive neutrophil phenotype is predicted to be highly detrimental in the setting of the COPD microenvironment.

The innate defense regulator peptides IDR-HH2, IDR-1002, and IDR-1018 modulate human neutrophil functions

Although HDPs were originally hypothesized to act as antimicrobial agents, they also have been shown to broadly modulate the immune response through the activation of different cell types. We recently developed a series of novel, synthetic peptides, termed IDRs, which are conceptually based on a natural HDP, bovine bactenecin. We showed that IDR-1 and IDR-1002 protect the host against bacterial infections through the induction of chemokines. The objective of this study was to investigate the effects of the IDRs on various functions of human neutrophils. Here, we demonstrated that IDR-HH2, IDR-1002, and IDR-1018 modulated the expression of neutrophil adhesion and activation markers. Moreover, these IDRs enhanced neutrophil adhesion to endothelial cells in a β₂ integrin-dependent manner and induced neutrophil migration and chemokine production. The IDR peptides also increased the release of the neutrophil-generated HDPs (antimicrobial), human α-defensins, and LL-37 and augmented neutrophil-mediated killing of Escherichia coli. Notably, the IDRs significantly suppressed LPS-mediated neutrophil degranulation, the release of ROS, and the production of the inflammatory cytokines TNF-α and IL-10, consistent with their ability to dampen inflammation. As evidenced by the inhibitory effects of MAPK-specific inhibitors, IDRs activated the MAPK pathway that was required for chemokine production. In conclusion, our study provides novel evidence regarding the contribution of the IDR peptides to the innate immune response through the modulation of neutrophil functions. The results described here may aid in the development of IDRs as novel, anti-infective and immunomodulatory agents.

Impairment of Mitochondrial–Nuclear Cross Talk in Neutrophils of Patients with Type 2 Diabetes Mellitus

Increased leukocyte apoptosis is intrinsically linked to disease patho-physiology, susceptibility to and severity of infections in type 2 diabetes mellitus (T2DM) patients. A consistent defect in neutrophil function is considered central to this increased risk for infections. Although redox imbalance is considered a potential mediator of these associated complications, detailed molecular evidence in clinical samples remains largely undetected. The study consisted of three groups (n=50 each) of Asian Indians: early diagnosed diabetic patients, cases with late-onset diabetic complications and age and gender-matched healthy controls. We evaluated mitochondrial oxidative stress, levels of nuclear DNA damage and apoptosis in peripheral blood neutrophils isolated from T2DM patients. We observed that in both early and late diabetic subjects, the HbA1c levels in neutrophils were altered considerably with respect to healthy controls. Increased oxidative stress observed in both early and late diabetics imply the disentanglement of fine equilibrium of mitochondria-nuclear cross talk which eventually effected the augmentation of downstream nuclear γH2AX activation and caspase-3 expression. It would be overly naïve to refute the fact that mitochondrial deregulation in neutrophils perturbs immunological balance in type 2 diabetic conditions. By virtue of our data, we posit that maneuvering mitochondrial function might offer a prospective and viable method to modulate neutrophil function in T2DM. Nevertheless, similar investigations from other ethnic groups in conjunction with experimental evidences would be a preeminent need. Obviously, our study might aid to comprehend this complex interplay between mitochondrial dysfunction and neutrophil homeostasis in T2DM.

A Novel Tetrapeptide Derivative Exhibits In Vitro Inhibition of Neutrophil-Derived Reactive Oxygen Species and Lysosomal Enzymes Release

Neutrophil infiltration plays a major role in the pathogenesis of myocardial injury. Oxidative injury is suggested to be a central mechanism of the cellular damage after acute myocardial infarction. This study is pertained to the prognostic role of a tetrapeptide derivative PEP1261 (BOC-Lys(BOC)-Arg-Asp-Ser(tBu)-OtBU), a peptide sequence (39–42) of lactoferrin, studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation, lysosomal enzymes release, and enhanced expression of C proteins. The groundwork experimentation was concerned with the isolation of neutrophils from the normal and acute myocardial infarct rats to find out the efficacy of PEP1261 in the presence of a powerful neutrophil stimulant, phorbol 12-myristate 13 acetate (PMA). Stimulation of neutrophils with PMA resulted in an oxidative burst of superoxide anion and enhanced release of lysosomal enzymes and expression of complement proteins. The present study further demonstrated that the free radicals increase the complement factors in the neutrophils confirming the role of ROS. PEP1261 treatment significantly reduced the levels of superoxide anion and inhibited the release of lysosomal enzymes in the stimulated control and infarct rat neutrophils. This study demonstrated that PEP1261 significantly inhibited the effect on the ROS generation as well as the mRNA synthesis and expression of the complement factors in neutrophils isolated from infarct heart.

Mycobacterium tuberculosisH37Rv is more effective compared to vaccine strains in modulating neutrophil functions: anin vitrostudy

Neutrophils are the primary cells contributing to initial defense against mycobacteria. Yet, little is known about the potential of various mycobacterial strains to stimulate neutrophils. This study was focused to compare the differential capacity of vaccine strains, Mycobacterium bovis bacillus Calmette-Guerin (BCG) and Mycobacterium indicus pranii (Mw), and laboratory strain H37Rv to activate and enhance neutrophil functions. The expression of phenotypic markers like Fcγ receptor, toll-like receptor (TLR), and chemokine receptor; secretion of pro-inflammatory cytokines; and the rate of apoptosis were studied in infected neutrophils. Increased expression of CD32, CD64, TLR4, and CXCR3; increased TNF-α secretion; and downregulation of early apoptosis were observed in H37Rv-infected neutrophils. Among the vaccine strains, BCG increased the expression of only CD32 on neutrophils, while Mw was comparatively ineffective. To understand the paracrine role of neutrophils, the supernatants from infected neutrophils were used to stimulate monocytes and T helper cells. The secretory molecules from all infected neutrophils increased the expression of CCR5 on monocytes, whereas only H37Rv-infected supernatant increased the expression of CCR7 on monocytes and CD69 on T cells. Thus, H37Rv was more effective in activating neutrophils and in turn stimulating monocytes and T cells. By comparison, vaccine strains were less effective in modulating neutrophil functions.

Epigenetic Regulation of Galectin-3 Expression by β1 Integrins Promotes Cell Adhesion and Migration

Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-to-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examined 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering.

Mechanisms of Neutrophil-mediated Disease: Innovative Therapeutic Interventions

Over recent years, neutrophil functions have extensively been reevaluted. For instance, it has been found that extracellular traps generated by activated neutrophils are able to perform a very effective antimicrobial activity against a plethora of microorganisms. In addition, neutrophils possess the ability to interact with monocytes/macrophages either via release of their granule content or via efferocytosis. Efferocytosis is based on neutrophil clearance by macrophages, which, in turn, exert pro- or antinflammatory effects. Furthermore, alarmins released by neutrophils have been shown to mobilize and activate dendritic cells as well as macrophages for antigenpresentation to occur. Finally, other researchers have demonstrated that neutrophils act as suppressor cells by deprivation of arginine in T cells. From a clinical point of view, it is well known that neutrophils are present as activated effector cells in a number of diseases such as infectious disease, sepsis, autoimmune disease, cardiovascular disease and tumors. They play pro-inflammatory and angiogenetic effects in some cases, while in other cases they depress host immune response. In this review, also emphasis will be placed on neutrophil functional deficits which seem to participate to the pathogenesis of various diseases, e.g., viral hepatitis C infection and inflammatory bowel disease. Finally, novel therapeutic measures able to modulate neutrophil functions will be described with special reference to both immune agents and natural products.

Regulation of Neutrophil Function by Adenosine

Adenosine is an endogenously released purine nucleoside that signals via 4 widely expressed G protein-coupled receptors: A(1), A(2A), A(2B), and A(3). In the setting of inflammation, the generation and release of adenosine is greatly enhanced. Neutrophils play an important role in host defense against invading pathogens and are the cellular hallmark of acute inflammation. Neutrophils both release adenosine and can respond to it via expression of all 4 adenosine receptor subtypes. At low concentrations, adenosine can act via the A(1) and A(3) adenosine receptor subtypes to promote neutrophil chemotaxis and phagocytosis. At higher concentrations, adenosine acts at the lower-affinity A(2A) and A(2B) receptors to inhibit neutrophil trafficking and effector functions such as oxidative burst, inflammatory mediator production, and granule release. Modulation of neutrophil function by adenosine is relevant in a broad array of disease models, including ischemia reperfusion injury, sepsis, and noninfectious acute lung injury. This review will summarize relevant research in order to provide a framework for understanding how adenosine directly regulates various elements of neutrophil function.

Coagulation Factors XIa and XIIa Modulate Neutrophil Elastase Release,

Abstract 3220Neutrophils play a vital role in innate immunity. Activated neutrophils can release proteolytic enzymes capable of neutralizing microbes and contributing importantly to host-defense. In severe sepsis, microbial components and pro-inflammatory cytokines can contribute to excess systemic neutrophil activation, resulting in tissue damage and organ failure. Thus, regulation of neutrophil activation and factor release is critical during pathologic conditions.Recent data indicate that components of the contact system modulate numerous inflammatory mediators during severe sepsis, but the exact role of the contact pathway in host-defense is not well understood. Inhibition of factor XII (FXII) in septic baboons reduces circulating neutrophil elastase (NE), a potent cytolytic enzyme that is increased during sepsis and implicated in organ failure. In vitro studies also indicate that both plasma kallikrein and FXIIa are capable of directly inducing NE release. While it is apparent that factors of the contact system interact with neutrophils, the molecular mechanisms by which these factors modulate neutrophil function have not been established. We therefore examined factor XI (FXI) neutrophil interactions and the cellular signaling pathways regulating FXIIa neutrophil stimulation.Human neutrophils were isolated from peripheral blood and resuspended in HBSS at a concentration of 0.5 ×106/ml. Cells were treated with FXI, FXIa, FXII, or FXIIa with or without fMLP (1 μM) stimulation, and the release of NE was assayed in the cell supernatants via ELISA. FXI, FXIa or FXII had no direct stimulatory effect on NE release compared to vehicle. While neither FXI nor FXII had any inhibitory effect on fMLP induced NE release, FXIa (10 μg/ml) modestly reduced fMLP-induced NE release by 20% (n=3). FXIIa (3, 10, 30 μg/ml) dose-dependently increased NE release in the presence of cytochalasin B (5 μg/ml), consistent with published data.To examine the mechanism by which FXIIa induces NE release, neutrophils were pretreated with signaling inhibitors and subsequently activated with FXIIa (30 μg/ml). Mammalian target of rapamycyin (mTOR) is a downstream serine/threonine kinase of the PI3K/AKT pathway that integrates signals from the microenvironment such as cytokines and growth factors. It is known that inhibition of mTORC2 abrogates neutrophil polarization and directed migration, thus we examined the role of rapamycin complex 1 and 2 (mTORC1/2) in mediating NE release. Pretreatment of cells with RAD001 (20 nM), an mTORC1 inhibitor had no effect on FXIIa-induced NE release, whereas the combined mTORC1/mTORC2 inhibitor, pp242 (100 nM) abrogated FXIIa-induced NE release, suggesting that components of the mTORC2 pathway contribute to NE release. Pretreatment with EHT 1864 (50 uM), a Rac inhibitor, significantly potentiated NE release induced by either fMLP or FXIIa, suggesting that Rac is also capable of modulating FXIIa signaling.Taken together, these results suggest that coagulation factors FXIa and FXIIa differentially modulate neutrophil function, and that the mTOR and Rac signaling pathways participate in FXIIa stimulated neutrophil activation. These data suggest that the contact pathway is involved in neutrophil stimulation through mTOR and Rac signaling, and thus modulating these pathways could be a potential therapeutic strategy for limiting excess neutrophil activation. Disclosures:Gruber:Aronora, LLC: Consultancy, Equity Ownership.

Effect of exogenous surfactant on phosphatidylinositol 3-kinase-Akt pathway and peroxisome proliferator activated receptor-γ during endotoxin induced acute respiratory distress syndrome

Lipopolysaccharide induced acute respiratory distress syndrome (ARDS) leads to an unacceptably high mortality. In this regard, the anti-inflammatory properties of surfactant may provide a therapeutic option. Phosphoinositide 3-kinase (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. This study explores the mechanisms of surfactant dependent protection by regulating PPAR-γ in a rat model of ARDS. Sprague-Dawley male rats were divided into four groups: buffer controls; rats challenged with LPS (055:B5 E. coli); challenged with LPS and treated with porcine surfactant; and challenged with LPS and treated with synthetic surfactant. Expression of PI3-K, Akt, GSK3-β, and PPAR-γ were studied by western immunoblot, immunofluorescence and by immunohistochemistry. In vivo endotoxin administration to rat resulted in activation of PI3-K and Akt in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in rat with surfactant administration. Similar results were also seen in PPAR-γ expression. These results show that PI3-K occupies a central position in regulating endotoxin-induced ALI involving inflammatory responses. Surfactant treatment conferred protection in rat model dependent on PPAR-γ and inhibition of PI3-K/Akt pathway.

Neutrophil activation and survival are modulated by interaction with NK cells

It is increasingly evident that neutrophils are able to cross-talk with other leukocytes to shape ongoing inflammatory and immune responses. In this study, we analyzed whether human NK cells may influence the survival and activation of neutrophils under co-culture conditions. We report that NK cells exposed to either IL-15 or IL-18 alone strongly protect the survival of neutrophils via the release of IFNγ and granulocyte macrophage colony-stimulating factor (GM-CSF) plus IFNγ, respectively, and cause a slight up-regulation of neutrophil CD64 and CD11b expression. In comparison, NK cells exposed to both IL-15 and IL-18 show a lesser ability to increase the survival of neutrophils but can more potently up-regulate CD64 and CD11b expression, as well as induce the de novo surface expression of CD69, in neutrophils. Analysis of the events occurring in neutrophil/NK co-cultures exposed to IL-15 plus IL-18 revealed that (i) neutrophil survival is positively affected by NK-derived GM-CSF but negatively influenced by a CD18-dependent neutrophil/NK contact, (ii) NK-derived IFNγ is almost entirely responsible for the induction of CD64, (iii) both soluble factors (primarily GM-CSF) and direct cell-cell contact up-regulate CD11b and CD69 and (iv) NK-derived GM-CSF induces the expression of biologically active heparin-binding EGF-like growth factor (HB-EGF) in neutrophils. Finally, we demonstrate that NK cells can also express HB-EGF when stimulated with either IL-2 or IL-15, yet independently of endogenous GM-CSF. Altogether, our results define a novel interaction within the innate immune system whereby NK cells, by directly modulating neutrophil functions, might contribute to the pathogenesis of inflammatory diseases.

Bone marrow MyD88 signaling modulates neutrophil function and ischemic myocardial injury

Myeloid differentiation factor 88 (MyD88), an adaptor critical for innate immune function, plays a role in neutrophil recruitment and myocardial injury after transient ischemia. However, how MyD88 signaling modulates neutrophil function and myocardial injury remains unclear. In an in vivo model of neutrophil migration and a chimeric model of MyD88 deletion, we demonstrated that Gr-1-positive (Gr-1(+)) neutrophil migration was significantly decreased by 68% in MyD88-deficient (Myd88(-/-)) mice and by 33% in knockout→wild-type (KO→WT; donor→recipient) chimeric mice, which lacked MyD88 in bone marrow cells but maintained normal MyD88 expression in the heart. This marked attenuation in neutrophil migration was associated with decreased peritoneal neutrophil CXCR2 expression and lower peritoneal KC, a neutrophil chemoattractant, in MyD88(-/-) mice. Moreover, in vitro, KC induces significantly more downregulation of CXCR2 expression in MyD88(-/-) than WT neutrophils. In an in vivo model of myocardial ischemia-reperfusion (I/R) injury, KO→WT chimeric mice had significantly smaller infarct sizes compared with the WT→WT mice. While there was a marked increase in proinflammatory cytokine/chemokine expression in the myocardium following I/R, there was no significant difference between WT→WT and KO→WT mice. In contrast, Gr-1(+) neutrophil recruitment in the myocardium was markedly attenuated in MyD88(-/-) mice. Deletion of Toll-interleukin-1 receptor (TIR)-domain-containing adaptor protein-inducing interferon-β-mediated transcription factor (Trif), another innate immune adaptor, had no effect on the KC-mediated CXCR2 downregulation or on myocardial neutrophil recruitment after I/R. Taken together, these findings suggest that MyD88 signaling is essential for maintaining neutrophil migratory function and chemokine receptor expression. MyD88 signaling in bone marrow-derived circulating cells may play a specific and critical role in the development of myocardial I/R-induced injury.