Neutrophil-endothelial Cell Interactions Research Articles

The critical role of neutrophil-endothelial cell interactions in sepsis: new synergistic approaches employing organ-on-chip, omics, immune cell phenotyping and in silico modeling to identify new therapeutics.

Sepsis is a global health concern accounting for more than 1 in 5 deaths worldwide. Sepsis is now defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis can develop from bacterial (gram negative or gram positive), fungal or viral (such as COVID) infections. However, therapeutics developed in animal models and traditional in vitro sepsis models have had little success in clinical trials, as these models have failed to fully replicate the underlying pathophysiology and heterogeneity of the disease. The current understanding is that the host response to sepsis is highly diverse among patients, and this heterogeneity impacts immune function and response to infection. Phenotyping immune function and classifying sepsis patients into specific endotypes is needed to develop a personalized treatment approach. Neutrophil-endothelium interactions play a critical role in sepsis progression, and increased neutrophil influx and endothelial barrier disruption have important roles in the early course of organ damage. Understanding the mechanism of neutrophil-endothelium interactions and how immune function impacts this interaction can help us better manage the disease and lead to the discovery of new diagnostic and prognosis tools for effective treatments. In this review, we will discuss the latest research exploring how in silico modeling of a synergistic combination of new organ-on-chip models incorporating human cells/tissue, omics analysis and clinical data from sepsis patients will allow us to identify relevant signaling pathways and characterize specific immune phenotypes in patients. Emerging technologies such as machine learning can then be leveraged to identify druggable therapeutic targets and relate them to immune phenotypes and underlying infectious agents. This synergistic approach can lead to the development of new therapeutics and the identification of FDA approved drugs that can be repurposed for the treatment of sepsis.

P204 Biomechanical Phenotype of Circulating Neutrophils Is Altered in ANCA Associated Vasculitis

Abstract Background/Aims Real Time-Deformability Cytometry (RT-DC) is a novel technique able to identify morpho-rheological characteristics of individual cells such as size, deformability, and elasticity using only 50µl of whole blood. Cells in suspension flow through a microfluidic channel while hydrodynamic force is applied, leading to reversible deformation of individual cells from shear stress and pressure gradients. Cell brightness and area can be used to identify individual cell types. The morpho-rheological characteristics of immune cells in ANCA associated vasculitis (AAV) are unknown. Methods Whole blood from healthy controls (HC), patients with active AAV, or patients with AAV in remission was analysed using RT-DC. The diagnosis of AAV was based upon positive ANCA testing, with either (i) pauci-immune glomerulonephritis, or (ii) typical clinical features of extra-renal AAV. Patients with active disease may have received steroids prior to sampling but those who received other immunosuppression or cytotoxics were excluded. Remission was defined as BVAS of 0 with prednisolone dose ≤7.5mg/day. Blood was collected into K2 EDTA and diluted 1:20 in 1xPBS/0.6% methylcellulose. Cell suspensions were flowed across a Flic20 polydimethylsiloxane microfluidic chip, containing reservoirs connected by a measurement channel with a 20x20 μm2 cross-section. RT-DC measurements were collected at a flow rate of 0.12μL/s using a high-speed CMOS camera to capture images at a rate of 2000 frames/second. ShapeOut software was used to calculate cell size, deformation, and elasticity. Results There was no difference in neutrophil size between patients with active AAV (n = 15), patients with AAV in remission (n = 31), and HC (n = 15). Neutrophils from patients with active AAV were significantly stiffer than patients in remission or HC, displaying decreased deformation (median 0.084, 0.085, and 0.078 au respectively, p = 0.0068) and increased elasticity (median 0.973, 0.968 and 1.006 au respectively, p = 0.0196). In patients with active AAV, there was strong inverse correlation between neutrophil deformation and disease activity measured by BVAS score (r=-0.573, p = 0.032). Neutrophils isolated from healthy donors exhibited a trend towards decreased deformability when primed with TNF and stimulated with MPO-ANCA IgG compared to unstimulated or cells treated with healthy donor IgG (median 0.086, 0.085, and 0.083 for unstimulated, control IgG, and MPO-ANCA IgG respectively, ns). Conclusion Neutrophils from patients display distinct physical properties in active AAV which correlate with disease activity. Similar results are seen when neutrophils are stimulated in vitro with MPO-ANCA IgG. This phenotype of increased cell stiffness may lead to increased neutrophil retention in pulmonary and renal microvasculature, thus increasing the potential for neutrophil-endothelial cell interactions and microvascular damage. Morphorheological parameters can be rapidly measured using a small volume of whole blood; thus, RT-DC may be a useful technique to aid identification of disease activity in AAV and to guide treatment. Disclosure N. Pisacano: None. S.P. McAdoo: None. J. Guck: None. C.D. Pusey: None. E.R. Chilvers: None. A.C. Cowburn: None. K.M. Lodge: None. M.F. Prendecki: None.

Targeting PDE4B (Phosphodiesterase-4 Subtype B) for Cardioprotection in Acute Myocardial Infarction via Neutrophils and Microcirculation.

Timely and complete restoration of blood flow is the most effective intervention for patients with acute myocardial infarction. However, the efficacy is limited by myocardial ischemia-reperfusion (MI/R) injury. PDE4 (phosphodiesterase-4) hydrolyzes intracellular cyclic adenosine monophosphate and it has 4 subtypes A-D. This study aimed to delineate the role of PDE4B (phosphodiesterase-4 subtype B) in MI/R injury. Mice were subjected to 30-minute coronary artery ligation, followed by 24-hour reperfusion. Cardiac perfusion was assessed by laser Doppler flow. Vasomotor reactivities were determined in mouse and human coronary (micro-)arteries. Cardiac expression of PDE4B, but not other PDE4 subtypes, was increased in mice following reperfusion. PDE4B was detected primarily in endothelial and myeloid cells of mouse and human hearts. PDE4B deletion strikingly reduced infarct size and improved cardiac function 24-hour or 28-day after MI/R. PDE4B in bone marrow-derived cells promoted MI/R injury and vascular PDE4B further exaggerated this injury. Mechanistically, PDE4B mediated neutrophil-endothelial cell interaction and PKA (protein kinase A)-dependent expression of cell adhesion molecules, neutrophil cardiac infiltration, and release of proinflammatory cytokines. Meanwhile, PDE4B promoted coronary microcirculatory obstruction and vascular permeability in MI/R, without affecting flow restriction-induced thrombosis. PDE4B blockade increased flow-mediated vasodilatation and promoted endothelium-dependent dilatation of coronary arteries in a PKA- and nitric oxide-dependent manner. Furthermore, postischemia administration with piclamilast, a PDE4 pan-inhibitor, improved cardiac microcirculation, suppressed inflammation, and attenuated MI/R injury in mice. Incubation with sera from patients with acute myocardial infarction impaired acetylcholine-induced relaxations in human coronary microarteries, which was abolished by PDE4 inhibition. Similar protection against MI/R-related coronary injury was recapitulated in mice with PDE4B deletion or inhibition, but not with the pure vasodilator, sodium nitroprusside. PDE4B is critically involved in neutrophil inflammation and microvascular obstruction, leading to MI/R injury. Selective inhibition of PDE4B might protect cardiac function in patients with acute myocardial infarction designated for reperfusion therapy.

Experimental Approaches to Evaluate Leukocyte-Endothelial Cell Interactions in Sepsis and Inflammation.

Sepsis is a life-threatening syndrome of organ dysfunction caused by a dysregulated host response to infection characterized by excessive neutrophil infiltration into vital organs. In sepsis, patients often die of organ failure and therapies directed against endothelial cell dysfunction and tissue damage are important targets for treatment of this disease. Novel approaches are required to understand the underlying pathophysiology of neutrophil dysregulation and neutrophil-endothelial cell interactions that play a critical role in the early course of organ damage and disruption of endothelial protective barrier. Here, we review methodologies that our laboratories have employed to study neutrophil-endothelial interaction and endothelial barrier function in in vivo and in vitro models of sepsis. We will focus on in vivo rodent models of sepsis and in vitro tools that use human cell culture models under static conditions and the more physiologically relevant biomimetic microfluidic assays. This Methods paper is based on our presentation in the Master Class Symposium at the 41st Annual Conference on Shock 2018.

Neutrophil-endothelial interactions of murine cells is not a good predictor of their interactions in human cells.

All drugs recently developed in rodent models to treat inflammatory disease have failed in clinical trials. We therefore used our novel biomimetic microfluidic assay (bMFA) to determine whether the response of murine cells to inflammatory activation or anti-inflammatory treatment is predictive of the response in human cells. Under physiologically relevant flow conditions, permeability and transendothelial electrical resistance (TEER) of human or mouse lung microvascular endothelial cells (HLMVEC or MLMVEC), and neutrophil-endothelial cell interaction was measured. The differential impact of a protein kinase C-delta TAT peptide inhibitor (PKCδ-i) was also quantified. Permeability of HLMVEC and MLMVEC was similar under control conditions but tumor necrosis factor α (TNF-α) and PKCδ-i had a significantly higher impact on permeability of HLMVEC. TEER across HLMVEC was significantly higher than MLMVEC, but PKCδ-i returned TEER to background levels only in human cells. The kinetics of N-formylmethionyl-leucyl-phenylalanine (fMLP)-mediated neutrophil migration was significantly different between the two species and PKCδ-i was significantly more effective in attenuating human neutrophil migration. However, human and mouse neutrophil adhesion patterns to microvascular endothelium were not significantly different. Surprisingly, while intercellular adhesion molecule 1 (ICAM-1) was significantly upregulated on activated HLMVEC, it was not significantly upregulated on activated MLMVEC. Responses to activation and anti-inflammatory treatment in mice may not always be predictive of their response in humans.

Repurposing pyridoxamine for therapeutic intervention of intravascular cell-cell interactions in mouse models of sickle cell disease.

Adherent neutrophils on vascular endothelium positively contribute to cell-cell aggregation and vaso-occlusion in sickle cell disease (SCD). In the present study, we demonstrated that pyridoxamine, a derivative of vitamin B6, might be a therapeutic agent to alleviate intravascular cell-cell aggregation in SCD. Using real-time intravital microscopy, we found that one oral administration of pyridoxamine, in a dose-dependent manner, increased the rolling influx of neutrophils and reduced neutrophil adhesion to endothelial cells in cremaster microvessels of SCD mice challenged with hypoxia-reoxygenation. Short-term treatment also mitigated neutrophil-endothelial cell and neutrophil-platelet interactions in the microvessels and improved the survival of SCD mice challenged with tumor necrosis factor-α. The inhibitory effects of pyridoxamine on intravascular cell-cell interactions were enhanced by co-treatment with hydroxyurea. We observed that long-term (5.5 months) oral treatment with pyridoxamine significantly reduced the adhesive function of neutrophils and platelets, and down-regulated the expression of E-selectin and intercellular adhesion molecule-1 on the vascular endothelium in tumor necrosis factor-α-challenged SCD mice. Ex vivo studies revealed that the surface amount of αMβ2 integrin was significantly decreased in stimulated neutrophils isolated from SCD mice treated with pyridoxamine-containing water. Studies using platelets and neutrophils from SCD mice and patients suggested that treatment with pyridoxamine reduced the activation state of platelets and neutrophils. These results suggest that pyridoxamine may be a novel therapeutic and a supplement to hydroxyurea to prevent and treat vaco-occlusion events in SCD.

Protein Kinase C-Delta (PKCδ) Tyrosine Phosphorylation is a Critical Regulator of Neutrophil-Endothelial Cell Interaction in Inflammation.

Neutrophil dysfunction plays an important role in inflammation-induced tissue injury. Previously, we identified protein kinase C-δ (PKCδ) as a critical controller of neutrophil activation and trafficking but how PKCδ is regulated in inflammation has not been delineated. PKCδ activity is regulated by tyrosine phosphorylation on multiple sites. Tyrosine155 is a key regulator of apoptosis and gene expression, but its role in proinflammatory signaling is not known. In-vitro studies - superoxide anion (O2) and neutrophil extracellular traps (NETs) were measured in bone marrow neutrophils (BMN) isolated from wild type (WT) and PKCδY155F knock-in mice (PKCδ tyrosine 155 → phenylalanine). Our novel 3D biomimetic microfluidic assay (bMFA) was used to delineate PKCδ-mediated regulation of individual steps in neutrophil adhesion and migration using WT and PKCδY155F BMN and mouse lung microvascular endothelial cells (MLMVEC). In-vivo studies - WT and PKCδY155F knock-in mice underwent sham or cecal ligation and puncture surgery and the lungs harvested 24 h post-surgery. In vitro - PKCδY155F BMN had significantly reduced O2 and NETs release compared with WT. WT BMN, but not PKCδY155F BMN, demonstrated significant adhesion and migration across tumor necrosis factor-activated MLMVEC in bMFA. PKCδ inhibition significantly reduced WT BMN adhesion and migration under low shear and near bifurcations, but had no effect on PKCδY155F BMN. In vivo - mutation of PKCδ tyrosine 155 significantly decreased neutrophil migration into the lungs of septic mice. PKCδ tyrosine 155 is a key phosphorylation site controlling proinflammatory signaling and neutrophil-endothelial cell interactions. These studies provide mechanistic insights into PKCδ regulation during inflammation.

The Role of Tyrosine Phosphorylation of Protein Kinase C Delta in Infection and Inflammation.

Protein Kinase C (PKC) is a family composed of phospholipid-dependent serine/threonine kinases that are master regulators of inflammatory signaling. The activity of different PKCs is context-sensitive and these kinases can be positive or negative regulators of signaling pathways. The delta isoform (PKCδ) is a critical regulator of the inflammatory response in cancer, diabetes, ischemic heart disease, and neurodegenerative diseases. Recent studies implicate PKCδ as an important regulator of the inflammatory response in sepsis. PKCδ, unlike other members of the PKC family, is unique in its regulation by tyrosine phosphorylation, activation mechanisms, and multiple subcellular targets. Inhibition of PKCδ may offer a unique therapeutic approach in sepsis by targeting neutrophil-endothelial cell interactions. In this review, we will describe the overall structure and function of PKCs, with a focus on the specific phosphorylation sites of PKCδ that determine its critical role in cell signaling in inflammatory diseases such as sepsis. Current genetic and pharmacological tools, as well as in vivo models, that are used to examine the role of PKCδ in inflammation and sepsis are presented and the current state of emerging tools such as microfluidic assays in these studies is described.

Microvascular Mechanisms of Polyphosphate-Induced Neutrophil-Endothelial Cell Interactions in vivo

Background: Polyphosphates (PolyPs) have been reported to exert pro-inflammatory effects. However, the molecular mechanisms regulating PolyP-provoked tissue accumulation of leukocytes are not known. The aim of the present investigation was to determine the role of specific adhesion molecules in PolyP-mediated leukocyte recruitment. Methods: PolyPs and TNF-α were intrascrotally administered, and anti-P-selectin, anti-E-selectin, anti-P-selectin glycoprotein ligand-1 (PSGL-1), anti-membrane-activated complex-1 (Mac-1), anti-lymphocyte function antigen-1 (LFA-1), and neutrophil depletion antibodies were injected intravenously or intraperitoneally. Intravital microscopy of the mouse cremaster microcirculation was used to examine leukocyte-endothelium interactions and recruitment in vivo. Results: Intrascrotal injection of PolyPs increased leukocyte accumulation. Depletion of neutrophils abolished PolyP-induced leukocyte-endothelium interactions, indicating that neutrophils were the main leukocyte subtype responding to PolyP challenge. Immunoneutralization of P-selectin and PSGL-1 abolished PolyP-provoked neutrophil rolling, adhesion, and emigration. Moreover, immunoneutralization of Mac-1 and LFA-1 had no impact on neutrophil rolling but markedly reduced neutrophil adhesion and emigration evoked by PolyPs. Conclusion: These results suggest that P-selectin and PSGL-1 exert important roles in PolyP-induced inflammatory cell recruitment by mediating neutrophil rolling. In addition, our data show that Mac-1 and LFA-1 are necessary for supporting PolyP-triggered firm adhesion of neutrophils to microvascular endothelium. These novel findings define specific molecules as potential targets for pharmacological intervention in PolyP-dependent inflammatory diseases.

Synthesis and biological evaluation of sphingosine kinase 2 inhibitors with anti-inflammatory activity.

The synthesis of inhibitors of SphK2 with novel structural scaffolds is reported. These compounds were designed from a molecular modeling study, in which the molecular interactions stabilizing the different complexes were taken into account. Particularly interesting is that 7-bromo-2-(2-phenylethyl)-2,3,4,5-tetrahydro-1,4-epoxynaphtho[1,2-b]azepine, which is a selective inhibitor of SphK2, does not exert any cytotoxic effects and has a potent anti-inflammatory effect. It was found to inhibit mononuclear cell adhesion to the dysfunctional endothelium with minimal impact on neutrophil-endothelial cell interactions. The information obtained from our theoretical and experimental study can be useful in the search for inhibitors of SphK2 that play a prominent role in different diseases, especially in inflammatory and cardiovascular disorders.

Ability of Platelet-Derived Extracellular Vesicles to Promote Neutrophil-Endothelial Cell Interactions

We tested the ability of platelet-derived extracellular vesicles (PEV) to promote adhesion of flowing neutrophils to endothelial cells (EC). PEV were collected from platelets stimulated with collagen-related peptide, and differential centrifugation was used to collect larger vesicles enriched for platelet membrane microvesicles (PMV) or smaller vesicles enriched for platelet exosomes (Pexo). Vesicle binding and resultant activation of neutrophils and EC were assessed by flow cytometry. Flow-based adhesion assays assessed binding of neutrophils directly to deposited vesicles or to EC, after neutrophils or EC had been treated with vesicles. PEV bound efficiently to neutrophils or EC, with resultant upregulation of activation markers. Binding was Ca++-dependent and dominantly mediated by CD62P for neutrophils or by integrins for EC. Deposited PEV supported mainly transient attachments of flowing neutrophils through CD62P and some stable adhesion through CXC-chemokines. Neutrophil adhesion to EC was promoted when either cell was pre-treated with PEV, although the effect was less prominent when EC were pre-activated with tumor necrosis factor-α. The pro-adhesive effects on neutrophils could largely be attributed to the larger PMV rather than Pexo. Thus, surface-bound PEV can capture flowing neutrophils, while PEV also activate neutrophils and EC to promote interactions. PEV may potentiate inflammatory responses after tissue injury.

PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.

In the event of a radiologic catastrophe, endothelial cell and neutrophil dysfunction play important roles in tissue injury. Clinically available therapeutics for radiation-induced vascular injury are largely supportive. PKCδ was identified as a critical regulator of the inflammatory response, and its inhibition was shown to protect critical organs during sepsis. We used a novel biomimetic microfluidic assay (bMFA) to interrogate the role of PKCδ in radiation-induced neutrophil-endothelial cell interaction and endothelial cell function. HUVECs formed a complete lumen in bMFA and were treated with 0.5, 2, or 5 Gy ionizing radiation (IR). At 24 h post-IR, the cells were treated with a PKCδ inhibitor for an additional 24 h. Under physiologic shear flow, the role of PKCδ on endothelium function and neutrophil adherence/migration was determined. PKCδ inhibition dramatically attenuated IR-induced endothelium permeability increase and significantly decreased neutrophil migration across IR-treated endothelial cells. Moreover, neutrophil adhesion to irradiated endothelial cells was significantly decreased after PKCδ inhibition in a flow-dependent manner. PKCδ inhibition downregulated IR-induced P-selectin, intercellular adhesion molecule 1, and VCAM-1 but not E-selectin overexpression. PKCδ is an important regulator of neutrophil-endothelial cell interaction post-IR, and its inhibition can serve as a potential radiation medical countermeasure.-Soroush, F., Tang, Y., Zaidi, H. M., Sheffield, J. B., Kilpatrick, L. E., Kiani, M. F. PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.

Small GTPase-dependent regulation of leukocyte-endothelial interactions in inflammation.

Inflammation is a complex biological response that serves to protect the body's tissues following harmful stimuli such as infection, irritation or injury and initiates tissue repair. At the start of an inflammatory response, pro-inflammatory mediators induce changes in the endothelial lining of the blood vessels and in leukocytes. This results in increased vascular permeability and increased expression of adhesion proteins, and promotes adhesion of leukocytes, especially neutrophils to the endothelium. Adhesion is a prerequisite for neutrophil extravasation and chemoattractant-stimulated recruitment to inflammatory sites, where neutrophils phagocytose and kill microbes, release inflammatory mediators and cross-talk with other immune cells to co-ordinate the immune response in preparation for tissue repair. Many signalling proteins are critically involved in the complex signalling processes that underpin the inflammatory response and cross-talk between endothelium and leukocytes. As key regulators of cell-cell and cell-substratum adhesion, small GTPases (guanosine triphosphatases) act as important controls of neutrophil-endothelial cell interactions as well as neutrophil recruitment to sites of inflammation. Here, we summarise key processes that are dependent upon small GTPases in leukocytes during these early inflammatory events. We place a particular focus on the regulation of integrin-dependent events and their control by Rho and Rap family GTPases as well as their regulators during neutrophil adhesion, chemotaxis and recruitment.

Myeloperoxidase Negatively Regulates Neutrophil-Endothelial Cell Interactions by Impairing αMβ2 Integrin Function in Sterile Inflammation.

Interactions of neutrophils with endothelial cells (ECs) and platelets contribute to tissue damage and vascular occlusion under sterile inflammatory conditions. However, the molecular mechanisms regulating the cell–cell interactions remain poorly understood. Previous studies suggest that reactive oxygen species, such as hydrogen peroxide (H2O2), produced from NADPH oxidase 2 play a critical role in platelet–neutrophil interactions by regulating the function of neutrophil αMβ2 integrin during sterile inflammation. In this study, we further demonstrate a crucial role for myeloperoxidase (MPO) in regulating the adhesive function of neutrophils through αMβ2 integrin. Using real-time fluorescence intravital microscopy and in vitro assays, we showed that loss of MPO promoted neutrophil–EC interactions and neutrophil emigration but did not affect neutrophil–platelet interactions under inflammatory conditions. Using genetic and pharmacologic approaches, we found that following agonist stimulation, MPO knockout (KO) neutrophils exhibited a significant increase in extracellular H2O2 and surface level of αMβ2 integrin and that these effects were dependent on MPO activity. Our in vivo studies using an ischemia/reperfusion-induced hepatic inflammation model revealed that compared to wild-type mice, neutrophils from MPO KO mice—displayed a pro-migratory phenotype while ameliorating tissue damage. These results suggest that MPO plays a negative role in the adhesive and migratory function of neutrophils by impairing αMβ2 integrin function under sterile inflammatory conditions.

Fasudil inhibits neutrophil-endothelial cell interactions by regulating the expressions of GRP78 and BMPR2

Regulation of leukocyte-endothelial cell interactions and of vascular permeability plays a critical role in the maintenance of functional pulmonary microvascular barriers. Little is yet known about the effect of the Rho-associated protein kinase (ROCK) inhibitor fasudil on leukocyte-endothelial cell interactions or the underlying mechanism. In the present study, as evaluated using co-culture systems of neutrophils and human pulmonary microvascular endothelial cells (HPMECs), fasudil dose-dependently suppressed neutrophil chemotaxis by decreasing the production of chemotactic factors in lipopolysaccharide (LPS)-treated HPMECs. The inhibitory role of fasudil in neutrophil chemotaxis was mediated by down-regulation of the chaperone glucose-regulated protein 78 (GRP78), since the inhibition was abolished by 4-phenyl butyric acid (a chemical chaperone mimicking GRP78). In addition, fasudil inhibited LPS-induced neutrophil-endothelial adhesion by reducing the expression of intercellular adhesion molecule (ICAM)-1. By use of lentiviral transfection in HPMECs, bone morphogenic protein receptor 2 (BMPR2) overexpression suppressed the LPS-induced increase of both ICAM-1 expression and neutrophil-endothelial adhesion, whereas knocking down BMPR2 abolished the inhibitory role of fasudil in both ICAM-1 expression and neutrophil-endothelial adhesion. Moreover, fasudil alleviated LPS-induced hyperpermeability of HPMEC monolayers by leading to the recovery of intercellular junctions, thereafter reduced neutrophil transendothelial cell migration. Therefore, fasudil inhibited leukocyte-endothelial cell interactions and vascular hyperpermeability through modulation of GRP78 and BMPR2 expression, suggesting a potential role for ROCK as a switch for inhibiting leukocyte-endothelial cell interactions.

Activation of the sympathetic nervous system modulates neutrophil function.

Emerging evidence has revealed that noradrenaline (NA), the main neurotransmitter of the sympathetic nervous system (SNS), regulates a variety of immune functions via binding to adrenergic receptors present on immune cells. In this study, we examined the role of NA in the regulation of neutrophil functions. Neutrophils were isolated from the bone marrow of naïve mice and treated with NA at various concentrations to assess the effect on various neutrophil functions. Additionally, we performed cremaster intravital microscopy to examine neutrophil‐endothelial cell interactions following NA superfusion in vivo. In a separate group of animals, mice were subjected to an experimental model of stroke and at 4 and 24 h neutrophils were isolated for assessment on their ability to migrate toward various chemokines. Treatment of neutrophils with NA for 4 h significantly impaired neutrophil chemotaxis and induced an N2 neutrophil phenotype with reduced expression of the genes critical for cytoskeleton remodeling and inflammation. Prolonged NA administration promoted neutrophils to release myeloperoxidase and IL‐6, but suppressed the production of interferon‐γ and IL‐10, reduced neutrophil activation and phagocytosis. Superfusion of NA over the cremaster muscle almost completely inhibited fMLP‐induced neutrophil adhesion/arrest and transmigration. Furthermore, using a mouse model of stroke, a pathological condition in which SNS activation is evident, neutrophils isolated from poststroke mice showed markedly reduced chemotaxis toward all of the chemokines tested. The findings from our study indicate that neutrophil chemotaxis, activation, and phagocytosis can all be negatively regulated in an NA‐dependent manner. A better understanding of the relationship between sympathetic activation and neutrophil function will be important for the development of effective antibacterial interventions.

Metabolic Remodeling of Neutrophils at Inflammatory Site Drives Invadosome Formation Favoring Transcellular Migration

Neutrophils traffic in and out of underlying vascular bed during hematopoiesis and immunosurveillance. However, during inflammatory conditions such as ischemia reperfusion injury or atherosclerosis, excessive neutrophil infiltration into tissue drives disease pathogenesis. Yet, the relationship between neutrophil transmigration and inflammation is ill-defined. Neutrophil extravasation can occur either between two endothelial cells (paracellular) or directly through an endothelial cell body (transcellular). During transcellular migration, neutrophils interact with underlying endothelial cells (EC) via invadosomal structures, which forms a ‘pore’ into endothelial cell membrane, thus facilitating neutrophil migration through EC body. We have recently reported that deficiency in Rap1b, a member of Ras superfamily of GTPase, enhanced neutrophil transcellular migration, invadosomal structures and metalloproteinase (MMP) release (Kumar et al, JEM, 2014), in a manner dependent on high Akt activity. Further, Rap1-deficiency increased neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock, suggesting mode of neutrophil migration may influence inflammatory outcome. Here, to further understand which factors drive neutrophil transcellular migration, we analyzed protein content of Rap1b-/- invadosomal structures during transcellular diapedesis. For this, neutrophils were stimulated in transwell filters of 1µM pore size, with FMLP placed in the lower chamber, allowing only invadosomal protrusions into the pores. After removing the cell body from top of the filter, mass spectrometric analysis was performed on the invadosomal fraction. About 680 proteins were identified in protrusions isolated from WT or Rap1b-/- neutrophils. As expected, majority of them were cytoskeleton and adhesion proteins. Interestingly, Rap1b-/- invadosomal structures contained more enzymes of glycolytic pathways, including HK1, Lactate dehydrogenase A (LDHA) and phosphoglycerate kinase1 (PGK1). Immunofluorescent staining and western blotting confirmed this observation. Importantly, glycolytic enzymes were present at the tip of the protrusions in colocalization with F-actin suggesting site specific glycolytic activity, raising the hypothesis that metabolic remodeling may influence the route of neutrophil migration. LDHA converts pyruvate to lactate and subsequent milieu acidification, which can then cause MMP activation. Consistently, Rap1b-/- neutrophils exhibited increased uptake of glucose analogue (2-NBDG) and concurrent intracellular acidification, as detected by pH sensitive dye. To investigate the importance of LDHA activity during transcellular migration, Rap1b-/- neutrophils were treated with a specific pharmacological inhibitor of LDHA, namely FX11. In vitro, FX11 treatment significantly decreased transcellular migration of Rap1b-/- neutrophils. It also reduced invadosome formation of Rap1b-/- neutrophils within transwell pores, as well as neutrophil acidity and MMP activity. Furthermore, during neutrophil-endothelial cell interactions in vitro, Rap1b-/- neutrophils caused F-actin depolymerization in EC, likely facilitating transcellular passage; this was inhibited by FX11. To examine its effect in vivo, under same inflammatory microenvironment, Rap1b-/- and WT neutrophils were tagged with cell tracker dyes and transferred to recipient mice, treated with FX11 or DMSO control. Ear microvasculature was stimulated with FMLP and labeled with PECAM antibody to visualize EC junctions. Rap1b-/- neutrophils migrated out of vessels at higher frequency than WT cells, which was abrogated by FX11 treatment. Moreover, treatment with FX11 reduced the number of Rap1b-/- neutrophils located away from EC junction (transcellular route), in vivo. These results suggest enhanced local glycolytic metabolism and LDHA activity could act as critical regulators of transcellular migration. Increase in extracellular acidification mediated by LDHA activity, could affect endothelial permeability and alter neutrophil migratory behavior affecting outcome of inflammation. Since milieu acidification plays a major role in ischemic damage to the heart, these findings may be clinically important for our understanding of hyperinflammatory disorders. DisclosuresNo relevant conflicts of interest to declare.

Mast cells mediate early neutrophil recruitment and exhibit anti-inflammatory properties via the formyl peptide receptor 2/lipoxin A4 receptor.

In recent years, studies have focused on the resolution of inflammation, which can be achieved by endogenous anti-inflammatory agonists such as Annexin A1 (AnxA1). Here, we investigated the effects of mast cells (MCs) on early LPS-induced neutrophil recruitment and the involvement of the AnxA1-formyl peptide receptor 2/ALX (FPR2/ALX or lipoxin A4 receptor) pathway. Intravital microscopy (IVM) was used to visualize and quantify the effects of LPS (10μg per mouse i.p.) on murine mesenteric cellular interactions. Furthermore, the role that MCs play in these inflammatory responses was determined in vivo and in vitro, and effects of AnxA1 mimetic peptide Ac2-26 were assessed. LPS increased both neutrophil endothelial cell interactions within the mesenteric microcirculation and MC activation (determined by IVM and ruthenium red dye uptake), which in turn lead to the early stages of neutrophil recruitment. MC recruitment of neutrophils could be blocked by preventing the pro-inflammatory activation (using cromolyn sodium) or enhancing an anti-inflammatory phenotype (using Ac2-26) in MCs. Furthermore, MCs induced neutrophil migration in vitro, and MC stabilization enhanced the release of AnxA1 from neutrophils. Pharmacological approaches (such as the administration of FPR pan-antagonist Boc2, or the FPR2/ALX antagonist WRW4) revealed neutrophil FPR2/ALX to be important in this process. Data presented here provide evidence for a role of MCs, which are ideally positioned in close proximity to the vasculature, to act as sentinel cells in neutrophil extravasation and resolution of inflammation via the AnxA1-FPR2/ALX pathway.

Patent highlights December 2016-January 2017.

Patent highlights December 2016-January 2017.

Specific Inhibition of AKT with ARQ 092, an Orally-Available Selective AKT Inhibitor, Attenuates Acute Vaso-Occlusive Events in Sickle Cell Disease

▪Previous studies have identified the Ser/Thr protein kinase, AKT, as a therapeutic target for thromboinflammatory diseases. Recently, we have shown that the basal levels of AKT phosphorylation were significantly increased in neutrophils and platelets isolated from sickle cell disease (SCD) patients compared with those cells from healthy donors and that specific inhibition of AKT2 impaired neutrophil-endothelial cell and neutrophil-platelet interactions in venules of Berkeley (SCD) mice (Li et al. J Clin Invest 2014; Barazia et al. Blood 2015). Although our results suggest that AKT2 inhibition may be a novel therapy to treat a vaso-occlusive crisis in SCD patients, no AKT2 specific inhibitor is available in clinic. Using ARQ 092, an orally-available, allosteric AKT inhibitor that is currently in Phase I clinical trials in oncology indications and AKT-driven overgrowth disease, Proteus syndrome, here we report that specific inhibition of AKT attenuates the adhesive function of neutrophils and platelets from sickle cell disease (SCD) patients in vitro and heterotypic cell-cell interactions in the Berkeley mouse model of SCD in vivo. Studies using neutrophils and platelets isolated from SCD patients revealed that treatment with 50-500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Consistently, treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Using in vivo real-time fluorescence intravital microscopy, we demonstrated that short-term oral administration of ARQ 092 (100 mg/kg body weight) or hydroxyurea (HU, 250 mg/kg body weight), a main therapy for the treatment of SCD, diminishes neutrophil-endothelial cell and neutrophil-platelet interactions in venules of Berkeley mice challenged with TNF-α to induce a vaso-occlusive crisis. Co-administration of HU and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) in the cremaster vessel wall, and improved survival in these mice. Ex vivo studies in Berkeley mice suggested that co-administration of HU and ARQ 092 efficiently blocks neutrophil and platelet activation and reactive oxygen species generation. The synergistic effects of both inhibitors resulted from increased NO production by HU and specific inhibition of AKT by ARQ 092. Our results provide important evidence that ARQ 092 has potential as a novel drug to be developed to prevent or treat the acute vaso-occlusive complications in SCD patients. DisclosuresAbbadessa:ArQule, Inc: Employment. Yu:ArQule, Inc: Employment. Schwartz:ArQule, Inc: Employment.