Neutrophil Transmigration Research Articles

Pneumolysin Induces 12-Lipoxygenase-Dependent Neutrophil Migration during Streptococcus pneumoniae Infection.

Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the α-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX-dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.

A novel 4-dimensional live-cell imaging system to study leukocyte-endothelial dynamics in ANCA-associated vasculitis

Neutrophils, monocytes and the endothelium are critical to ANCA-associated vasculitis (AAV) pathogenesis. This study aimed to develop a 4-dimensional (4D) live-cell imaging system that would enable investigation of spatial and temporal dynamics of these cells in health and disease. We further aimed to validate this system using autologous donor serum from AAV patients and polyclonal ANCA IgG, as well as exploring its potential in the pre-clinical testing of putative therapeutic compounds. Neutrophils and monocytes were isolated from peripheral venous blood of AAV patients or healthy controls and co-incubated on an endothelial monolayer in the presence of autologous serum. Alternatively, polyclonal ANCA IgG was used, following TNF-α priming, and imaged in 4-dimensions for 3 h using a spinning disc confocal microscope. Volocity 6.3® analysis software was used for quantification of leukocyte dynamics. The use of autologous serum resulted in increased neutrophil degranulation (p = .002), transmigration (p = .0096) and monocyte transcellular transmigration (p = .0013) in AAV patients. Polyclonal MPO-ANCA IgG induced neutrophil degranulation (p < .001) in this system. C5aR1 antagonism reduced neutrophil degranulation (p < .0002). We have developed a novel 4D in vitro system that allows accurate quantification of multiple neutrophil- and monocyte-endothelial interactions in AAV in a single assay. This system has the potential to highlight dynamics key to pathophysiology of disease, as well investigating the impact of potential therapeutics on these functions.

Exhaled Volatile Organic Compounds Precedes Pulmonary Injury in a Swine Pulmonary Oxygen Toxicity Model.

PurposeInspiring high partial pressure of oxygen (FiO2 > 0.6) for a prolonged duration can lead to lung damage termed pulmonary oxygen toxicity (PO2T). While current practice is to limit oxygen exposure, there are clinical and military scenarios where higher FiO2 levels and partial pressures of oxygen are required. The purpose of this study is to develop a non-invasive breath-based biomarker to detect PO2T prior to the onset of clinical symptoms.MethodsMale Yorkshire swine (20–30 kg) were placed into custom airtight runs and randomized to air (0.209 FiO2, n = 12) or oxygen (>0.95 FiO2, n = 10) for 72 h. Breath samples, arterial blood gases, and vital signs were assessed every 12 h. After 72 h of exposure, animals were euthanized and the lungs processed for histology and wet-dry ratios.ResultsSwine exposed to hyperoxia developed pulmonary injury consistent with PO2T. Histology of oxygen-exposed swine showed pulmonary lymphatic congestion, epithelial sloughing, and neutrophil transmigration. Pulmonary injury was also evidenced by increased interstitial edema and a decreased PaO2/FiO2 ratio in the oxygen group when compared to the air control group. Breath volatile organic compound (VOC) sample analysis identified six VOCs that were combined into an algorithm which generated a breath score predicting PO2T with a ROC/AUC curve of 0.72 defined as a of PaO2/FiO2 ratio less than 350 mmHg.ConclusionExposing swine to 72 h of hyperoxia induced a pulmonary injury consistent with human clinical endpoints of PO2T. VOC analysis identified six VOCs in exhaled breath that preceded PO2T. Results show promise that a simple, non-invasive breath test could potentially predict the risk of pulmonary injury in humans exposed to high partial pressures of oxygen.

Kinin B1 Receptor Is Important in the Pathogenesis of Myeloperoxidase-Specific ANCA GN.

Myeloperoxidase-specific ANCA (MPO-ANCA) are implicated in the pathogenesis of vasculitis and GN. Kinins play a major role during acute inflammation by regulating vasodilatation and vascular permeability and by modulating adhesion and migration of leukocytes. Kinin system activation occurs in patients with ANCA vasculitis. Previous studies in animal models of GN and sclerosing kidney diseases have demonstrated protective effects of bradykinin receptor 1 (B1R) blockade via interference with myeloid cell trafficking. To investigate the role of B1R in a murine model of MPO-ANCA GN, we evaluated effects of B1R genetic ablation and pharmacologic blockade. We used bone marrow chimeric mice to determine the role of B1R in bone marrow-derived cells (leukocytes) versus nonbone marrow-derived cells. We elucidated mechanisms of B1R effects using in vitro assays for MPO-ANCA-induced neutrophil activation, endothelial adherence, endothelial transmigration, and neutrophil adhesion molecule surface display. B1R deficiency or blockade prevented or markedly reduced ANCA-induced glomerular crescents, necrosis, and leukocyte influx in mice. B1R was not required for in vitro MPO-ANCA-induced neutrophil activation. Leukocyte B1R deficiency, but not endothelial B1R deficiency, decreased glomerular neutrophil infiltration induced by MPO-ANCA in vivo. B1R enhanced ANCA-induced neutrophil endothelial adhesion and transmigration in vitro. ANCA-activated neutrophils exhibited changes in Mac-1 and LFA-1, important regulators of neutrophil endothelial adhesion and transmigration: ANCA-activated neutrophils increased surface expression of Mac-1 and increased shedding of LFA-1, whereas B1R blockade reduced these effects. The leukocyte B1R plays a critical role in the pathogenesis of MPO-ANCA-induced GN in a mouse model by modulating neutrophil-endothelial interaction. B1R blockade may have potential as a therapy for ANCA GN and vasculitis.

Epithelial membrane protein 2 governs transepithelial migration of neutrophils into the airspace.

Whether respiratory epithelial cells regulate the final transit of extravasated neutrophils into the inflamed airspace or are a passive barrier is poorly understood. Alveolar epithelial type 1 (AT1) cells, best known for solute transport and gas exchange, have few established immune roles. Epithelial membrane protein 2 (EMP2), a tetraspan protein that promotes recruitment of integrins to lipid rafts, is highly expressed in AT1 cells but has no known function in lung biology. Here, we show that Emp2-/- mice exhibit reduced neutrophil influx into the airspace after a wide range of inhaled exposures. During bacterial pneumonia, Emp2-/- mice had attenuated neutrophilic lung injury and improved survival. Bone marrow chimeras, intravital neutrophil labeling, and in vitro assays suggested that defective transepithelial migration of neutrophils into the alveolar lumen occurs in Emp2-/- lungs. Emp2-/- AT1 cells had dysregulated surface display of multiple adhesion molecules, associated with reduced raft abundance. Epithelial raft abundance was dependent upon putative cholesterol-binding motifs in EMP2, whereas EMP2 supported adhesion molecule display and neutrophil transmigration through suppression of caveolins. Taken together, we propose that EMP2-dependent membrane organization ensures proper display on AT1 cells of a suite of proteins required to instruct paracellular neutrophil traffic into the alveolus.

Analysis of Neutrophil Transmigration Through Epithelial Cell Monolayers.

Transmigration of neutrophils through an epithelial layer, such as in the intestine or lung, is a necessary response to a perceived attack at the mucosal surface of that tissue. This process is dynamically regulated by a number of interactive events between the neutrophil and other cell types and allows for an effective and localized neutrophil response. However, in certain inflammatory diseases, including inflammatory bowel disease and chronic obstructive pulmonary disease (COPD), persistent neutrophil accumulation can contribute to disease pathology. Elucidating the mechanisms of this aberrant neutrophil accumulation is crucial for understanding and ameliorating these disease processes. The method we describe here is a controlled model system that allows for the investigation of the interactive signals involved in neutrophil transmigration through epithelial barriers, and possible mechanisms of deregulation of this process.

Neutrophil-Mediated Transport is Crucial for Short-Circulating Magnetic Nanoparticles Delivery to Tumors

Recently neutrophil-based nanoparticles (NP) drug delivery systems have gained considerable attention in cancer therapy. Numerous studies have been conducted to identify optimal NP parameters for passive tumor targeting, while there is a fundamental dearth of knowledge about the factors governing cell-mediated delivery. Here, using intravital microscopy and magnetic resonance imaging we describe accumulation dynamics of 140 nm magnetic cubes and clusters in murine breast cancer (4T1) and colon cancer (CT26) models. Notwithstanding rapid clearance from the blood flow, NP readily accumulated in tumors at later time points. Both NP types were captured mostly by intravascular neutrophils immediately after injection and transmigration of NP-bound neutrophils through the vessel wall was first shown in real-time. A dramatic drop in NP accumulation upon Ly6G and Gr1 depletion further confirmed neutrophils role as biocarrier for targeting tumors. Of note, for shorter circulating NP cell-dependent delivery route was more impactful, while the accumulation of longer circulating counterpart was less compromised by neutrophil depletion. Neutrophil-mediated transport was also shown to depend on tumor type being more efficient in neutrophil-rich tumors. Revealing NP characteristics and host factors influencing neutrophil-based tumor targeting will help to rationally design novel drug delivery systems for improved cancer treatment.

CD47: role in the immune system and application to cancer therapy.

CD47 is a widely expressed cellular receptor well known for its immunoregulatory functions. By interacting with its ligands, including thrombospondin-1 (TSP-1), signal regulatory protein α (SIRPα), integrins, and SH2-domain bearing protein tyrosine phosphatase substrate-1 (SHPS-1), it modulates cellular phagocytosis by macrophages, transmigration of neutrophils and activation of dendritic cells, T cells and B cells. Ample studies have shown that various types of cancer express high levels of CD47 to escape from the immune system. Based on this observation, CD47 is currently considered as a prominent target in cancer therapy. Here, we review the role of CD47 in the maintenance of immune system homeostasis. We also depict three emerging CD47-targeting strategies for cancer therapy, including the use of mimicry peptides, antibodies, and gene silencing strategies. Among these approaches, the most advanced one is the use of anti-CD47 antibodies, which enhances cancer cell phagocytosis via inhibition of the CD47-SIRPα axis. These antibodies can also achieve higher anti-cancer efficacies when combined with chemotherapy and immunotherapy and hold promise for improving the survival of patients with cancer.

Prokaryotic RNA activates endothelial cells promoting neutrophil transmigration.

Endothelial cell (EC)-neutrophil (PMN) interactions are crucial in the resolution of bacterial infections. Prokaryotic RNA (pRNA) has been reported as a pathogen-associated molecular pattern that is released from bacteria upon death and is able to activate PMN. In this work, we studied the effects of pRNA on EC and investigated whether these effects could modulate EC-PMN interaction. For this purpose, we purified total pRNA from Escherichia coli and used it as a stimulus for Human Umbilical Vein Endothelial Cells (HUVEC). We found that the incubation of pRNA with HUVEC caused the increase of surface intercellular adhesion molecule 1 (ICAM-1 or CD54) expression on HUVEC, and the secretion of IL-8 and von Willebrand factor, characteristics consistent with HUVEC activation, without causing toxic effects. Moreover, pRNA-treated HUVEC also induced PMN adhesion and the conditioned medium obtained from treated-HUVEC was chemotactic for PMN and caused their activation, as determined by CD11b upregulation. As reported previously, the degradation products of pRNA induced similar biological effects. The treatment of HUVEC with endocytosis inhibitors revealed that the entry of pRNA partially relied on a clathrin-dependent mechanism, whereas the effects of degradation products could not be inhibited by any of the inhibitors tested. Using a transwell system, we found that pRNA or degraded pRNA were also able to stimulate HUVEC when recognized from the basolateral side. Ourresults indicate that pRNA activates EC, resulting in the modulation ofEC-PMN interaction by inducing PMN chemotaxis, adhesion and activation. In the context of infection, pRNA sensed by EC and PMN could favor bacterial clearance.

Leukocyte Adhesion Deficiency Type 1: A Case Series and Review of the Literature

Leukocyte adhesion deficiency type-1 (LAD-1) is a rare primary immunodeficiency that is characterised by compromised neutrophil adhesion and transmigration to infection or inflammation sites. In this article, the authors report the cases of five patients with LAD-1 deficiency. The aim of this study is the demonstration of the wide variety of manifestations in these patients with a review of the literature. Patients were both male and female, with their ages ranging from 1 month to 10 years old. Omphalitis was the most common presentation in this series, followed by pneumonia and gingivitis. Impaired wound healing and cellulitis were other common findings in these patients. Two of the patients did not show delayed separation of the umbilical cord. The findings indicate that LAD-1 remains a life-threatening condition with omphalitis, oral, skin, respiratory tract, and ear infections as the most common complications. Early identification of these patients is essential in ensuring a definitive diagnosis and early implementation of haematopoietic stem cell transplantation.

PB2221 OVEREXPRESSION OF PRV1 GENE IN MYELOID NEOLASMS AND ASSOCIATION WITH OTHER MOELCUALR ABNORMALITIES

Background:The polycythemia rubra vera 1 gene(PRV1) gene encodes a cell surface glycoprotein that plays a role in neutrophil activation and transmigration. PRV1 overexpression was found in significant proportion of patients(pts) with BCR‐ABL1‐negative myeloproloferative neoplasms(MPN). In the pre‐JAK2 mutation era testing of PRV1expression was recommented to differentiate polycythemia vera(PV) and essential thrombocytemia(ET) from secondary erythrocytosis or thombocytosis, respectively. The available data concerning the incidence of PRV1 overexpression, as well as its association with JAK2 gene mutation status is highly variable.Aims:To determine the incidence of PRV1 overexpression in different myeloid neoplasms and its association with other molecular abnormalities.Methods:Overall 167 pts with MPN were included in this study:43 with ET; 30 – PV; 15 ‐ primary myelofibrosis(PMF); 22 ‐ unspecified MPN(uPMN), 53 ‐ chronic myeloid leukemia(CML), including 18 pts at diagnosis and 35 pts on therapy with TKI; 4 pts with chronic myelomonocytic leukemia(CMML). PRV1 expression level was evaluated by reverse‐transcription polymerase chain reaction(RT‐PCR), with co‐amplification of b‐actin RNA as an internal control using total RNA from peripheral blood mononuclear cells. Semi‐quantitative characterization of PRV1 expression was done using 3grade score system: negative (‐): no product of PRV1 amplification; moderately elevated expression (+): PRV1 product with lower intensity than b‐actin product; strong overexpression(++): product of PRV1 amplification with intensity equivalent or higher than that of the b‐actin. To determine the association of PRV1 expression with JAK2 mutation status PRV1 expression was assessed in serial dilutions of homozygous JAK2V617F RNA in JAK2 wild type RNA. The PRV1 expression was clearly visible in up to 25% JAK2V617F mutation load. All pts were tested for the presence of BCR‐ABL1, JAK2V617F, and some cases for CALR and/or MPL W515L/K mutations.Results:Overall, PRV1 overexpression was found in 80.5% of pts with MPN (Table 1): 83.6%(n = 36) in ET; 93.3%(n = 28) in PV; 73.3%(n = 11) in PMF; 61.1%(n = 11) in CML(including 1 pt with co‐expression of BCR‐ABL1 and JAK2V617F); 95.5%(n = 21) in uMPN. PRV1 overexpression was significantly associated with JAK2V617F (+) status in BCR‐ABL1(‐) MPN with frequency of ranging between 73.3% and 95.5%. Interestingly, 3 JAK2V617F (‐) pts with PMF and PRV1 overexpression were found positive for CALR(n = 2) or MPL(n = 1) mutations.It was found higher prevalence of pts with strong overexpression PRV1(++) ‐ 67.9%(n = 19) in PV, 71.4%(n = 15) in uMPN, 77.8%(n = 7/9) in CML, with highest value in PMF ‐ 81.8%(n = 9). In contrast, in ET only 58.3%(n = 21) were PRV1(++). In CMML 1 pt was PRV1(++) and the second was PRV1(+). Strong PRV1 expression clearly correlated with JAK2V617F mutation load: PRV1(++) was found 24/28(85.7%) of homozygous pts, compared to 20/46 (43.5%) in heterozygous pts and 21/38 (55.3%) in JAK2 V617F(‐) pts. In CML pts on TKI therapy, PRV1 overexpression was found in 6/35 (17.1%) ‐ cases without any molecular response.Summary/Conclusion:PRV1 overexpression was found in a significant proportion of pts with BCR‐ABL1‐pos and BCR‐ABL1‐neg MPN, with highest frequency in PV and lowest in CML. Despite the clear association of PRV1 overexpression and JAK2V617F status, overexpression was also found in the absence of mutation suggesting alternative underlying mechanisms of activation of PRV1.image

Pentraxin 3 regulates neutrophil infiltration to the brain during neuroinflammation

Introduction: The acute phase protein pentraxin 3 (PTX3) is known for its anti-inflammatory effects through downregulating neutrophil transmigration during peripheral inflammation. Furthermore, we have previously demonstrated a neuroprotective and neuroreparative effect of PTX3 after cerebral ischaemia. Here we investigated, to our knowledge for the first time, the role of PTX3 in neutrophil transmigration and neurotoxicity following lipopolysaccharide (LPS)-induced cerebral inflammation and cerebral ischaemia. Methods: Neutrophil transmigration through interleukin-1β (IL-1β) activated brain endothelium and neurotoxicity of neutrophils isolated from wild-type (WT) or PTX3 knock-out (KO) mice was assessed in vitro. Primary cortical neuronal death after treatment with transmigrated neutrophils was quantified by lactate dehydrogenase (LDH) assay. Cerebral inflammation or ischemia was induced in WT and PTX3 KO mice via intrastriatal LPS injection or by transient middle cerebral artery occlusion (MCAo) respectively. Subsequent neutrophil infiltration in the brain was assessed by immunohistochemistry and the expression of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1β by enzyme-linked immunosorbent assay (ELISA). Results: Neutrophils isolated from WT mice after intrastriatal LPS injection transmigrated significantly more through IL-1β activated brain endothelium compared to neutrophils from PTX3 KO mice. Transmigrated WT and PTX3 KO neutrophils were significantly more neurotoxic than corresponding non-transmigrated neutrophils; however, no significant differences in neurotoxicity between genotypes were observed. PTX3 reduced the number of transmigrated neutrophils to the brain after intrastriatal LPS injection. Furthermore, PTX3 KO mice showed significantly increased levels of neutrophils in the brain after LPS administration or in the ischaemic hemisphere after MCAo, compared to WT mice. Conclusion: Our study shows that PTX3 regulates neutrophil transmigration in the CNS during neuroinflammation, demonstrating the potential of PTX3 as an effective therapeutic target in neuroinflammatory conditions.

WS03-5 In vitro neutrophil transmigration models to study neutrophilepithelial interactions in cystic fibrosis airways of young children

WS03-5 In vitro neutrophil transmigration models to study neutrophilepithelial interactions in cystic fibrosis airways of young children

Pentraxin 3 regulates neutrophil infiltration to the brain during neuroinflammation.

Introduction: The acute phase protein pentraxin 3 (PTX3) is known for its anti-inflammatory effects through downregulating neutrophil transmigration during peripheral inflammation. Furthermore, we have previously demonstrated a neuroprotective and neuroreparative effect of PTX3 after cerebral ischaemia. Here we investigated, to our knowledge for the first time, the role of PTX3 in neutrophil transmigration and neurotoxicity following lipopolysaccharide (LPS)-induced cerebral inflammation and cerebral ischaemia. Methods: Neutrophil transmigration through interleukin-1β (IL-1β) activated brain endothelium and neurotoxicity of neutrophils isolated from wild-type (WT) or PTX3 knock-out (KO) mice was assessed in vitro. Primary cortical neuronal death after treatment with transmigrated neutrophils was quantified by lactate dehydrogenase (LDH) assay. Cerebral inflammation or ischemia was induced in WT and PTX3 KO mice via intrastriatal LPS injection or by transient middle cerebral artery occlusion (MCAo) respectively. Subsequent neutrophil infiltration in the brain was assessed by immunohistochemistry and the expression of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1β by enzyme-linked immunosorbent assay (ELISA). Results: Neutrophils isolated from WT mice after intrastriatal LPS injection transmigrated significantly more through IL-1β activated brain endothelium compared to neutrophils from PTX3 KO mice. Transmigrated WT and PTX3 KO neutrophils were significantly more neurotoxic than corresponding non-transmigrated neutrophils; however, no significant differences in neurotoxicity between genotypes were observed. PTX3 reduced the number of transmigrated neutrophils to the brain after intrastriatal LPS injection. Furthermore, PTX3 KO mice showed significantly increased levels of neutrophils in the brain after LPS administration or in the ischaemic hemisphere after MCAo, compared to WT mice. Conclusion: Our study shows that PTX3 regulates neutrophil transmigration in the CNS during neuroinflammation, demonstrating the potential of PTX3 as an effective therapeutic target in neuroinflammatory conditions.

Harmful neutrophil subsets in patients with ischemic stroke: Association with disease severity.

ObjectiveTo better understand the functional state of circulating neutrophils in patients with ischemic stroke (IS) for planning future clinical trials.MethodsWe analyzed by flow cytometry activation state of circulating neutrophils and the distribution of neutrophil peripheral subsets in 41 patients with acute IS less than 6 hours before admission and compared them with 22 age-matched healthy controls.ResultsOur results demonstrated continuous basal hyperactivation of circulating neutrophils during acute IS, characterized by lower l-selectin expression and higher CD11b expression at the cell surface, increased ROS production by neutrophils, and greater circulating levels of neutrophil elastase. Neutrophil hyperactivation was associated with deregulation of the equilibrium between apoptotic and necrotic. Patients also had higher percentages than controls of the overactive senescent (CXCR4bright/CD62Ldim) neutrophil subset and increased percentage of neutrophils with a reverse transendothelial migration (CD54highCXCR1low) phenotype. Importantly, neutrophil alterations were associated with the clinical severity of the stroke, evaluated by its NIH Stroke Scale score.ConclusionAltogether, our results indicate that during acute IS, the inflammatory properties of circulating neutrophils rise, associated with the expansion of harmful neutrophil subsets. These changes in neutrophil homeostasis, associated with disease severity, may play an instrumental role by contributing to systemic inflammation and to the blood-brain barrier breakdown. Our findings highlight new potential therapeutic approaches of stroke by rebalancing the ratio of senescent to immunosuppressive neutrophils or decreasing reverse neutrophil transmigration or both.

Modeling the pasture-associated severe equine asthma bronchoalveolar lavage fluid proteome identifies molecular events mediating neutrophilic airway inflammation.

Background: Pasture-associated severe equine asthma is a warm season, environmentally-induced respiratory disease characterized by reversible airway obstruction, persistent and non-specific airway hyper-responsiveness, and chronic neutrophilic airway inflammation. During seasonal exacerbation, signs vary from mild to life-threatening episodes of wheezing, coughing, and chronic debilitating labored breathing.Purpose: In human asthma, neutrophilic airway inflammation is associated with more severe and steroid-refractory asthma phenotypes, highlighting a need to decipher the mechanistic basis of this disease characteristic. We hypothesize that the collective biological activities of proteins in bronchoalveolar lavage fluid (BALF) of horses with pasture-associated severe asthma predict changes in neutrophil functions that contribute to airway neutrophilic inflammation.Methods: Using shotgun proteomics, we identified 1,003 unique proteins in cell-free BALF from six horses experiencing asthma exacerbation and six control herdmates. Contributions of each protein to ten neutrophil functions were modeled using manual biocuration to determine each protein’s net effect on the respective neutrophil functions.Results: A total of 417 proteins were unique to asthmatic horses, 472 proteins were unique to control horses (p<0.05), and 114 proteins were common in both groups. Proteins whose biological activities are responsible for increasing neutrophil migration, chemotaxis, cell spreading, transmigration, and infiltration, which would collectively bring neutrophils to airways, were over-represented in the BALF of asthmatic relative to control horses. By contrast, proteins whose biological activities support neutrophil activation, adhesion, phagocytosis, respiratory burst, and apoptosis, which would collectively shorten neutrophil lifespan, were under-represented in BALF of asthmatic relative to control horses. Interaction networks generated using Ingenuity® Pathways Analysis further support the results of our biocuration.Conclusion: Congruent with our hypothesis, the collective biological functions represented in differentially expressed proteins of BALF from horses with pasture-associated severe asthma support neutrophilic airway inflammation. This illustrates the utility of systems modeling to organize functional genomics data in a manner that characterizes complex molecular events associated with clinically relevant disease.

Lung-Derived SOD3 Attenuates Neurovascular Injury After Transient Global Cerebral Ischemia.

BackgroundSystemic innate immune priming is a recognized sequela of post‐ischemic neuroinflammation and contributor to delayed neurodegeneration. Given mounting evidence linking acute stroke with reactive lung inflammation, we asked whether enhanced expression of the endogenous antioxidant extracellular superoxide dismutase 3 (SOD3) produced by alveolar type II pneumocytes would protect the lung from transient global cerebral ischemia and the brain from the delayed effects of ischemia‐reperfusion.Methods and ResultsFollowing 15 minutes of global cerebral ischemia or sham conditions, transgenic SOD3 and wild‐type mice were followed daily for changes in weight, core temperature, and neurological function. Three days after reperfusion, arterial and venous samples were collected for complete blood counts, flow cytometry, and SOD3 protein blotting, and immunohistochemistry was performed on lung and brain tissue to assess tissue injury, blood‐brain barrier permeability, and neutrophil transmigration. Relative to ischemic controls, transgenic SOD3 mice performed better on functional testing and exhibited reduced peripheral neutrophil activation, lung inflammation, and blood‐brain barrier leak. Once released from the lung, SOD3 was predominantly not cell associated and depleted in the venous phase of circulation.ConclusionsIn addition to reducing the local inflammatory response to cerebral ischemia, targeted enrichment of SOD3 within the lung confers distal neuroprotection against ischemia‐reperfusion injury. These data suggest that therapies geared toward enhancing adaptive lung‐neurovascular coupling may improve outcomes following acute stroke and cardiac arrest.

The secreted Ly6/uPAR-related protein-1 suppresses neutrophil binding, chemotaxis, and transmigration through human umbilical vein endothelial cells

The secreted Ly-6/uPAR Related Protein-1 (SLURP1) is an immunomodulatory protein that promotes corneal immune- and angiogenic-privilege. Here, we have examined the influence of SLURP1 on neutrophil-vascular endothelial cell interactions using human umbilical vein endothelial cells (HUVEC) and differentiated neutrophil-like HL-60 (dHL-60) cells, or primary human neutrophils. SLURP1 blocked the tumor necrosis factor-alpha (TNF-α)-activated dHL-60 cells (i) binding to TNF-α-activated HUVEC with a concurrent reduction in endothelial cell adhesion molecule E-selectin, (ii) transmigration through TNF-α-activated confluent HUVEC monolayer by stabilizing VE-cadherin and β-catenin on endothelial cell cytoplasmic membranes, (iii) chemotaxis towards chemoattractant formyl Met-Leu-Phe (fMLP) coupled with their decreased polarization, and (iv) TNF-α-stimulated matrix metalloproteinase-9 (MMP9) expression and activity. SLURP1 also suppressed the primary human neutrophil chemotaxis, and interaction with HUVEC. Furthermore, SLURP1 suppressed fMLP-induced phosphorylation of protein kinase-B (AKT) in dHL-60 cells. Collectively, these results provide evidence that SLURP1 suppresses neutrophil (i) docking on HUVEC cells by decreasing endothelial cell adhesion molecule E-Selectin production, (ii) transmigration through HUVEC monolayer by stabilizing endothelial cell membrane localization of VE-cadherin and β-catenin complex and promoting their barrier function, and (iii) chemotaxis by modulating their polarization and TNF-α-stimulated MMP9 production.

Efficacy and Safety of Danirixin (GSK1325756) Co-administered With Standard-of-Care Antiviral (Oseltamivir): A Phase 2b, Global, Randomized Study of Adults Hospitalized With Influenza.

BackgroundExcessive neutrophil migration has been correlated with influenza symptom severity. Danirixin (GSK1325756), a selective and reversible antagonist of C-X-C chemokine receptor 2, decreases neutrophil activation and transmigration to areas of inflammation. This study evaluated the efficacy and safety of intravenous (IV) danirixin co-administered with oseltamivir for the treatment of adults hospitalized with influenza.MethodsIn this phase 2b, double-blind, 3-arm study (NCT02927431), influenza-positive participants were randomized 2:2:1 to receive danirixin 15mg intravenously (IV) twice daily (bid) + oral oseltamivir 75mg bid (OSV), danirixin 50mg IV bid + OSV, or placebo IV bid + OSV, for up to 5 days. The primary endpoint was time to clinical response (TTCR).ResultsIn total, 10 participants received study treatment (danirixin 15mg + OSV, n = 4; danirixin 50mg + OSV, n = 4; placebo + OSV, n = 2) before the study was terminated early due to low enrollment. All participants achieved a clinical response. Median (95% confidence interval) TTCR was 4.53 days (2.95, 5.71) for danirixin 15mg + OSV, 4.76 days (2.71, 5.25) for danirixin 50mg + OSV, and 1.33 days (0.71, 1.95) for placebo + OSV. Adverse events (AEs) were generally of mild or moderate intensity; no serious AEs were considered treatment-related. Interleukin-8 levels increased in nasal samples (using synthetic absorptive matrix strips) and decreased serum neutrophil-elastase–mediated degradation of elastin decreased in danirixin-treated participants, suggesting effective target engagement.ConclusionsInterpretation of efficacy results is restricted by the low participant numbers. The safety and tolerability profile of danirixin was consistent with previous studies.Clinical trial informationThe registration data for the trial are in the ClinicalTrials.gov database, number NCT02927431, and in the EU Clinical Trials Register (https://www.clinicaltrialsregister.eu/) as GSK study 201023, EudraCT 2016-002512-40. Anonymized individual participant data and study documents can be requested for further research from www.clinicalstudydatarequest.com.

Randomized, Double-Blind, Placebo-Controlled Study of the Safety, Tolerability, and Clinical Effect of Danirixin in Adults With Acute, Uncomplicated Influenza.

BackgroundDanirixin (DNX), a selective and reversible CXC chemokine receptor 2 antagonist, inhibits neutrophil transmigration and activation. This study assessed the safety, tolerability, and clinical effect of DNX with and without oseltamivir (OSV) in adults with acute, uncomplicated influenza.MethodsThis was a placebo-controlled, double-blind, Phase IIa study. Participants (18–64 years) with influenza-like symptoms (onset ≤48 hours) and positive influenza rapid antigen test were randomized 2:1:2:1 to DNX, placebo, DNX+OSV, or OSV (75 mg each, administered twice daily for 5 days) and followed for 28 days. Primary endpoints included frequency of adverse events (AEs) and serious AEs (SAEs). The effect of DNX on virologic response and clinical effect on influenza symptoms were secondary endpoints.ResultsA total of 45 participants were enrolled, 35 of whom were confirmed influenza positive by polymerase chain reaction analysis. The highest incidence of AEs was in the placebo group (4 of 7, 57%), followed by the DNX+OSV (7 of 16, 44%), DNX (3 of 15, 20%), and OSV (0 of 7, 0%) groups. One SAE (T-wave abnormality) was reported in the DNX group (unrelated to treatment). No differences in viral load assessments were observed among treatment groups.ConclusionsDanirixin treatment was well tolerated and did not impede viral clearance.