Incidence and recognition of acute respiratory distress syndrome in a UK intensive care unit.

The reported incidence of ARDS is highly variable (2.5%-19% of intensive care unit (ICU) patients) and varies depending on study patient population used. We undertook a 6-month, prospective study to determine the incidence and outcome of ARDS in a UK adult University Hospital ICU. 344 patients were admitted during the study period, of these 43 (12.5%) were determined to have ARDS. Patients with ARDS had increased mortality at 28 days and 2 years post-diagnosis, and there was under-recognition of ARDS in both medical records and death certificattion. Our findings have implications for critical care resource planning.This is the final version of the article. It first appeared from BMJ Thorax via ://dx.doi.org/10.1136/thoraxjnl-2016-20840

Real-time deformability cytometry reveals sequential contraction and expansion during neutrophil priming.

It has become increasingly apparent that the biomechanical properties of neutrophils impact on their trafficking through the circulation and in particularly through the pulmonary capillary bed. The retention of polarized or shape-changed neutrophils in the lungs was recently proposed to contribute to acute respiratory distress syndrome pathogenesis. Accordingly, this study tested the hypothesis that neutrophil priming is coupled to morpho-rheological (MORE) changes capable of altering cell function. We employ real-time deformability cytometry (RT-DC), a recently developed, rapid, and sensitive way to assess the distribution of size, shape, and deformability of thousands of cells within seconds. During RT-DC analysis, neutrophils can be easily identified within anticoagulated "whole blood" due to their unique granularity and size, thus avoiding the need for further isolation techniques, which affect biomechanical cell properties. Hence, RT-DC is uniquely suited to describe the kinetics of MORE cell changes. We reveal that, following activation or priming, neutrophils undergo a short period of cell shrinking and stiffening, followed by a phase of cell expansion and softening. In some contexts, neutrophils ultimately recover their un-primed mechanical phenotype. The mechanism(s) underlying changes in human neutrophil size are shown to be Na+ /H+ antiport-dependent and are predicted to have profound implications for neutrophil movement through the vascular system in health and disease

In vivo imaging of hepatic neutrophil migration in severe alcoholic hepatitis with 111In-radiolabelled leucocytes.

The study's aim was to image severe alcoholic hepatitis (SAH) using 111In-labelled leucocytes with two objectives in mind: firstly for non-invasive diagnosis and secondly to provide a platform for experimental therapies aiming to inhibit intrahepatic neutrophil migration. 111In-leucocyte scintigraphy was performed 30 min and 24 h post-injection in 19 patients with SAH, 14 abstinent patients with alcohol-related cirrhosis and 11 normal controls. Eleven with SAH and seven with cirrhosis also had 99mTc-nanocolloid scintigraphy. Change in hepatic 111In radioactivity was expressed as decay-corrected 24 h:30 min count ratio and, in SAH, compared with histological grading of steatohepatitis and expression of granulocyte marker, CD15. Hepatic microautoradiography on biopsy specimens obtained 24 h post-injection of 111In-leucocytes was performed in one patient. Median 24 h:30 min hepatic 111In activity ratio was higher in SAH (2.5 (interquartile range (IQR): 1.7-4.0) compared with cirrhotics and normal controls (1.0 (0.8-1.1) and 0.8 (0.7-0.9) respectively, P<0.0001). In SAH, it correlated with CD15 expression (r = 0.62, P=0.023) and was higher in marked compared with mild/moderate steatohepatitis (4.0 (3.0-4.6) compared with 1.8 (1.5-2.6), P=0.006). Hepatic-to-splenic 99mTc count rate ratio was reduced in SAH (0.5 (0.4-1.4)) compared with cirrhotics (2.3( 0.6-3.0)) and three historic normal controls (4.2 (3.8-5.0); P=0.003), consistent with impaired hepatic reticuloendothelial function. Scintigraphic findings in SAH included prominent lung radioactivity at 30 min, likely the result of neutrophil primimg. Microautoradiography demonstrated cell-associated 111In in areas of parenchymal neutrophil infiltration. In conclusion, 111In-leucocyte scintigraphy can non-invasively diagnose SAH and could provide a platform for evaluation of novel treatments aiming to inhibit intrahepatic neutrophil migration.The study was funded by a project grant from Brighton and Sussex Medical Schoo

Characterisation of secondary metabolites associated with neutrophil apoptosis

We studied changes in secondary metabolites in human neutrophils undergoing constitutive or tumour necrosis factor (TNFalpha) stimulated apoptosis by a combination of high-performance liquid chromatography (HPLC) and NMR spectroscopy. Our results show that in contrast to freshly isolated neutrophils, neutrophil cells aged for 20 h in vitro had marked differences in the levels of a number of endogenous metabolites including lactate, amino acids and phosphocholine (PCho). There was no change in the concentration of taurine or glutamate and the ATP/ADP ratio was not affected. Levels of glutamine and lactate actually decreased. Identical changes were also observed in neutrophils stimulated to undergo apoptosis over a shorter time period (6 h) in the presence of TNFalpha and the phosphatidylinositol-3-kinase inhibitor wortmannin (WM). The changes in the concentration of PCho suggest possible activation of phospholipase associated with apoptosis or a selective failure of phosphatidycholine synthesis. The increased levels of apoptosis obtained with WM+TNFalpha, compared to TNFalpha by itself, suggest a synergistic effect by these compounds. The acceleration in rate of apoptosis probably arises from suppression by WM of pathway(s) that normally delay the onset of apoptosis. Changes in PCho and other endogenous metabolites, if proven to be characteristic of apoptosis in other cell systems, may permit non-invasive quantification of apoptosis.

Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus

Funder: NHLI FoundationFunder: NIHR Imperial Biomedical Research Centre; FundRef: http://dx.doi.org/10.13039/501100013342Funder: National Heart Lung and Blood InstituteFunder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265Funder: National Institute of Biomedical Imaging and Bioengineering; FundRef: http://dx.doi.org/10.13039/100000070Funder: Gates Cambridge ScholarshipFunder: NIH/OXCAM FellowshipObjectives: Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE. Methods: Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively. Results: Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM. Conclusions: Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus.

Bicomponent pore-forming leukocidins are a family of potent toxins secreted by Staphylococcus aureus, which target white blood cells preferentially and consist of an S- and an F-component. The S-component recognizes a receptor on the host cell, enabling high-affinity binding to the cell surface, after which the toxins form a pore that penetrates the cell lipid bilayer. Until now, six different leukocidins have been described, some of which are host and cell specific. Here, we identify and characterise a novel S. aureus leukocidin; LukPQ. LukPQ is encoded on a 45 kb prophage (ΦSaeq1) found in six different clonal lineages, almost exclusively in strains cultured from equids. We show that LukPQ is a potent and specific killer of equine neutrophils and identify equine-CXCRA and CXCR2 as its target receptors. Although the S-component (LukP) is highly similar to the S-component of LukED, the species specificity of LukPQ and LukED differs. By forming non-canonical toxin pairs, we identify that the F-component contributes to the observed host tropism of LukPQ, thereby challenging the current paradigm that leukocidin specificity is driven solely by the S-component

Neutrophil GM-CSF receptor dynamics in acute lung injury.

GM-CSF is important in regulating acute, persistent neutrophilic inflammation in certain settings, including lung injury. Ligand binding induces rapid internalization of the GM-CSF receptor (GM-CSFRα) complex, a process essential for signaling. Whereas GM-CSF controls many aspects of neutrophil biology, regulation of GM-CSFRα expression is poorly understood, particularly the role of GM-CSFRα in ligand clearance and whether signaling is sustained despite major down-regulation of GM-CSFRα surface expression. We established a quantitative assay of GM-CSFRα surface expression and used this, together with selective anti-GM-CSFR antibodies, to define GM-CSFRα kinetics in human neutrophils, and in murine blood and alveolar neutrophils in a lung injury model. Despite rapid sustained ligand-induced GM-CSFRα loss from the neutrophil surface, which persisted even following ligand removal, pro-survival effects of GM-CSF required ongoing ligand-receptor interaction. Neutrophils recruited to the lungs following LPS challenge showed initially high mGM-CSFRα expression, which along with mGM-CSFRβ declined over 24 hr; this was associated with a transient increase in bronchoalveolar lavage fluid (BALF) mGM-CSF concentration. Treating mice in an LPS challenge model with CAM-3003, an anti-mGM-CSFRα mAb, inhibited inflammatory cell influx into the lung and maintained the level of BALF mGM-CSF. Consistent with neutrophil consumption of GM-CSF, human neutrophils depleted exogenous GM-CSF, independent of protease activity. These data show that loss of membrane GM-CSFRα following GM-CSF exposure does not preclude sustained GM-CSF/GM-CSFRα signaling and that this receptor plays a key role in ligand clearance. Hence neutrophilic activation via GM-CSFR may play an important role in neutrophilic lung inflammation even in the absence of high GM-CSF levels or GM-CSFRα expression

The Aminopeptidase CD13 Induces Homotypic Aggregation in Neutrophils and Impairs Collagen Invasion.

Aminopeptidase N (CD13) is a widely expressed cell surface metallopeptidase involved in the migration of cancer and endothelial cells. Apart from our demonstration that CD13 modulates the efficacy of tumor necrosis factor-α-induced apoptosis in neutrophils, no other function for CD13 has been ascribed in this cell. We hypothesized that CD13 may be involved in neutrophil migration and/or homotypic aggregation. Using purified human blood neutrophils we confirmed the expression of CD13 on neutrophils and its up-regulation by pro-inflammatory agonists. However, using the anti-CD13 monoclonal antibody WM-15 and the aminopeptidase enzymatic inhibitor bestatin we were unable to demonstrate any direct involvement of CD13 in neutrophil polarisation or chemotaxis. In contrast, IL-8-mediated neutrophil migration in type I collagen gels was significantly impaired by the anti-CD13 monoclonal antibodies WM-15 and MY7. Notably, these antibodies also induced significant homotypic aggregation of neutrophils, which was dependent on CD13 cross-linking and was attenuated by phosphoinositide 3-kinase and extracellular signal-related kinase 1/2 inhibition. Live imaging demonstrated that in WM-15-treated neutrophils, where homotypic aggregation was evident, the number of cells entering IL-8 impregnated collagen I gels was significantly reduced. These data reveal a novel role for CD13 in inducing homotypic aggregation in neutrophils, which results in a transmigration deficiency; this mechanism may be relevant to neutrophil micro-aggregation in vivo.This work was funded by a Medical Research Council Research Training Fellowship to CAF (G0900329), Addenbrooke’s Charitable Trust (ACT), CUHNHSFT, Papworth Hospital NHS Foundation Trust and the NIHR Cambridge Biomedical Research Centre. CAF received a Raymond and Beverly Sackler Studentship.This is the final version of the article. It first appeared from the Public Library of Science via http://dx.doi.org/10.1371/journal.pone.016010

Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses.

Fully activated innate immune cells are required for effective responses to infection, but their prompt deactivation and removal are essential for limiting tissue damage. Here, we have identified a critical role for the prolyl hydroxylase enzyme Phd2 in maintaining the balance between appropriate, predominantly neutrophil-mediated pathogen clearance and resolution of the innate immune response. We demonstrate that myeloid-specific loss of Phd2 resulted in an exaggerated inflammatory response to Streptococcus pneumonia, with increases in neutrophil motility, functional capacity, and survival. These enhanced neutrophil responses were dependent upon increases in glycolytic flux and glycogen stores. Systemic administration of a HIF-prolyl hydroxylase inhibitor replicated the Phd2-deficient phenotype of delayed inflammation resolution. Together, these data identify Phd2 as the dominant HIF-hydroxylase in neutrophils under normoxic conditions and link intrinsic regulation of glycolysis and glycogen stores to the resolution of neutrophil-mediated inflammatory responses. These results demonstrate the therapeutic potential of targeting metabolic pathways in the treatment of inflammatory disease.This work was principally supported by a Wellcome Trust Senior Clinical Fellowship award (098516 to SRW), Medical Research Council (MRC) Clinical Training Fellowship awards (G0802255 to AART; MR/K023845/1 to RSD), an Academy of Medical Sciences (AMS) starter grant (to AART), a Wellcome Trust Senior Clinical Fellowship award (076945 to DHD), British Lung Foundation Fellowship (F05/7 to HMM), and a Engineering and Physical Sciences Research Council and Medical Research Council grant (EP/L016559/1, JAW). The MRC /University of Edinburgh Centre for Inflammation Research is supported by an MRC Centre Grant. The work of PC is supported by long-term structural funding-Methusalem funding from the Flemish Government. CJS thanks the Wellcome Trust and Cancer Research UK for support

Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity

The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91$\textit{phox}$ and p22$\textit{phox}$ subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene $\textit{bc017643}$, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91$\textit{phox}$ and p22$\textit{phox}$. Consequently, $\textit{Eros}$-deficient mice quickly succumb to infection. $\textit{Eros}$ also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. $\textit{Eros}$ is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.D.C. Thomas was funded by a Wellcome Trust/CIMR Next Generation Fellowship, a National Institute for Health Research (NIHR) Clinical Lectureship, and a Starter Grant for Clinical Lecturers (Academy of Medical Sciences). K.G.C. Smith was funded by funded by the Medical Research Council (program grant MR/L019027) and is a Wellcome Investigator and a NIHR Senior Investigator. S. Clare and G. Dougan were funded by the Wellcome Trust (grant 098051). The Cambridge Institute for Medical Research is in receipt of a Wellcome Trust Strategic Award (079895). J.C.L is funded by a Wellcome Intermediate Clinical Fellowship 105920/2/14/2