A fluorogenic peptide-based smartprobe for the detection of neutrophil extracellular traps and inflammation.

A highly sensitive optical probe for the detection of activated neutrophils and Neutrophil Extracellular Traps (NETs) is reported. It is based on a triple-quenched, super-silent tri-branched probe that generates >20 fold increase in fluorescence upon cleavage. The probe was highly specific for human neutrophil elastase, a protease that mediates a variety of inflammatory diseases, and detected NETosis and neutrophil activation in in vitro differentiated neutrophils and isolated human neutrophils.

Mcl-1 protects eosinophils from apoptosis and exacerbates allergic airway inflammation.

Eosinophils are key effector cells in allergic diseases. Here we investigated Mcl-1 (an anti-apoptotic protein) in experimental allergic airway inflammation using transgenic overexpressing human Mcl-1 mice (hMcl-1) and reducing Mcl-1 by a cyclin-dependent kinase inhibitor. Overexpression of Mcl-1 exacerbated allergic airway inflammation, with increased bronchoalveolar lavage fluid cellularity, eosinophil numbers and total protein, and an increase in airway mucus production. Eosinophil apoptosis was suppressed by Mcl-1 overexpression, with this resistance to apoptosis attenuated by cyclin-dependent kinase inhibition which also rescued Mcl-1-exacerbated allergic airway inflammation. We propose that targeting Mcl-1 may be beneficial in treatment of allergic airway disease.

Purine metabolism controls innate lymphoid cell function and protects against intestinal injury.

Inflammatory bowel disease (IBD) is a condition of chronic inflammatory intestinal disorder with increasing prevalence but limited effective therapies. The purine metabolic pathway is involved in various inflammatory processes including IBD. However, the mechanisms through which purine metabolism modulates IBD remain to be established. Here, we found that mucosal expression of genes involved in the purine metabolic pathway is altered in patients with active ulcerative colitis (UC), which is associated with elevated gene expression signatures of the group 3 innate lymphoid cell (ILC3)-interleukin (IL)-22 pathway. In mice, blockade of ectonucleotidases (NTPDases), critical enzymes for purine metabolism by hydrolysis of extracellular adenosine 5'-triphosphate (eATP) into adenosine, exacerbates dextran-sulfate sodium-induced intestinal injury. This exacerbation of colitis is associated with reduction of colonic IL-22-producing ILC3s, which afford essential protection against intestinal inflammation, and is rescued by exogenous IL-22. Mechanistically, activation of ILC3s for IL-22 production is reciprocally mediated by eATP and adenosine. These findings reveal that the NTPDase-mediated balance between eATP and adenosine regulates ILC3 cell function to provide protection against intestinal injury and suggest potential therapeutic strategies for treating IBD by targeting the purine-ILC3 axis.

Mer-mediated eosinophil efferocytosis regulates resolution of allergic airway inflammation.

BACKGROUND: Eosinophils play a central role in propagation of allergic diseases, including asthma. Both recruitment and retention of eosinophils regulate pulmonary eosinophilia, but the question of whether alterations in apoptotic cell clearance by phagocytes contributes directly to resolution of allergic airway inflammation remains unexplored. OBJECTIVES: In this study we investigated the role of the receptor tyrosine kinase Mer in mediating apoptotic eosinophil clearance and allergic airway inflammation resolution in vivo to establish whether apoptotic cell clearance directly affects the resolution of allergic airway inflammation. METHODS: Alveolar and bone marrow macrophages were used to study Mer-mediated phagocytosis of apoptotic eosinophils. Allergic airway inflammation resolution was modeled in mice by using ovalbumin. Fluorescently labeled apoptotic cells were administered intratracheally or eosinophil apoptosis was driven by administration of dexamethasone to determine apoptotic cell clearance in vivo. RESULTS: Inhibition or absence of Mer impaired phagocytosis of apoptotic human and mouse eosinophils by macrophages. Mer-deficient mice showed delayed resolution of ovalbumin-induced allergic airway inflammation, together with increased airway responsiveness to aerosolized methacholine, increased bronchoalveolar lavage fluid protein levels, altered cytokine production, and an excess of uncleared dying eosinophils after dexamethasone treatment. Alveolar macrophage phagocytosis was significantly Mer dependent, with the absence of Mer attenuating apoptotic cell clearance in vivo to enhance inflammation in response to apoptotic cells. CONCLUSIONS: We demonstrate that Mer-mediated apoptotic cell clearance by phagocytes contributes to resolution of allergic airway inflammation, suggesting that augmenting apoptotic cell clearance is a potential therapeutic strategy for treating allergic airway inflammation.

Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation. METHODS: Blood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR. RESULTS: CF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF. CONCLUSIONS: CF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.

Models for the Study of the Cross Talk Between Inflammation and Cell Cycle.

Cyclin-dependent kinases (CDKs) have been traditionally associated with the cell cycle. However, it is now known that CDK7 and CDK9 regulate transcriptional activity via phosphorylation of RNA polymerase II and subsequent synthesis of, for example, inflammatory mediators and factors that influence the apoptotic process; including apoptosis of granulocytes such as neutrophils and eosinophils. Successful resolution of inflammation and restoration of normal tissue homeostasis requires apoptosis of these inflammatory cells and subsequent clearance of apoptotic bodies by phagocytes such as macrophages. It is believed that CDK7 and CDK9 influence resolution of inflammation since they are involved in the transcription of anti-apoptotic proteins such as Mcl-1 which is especially important in granulocyte survival.This chapter describes various in vitro and in vivo models used to investigate CDKs and their inhibitors in granulocytes and particularly the role of CDKs in the apoptosis pathway. This can be performed in vitro by isolation and use of primary granulocytes and in vivo using animal models of inflammatory disease in rodents and zebrafish. Some of the methods described here to assess the role of CDKs in inflammation and apoptosis include flow cytometry and western blotting, together with imaging and quantification of apoptosis in fixed tissue, as well as in vivo models of inflammation.

Mechanism of neutrophil activation and toxicity elicited by engineered nanomaterials.

The effects of nanomaterials (NMs) on biological systems, especially their ability to stimulate inflammatory responses requires urgent investigation. We evaluated the response of the human differentiated HL60 neutrophil-like cell line to NMs. It was hypothesised that NM physico-chemical characteristics would influence cell responsiveness by altering intracellular Ca2+ concentration [Ca2+]i and reactive oxygen species production. Cells were exposed (1.95-125 µg/ml, 24 h) to silver (Ag), zinc oxide (ZnO), titanium dioxide (TiO2), multi-walled carbon nanotubes (MWCNTs) or ultrafine carbon black (ufCB) and cytotoxicity assessed (alamar blue assay). Relatively low (TiO2, MWCNTs, ufCB) or high (Ag, ZnO) cytotoxicity NMs were identified. Sub-lethal impacts of NMs on cell function were investigated for selected NMs only, namely TiO2, Ag and ufCB. Only Ag stimulated cell activation. Within minutes, Ag stimulated an increase in [Ca2+]i (in Fura-2 loaded cells), and a prominent inward ion current (assessed by electrophysiology). Within 2-4 h, Ag increased superoxide anion release and stimulated cytokine production (MCP-1, IL-8) that was diminished by Ca2+ inhibitors or trolox. Light microscopy demonstrated that cells had an activated phenotype. In conclusion NM toxicity was ranked; Ag>ufCB>TiO2, and the battery of tests used provided insight into the mechanism of action of NM toxicity to guide future testing strategies.