PAR4 Antagonism in Patients With Coronary Artery Disease Receiving Antiplatelet Therapies.

BACKGROUND: BMS-986141 is a novel potent highly selective antagonist of PAR (protease-activated receptor) type 4. PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people. We sought to determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy. METHODS: Forty-five patients with stable coronary heart disease and 10 healthy volunteers completed a phase 2a open-label 4-arm single-center study. Patients were allocated to 1 of 3 treatment arms for 7 days: (1) ticagrelor (90 mg BID), (2) aspirin (75 mg QD), or (3) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before and 2 and 24 hours after a single oral 4-mg dose of BMS-986141 on the first study visit day in all participants. RESULTS: BMS-986141 demonstrated highly selective inhibition of PAR4-AP (agonist peptide)-induced platelet aggregation, P-selectin expression, and platelet-monocyte aggregate expression (P≤0.001 for all), which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (-21%; P=0.001) and in patients receiving ticagrelor alone (-28%; P=0.001), aspirin alone (-23%; P=0.018), or both in combination (-24%; P≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP-induced platelet responses (P≤0.001 for all) and total thrombus area under high shear stress conditions (P≤0.01 for all). CONCLUSIONS: PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin, or their combination, in patients with stable coronary heart disease. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT05093790.

Metabolic regulation by prostaglandin E2 impairs lung group 2 innate lymphoid cell responses.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E2 (PGE2 ). However, the respective roles for the PGE2 receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. METHODS: The roles of PGE2 receptors EP2 and EP4 on ILC2-mediated lung inflammation were investigated using genetically modified mouse lines and pharmacological approaches in IL-33-induced lung allergy model. The effects of PGE2 receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures. RESULTS: Deficiency of EP2 rather than EP4 augments IL-33-induced mouse lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 and EP2 or inhibition of phosphodiesterase markedly restricts IL-33-induced lung ILC2 responses. Mechanistically, PGE2 directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice where endogenous PG synthesis or EP2 signaling is blocked but not in mice with intact PGE2 -EP2 signaling. CONCLUSION: We have defined a mechanism for optimal suppression of mouse lung ILC2 responses by endogenous PGE2 -EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE2 -EP4-cAMP and energy metabolic pathways may provide novel opportunities for treating the ILC2-initiated lung inflammation in asthma and NERD.

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.

Single-cell transcriptome analyses reveal novel targets modulating cardiac neovascularization by resident endothelial cells following myocardial infarction.

AIMS: A better understanding of the pathways that regulate regeneration of the coronary vasculature is of fundamental importance for the advancement of strategies to treat patients with heart disease. Here, we aimed to investigate the origin and clonal dynamics of endothelial cells (ECs) associated with neovascularization in the adult mouse heart following myocardial infarction (MI). Furthermore, we sought to define murine cardiac endothelial heterogeneity and to characterize the transcriptional profiles of pro-angiogenic resident ECs in the adult mouse heart, at single-cell resolution. METHODS AND RESULTS: An EC-specific multispectral lineage-tracing mouse (Pdgfb-iCreERT2-R26R-Brainbow2.1) was used to demonstrate that structural integrity of adult cardiac endothelium following MI was maintained through clonal proliferation by resident ECs in the infarct border region, without significant contributions from bone marrow cells or endothelial-to-mesenchymal transition. Ten transcriptionally discrete heterogeneous EC states, as well as the pathways through which each endothelial state is likely to enhance neovasculogenesis and tissue regeneration following ischaemic injury were defined. Plasmalemma vesicle-associated protein (Plvap) was selected for further study, which showed an endothelial-specific and increased expression in both the ischaemic mouse and human heart, and played a direct role in regulating human endothelial proliferation in vitro. CONCLUSION: We present a single-cell gene expression atlas of cardiac specific resident ECs, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. These data provide a rich resource that could assist in the development of new therapeutic interventions to augment endogenous myocardial perfusion and enhance regeneration in the injured heart.

The toxicology of chrysotile-containing brake debris: implications for mesothelioma.

The global use of "asbestos" in various commercial products has led to a wide range and pervasive legacy of disease. One such use of chrysotile asbestos was brake pads and was utilized commonly in automobiles and heavy vehicles. The result of incorporation of chrysotile into brake pads is associated with the exposure of mechanics fitting and servicing vehicles to liberated chrysotile fibers. Despite the proven exposure, the relative risk of malignant mesothelioma (MM) in this occupational population is broadly seen as low. The toxicity of particulates, including fibers such as chrysotile, is driven by a combination of dose and physicochemical properties. As such, it is plausible that chrysotile released from brake pads may have undergone modification, thereby altering the pathogenicity profile. The impact of high sheer stress causing shortening of long fibers, heat modification, binding of resin matrix to the fiber surface on the relative toxicity of brake debris with regards to MM is considered. It is apparent that released chrysotile can undergo significant modification, reducing the long fiber dose although not all modifications may lead to reduced toxicity.

Assessment of the physicochemical properties of chrysotile-containing brake debris pertaining to toxicity.

Grinding and drilling of chrysotile asbestos-containing brake pads during the 20th century led to release of chrysotile, resulting in varying levels of workplace exposures of mechanics. Despite exposures, excess risk of mesothelioma remains in doubt. Objectives: The toxicity of particulates is primarily derived through a combination of physicochemical properties and dose and as such this study aimed to determine properties of asbestos-containing brake debris (BD) which may influence pathogenicity and potential of mesothelioma. Materials and Methods: Chrysotile-containing brake pads were ground - to reflect occupational activities, aerosolized, and size-fractionated to isolate respirable fractions. Analysis of morphology, biodurability, surface charge, and interactions with macrophages were undertaken. Results: The respirable fraction of BD contained ∼15-17% free chrysotile fibers thereby constituting a small but relevant potential long fiber dose. Acellular biodurability studies showed rapid dissolution and fragmentation of chrysotile fibers that was consistent for pure chrysotile control and BD samples. Conclusions: The long, free, respirable chrysotile fibers were present in BD, yet were of low bio-durability; incubation in artificial lysosomal fluid led to destruction of free fibers.

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.

Facilitation of IL-22 production from innate lymphoid cells by prostaglandin E2 prevents experimental lung neutrophilic inflammation.

Acute lung injury is a neutrophil-dominant, life-threatening disease without effective therapies and better understanding of the pathophysiological mechanisms involved is an urgent need. Here we show that interleukin (IL)-22 is produced from innate lymphoid cells (ILC) and is responsible for suppression of experimental lung neutrophilic inflammation. Blocking prostaglandin E2 (PGE2) synthesis reduces lung ILCs and IL-22 production, resulting in exacerbation of lung neutrophilic inflammation. In contrast, activation of the PGE2 receptor EP4 prevents acute lung inflammation. We thus demonstrate a mechanism for production of innate IL-22 in the lung during acute injury, highlighting potential therapeutic strategies for control of lung neutrophilic inflammation by targeting the PGE2/ILC/IL-22 axis.

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.

Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell-Derived Hepatocytes, HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for Drug-Induced Liver Injury.

Reliable and versatile hepatic in vitro systems for the prediction of drug pharmacokinetics and toxicity are essential constituents of preclinical safety assessment pipelines for new medicines. Here, we compared three emerging cell systems-hepatocytes derived from induced pluripotent stem cells, HepaRG cells, and three-dimensional primary human hepatocyte (PHH) spheroids-at transcriptional and functional levels in a multicenter study to evaluate their potential as predictive models for drug-induced hepatotoxicity. Transcriptomic analyses revealed widespread gene expression differences between the three cell models, with 8148 of 17,462 analyzed genes (47%) being differentially expressed. Expression levels of genes involved in the metabolism of endogenous as well as xenobiotic compounds were significantly elevated in PHH spheroids, whereas genes involved in cell division and endocytosis were significantly upregulated in HepaRG cells and hepatocytes derived from induced pluripotent stem cells, respectively. Consequently, PHH spheroids were more sensitive to a panel of drugs with distinctly different toxicity mechanisms, an effect that was amplified by long-term exposure using repeated treatments. Importantly, toxicogenomic analyses revealed that transcriptomic changes in PHH spheroids were in compliance with cholestatic, carcinogenic, or steatogenic in vivo toxicity mechanisms at clinically relevant drug concentrations. Combined, the data reveal important phenotypic differences between the three cell systems and suggest that PHH spheroids can be used for functional investigations of drug-induced liver injury in vivo in humans.

Platelet activation independent of pulmonary inflammation contributes to diesel exhaust particulate-induced promotion of arterial thrombosis.

BACKGROUND: Accelerated thrombus formation induced by exposure to combustion-derived air pollution has been linked to alterations in endogenous fibrinolysis and platelet activation in response to pulmonary and systemic inflammation. We hypothesised that mechanisms independent of inflammation contribute to accelerated thrombus formation following exposure to diesel exhaust particles (DEP). METHODS: Thrombosis in rats was assessed 2, 6 and 24 h after administration of DEP, carbon black (CB; control carbon nanoparticle), DQ12 quartz microparticles (to induce pulmonary inflammation) or saline (vehicle) by either intra-tracheal instillation (0.5 mg, except Quartz; 0.125 mg) or intravenous injection (0.5 mg/kg). Thrombogenicity was assessed by carotid artery occlusion, fibrinolytic variables and platelet-monocyte aggregates. Measures of inflammation were determined in plasma and bronchoalveolar lavage fluid. Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI)-1 were measured following direct in vitro exposure of human umbilical vein endothelial cells (HUVECs) to DEP (10-150 µg/mL). RESULTS: Instillation of DEP reduced the time to thrombotic occlusion in vivo, coinciding with the peak of DEP-induced pulmonary inflammation (6 h). CB and DQ12 produced greater inflammation than DEP but did not alter time to thrombotic occlusion. Intravenous DEP produced an earlier (2 h) acceleration of thrombosis (as did CB) without pulmonary or systemic inflammation. DEP inhibited t-PA and PAI-1 release from HUVECs, and reduced the t-PA/PAI-1 ratio in vivo; similar effects in vivo were seen with CB and DQ12. DEP, but not CB or DQ12, increased platelet-monocyte aggregates. CONCLUSION: DEP accelerates arterial thrombus formation through increased platelet activation. This effect is dissociated from pulmonary and systemic inflammation and from impaired fibrinolytic function.

Wogonin induces eosinophil apoptosis and attenuates allergic airway inflammation.

RATIONALE: Eosinophils are key effector cells in allergic diseases, including allergic rhinitis, eczema, and asthma. Their tissue presence is regulated by both recruitment and increased longevity at inflamed sites. OBJECTIVES: To investigate the ability of the flavone wogonin to induce eosinophil apoptosis in vitro and attenuate eosinophil-dominant allergic inflammation in vivo in mice. METHODS: Human and mouse eosinophil apoptosis in response to wogonin was investigated by cellular morphology, flow cytometry, mitochondrial membrane permeability, and pharmacological caspase inhibition. Allergic lung inflammation was modeled in mice sensitized and challenged with ovalbumin. Bronchoalveolar lavage (BAL) and lung tissue were examined for inflammation, mucus production, and inflammatory mediator production. Airway hyperresponsiveness to aerosolized methacholine was measured. MEASUREMENTS AND MAIN RESULTS: Wogonin induced time- and concentration-dependent human and mouse eosinophil apoptosis in vitro. Wogonin-induced eosinophil apoptosis occurred with activation of caspase-3 and was inhibited by pharmacological caspase inhibition. Wogonin administration attenuated allergic airway inflammation in vivo with reductions in BAL and interstitial eosinophil numbers, increased eosinophil apoptosis, reduced airway mucus production, and attenuated airway hyperresponsiveness. This wogonin-induced reduction in allergic airway inflammation was prevented by concurrent caspase inhibition in vivo. CONCLUSIONS: Wogonin induces eosinophil apoptosis and attenuates allergic airway inflammation, suggesting that it has therapeutic potential for the treatment of allergic inflammation in humans.

The biologically effective dose in inhalation nanotoxicology.

In all branches of toxicology, the biologically effective dose (BED) is the fraction of the total dose of a toxin that actually drives any toxic effect. Knowledge of the BED has a number of applications including in building structure-activity relationships, the selection of metrics, the design of safe particles, and the determination of when a nanoparticle (NP) can be considered to be "new" for regulatory purposes. In particle toxicology, we define the BED as "the entity within any dose of particles in tissue that drives a critical pathophysiogically relevant form of toxicity (e.g., oxidative stress, inflammation, genotoxicity, or proliferation) or a process that leads to it." In conventional chemical toxicology, researchers generally use the mass as the metric to describe dose (such as mass per unit tissue or cells in culture) because of its convenience. Concentration, calculated from mass, may also figure in any description of dose. In the case of a nanoparticle dose, researchers use either the mass or the surface area. The mass of nanoparticles is not the only driver of their activity: the surfaces of insoluble particles interact with biological systems, and soluble nanoparticles can release factors that interact with these systems. Nanoparticle shape can modify activity. In this Account, we describe the current knowledge of the BED as it pertains to different NP types. Soluble toxins released by NPs represent one potential indicator of BED for wholly or partially soluble NPs composed of copper or zinc. Rapid dissolution of these NPs into their toxic ions in the acidic environment of the macrophage phagolysosome causes those ions to accumulate, which leads to lysosome destabilization and inflammation. In contrast, soluble NPs that release low toxicity ions, such as magnesium oxide NPs, are not inflammogenic. For insoluble NPs, ζ potential can serve as a BED measurement because the exposure of the particle surface to the acidic milieu of the phagolysosome and interactions with the lysosomal membrane can compromise the integrity of the NPs. Researchers have explored oxidative potential of NPs most extensively as an indicator of the BED: the ability of an NP to cause oxidative stress in cells is a key factor in determining cell toxicity, inflammogenicity, and oxidative DNA adduct formation. Finally we discuss BEDs for high aspect ratio nanoparticles because long fibers or nanoplatelets can cause inflammation and further effects. These consequences arise from the paradoxically small aerodynamic diameter of fibers or thin platelets. As a result, these NPs can deposit beyond the ciliated airways where their extended dimensions prevent them from being fully phagocytosed by macrophages, leading to frustrated phagocytosis. Although knowledge is accumulating on the BED for NPs, many questions and challenges remain in understanding and utilizing this important nanotoxicological parameter.

Flavones induce neutrophil apoptosis by down-regulation of Mcl-1 via a proteasomal-dependent pathway.

Neutrophil apoptosis and subsequent nonphlogistic clearance by surrounding phagocytes are key to the successful resolution of neutrophilic inflammation, with dysregulated apoptosis reported in multiple human inflammatory diseases. Enhancing neutrophil apoptosis has proresolution and anti-inflammatory effects in preclinical models of inflammation. Here we investigate the ability of the flavones apigenin, luteolin, and wogonin to induce neutrophil apoptosis in vitro and resolve neutrophilic inflammation in vivo. Human neutrophil apoptosis was assessed morphologically and by flow cytometry following incubation with apigenin, luteolin, and wogonin. All three flavones induced time- and concentration-dependent neutrophil apoptosis (apigenin, EC=12.2 µM; luteolin, EC=14.6 µM; and wogonin, EC=28.9 µM). Induction of apoptosis was caspase dependent, as it was blocked by the broad-spectrum caspase inhibitor Q-VD-OPh and was associated with both caspase-3 and caspase-9 activation. Flavone-induced apoptosis was preceded by down-regulation of the prosurvival protein Mcl-1, with proteasomal inhibition preventing flavone-induced Mcl-1 down-regulation and apoptosis. The flavones abrogated the survival effects of mediators that prolong neutrophil life span, including lipoteichoic acid, peptidoglycan, dexamethasone, and granulocyte-macrophage colony stimulating factor, by driving apoptosis. Furthermore, wogonin enhanced resolution of established neutrophilic inflammation in a zebrafish model of sterile tissue injury. Wogonin-induced resolution was dependent on apoptosis in vivo as it was blocked by caspase inhibition. Our data show that the flavones induce neutrophil apoptosis and have potential as neutrophil apoptosis-inducing anti-inflammatory, proresolution agents.

Targeting granulocyte apoptosis: mechanisms, models, and therapies.

The inflammatory process is a complex series of tightly controlled cellular and biochemical events initiated by the immune system, which has evolved to eliminate or contain infectious agents and to repair damaged tissue. Apoptosis is essential for the clearance of potentially injurious inflammatory cells, such as neutrophils, eosinophils, and basophils, and the subsequent efficient resolution of inflammation. In this review, we aim to cover key features of the granulocyte life-cycle ranging from their differentiation within the bone marrow to their maturation and ultimate clearance, with a focus on granulocyte apoptosis and macrophage efferocytosis. We further aim to discuss current and emerging models of inflammation and suggest novel ways of terminating or resolving deleterious inflammatory responses with a specific view to the translation of these strategies into fully realized, pro-resolution therapies.

Formylated Peptide Receptor-1-Mediated Gut Inflammation as a Therapeutic Target in Inflammatory Bowel Disease.

Background: Formylated peptide receptor (FPR)-1 is a G-coupled receptor that senses foreign bacterial and host-derived mitochondrial formylated peptides (FPs), leading to innate immune system activation. Aim: We sought to investigate the role of FPR1-mediated inflammation and its potential as a therapeutic target in inflammatory bowel disease (IBD). Methods: We characterized FPR1 gene and protein expression in 8 human IBD (~1000 patients) datasets with analysis on disease subtype, mucosal inflammation, and drug response. We performed in vivo dextran-sulfate sodium (DSS) colitis in C57/BL6 FPR1 knockout mice. In ex vivo studies, we studied the role of mitochondrial FPs and pharmacological blockade of FPR1 using cyclosporin H in human peripheral blood neutrophils. Finally, we assess mitochondrial FPs as a potential mechanistic biomarker in the blood and stools of patients with IBD. Results: Detailed in silico analysis in human intestinal biopsies showed that FPR1 is highly expressed in IBD (n = 207 IBD vs 67 non-IBD controls, P < .001), and highly correlated with gut inflammation in ulcerative colitis (UC) and Crohn's disease (CD) (both P < .001). FPR1 receptor is predominantly expressed in leukocytes, and we showed significantly higher FPR1+ve neutrophils in inflamed gut tissue section in IBD (17 CD and 24 UC; both P < .001). Further analysis in 6 independent IBD (data available under Gene Expression Omnibus accession numbers GSE59071, GSE206285, GSE73661, GSE16879, GSE92415, and GSE235970) showed an association with active gut inflammation and treatment resistance to infliximab, ustekinumab, and vedolizumab. FPR1 gene deletion is protective in murine DSS colitis with lower gut neutrophil inflammation. In the human ex vivo neutrophil system, mitochondrial FP, nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) is a potent activator of neutrophils resulting in higher CD62L shedding, CD63 expression, reactive oxygen species production, and chemotactic capacity; these effects are inhibited by cyclosporin H. We screened for mitochondrial ND6 in IBD (n = 54) using ELISA and detected ND6 in stools with median values of 2.2 gg/mL (interquartile range [IQR] 0.0-4.99; range 0-53.3) but not in blood. Stool ND6 levels, however, were not significantly correlated with paired stool calprotectin, C-reactive protein, and clinical IBD activity. Conclusions: Our data suggest that FPR1-mediated neutrophilic inflammation is a tractable target in IBD; however, further work is required to clarify the clinical utility of mitochondrial FPs as a potential mechanistic marker for future stratification.

First-in-human controlled inhalation of thin graphene oxide nanosheets to study acute cardiorespiratory responses.

Graphene oxide nanomaterials are being developed for wide-ranging applications but are associated with potential safety concerns for human health. We conducted a double-blind randomized controlled study to determine how the inhalation of graphene oxide nanosheets affects acute pulmonary and cardiovascular function. Small and ultrasmall graphene oxide nanosheets at a concentration of 200 µg m-3 or filtered air were inhaled for 2 h by 14 young healthy volunteers in repeated visits. Overall, graphene oxide nanosheet exposure was well tolerated with no adverse effects. Heart rate, blood pressure, lung function and inflammatory markers were unaffected irrespective of graphene oxide particle size. Highly enriched blood proteomics analysis revealed very few differential plasma proteins and thrombus formation was mildly increased in an ex vivo model of arterial injury. Overall, acute inhalation of highly purified and thin nanometre-sized graphene oxide nanosheets was not associated with overt detrimental effects in healthy humans. These findings demonstrate the feasibility of carefully controlled human exposures at a clinical setting for risk assessment of graphene oxide, and lay the foundations for investigating the effects of other two-dimensional nanomaterials in humans. Clinicaltrials.gov ref: NCT03659864.

Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis.

Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders.

Predictive value of in vitro assays depends on the mechanism of toxicity of metal oxide nanoparticles.

BACKGROUND: Hazard identification for risk assessment of nanoparticles (NPs) is mainly composed of in vitro cell-based assays and in vivo animal experimentation. The rapidly increasing number and functionalizations of NPs makes in vivo toxicity tests undesirable on both ethical and financial grounds, creating an urgent need for development of in vitro cell-based assays that accurately predict in vivo toxicity and facilitate safe nanotechnology. METHODS: In this study, we used 9 different NPs (CeO2, TiO2, carbon black, SiO2, NiO, Co3O4, Cr2O3, CuO, and ZnO). As an in vivo toxicity endpoint, the acute lung inflammogenicity in a rat instillation model was compared with the in vitro toxicity endpoints comprising cytotoxicity, pro-inflammatory cytokine expression, or haemolytic potential. For in vitro assays, 8 different cell-based assays were used including epithelial cells, monocytic/macrophage cells, human erythrocytes, and combined culture. RESULTS: ZnO and CuO NPs acting via soluble toxic ions showed positive results in most of assays and were consistent with the lung inflammation data. When compared in in vitro assays at the same surface area dose (30 cm2/mL), NPs that were low solubility and therefore acting via surface reactivity had no convincing activity, except for CeO2 NP. Cytotoxicity in differentiated peripheral blood mononuclear cells was the most accurate showing 89% accuracy and 11% false negativity in predicting acute lung inflammogenicity. However, the haemolysis assay showed 100% consistency with the lung inflammation if any dose, having statistical significance was considered positivity. Other cell-based in vitro assays showed a poorer correlation with in vivo inflammogenicity. CONCLUSIONS: Based on the toxicity mechanisms of NPs, two different approaches can be applied for prediction of in vivo lung inflammogenicity. Most in vitro assays were good at detecting NPs that act via soluble ions (i.e., ZnO and CuO NP). However, in vitro assays were limited in detecting NPs acting via surface reactivity as their mechanism of toxicity, except for the haemolysis assay.

Diesel exhaust particulate increases the size and complexity of lesions in atherosclerotic mice.

OBJECTIVE: Diesel exhaust particulate (DEP), a major component of urban air pollution, has been linked to atherogenesis and precipitation of myocardial infarction. We hypothesized that DEP exposure would increase and destabilise atherosclerotic lesions in apolipoprotein E deficient (ApoE-/-) mice. METHODS: ApoE-/- mice were fed a 'Western diet' (8 weeks) to induce 'complex' atherosclerotic plaques, with parallel experiments in normal chow fed wild-type mice. During the last 4 weeks of feeding, mice received twice weekly instillation (oropharyngeal aspiration) of 35 µL DEP (1 mg/mL, SRM-2975) or vehicle (saline). Atherosclerotic burden was assessed by en-face staining of the thoracic aorta and histological examination of the brachiocephalic artery. RESULTS: Brachiocephalic atherosclerotic plaques were larger in ApoE-/- mice treated with DEP (59 ± 10%) than in controls (32 ± 7%; P = 0.017). In addition, DEP-treated mice had more plaques per section of artery (2.4 ± 0.2 vs 1.8 ± 0.2; P = 0.048) and buried fibrous layers (1.2 ± 0.2 vs 0.4 ± 0.1; P = 0.028). These changes were associated with lung inflammation and increased antioxidant gene expression in the liver, but not with changes in endothelial function, plasma lipids or systemic inflammation. CONCLUSIONS: Increased atherosclerosis is caused by the particulate component of diesel exhaust producing advanced plaques with a potentially more vulnerable phenotype. These results are consistent with the suggestion that removal of the particulate component would reduce the adverse cardiovascular effects of diesel exhaust.