ABSTRACT
Despite the prevalence and clinical importance of influenza, its long-term effect on lung immunity is unclear. Here we describe that following viral clearance and clinical recovery, at 1 month after infection with influenza, mice are better protected from Streptococcus pneumoniae infection due to a population of monocyte-derived alveolar macrophages (AMs) that produce increased interleukin-6. Influenza-induced monocyte-derived AMs have a surface phenotype similar to resident AMs but display a unique functional, transcriptional and epigenetic profile that is distinct from resident AMs. In contrast, influenza-experienced resident AMs remain largely similar to naive AMs. Thus, influenza changes the composition of the AM population to provide prolonged antibacterial protection. Monocyte-derived AMs persist over time but lose their protective profile. Our results help to understand how transient respiratory infections, a common occurrence in human life, can constantly alter lung immunity by contributing monocyte-derived, recruited cells to the AM population.
Subject(s)
Immunity, Innate/immunology , Macrophages, Alveolar/immunology , Orthomyxoviridae Infections/immunology , Pneumococcal Infections/immunology , Animals , MiceABSTRACT
Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health1. Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells2, we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a ß-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies.
Subject(s)
Escherichia coli , Gastrointestinal Microbiome , Gene Editing , Klebsiella pneumoniae , beta-Lactamases , Animals , Mice , Escherichia coli/genetics , Gene Editing/methods , Klebsiella pneumoniae/genetics , beta-Lactamases/genetics , beta-Lactamases/metabolism , Female , Bacteriophages/genetics , CRISPR-Cas Systems/genetics , Male , Gastrointestinal Tract/microbiology , Genes, Bacterial/genetics , Genetic Vectors/geneticsABSTRACT
Single nucelotide polymorphisms (SNPs) at the FAM13A locus are among the most commonly reported risk alleles associated with chronic obstructive pulmonary disease (COPD) and other respiratory diseases; however, the physiological role of FAM13A is unclear. In humans, two major protein isoforms are expressed at the FAM13A locus: "long" and "short," but their functions remain unknown, partly because of a lack of isoform conservation in mice. We performed in-depth characterization of organotypic primary human airway epithelial cell subsets and show that multiciliated cells predominantly express the FAM13A long isoform containing a putative N-terminal Rho GTPase-activating protein (RhoGAP) domain. Using purified proteins, we directly demonstrate the RhoGAP activity of this domain. In Xenopus laevis, which conserve the long-isoform, Fam13a deficiency impaired cilia-dependent embryo motility. In human primary epithelial cells, long-isoform deficiency did not affect multiciliogenesis but reduced cilia coordination in mucociliary transport assays. This is the first demonstration that FAM13A isoforms are differentially expressed within the airway epithelium, with implications for the assessment and interpretation of SNP effects on FAM13A expression levels. We also show that the long FAM13A isoform coordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance.
Subject(s)
Cilia , GTPase-Activating Proteins , Mucociliary Clearance , Protein Isoforms , Xenopus laevis , Cilia/metabolism , Humans , Animals , Protein Isoforms/metabolism , Protein Isoforms/genetics , GTPase-Activating Proteins/metabolism , GTPase-Activating Proteins/genetics , Epithelial Cells/metabolism , Respiratory Mucosa/metabolism , Cells, CulturedABSTRACT
BACKGROUND: Chronic sputum production impacts on quality of life and is a feature of many respiratory diseases. Identification of the genetic variants associated with chronic sputum production in a disease agnostic sample could improve understanding of its causes and identify new molecular targets for treatment. METHODS: We conducted a genome-wide association study (GWAS) of chronic sputum production in UK Biobank. Signals meeting genome-wide significance (p<5×10-8) were investigated in additional independent studies, were fine-mapped and putative causal genes identified by gene expression analysis. GWASs of respiratory traits were interrogated to identify whether the signals were driven by existing respiratory disease among the cases and variants were further investigated for wider pleiotropic effects using phenome-wide association studies (PheWASs). RESULTS: From a GWAS of 9714 cases and 48 471 controls, we identified six novel genome-wide significant signals for chronic sputum production including signals in the human leukocyte antigen (HLA) locus, chromosome 11 mucin locus (containing MUC2, MUC5AC and MUC5B) and FUT2 locus. The four common variant associations were supported by independent studies with a combined sample size of up to 2203 cases and 17 627 controls. The mucin locus signal had previously been reported for association with moderate-to-severe asthma. The HLA signal was fine-mapped to an amino acid change of threonine to arginine (frequency 36.8%) in HLA-DRB1 (HLA-DRB1*03:147). The signal near FUT2 was associated with expression of several genes including FUT2, for which the direction of effect was tissue dependent. Our PheWAS identified a wide range of associations including blood cell traits, liver biomarkers, infections, gastrointestinal and thyroid-associated diseases, and respiratory disease. CONCLUSIONS: Novel signals at the FUT2 and mucin loci suggest that mucin fucosylation may be a driver of chronic sputum production even in the absence of diagnosed respiratory disease and provide genetic support for this pathway as a target for therapeutic intervention.
Subject(s)
Genome-Wide Association Study , Sputum , Humans , Sputum/metabolism , HLA-DRB1 Chains , Quality of Life , Proteins , Mucins , Mucus/metabolism , Genetic Predisposition to Disease , Polymorphism, Single NucleotideABSTRACT
Human rhinovirus (RV) is a major risk factor for chronic obstructive pulmonary disease (COPD) and asthma exacerbations. The exploration of RV pathogenesis has been hampered by a lack of disease-relevant model systems. We performed a detailed characterization of host responses to RV infection in human lung tissue ex vivo and investigated whether these responses are disease relevant for patients with COPD and asthma. In addition, impact of the viral replication inhibitor rupintrivir was evaluated. Human precision-cut lung slices (PCLS) were infected with RV1B with or without rupintrivir. At Days 1 and 3 after infection, RV tissue localization, tissue viability, and viral load were determined. To characterize host responses to infection, mediator and whole genome analyses were performed. RV successfully replicated in PCLS airway epithelial cells and induced both antiviral and proinflammatory cytokines such as IFNα2a, CXCL10, CXCL11, IFN-γ, TNFα, and CCL5. Genomic analyses revealed that RV not only induced antiviral immune responses but also triggered changes in epithelial cell-associated pathways. Strikingly, the RV response in PCLS was reflective of gene expression changes described in patients with COPD and asthma. Although RV-induced host immune responses were abrogated by rupintrivir, RV-triggered epithelial processes were largely refractory to antiviral treatment. Detailed analysis of RV-infected human PCLS and comparison with gene signatures of patients with COPD and asthma revealed that the human RV PCLS model represents disease-relevant biological mechanisms that can be partially inhibited by a well-known antiviral compound and provide an outstanding opportunity to evaluate novel therapeutics.
Subject(s)
Asthma/genetics , Host-Pathogen Interactions/genetics , Lung/virology , Picornaviridae Infections/genetics , Pulmonary Disease, Chronic Obstructive/genetics , Aged , Antiviral Agents/pharmacology , Asthma/pathology , Bronchi/pathology , Bronchi/physiology , Epithelial Cells/pathology , Epithelial Cells/virology , Female , Gene Expression Profiling , Genome, Human , Humans , Isoxazoles/pharmacology , Lung/physiology , Male , Middle Aged , Phenylalanine/analogs & derivatives , Phenylalanine/pharmacology , Picornaviridae Infections/drug therapy , Picornaviridae Infections/pathology , Pulmonary Disease, Chronic Obstructive/pathology , Pyrrolidinones/pharmacology , Rhinovirus/pathogenicity , Valine/analogs & derivatives , Valine/pharmacologyABSTRACT
BACKGROUND: Phosphoinositide-3-kinase-delta (PI3Kδ) inhibition is a promising therapeutic approach for inflammatory conditions due to its role in leucocyte proliferation, migration and activation. However, the effect of PI3Kδ inhibition on group 2 innate lymphoid cells (ILC2s) and inflammatory eosinophils remains unknown. Using a murine model exhibiting persistent airway inflammation we sought to understand the effect of PI3Kδ inhibition, montelukast and anti-IL5 antibody treatment on IL33 expression, group-2-innate lymphoid cells, inflammatory eosinophils, and goblet cell metaplasia. RESULTS: Mice were sensitised to house dust mite and after allowing inflammation to resolve, were re-challenged with house dust mite to re-initiate airway inflammation. ILC2s were found to persist in the airways following house dust mite sensitisation and after re-challenge their numbers increased further along with accumulation of inflammatory eosinophils. In contrast to montelukast or anti-IL5 antibody treatment, PI3Kδ inhibition ablated IL33 expression and prevented group-2-innate lymphoid cell accumulation. Only PI3Kδ inhibition and IL5 neutralization reduced the infiltration of inflammatory eosinophils. Moreover, PI3Kδ inhibition reduced goblet cell metaplasia. CONCLUSIONS: Hence, we show that PI3Kδ inhibition dampens allergic inflammatory responses by ablating key cell types and cytokines involved in T-helper-2-driven inflammatory responses.
Subject(s)
Class I Phosphatidylinositol 3-Kinases/metabolism , Eosinophils/immunology , Hypersensitivity/immunology , Inflammation/immunology , Interleukin-33/metabolism , Lymphocytes/immunology , Respiratory System/immunology , Acetates/therapeutic use , Animals , Antigens, Dermatophagoides/immunology , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Cyclopropanes/therapeutic use , Cytokines/metabolism , Female , Goblet Cells/drug effects , Goblet Cells/pathology , Hypersensitivity/drug therapy , Inflammation/drug therapy , Interleukin-5/antagonists & inhibitors , Mice , Mice, Inbred BALB C , Pyroglyphidae , Quinolines/therapeutic use , Sulfides/therapeutic use , Th2 Cells/immunologyABSTRACT
Defective phagocytosis has been shown in chronic obstructive pulmonary disease (COPD) bronchoalveolar lavage and blood monocyte-derived macrophages. Phagocytic capabilities of sputum macrophages and neutrophils in COPD are unknown. We investigated phagocytosis in these cells from COPD patients and controls. Phagocytosis of Streptococcus pneumoniae or fluorescently labelled non-typeable Haemophilus influenzae (NTHi) by sputum macrophages and neutrophils was determined by gentamycin protection assay (COPD; n = 5) or flow cytometry in 14 COPD patients, 8 healthy smokers (HS) and 9 healthy never-smokers (HNS). Sputum macrophages and neutrophils were differentiated by adherence for the gentamycin protection assay or receptor expression (CD206 and CD66b, respectively), by flow cytometry. The effects of NTHi on macrophage expression of CD206 and CD14 and neutrophil expression of CD16 were determined by flow cytometry. There was greater uptake of S. pneumoniae [~10-fold more colony-forming units (CFU)/ml] by sputum neutrophils compared to macrophages in COPD patients. Flow cytometry showed greater NTHi uptake by neutrophils compared to macrophages in COPD (67 versus 38%, respectively) and HS (61 versus 31%, respectively). NTHi uptake by macrophages was lower in HS (31%, p = 0.019) and COPD patients (38%, p = 0.069) compared to HNS (57%). NTHi uptake by neutrophils was similar between groups. NTHi exposure reduced CD206 and CD14 expression on macrophages and CD16 expression on neutrophils. Sputum neutrophils showed more phagocytic activity than macrophages. There was some evidence that bacterial phagocytosis was impaired in HS sputum macrophages, but no impairment of neutrophils was observed in HS or COPD patients. These results highlight the relative contributions of neutrophils and macrophages to bacterial clearance in COPD.
Subject(s)
Haemophilus influenzae/immunology , Macrophages/immunology , Neutrophils/immunology , Phagocytosis , Pulmonary Disease, Chronic Obstructive/immunology , Sputum/immunology , Streptococcus mutans/immunology , Adult , Aged , Antigens, CD/immunology , Female , Flow Cytometry , Humans , Macrophages/microbiology , Male , Middle Aged , Neutrophils/microbiology , Pulmonary Disease, Chronic Obstructive/microbiology , Sputum/microbiologyABSTRACT
Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.
Subject(s)
Macrophages , Microscopy , Cell Membrane , Lung , MicrotubulesABSTRACT
In vitro data for low-dose inhaled phosphoinositide 3-kinase delta inhibitor nemiralisib revealed that it was a substrate and a potent metabolism-dependent inhibitor of cytochrome P450 (P450) 3A4 and a P-glycoprotein (P-gp) substrate. An integrated in silico, in vitro, and clinical approach including a clinical drug interaction study as well as a bespoke physiologically based pharmacokinetic (PBPK) model was used to assess the drug-drug interaction (DDI) risk. Inhaled nemiralisib (100 µg, single dose) was coadministered with itraconazole, a potent P4503A4/P-gp inhibitor, following 200 mg daily administrations for 10 days in 20 male healthy subjects. Systemic exposure to nemiralisib (AUC0-inf) increased by 2.01-fold versus nemiralisib alone. To extrapolate the clinical data to other P4503A4 inhibitors, an inhaled PBPK model was developed using Simcyp software. Retrospective simulation of the victim risk showed good agreement between simulated and observed data (AUC0-inf ratio 2.3 vs. 2.01, respectively). Prospective DDI simulations predicted a weak but manageable drug interaction when nemiralisib was coadministered with other P4503A4 inhibitors, such as the macrolides clarithromycin and erythromycin (simulated AUC0-inf ratio of 1.7), both common comedications in the intended patient populations. PBPK and static mechanistic models were also used to predict a negligible perpetrator DDI effect for nemiralisib on other P4503A4 substrates, including midazolam (a sensitive probe substrate of P4503A4) and theophylline (a narrow therapeutic index drug and another common comedication). In summary, an integrated in silico, in vitro, and clinical approach including an inhalation PBPK model has successfully discharged any potential patient DDI risks in future nemiralisib clinical trials. SIGNIFICANCE STATEMENT: This paper describes the integration of in silico, in vitro, and clinical data to successfully discharge potential drug-drug interaction risks for a low-dose inhaled drug. This work featured assessment of victim and perpetrator risks of drug transporters and cytochrome P450 enzymes, utilizing empirical and mechanistic approaches combined with clinical data (drug interaction and human absorption, metabolism, and pharmacokinetics) and physiologically based pharmacokinetic modeling approaches to facilitate bespoke risk assessment in target patient populations.
Subject(s)
Cytochrome P-450 CYP3A Inhibitors/pharmacokinetics , Cytochrome P-450 CYP3A/metabolism , Indazoles/pharmacokinetics , Indoles/pharmacokinetics , Itraconazole/pharmacokinetics , Oxazoles/pharmacokinetics , Phosphoinositide-3 Kinase Inhibitors/pharmacokinetics , Piperazines/pharmacokinetics , Administration, Inhalation , Adolescent , Adult , Aged , Area Under Curve , Clarithromycin/administration & dosage , Clarithromycin/pharmacokinetics , Computer Simulation , Cross-Over Studies , Cytochrome P-450 CYP3A Inhibitors/administration & dosage , Drug Interactions , Erythromycin/administration & dosage , Erythromycin/pharmacokinetics , Healthy Volunteers , Humans , Indazoles/administration & dosage , Indoles/administration & dosage , Itraconazole/administration & dosage , Male , Microsomes, Liver , Midazolam/administration & dosage , Midazolam/pharmacokinetics , Middle Aged , Models, Biological , Oxazoles/administration & dosage , Phosphoinositide-3 Kinase Inhibitors/administration & dosage , Piperazines/administration & dosage , Prospective Studies , Retrospective Studies , Young AdultABSTRACT
RATIONALE: There is a need to develop imaging protocols which assess neutrophilic inflammation in the lung. AIM: To quantify whole lung neutrophil accumulation in (1) healthy volunteers (HV) following inhaled lipopolysaccharide (LPS) or saline and (2) patients with COPD using radiolabelled autologous neutrophils and single-photon emission computed tomography/CT (SPECT/CT). METHODS: 20 patients with COPD (Global initiative for chronic obstructive lung disease (GOLD) stages 2-3) and 18 HVs were studied. HVs received inhaled saline (n=6) or LPS (50 µg, n=12) prior to the injection of radiolabelled cells. Neutrophils were isolated using dextran sedimentation and Percoll plasma gradients and labelled with 99mTechnetium (Tc)-hexamethylpropyleneamine oxime. SPECT was performed over the thorax/upper abdomen at 45 min, 2 hours, 4 hours and 6 hours. Circulating biomarkers were measured prechallenge and post challenge. Blood neutrophil clearance in the lung was determined using Patlak-Rutland graphical analysis. RESULTS: There was increased accumulation of 99mTc-neutrophils in the lungs of patients with COPD and LPS-challenged subjects compared with saline-challenged subjects (saline: 0.0006±0.0003 mL/min/mL lung blood distribution volume [mean ±1 SD]; COPD: 0.0022±0.0010 mL/min/mL [p<0.001]; LPS: 0.0025±0.0008 mL/min/mL [p<0.001]). The accumulation of labelled neutrophils in 10 patients with COPD who underwent repeat radiolabelling/imaging 7-10 days later was highly reproducible (0.0022±0.0010 mL/min/mL vs 0.0023±0.0009 mL/min/mL). Baseline interleukin (IL)-6 levels in patients with COPD were elevated compared with HVs (1.5±1.06 pg/mL [mean ±1 SD] vs 0.4±0.24 pg/mL). LPS challenge increased the circulating IL-6 levels (7.5±2.72 pg/mL) 9 hours post challenge. CONCLUSIONS: This study shows the ability to quantify 'whole lung' neutrophil accumulation in HVs following LPS inhalation and in subjects with COPD using autologous radiolabelled neutrophils and SPECT/CT imaging. Moreover, the reproducibility observed supports the feasibility of using this approach to determine the efficacy of therapeutic agents aimed at altering neutrophil migration to the lungs.
Subject(s)
Lung/diagnostic imaging , Neutrophils/physiology , Pulmonary Disease, Chronic Obstructive/diagnostic imaging , Aged , Biomarkers/blood , Female , Humans , Interleukin-6/blood , Lipopolysaccharides , Male , Middle Aged , Neutrophil Infiltration/drug effects , Neutrophil Infiltration/physiology , Pulmonary Disease, Chronic Obstructive/pathology , Reproducibility of Results , Single Photon Emission Computed Tomography Computed Tomography/methods , TechnetiumABSTRACT
This study describes the pharmacokinetic (PK) and pharmaco-dynamic (PD) profile of N-(5-(4-(5-(((2R,6S)-2,6-dimethylmorpholino)methyl)oxazol-2-yl)-1H-indazol-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide (GSK2292767A), a novel low-solubility inhaled phosphoinositide 3-kinase delta (PI3Kδ) inhibitor developed as an alternative to 2-(6-(1H-indol-4-yl)-1H-indazol-4-yl)-5-((4-isopropylpiperazin-1-yl)methyl)oxazole (nemiralisib), which is a highly soluble inhaled inhibitor of PI3Kδ with a lung profile consistent with once-daily dosing. GSK2292767A has a similar in vitro cellular profile to nemiralisib and reduces eosinophilia in a murine PD model by 63% (n = 5, P < 0.05). To explore whether a low-soluble compound results in effective PI3Kδ inhibition in humans, a first time in human study was conducted with GSK2292767A in healthy volunteers who smoke. GSK2292767A was generally well tolerated, with headache being the most common reported adverse event. PD changes in induced sputum were measured in combination with drug concentrations in plasma from single (0.05-2 mg, n = 37), and 14-day repeat (2 mg, n = 12) doses of GSK2292767A. Trough bronchoalveolar lavage (BAL) for PK was taken after 14 days of repeat dosing. GSK2292767A displayed a linear increase in plasma exposure with dose, with marginal accumulation after 14 days. Induced sputum showed a 27% (90% confidence interval 15%, 37%) reduction in phosphatidylinositol-trisphosphate (the product of phosphoinositide 3-kinase activation) 3 hours after a single dose. Reduction was not maintained 24 hours after single or repeat dosing. BAL analysis confirmed the presence of GSK2292767A in lung at 24 hours, consistent with the preclinical lung retention profile. Despite good lung retention, target engagement was only present at 3 hours. This exposure-response disconnect is an important observation for future inhaled drug design strategies considering low solubility to drive lung retention.
Subject(s)
Indazoles/pharmacology , Indazoles/pharmacokinetics , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors/pharmacokinetics , Sulfonamides/pharmacology , Sulfonamides/pharmacokinetics , Translational Research, Biomedical , Administration, Inhalation , Adult , Animals , Bronchoalveolar Lavage , Eosinophilia/drug therapy , Female , Humans , Indazoles/administration & dosage , Indazoles/adverse effects , Lung/metabolism , Male , Mice , Middle Aged , Permeability , Phosphoinositide-3 Kinase Inhibitors/administration & dosage , Phosphoinositide-3 Kinase Inhibitors/adverse effects , Safety , Solubility , Sputum/drug effects , Sputum/metabolism , Sulfonamides/administration & dosage , Sulfonamides/adverse effectsABSTRACT
An innovative open-label, crossover clinical study was used to investigate the excretion balance, pharmacokinetics, and metabolism of nemiralisib-an inhaled phosphoinositide 3-kinase delta inhibitor being developed for respiratory diseases. Six healthy men received a single intravenous microtracer of 10 µg [14C]nemiralisib with a concomitant inhaled nonradiolabeled 1000 µg dose followed by an oral 800 µg dose of [14C]nemiralisib 14 days later. Complementary methods including accelerator mass spectrometry allowed characterization of a range of parameters including oral absorption (Fabs), proportion of nemiralisib escaping gut wall metabolism (Fg), hepatic extraction (Eh), fraction of dose absorbed from inhaled dose (Flung), and renal clearance. Intravenous pharmacokinetics of nemiralisib were characterized by low blood clearance (10.0 l/h), long terminal half-life (55 hours), and high volume of distribution at steady state (728 l). Nemiralisib exhibited moderate inhaled and oral bioavailability (38% and 35%) while Flung was 29%. Absorption and first-pass parameters were corrected for blood renal clearance and compared with values without correction. Any swallowed nemiralisib was relatively well absorbed (Fabs, 0.48) with a high fraction escaping gut wall metabolism and low extraction by the liver (Fg and Eh being 0.83 and 0.10, respectively). There were no major human plasma metabolites requiring further qualification in animal studies. Both unchanged nemiralisib and its oxidative/conjugative metabolites were secreted in bile, with nemiralisib likely subject to further metabolism through enterohepatic recirculation. Direct renal clearance and metabolism followed by renal clearance were lesser routes of elimination. SIGNIFICANCE STATEMENT: A number of innovative features have been combined into one small clinical study enabling a comprehensive description of the human pharmacokinetics and metabolism of an inhaled molecule. Design elements included an intravenous 14C tracer administration concomitant with an inhalation dose that enabled derivation of parameters such as fraction absorbed (Fabs), the proportion of drug escaping first-pass extraction through the gut wall and liver (Fg and Fh) and hepatic extraction (Eh). Entero-test bile sampling enabled characterization of biliary elimination pathways.
Subject(s)
Drug Monitoring/methods , Indazoles/pharmacokinetics , Indoles/pharmacokinetics , Oxazoles/pharmacokinetics , Piperazines/pharmacokinetics , Protein Kinase Inhibitors/pharmacokinetics , Administration, Inhalation , Administration, Intravenous , Administration, Oral , Adult , Biological Availability , Carbon Radioisotopes , Cross-Over Studies , Feces/chemistry , Healthy Volunteers , Humans , Indazoles/administration & dosage , Indazoles/blood , Indazoles/urine , Indoles/administration & dosage , Indoles/blood , Indoles/urine , Injections, Intravenous , Male , Metabolic Clearance Rate , Middle Aged , Oxazoles/administration & dosage , Oxazoles/blood , Oxazoles/urine , Piperazines/administration & dosage , Piperazines/blood , Piperazines/urine , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/blood , Protein Kinase Inhibitors/urine , Tissue DistributionABSTRACT
BACKGROUND: Little is known about the interactions between the lung microbiome and host response in chronic obstructive pulmonary disease (COPD). METHODS: We performed a longitudinal 16S ribosomal RNA gene-based microbiome survey on 101 sputum samples from 16 healthy subjects and 43 COPD patients, along with characterization of host sputum transcriptome and proteome in COPD patients. RESULTS: Dysbiosis of sputum microbiome was observed with significantly increased relative abundance of Moraxella in COPD versus healthy subjects and during COPD exacerbations, and Haemophilus in COPD ex-smokers versus current smokers. Multivariate modeling on sputum microbiome, host transcriptome and proteome profiles revealed that significant associations between Moraxella and Haemophilus, host interferon and pro-inflammatory signaling pathways and neutrophilic inflammation predominated among airway host-microbiome interactions in COPD. While neutrophilia was positively correlated with Haemophilus, interferon signaling was more strongly linked to Moraxella. Moreover, while Haemophilus was significantly associated with host factors both in stable state and during exacerbations, Moraxella-associated host responses were primarily related to exacerbations. CONCLUSIONS: Our study highlights a significant airway host-microbiome interplay associated with COPD inflammation and exacerbations. These findings indicate that Haemophilus and Moraxella influence different components of host immune response in COPD, and that novel therapeutic strategies should consider targeting these bacteria and their associated host pathways in COPD.
Subject(s)
Host Microbial Interactions/physiology , Lung/microbiology , Lung/physiology , Microbiota/physiology , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/microbiology , Aged , Female , Gene Expression Profiling/methods , Haemophilus influenzae/genetics , Humans , Longitudinal Studies , Male , Middle Aged , Moraxella/genetics , Sputum/microbiology , Sputum/physiologyABSTRACT
Phosphoinositide 3-kinase δ (PI3Kδ) is a lipid kinase involved in leukocyte recruitment and activation. Activation of PI3Kδ has been linked to airway inflammation and asthma pathogenesis. This randomized, double-blind, placebo-controlled, crossover study investigated the efficacy, safety, tolerability, and pharmacokinetics of a PI3Kδ inhibitor, nemiralisib (GSK2269557), in patients with persistent, uncontrolled asthma. Patients (n = 50) received once-daily inhaled nemiralisib (1000 µg) or placebo for 28 days, with a crossover to the alternative treatment following a 4-week washout period. Spirometry demonstrated no discernible difference in trough forced expiratory volume in 1 second (FEV1) from baseline (adjusted posterior median 7 ml; 95% credible interval -83, 102 ml) between nemiralisib and placebo treatment at day 28 (primary endpoint). These results were supported by most secondary endpoints, including weighted mean FEV1 (0-4 hours) and change in trough forced vital capacity at day 28. Nemiralisib was generally well-tolerated, with few side effects except for post-inhalation cough (nemiralisib: 35%; placebo: 9%). At day 14, sputum interleukin (IL)-5, IL-13, IL-6, and IL-8 levels were reduced by a median of 17%, 7%, 15%, and 8%, respectively, when comparing nemiralisib with placebo [n = 15 (IL-5, IL-8) or 16 (IL-6, IL-13); posterior probability of a true ratio >0%: 78%, 64%, 76%, and 63%, respectively]. These results suggest that nemiralisib inhibited PI3Kδ locally; however, this did not translate into meaningful clinical improvement. Further studies will investigate the potential efficacy of nemiralisib in patients with asthma with other specific more severe phenotypes, including those who are colonized with bacteria and frequently exacerbate.
Subject(s)
Asthma/drug therapy , Bronchodilator Agents/administration & dosage , Indazoles/administration & dosage , Indoles/administration & dosage , Oxazoles/administration & dosage , Piperazines/administration & dosage , Administration, Inhalation , Adrenergic beta-2 Receptor Agonists/administration & dosage , Adult , Aged , Asthma/metabolism , Cross-Over Studies , Double-Blind Method , Female , Forced Expiratory Volume/drug effects , Humans , Male , Middle Aged , Phosphatidylinositol 3-Kinases/metabolism , Vital Capacity/drug effects , Young AdultABSTRACT
Lymphocyte numbers are increased in the lungs of chronic obstructive pulmonary disease (COPD) patients. Phosphatidylinositol-3-kinase delta (PI3Kδ) is involved in lymphocyte activation. We investigated the effect of PI3Kδ inhibition on cytokine release from COPD lymphocytes. We also evaluated phosphorylated ribosomal S6 protein (rS6) as a potential biomarker of PI3Kδ activation. Peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage (BAL) cells isolated from healthy never smokers (HNS), smokers (S) and COPD patients were stimulated to induce a T cell receptor response. The effects of a PI3Kδ specific inhibitor (GSK045) on cytokine release and rS6 phosphorylation were measured by Luminex and flow cytometry respectively. The effects of GSK045 on cytokine production from PHA stimulated chopped lung samples were investigated. GSK045 reduced cytokine release from PBMCs, BAL cells and chopped lung. Inhibition was greatest in the chopped lung model, with approximately 80% inhibition of interferon (IFN) γ, interleukin (IL)-2, IL-17 and IL-10. PI3Kδ inhibition suppressed rS6 phosphorylation in unstimulated airway T-lymphocytes by up to 60%. Inhibition of PI3Kδ suppressed T cell cytokine production in COPD patients. rS6 phosphorylation shows potential as a biomarker to assess PI3Kδ activity.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Enzyme Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Pulmonary Disease, Chronic Obstructive/immunology , T-Lymphocytes/drug effects , Anti-Inflammatory Agents/therapeutic use , Biomarkers/metabolism , Bronchoalveolar Lavage , Cytokines/biosynthesis , Enzyme Inhibitors/therapeutic use , Female , Humans , Male , Middle Aged , Phosphorylation/drug effects , Pulmonary Disease, Chronic Obstructive/drug therapy , Ribosomal Protein S6/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
RATIONALE: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied. OBJECTIVES: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition. METHODS: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays. MEASUREMENTS AND MAIN RESULTS: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures. CONCLUSIONS: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.
Subject(s)
Neutrophils/drug effects , Phosphoinositide-3 Kinase Inhibitors , Respiratory Distress Syndrome/immunology , Bronchoalveolar Lavage Fluid/cytology , CD11b Antigen/metabolism , Cytokines/metabolism , Female , Flow Cytometry , Gene Expression Profiling , Humans , L-Selectin/metabolism , Male , Middle Aged , Neutrophils/metabolism , Neutrophils/physiology , Oligonucleotide Array Sequence Analysis , Phenotype , Respiratory Distress Syndrome/drug therapyABSTRACT
BACKGROUND: Acute respiratory distress syndrome (ARDS) is characterised by diffuse neutrophil-mediated alveolar inflammation. Recently, we demonstrated that blood polymorphonuclear leucocytes (PMNs) in ARDS are basally activated, and exhibit aberrant oxidative burst and survival responses. The molecular mechanisms governing ARDS PMN function and longevity are incompletely understood. We aimed to use genome-wide transcriptional profiling of ARDS blood PMNs to explore underlying disease mechanisms and identify therapeutic targets aimed at manipulating PMN function and longevity. METHODS: GeneChip Affymetrix oligonucleotide arrays were used to assess global transcriptional profiles in highly pure PMNs from ventilated patients fulfilling the Berlin ARDS definition (n=10), in freshly isolated PMNs from age-matched and sex-matched healthy volunteers (n=10), and in healthy volunteer PMNs exposed in vitro to recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (1 ng/mL for 6 h). Ingenuity Pathway Analysis software was used to map probes identified as important onto specific pathways. FINDINGS: Transcriptomic analysis showed that 1319 genes were altered in ARDS PMNs relative to healthy volunteer PMNs. Compared with well established reference databases, the gene expression profile in ARDS PMNs showed near-complete correlation to datasets derived from patients with sepsis and burns. Transcripts enriched in ARDS PMNs were differentially expressed in known functional network pathways associated with cancer, cellular compromise, apoptotic mechanisms, and chemotaxis. Of the observed gene changes, only 292 (22%) were seen in healthy volunteer PMNs after exposure to rhGM-CSF, of which 216 showed the same directional change as ARDS PMNs. INTERPRETATION: Existing genome-wide studies in ARDS use total blood leucocytes; our study is the first, to our knowledge, to use unbiased global genomic profiling of highly pure ARDS blood PMNs in parallel with age-matched and gender-matched healthy volunteer PMNs treated with rhGM-CSF. Collectively our results show that ARDS PMNs display important de-novo transcriptional activity. The global transcriptomic changes were consistent with the observed aberrant ARDS PMN survival and functional phenotype that we have previously reported, and show near-complete correlation to existing sepsis and burns datasets, but only limited transcriptomic overlap with healthy volunteer PMNs treated with rhGM-CSF. FUNDING: National Institute for Health Research, GlaxoSmithKline.
ABSTRACT
PURPOSE: We developed and tested a novel Quantitative Structure-Activity Relationship (QSAR) model to better understand the physicochemical drivers of pulmonary absorption, and to facilitate compound design through improved prediction of absorption. The model was tested using a large array of both existing and newly designed compounds. METHODS: Pulmonary absorption data was generated using the isolated perfused respiring rat lung (IPRLu) model for 82 drug discovery compounds and 17 marketed drugs. This dataset was used to build a novel QSAR model based on calculated physicochemical properties. A further 9 compounds were used to test the model's predictive capability. RESULTS: The QSAR model performed well on the 9 compounds in the "Test set" with a predicted versus observed correlation of R(2) = 0.85, and >65% of compounds correctly categorised. Calculated descriptors associated with permeability and hydrophobicity positively correlated with pulmonary absorption, whereas those associated with charge, ionisation and size negatively correlated. CONCLUSIONS: The novel QSAR model described here can replace routine generation of IPRLu model data for ranking and classifying compounds prior to synthesis. It will also provide scientists working in the field of inhaled drug discovery with a deeper understanding of the physicochemical drivers of pulmonary absorption based on a relevant respiratory compound dataset.
Subject(s)
Lung/metabolism , Models, Biological , Models, Molecular , Pharmaceutical Preparations/metabolism , Quantitative Structure-Activity Relationship , Respiration , Respiratory Tract Absorption/physiology , Animals , Drug Discovery , Hydrophobic and Hydrophilic Interactions , Ions , Male , Molecular Structure , Particle Size , Permeability , Pharmaceutical Preparations/chemistry , Rats , Surface PropertiesABSTRACT
BACKGROUND: CpG-containing oligodeoxynucleotides (CpG-ODNs) are potent inhibitors of T helper 2 mediated allergic airway disease in sensitised mice challenged with allergen. A single treatment has transient effects but a limited series of treatments has potential to achieve clinically meaningful sustained inhibition of allergic airway disease. OBJECTIVE: To optimise the treatment regimen for sustained efficacy and to determine the mechanisms of action in mice of an inhaled form of CpG-ODN being developed for human asthma treatment. METHODS: We set up a chronic allergic-asthma model using ragweed-sensitised mice exposed weekly to intranasal ragweed. Using this model, the effects of a limited series of weekly intranasal 1018 ISS (CpG-ODN; B-class) treatments were evaluated during treatment and for several weeks after treatments had stopped but weekly allergen exposures continued. Treatment efficacy was evaluated by measuring effects on lung T helper 2 cytokines and eosinophilia, and lung dendritic cell function and T-cell responses. RESULTS: Twelve intranasal 1018 ISS treatments induced significant suppression of bronchoalveolar lavage eosinophilia and interleukin 4, 5 and 13 levels. This suppression of allergic T helper 2 parameters was maintained through 13 weekly ragweed exposures administered after treatment cessation. Subsequent experiments demonstrated that at least five treatments were required for lasting suppression. Although CpG-ODN induced moderate T helper 1 responses, suppression of allergic airway disease did not require interferon γ but was associated with induction of a regulatory T-cell response. CONCLUSIONS: A short series of CpG-ODN treatments results in sustained suppression of allergic lung inflammation induced by a clinically relevant allergen.