The FAM13A Long Isoform Regulates Cilia Movement and Co-ordination in Airway Mucociliary Transport.

SNPs in the FAM13A locus are amongst 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 due to a lack of isoform conservation in mice. We performed in-depth characterisation 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 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 co-ordination 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 co-ordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

Genome-wide association study of chronic sputum production implicates loci involved in mucus production and infection.

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.

Exploring PI3Kδ Molecular Pathways in Stable COPD and Following an Acute Exacerbation, Two Randomized Controlled Trials.

Background: Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) exerts corrective effects on the dysregulated migration characteristics of neutrophils isolated from patients with chronic obstructive pulmonary disease (COPD). Objective: To develop novel, induced sputum endpoints to demonstrate changes in neutrophil phenotype in the lung by administering nemiralisib, a potent and selective inhaled PI3Kδ inhibitor, to patients with stable COPD or patients with acute exacerbation (AE) of COPD. Methods: In two randomized, double-blind, placebo-controlled clinical trials patients with A) stable COPD (N=28, randomized 3:1) or B) AECOPD (N=44, randomized 1:1) received treatment with inhaled nemiralisib (1mg). Endpoints included induced sputum at various time points before and during treatment for the measurement of transcriptomics (primary endpoint), inflammatory mediators, functional respiratory imaging (FRI), and spirometry. Results: In stable COPD patients, the use of nemiralisib was associated with alterations in sputum neutrophil transcriptomics suggestive of an improvement in migration phenotype; however, the same nemiralisib-evoked effects were not observed in AECOPD. Inhibition of sputum inflammatory mediators was also observed in stable but not AECOPD patients. In contrast, a placebo-corrected improvement in forced expiratory volume in 1 sec of 136 mL (95% Credible Intervals -46, 315mL) with a probability that the true treatment ratio was >0% (Pr(θ>0)) of 93% was observed in AECOPD. However, FRI endpoints remained unchanged. Conclusion: We provide evidence for nemiralisib-evoked changes in neutrophil migration phenotype in stable COPD but not AECOPD, despite improving lung function in the latter group. We conclude that induced sputum can be used for measuring evidence of alteration of neutrophil phenotype in stable patients, and our study provides a data set of the sputum transcriptomic changes during recovery from AECOPD.

Intranasal Bifidobacterium longum protects against viral-induced lung inflammation and injury in a murine model of lethal influenza infection.

BACKGROUND: Prophylactic strategies are urgently needed for prevention of severe inflammatory responses to respiratory viral infections. Bacterial-host interactions may modify the immune response to viral infections. METHODS: We examined the contribution of Intranasal administration of two different Bifidobacterium longum strains or its isolated cell wall in controlling viral induced inflammation using a murine model of influenza infection. We monitored mortality and morbidity over a 10-day period and viral load, differential broncho alveolar lavage (BAL) fluid inflammatory cell counts, Lung tissue histology, BAL and serum cytokines, markers of vascular damage and cell death were quantified. FINDINGS: Intranasal administration of Bifidobacterium longum35624® or its isolated cell wall prior to virus inoculation significantly reduced viral load within the lungs and significantly improved survival. Reduced viral load was associated with reduced lung injury as suggested by cell death and vascular leakage markers, a shift from neutrophil to macrophage recruitment, reduced inflammatory cytokine levels (including IL-6), reduced type 1 and 2 interferon levels, but increased levels of interferon-λ and surfactant protein D. These protective effects were maintained when the bifidobacterial cell wall preparation was administered 24 h after viral inoculation. The protective effects were also observed for the Bifidobacterium longumPB-VIR™ strain. INTERPRETATION: Exposure to these bifidobacterial strains protect against the inflammatory sequelae and damage associated with uncontrolled viral replication within the lung. FUNDING: This work has been funded, in part, by a research grant from GlaxoSmithKline, PrecisionBiotics Group Ltd., Swiss National Science Foundation grants (project numbers CRSII3_154488, 310030_144219, 310030_127356 and 310030_144219) and Christine Kühne - Center for Allergy Research and Education (CK-CARE).

Obesity and disease severity magnify disturbed microbiome-immune interactions in asthma patients.

In order to improve targeted therapeutic approaches for asthma patients, insights into the molecular mechanisms that differentially contribute to disease phenotypes, such as obese asthmatics or severe asthmatics, are required. Here we report immunological and microbiome alterations in obese asthmatics (n = 50, mean age = 45), non-obese asthmatics (n = 53, mean age = 40), obese non-asthmatics (n = 51, mean age = 44) and their healthy counterparts (n = 48, mean age = 39). Obesity is associated with elevated proinflammatory signatures, which are enhanced in the presence of asthma. Similarly, obesity or asthma induced changes in the composition of the microbiota, while an additive effect is observed in obese asthma patients. Asthma disease severity is negatively correlated with fecal Akkermansia muciniphila levels. Administration of A. muciniphila to murine models significantly reduces airway hyper-reactivity and airway inflammation. Changes in immunological processes and microbiota composition are accentuated in obese asthma patients due to the additive effects of both disease states, while A. muciniphila may play a non-redundant role in patients with a severe asthma phenotype.

Airway host-microbiome interactions in chronic obstructive pulmonary disease.

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.

Activated PI3 Kinase Delta Syndrome: From Genetics to Therapy.

Activated PI3 kinase delta syndrome (APDS) is a primary immunodeficiency caused by dominant mutations that increase activity of phosphoinositide-3-kinase δ (PI3Kδ). APDS can be caused by mutations in the PIK3CD gene that encodes PI3Kδ catalytic subunit p110δ (APDS1) or mutations in the PIK3R1 gene that encodes regulatory subunit p85α (APDS2). APDS research advanced rapidly after the initial discovery in 2013. More than 200 APDS patients have been identified around the world. Multiple novel APDS mutations were reported and molecular mechanisms leading to PI3Kδ activation have been elucidated. The finding of APDS significantly increased our understanding of the role of PI3Kδ in the human immune system. Perhaps most importantly, discovery of the molecular basis of this primary immunodeficiency suggested that APDS patients, who previously received only non-specific therapy, could be treated by a novel class of drugs that inhibits PI3Kδ activity. This led to the ongoing clinical trials of selective PI3Kδ inhibitors in APDS patients. Overall, the APDS story provides an excellent example of translational research, beginning with patients who had an unknown disease cause and leading to a novel specific knowledge-based treatment.

ChEMBL: a large-scale bioactivity database for drug discovery.

ChEMBL is an Open Data database containing binding, functional and ADMET information for a large number of drug-like bioactive compounds. These data are manually abstracted from the primary published literature on a regular basis, then further curated and standardized to maximize their quality and utility across a wide range of chemical biology and drug-discovery research problems. Currently, the database contains 5.4 million bioactivity measurements for more than 1 million compounds and 5200 protein targets. Access is available through a web-based interface, data downloads and web services at: https://www.ebi.ac.uk/chembldb.

Gene profiling and bioinformatic analysis of Schwann cell embryonic development and myelination.

To elucidate the molecular mechanisms involved in Schwann cell development, we profiled gene expression in the developing and injured rat sciatic nerve. The genes that showed significant changes in expression in developing and dedifferentiated nerve were validated with RT-PCR, in situ hybridisation, Western blot and immunofluorescence. A comprehensive approach to annotating micro-array probes and their associated transcripts was performed using Biopendium, a database of sequence and structural annotation. This approach significantly increased the number of genes for which a functional insight could be found. The analysis implicates agrin and two members of the collapsin response-mediated protein (CRMP) family in the switch from precursors to Schwann cells, and synuclein-1 and alphaB-crystallin in peripheral nerve myelination. We also identified a group of genes typically related to chondrogenesis and cartilage/bone development, including type II collagen, that were expressed in a manner similar to that of myelin-associated genes. The comprehensive function annotation also identified, among the genes regulated during nerve development or after nerve injury, proteins belonging to high-interest families, such as cytokines and kinases, and should therefore provide a uniquely valuable resource for future research.

Bioinformatic approaches to assigning protein function from novel sequence data.

The current pace of functional genomic initiatives and genome sequencing projects has provided researchers with a bewildering array of sequence and biological data to analyze. The disease system-driven approach to identifying key genes frequently identifies nucleotide and protein sequences for which the gene and protein function are not known in sufficient detail to allow informed follow-up. Using a range of bioinformatic tools and sequence-based clues, most of unassigned sequences can now be annotated. This chapter takes as an example an unannotated expressed sequence tag, describing how to identify its related gene, and how to annotate the encoded protein using sequence, profile, and structure-based annotation methodologies.

Localisation of NMU1R and NMU2R in human and rat central nervous system and effects of neuromedin-U following central administration in rats.

RATIONALE: Neuromedin-U (NmU) is an agonist at NMU1R and NMU2R. The brain distribution of NmU and its receptors, in particular NMU2R, suggests widespread central roles for NmU. In agreement, centrally administered NmU affects feeding behaviour, energy expenditure and pituitary output. Further central nervous system (CNS) roles for NmU warrant investigation. OBJECTIVES: To investigate the CNS role of NmU by mapping NMU1R and NMU2R mRNA and measuring the behavioural, endocrine, neurochemical and c-fos response to intracerebroventricular (i.c.v.) NmU. METHODS: Binding affinity and functional potency of rat NmU was determined at human NMU1R and NMU2R. Expression of NMU1R and NMU2R mRNA in rat and human tissue was determined using semi-quantitative reverse-transcription polymerase chain reaction. In in-vivo studies, NmU was administered i.c.v. to male Sprague-Dawley rats, and changes in grooming, motor activity and pre-pulse inhibition (PPI) were assessed. In further studies, plasma endocrine hormones, [DOPAC + HVA]/[dopamine] and [5-HIAA]/[5-HT] ratios and levels of Fos-like immunoreactivity (FLI) were measured 20 min post-NmU (i.c.v.). RESULTS: NmU bound to NMU1R ( K(I), 0.11+/-0.02 nM) and NMU2R ( K(I), 0.21+/-0.05 nM) with equal affinity and was equally active at NMU1R (EC(50), 1.25+/-0.05 nM) and NMU2R (EC(50), 1.10+/-0.20 nM) in a functional assay. NMU2R mRNA expression was found at the highest levels in the CNS regions of both rat and human tissues. NMU1R mRNA expression was restricted to the periphery of both species with the exception of the rat amygdala. NmU caused a marked increase in grooming and motor activity but did not affect PPI. Further, NmU decreased plasma prolactin but did not affect levels of corticosterone, luteinising hormone or thyroid stimulating hormone. NmU elevated levels of 5-HT in the frontal cortex and hypothalamus, with decreased levels of its metabolites in the hippocampus and hypothalamus, but did not affect dopamine function. NmU markedly increased FLI in the nucleus accumbens, frontal cortex and central amygdala. CONCLUSIONS: These data provide further evidence for widespread roles for NmU and its receptors in the brain.

Identification of human and mouse CatSper3 and CatSper4 genes: characterisation of a common interaction domain and evidence for expression in testis.

BACKGROUND: CatSper1 and CatSper2 are two recently identified channel-like proteins, which show sperm specific expression patterns. Through targeted mutagenesis in the mouse, CatSper1 has been shown to be required for fertility, sperm motility and for cAMP induced Ca2+ current in sperm. Both channels resemble a single pore forming repeat from a four repeat voltage dependent Ca2+ /Na+ channel. However, neither CatSper1 or CatSper2 have been shown to function as cation channels when transfected into cells, singly or in conjunction. As the pore forming units of voltage gated cation channels form a tetramer it has been suggested that the known CatSper proteins require additional subunits and/or interaction partners to function. RESULTS: Using in silico gene identification and prediction techniques, we have identified two further members of the CatSper family, CatSper3 and Catsper4. Each carries a single channel-forming domain with the predicted pore-loop containing the consensus sequence TxDxW. Each of the new CatSper genes has evidence for expression in the testis. Furthermore we identified coiled-coil protein-protein interaction domains in the C-terminal tails of each of the CatSper channels, implying that CatSper channels 1,2,3 and 4 may interact directly or indirectly to form a functional tetramer. CONCLUSIONS: The topological and sequence relationship of CatSper1 and CatSper2 to the four repeat Ca2+ /Na+ channels suggested other members of this family may exist. We have identified a further two novel CatSper genes, conserved in both the human and mouse genomes. Furthermore, all four of the CatSper proteins are predicted to contain a common coiled-coil protein-protein interaction domain in their C-terminal tail. Coupled with expression data this leads to the hypothesis that the CatSper proteins form a functional hetero-tetrameric channel in sperm.

Protein sequence analysis in silico: application of structure-based bioinformatics to genomic initiatives.

The current pace of high-throughput genome sequencing programs coupled with high-throughput functional genomic screens has provided researchers with a bewildering array of sequence and biological data to contend with. Identification of proteins of interest from a particular biological study requires the application of bioinformatic tools to process and prioritise the data. From a protein function standpoint, transfer of annotation from known proteins to a novel target is currently the only practical way to convert vast quantities of raw sequence data into meaningful information. New bioinformatics tools now provide more sophisticated methods to transfer functional annotation, integrating sequence, family profile and structural search methodology. The importance of these approaches to medical research is increasing as we move to annotate the proteome through functional and structural genomic efforts.