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Chronic airway inflammation is the main driver of pathogenesis in respiratory diseases, such as severe asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and bronchiectasis. While the role of common pathogens in airway inflammation is widely recognized, the influence of other microbiota members is still poorly understood. Here, we show that Rothia mucilaginosa, a common resident of the oral cavity that is also often detectable in the lower airways in chronic disease, has an inhibitory effect on pathogen- and LPS-induced pro-inflammatory responses, both in vitro (3-D cell culture model) and in vivo (mouse model). Furthermore, in a cohort of adults with bronchiectasis, the abundance of Rothia spp. was negatively correlated with pro-inflammatory markers (IL-8, IL-1β) and matrix metalloproteinases (MMP-1, MMP-8 and MMP-9) in sputum. Mechanistic studies revealed that R. mucilaginosa inhibits NF-κB pathway activation by reducing the phosphorylation of IκB-α and consequently the expression of NF-κB target genes. These findings indicate that the presence of R. mucilaginosa in the lower airways potentially mitigates inflammation, which could in turn influence severity and progression of chronic respiratory disorders.

Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival.Using an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression.Together, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes.

INTRODUCTION: Chlamydia trachomatis is the most commonly reported sexually transmitted disease and although infection during pregnancy is associated with neonatal complications, long-term respiratory consequences are unknown. We aimed to determine whether C. trachomatis infection during pregnancy is associated with asthma-related symptoms across childhood METHODS: This study among 2475 children and their mothers was embedded in a population-based prospective cohort study. Maternal urine samples were tested for C. trachomatis infection during pregnancy. Questionnaires provided information on childhood physician-attended lower respiratory tract infections and wheezing, and current asthma at age 10 years. Lung function was measured by spirometry at age 10 years. RESULTS: The prevalence of C. trachomatis infection during pregnancy was 3.2% (78 out of 2475). C. trachomatis infection during pregnancy was not associated with lower respiratory tract infections until age 6 years, but was associated with a higher odds of wheezing in children until age 10 years (OR 1.50 (95% CI 1.10-2.03)). C. trachomatis infection during pregnancy was associated with an increased odds of asthma (OR 2.29 (95% CI 1.02-5.13)), and with a lower forced expiratory volume in 1 s/forced vital capacity and forced expiratory flow at 75% of forced vital capacity (z-score difference -0.28 (95% CI -0.52- -0.04) and -0.24 (95% CI -0.46- -0.01), respectively) in children at age 10 years. The observed associations were only partly explained by mode of delivery, gestational age at birth or birthweight. CONCLUSIONS: C. trachomatis infection during pregnancy is associated with increased odds of wheezing, asthma and impaired lung function. The causality of the observed associations and potential underlying mechanisms need to be explored.

While immunoglobulin (Ig) E is a prominent biomarker for early-onset, its levels are often elevated in non-allergic late-onset asthma. However, the pattern of IgE expression in the latter is mostly polyclonal, with specific IgEs low or below detection level albeit with an increased total IgE. In late-onset severe asthma patients, specific IgE to Staphylococcal enterotoxins (se-IgE) can frequently be detected in serum, and has been associated with asthma, with severe asthma defined by hospitalisations, oral steroid use and decrease in lung function. Recently, se-IgE was demonstrated to even predict the development into severe asthma with exacerbations over the next decade. Staphylococcus aureus manipulates the airway mucosal immunology at various levels via its proteins, including superantigens, serine-protease-like proteins (Spls), or protein A (SpA) and possibly others. Release of IL-33 from respiratory epithelium and activation of innate lymphoid cells (ILCs) via its receptor ST2, type 2 cytokine release from those ILCs and T helper (Th) 2 cells, mast cell degranulation, massive local B-cell activation and IgE formation, and finally eosinophil attraction with consequent release of extracellular traps, adding to the epithelial damage and contributing to disease persistence via formation of Charcot-Leyden crystals are the most prominent hallmarks of the manipulation of the mucosal immunity by S. aureus In summary, S. aureus claims a prominent role in the orchestration of severe airway inflammation and in current and future disease severity. In this review, we discuss current knowledge in this field and outline the needs for future research to fully understand the impact of S. aureus and its proteins on asthma.

Observations in vivo in patients, supported by guinea-pig in vivo data, take centre stage in this perspective. Its objective is to highlight dichotomies between asthma features observed in vivo and accepted views involving cell/molecular biology research paradigms. For example, increased bronchial epithelial permeability is now considered a major paradigm and trait of asthma, yet, absorption of inhaled tracers has not been increased in vivo in asthma. Such maintained barrier function in exudative asthma reflects in vivo asymmetry of the epithelial lining as barrier between outside and inside world of molecules and cells. In desquamatory asthma, maintained epithelial tightness may be explained by in vivo demonstrations of exceedingly patchy epithelial loss, prompt creation of plasma-derived provisional barriers, and high-speed epithelial regeneration. Acknowledged protein/peptide secretion by epithelial cells in vitro is contrasted here with a dominant, unidirectional movement in vivo of plasma-derived proteins/peptides (including antimicrobial peptides) to the surface of an intact epithelial lining. Furthermore, longstanding claims that epithelium-produced adenosine is a mediator of asthma are eroded by observations in vivo in asthmatics. Notions concerning activation/fate of mucosal tissue eosinophils illustrate additional distinctions between accepted views and in vivo patient observations. Finally, in vitro-based paradigms preaching defect epithelial regeneration and increased permeability in pathogenesis of asthma are contrasted with experimental in vivo observations of exaggerated epithelial regeneration, which is multipathogenic in its own right. In conclusion, unexpected and challenging in vivo observations in recent decades underpin novel insights into mucosal mechanisms in asthma.

Treatment of Mycobacterium abscessus pulmonary disease (MAB-PD), caused by M. abscessus subsp. abscessus, M. abscessus subsp. massiliense or M. abscessus subsp. bolletii, is challenging.We conducted an individual patient data meta-analysis based on studies reporting treatment outcomes for MAB-PD to clarify treatment outcomes for MAB-PD and the impact of each drug on treatment outcomes. Treatment success was defined as culture conversion for ≥12 months while on treatment or sustained culture conversion without relapse until the end of treatment.Among 14 eligible studies, datasets from eight studies were provided and a total of 303 patients with MAB-PD were included in the analysis. The treatment success rate across all patients with MAB-PD was 45.6%. The specific treatment success rates were 33.0% for M. abscessus subsp. abscessus and 56.7% for M. abscessus subsp. massiliense For MAB-PD overall, the use of imipenem was associated with treatment success (adjusted odds ratio (aOR) 2.65, 95% CI 1.36-5.10). For patients with M. abscessus subsp. abscessus, the use of azithromycin (aOR 3.29, 95% CI 1.26-8.62), parenteral amikacin (aOR 1.44, 95% CI 1.05-1.99) or imipenem (aOR 7.96, 95% CI 1.52-41.6) was related to treatment success. For patients with M. abscessus subsp. massiliense, the choice among these drugs was not associated with treatment outcomes.Treatment outcomes for MAB-PD are unsatisfactory. The use of azithromycin, amikacin or imipenem was associated with better outcomes for patients with M. abscessus subsp. abscessus.

Bronchial thermoplasty is a treatment for asthma. It is currently unclear whether its histopathological impact is sufficiently explained by the proportion of airway wall that is exposed to temperatures necessary to affect cell survival.Airway smooth muscle and bronchial epithelial cells were exposed to media (37-70°C) for 10 s to mimic thermoplasty. In silico we developed a mathematical model of airway heat distribution post-thermoplasty. In vivo we determined airway smooth muscle mass and epithelial integrity pre- and post-thermoplasty in 14 patients with severe asthma.In vitro airway smooth muscle and epithelial cell number decreased significantly following the addition of media heated to ≥65°C. In silico simulations showed a heterogeneous heat distribution that was amplified in larger airways, with <10% of the airway wall heated to >60°C in airways with an inner radius of ∼4 mm. In vivo at 6 weeks post-thermoplasty, there was an improvement in asthma control (measured via Asthma Control Questionnaire-6; mean difference 0.7, 95% CI 0.1-1.3; p=0.03), airway smooth muscle mass decreased (absolute median reduction 5%, interquartile range (IQR) 0-10; p=0.03) and epithelial integrity increased (14%, IQR 6-29; p=0.007). Neither of the latter two outcomes was related to improved asthma control.Integrated in vitro and in silico modelling suggest that the reduction in airway smooth muscle post-thermoplasty cannot be fully explained by acute heating, and nor did this reduction confer a greater improvement in asthma control.

Current pulmonary nodule management guidelines are based on nodule volume doubling time, which assumes exponential growth behaviour. However, this is a theory that has never been validated in vivo in the routine-care target population. This study evaluates growth patterns of untreated solid and subsolid lung cancers of various histologies in a non-screening setting.Growth behaviour of pathology-proven lung cancers from two academic centres that were imaged at least three times before diagnosis (n=60) was analysed using dedicated software. Random-intercept random-slope mixed-models analysis was applied to test which growth pattern most accurately described lung cancer growth. Individual growth curves were plotted per pathology subgroup and nodule type.We confirmed that growth in both subsolid and solid lung cancers is best explained by an exponential model. However, subsolid lesions generally progress slower than solid ones. Baseline lesion volume was not related to growth, indicating that smaller lesions do not grow slower compared to larger ones.By showing that lung cancer conforms to exponential growth we provide the first experimental basis in the routine-care setting for the assumption made in volume doubling time analysis.

Epigenome-wide association studies have shown a consistent association between smoking exposure and hypomethylation in the aryl hydrocarbon receptor repressor (AHRR) gene (cg05575921). We tested the hypothesis that AHRR hypomethylation is associated with low lung function, steeper lung function decline, and respiratory symptoms in the general population.AHRR methylation extent was measured in 9113 individuals from the 1991-1994 examination of the Copenhagen City Heart Study, using bisulfite-treated leukocyte DNA. Spirometry at the time of blood sampling was available for all individuals. Lung function was measured again for 4532 of these individuals in 2001-2003.Cross-sectionally, a 10% lower methylation extent was associated with a 0.2 z-score (95% CI 0.1-0.2) lower forced expiratory volume in 1 s (FEV1) after multivariable adjustment including smoking. Hypomethylation was also associated with a lower z-score for both forced vital capacity (FVC) and FEV1/FVC. In prospective analyses, individuals in the lowest versus highest tertile of methylation extent had a steeper decline in FEV1/height3 (p for examination×methylation interaction=0.003) and FVC/height3 (p=0.01), but not FEV1/FVC (p=0.08). Multivariable-adjusted odds ratios per 10% lower methylation extent were 1.31 (95% CI 1.18-1.45) for chronic bronchitis and 1.21 (95% CI 1.13-1.30) for any respiratory symptoms.AHRR hypomethylation was associated with low lung function, steeper lung function decline, and respiratory symptoms.

Sarcoidosis is a granulomatous disease that mainly affects the lung. A role of microbial factors in disease pathogenesis is assumed, but has not been investigated systematically in a large cohort.This cross-sectional study compared the lung microbiota of 71 patients with sarcoidosis, 15 patients with idiopathic pulmonary fibrosis (non-infectious controls) and 10 healthy controls (HCs). Next-generation sequencing of 16S DNA was used on bronchoalveolar lavage samples to characterise the microbial composition, which was analysed for diversity and indicator species. Host genotypes for 13 known sarcoidosis risk variants were determined and correlated with microbial parameters.The microbial composition differed significantly between sarcoidosis and HC samples (redundancy analysis ANOVA, p=0.025) and between radiographic Scadding types. Atopobium spp. was detected in 68% of sarcoidosis samples, but not in HC samples. Fusobacterium spp. was significantly more abundant in sarcoidosis samples compared with those from HCs. Mycobacteria were found in two of 71 sarcoidosis samples. Host-genotype analysis revealed an association of the rs2076530 (BTNL2) risk allele with a decrease in bacterial burden (p=0.002).Our results indicate Scadding type-dependent microbiota in sarcoidosis BAL samples. Atopobium spp. and Fusobacterium spp. were identified as sarcoidosis-associated bacteria, which may enable new insights into the pathogenesis and treatment of the disease.

To characterise Pseudomonas aeruginosa populations during chronic lung infections of non-cystic fibrosis bronchiectasis patients, we used whole-genome sequencing to 1) assess the diversity of P. aeruginosa and the prevalence of multilineage infections; 2) seek evidence for cross-infection or common source acquisition; and 3) characterise P. aeruginosa adaptations.189 isolates, obtained from the sputa of 91 patients attending 16 adult bronchiectasis centres in the UK, were whole-genome sequenced.Bronchiectasis isolates were representative of the wider P. aeruginosa population. Of 24 patients from whom multiple isolates were examined, there were seven examples of multilineage infections, probably arising from multiple infection events. The number of nucleotide variants between genomes of isolates from different patients was in some cases similar to the variations observed between isolates from individual patients, implying the possible occurrence of cross-infection or common source acquisition.Our data indicate that during infections of bronchiectasis patients, P. aeruginosa populations adapt by accumulating loss-of-function mutations, leading to changes in phenotypes including different modes of iron acquisition and variations in biofilm-associated polysaccharides. The within-population diversification suggests that larger scale longitudinal surveillance studies will be required to capture cross-infection or common source acquisition events at an early stage.