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Netsanet A Negewo,1 Peter G Gibson,2– 4 Jodie L Simpson,1 Vanessa M McDonald,2– 5 Katherine J Baines1 1Immune Health Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia; 2Centre of Excellence in Treatable Traits, University of Newcastle, New Lambton Heights, NSW, Australia; 3Department of Respiratory and Sleep Medicine, John Hunter Hospital, Newcastle, NSW, Australia; 4Asthma and Breathing Research Centre, Hunter Medical Research Centre, New Lambton Heights, NSW, Australia; 5School of Nursing and Midwifery, The University of Newcastle, Callaghan, NSW, AustraliaCorrespondence: Katherine J Baines, Hunter Medical Research Institute, Level 2 East Wing, Locked Bag 1000, New Lambton Heights, NSW, 2305, Australia, Tel +61 2 40420090, Fax +61 2 40420046, Email katherine.baines@newcastle.edu.auPurpose: This study sought to characterize transcriptional phenotypes of COPD through unsupervised clustering of sputum gene expression profiles, and further investigate mechanisms underlying the characteristics of these clusters.Patients and methods: Induced sputum samples were collected from patients with stable COPD (n = 72) and healthy controls (n = 15). Induced sputum was collected for inflammatory cell counts, and RNA extracted. Transcriptional profiles were generated (Illumina Humanref-8 V2) and analyzed by GeneSpring GX14.9.1. Unsupervised hierarchical clustering and differential gene expression analysis were performed, and gene alterations validated in the ECLIPSE dataset (GSE22148).Results: We identified 2 main clusters (Cluster 1 [n = 35] and Cluster 2 [n = 37]), which further divided into 4 sub-clusters (Sub-clusters 1.1 [n = 14], 1.2 [n = 21], 2.1 [n = 20] and 2.2 [n = 17]). Compared with Cluster 1, Cluster 2 was associated with significantly lower lung function (p = 0.014), more severe disease (p = 0.009) and breathlessness (p = 0.035), and increased sputum neutrophils (p = 0.031). Sub-cluster 1.1 had significantly higher proportion of people with comorbid cardiovascular disease compared to the other 3 sub-clusters (92.5% vs 57.1%, 50% and 52.9%, p < 0.013). Through supervised analysis we determined that degree of airflow limitation (GOLD stage) was the predominant factor driving gene expression differences in our transcriptional clusters. There were 452 genes (adjusted p < 0.05 and ≥ 2 fold) altered in GOLD stage 3 and 4 versus 1 and 2, of which 281 (62%) were also found to be significantly expressed between these GOLD stages in the ECLIPSE data set (GSE22148). Differentially expressed genes were largely downregulated in GOLD stages 3 and 4 and connected in 5 networks relating to lipoprotein and cholesterol metabolism; metabolic processes in oxidation/reduction and mitochondrial function; antigen processing and presentation; regulation of complement activation and innate immune responses; and immune and metabolic processes.Conclusion: Severity of lung function drives 2 distinct transcriptional phenotypes of COPD and relates to immune and metabolic processes.Keywords: COPD, gene expression, inflammation, sputum

Katherine J Baines,1 Netsanet A Negewo,1 Peter G Gibson,1,2 Juan-Juan Fu,3 Jodie L Simpson,1 Peter AB Wark,1,2 Michael Fricker,1 Vanessa M McDonald1,2,4 1Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute, The University of Newcastle, Callaghan, NSW, Australia; 2Department of Respiratory and Sleep Medicine, John Hunter Hospital, Newcastle, NSW, Australia; 3Respiratory Group, Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan Province, People’s Republic of China; 4School of Nursing and Midwifery, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, AustraliaCorrespondence: Katherine J BainesHunter Medical Research Institute, Level 2 West Wing, Locked Bag 1000, New Lambton, NSW 2305, AustraliaEmail katherine.baines@newcastle.edu.auBackground: The 6 gene expression signature (6GS) predicts inflammatory phenotype, exacerbation risk, and corticosteroid responsiveness in asthma. In COPD, patterns of airway inflammation are similar, suggesting the 6GS may be useful. This study determines the diagnostic and prognostic ability of 6GS in predicting inflammatory phenotypes and exacerbation risk in COPD.Methods: We performed 2 studies: a cross-sectional phenotype prediction study in stable COPD (total N=132; n=34 eosinophilic (E)-COPD, n=42 neutrophilic (N)-COPD, n=39 paucigranulocytic (PG)-COPD, n=17 mixed-granulocytic (MG)-COPD) that assessed 6GS ability to discriminate phenotypes (eosinophilia≥ 3%; neutrophilia≥ 61%); and a prospective cohort study (total n=54, n=8 E-COPD; n=18 N-COPD; n=20 PG-COPD; n=8 MG-COPD, n=21 exacerbation prone (≥ 2/year)) that investigated phenotype and exacerbation prediction utility. 6GS was measured by qPCR and evaluated using multiple logistic regression and area under the curve (AUC). Short-term reproducibility (intra-class correlation) and phenotyping method agreement (κ statistic) were assessed.Results: In the phenotype prediction study, 6GS could accurately identify and discriminate patients with E-COPD from N-COPD (AUC=96.4%; p< 0.0001), PG-COPD (AUC=88.2%; p< 0.0001) or MG-COPD (AUC=86.2%; p=0.0001), as well as N-COPD from PG-COPD (AUC=83.6%; p< 0.0001) or MG-COPD (AUC=87.4%; p< 0.0001) and was reproducible. In the prospective cohort study, 6GS had substantial agreement for neutrophilic inflammation (82%, κ=0.63, p< 0.001) and moderate agreement for eosinophilic inflammation (78%, κ=0.42, p< 0.001). 6GS could significantly discriminate exacerbation prone patients (AUC=77.2%; p=0.034). Higher IL1B levels were associated with poorer lung function and increased COPD severity.Conclusion: 6GS can significantly and reproducibly discriminate COPD inflammatory phenotypes and predict exacerbation prone patients and may become a useful molecular diagnostic tool assisting COPD management.Keywords: COPD, airway markers, inflammation, molecular biology, eosinophil

Ravi Lokwani,1–3 Peter AB Wark,1–3 Katherine J Baines,1,3 Michael Fricker,1,3 Daniel Barker,3 Jodie L Simpson1–3 1Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute, University of Newcastle, Callaghan, NSW 2308, Australia; 2Department of Respiratory and Sleep Medicine, John Hunter Hospital, New Lambton Heights, NSW 2305, Australia; 3School of Medicine and Public Health, Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW 2308, AustraliaCorrespondence: Jodie L SimpsonLevel 2, East Wing, Hunter Medical Research Institute, Locked Bag, New Lambton 1000, NSW 2305, AustraliaTel +61 422220992Email jodie.simpson@newcastle.edu.auPurpose: To characterize neutrophils in obstructive airway disease by measuring their surface adhesion molecules and oxidative burst along with characterizing them into different subsets as per their adhesion molecule expression.Patients and methods: Peripheral blood from adults with COPD (n=17), asthma (n=20), and healthy participants (n=19) was examined for expression of CD16, CD62L, CD11b, CD11c, and CD54, and analyzed by flow cytometry. For oxidative burst and CD62L shedding analysis, CD16 and CD62L stained leukocytes were loaded with Dihydrorhodamine-123 (DHR-123) and stimulated with N-Formylmethionine-leucyl-phenylalanine (fMLF). Neutrophil subsets were characterized based on CD16 and CD62L expression. Marker surface expression was recorded on CD16+ neutrophils as median fluorescence intensity (MFI).Results: Neutrophil surface expression of CD62L was significantly reduced in COPD (median (IQR) MFI: 1156 (904, 1365)) compared with asthma (1865 (1157, 2408)) and healthy controls (2079 (1054, 2960)); p=0.028. COPD neutrophils also demonstrated a significant reduction in CD62L expression with and without fMLF stimulation. Asthma participants had a significantly increased proportion and number of CD62Lbright/CD16dim neutrophils (median: 5.4% and 0.14 × 109/L, respectively), in comparison with healthy (3.54% and 0.12 × 109/L, respectively); p

Katherine J Baines,1,2 Juan-juan Fu,3 Vanessa M McDonald,1,2 Peter G Gibson1,2 1Faculty of Health and Medicine, Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute, The University of Newcastle, 2Department of Respiratory and Sleep Medicine, John Hunter Hospital, New Lambton, NSW, Australia; 3Respiratory Group, Department of Integrated Traditional Chinese and West Medicine, West China Hospital, Sichuan University, People’s Republic of China Background: Exacerbations of asthma and COPD are a major cause of morbidity and mortality and are responsible for significant health care costs. This study further investigates interleukin (IL)-1 pathway activation and its relationship with exacerbations of asthma and COPD. Methods: In this prospective cohort study, 95 participants with stable asthma (n=35) or COPD (n=60) were recruited and exacerbations recorded over the following 12 months. Gene expressions of IL-1 pathway biomarkers, including the IL-1 receptors (IL1R1, IL1R2, and IL1RN), and signaling molecules (IRAK2, IRAK3, and PELI1), were measured in sputum using real-time quantitative polymerase chain reaction. Mediators were compared between the frequent (≥2 exacerbations in the 12 months) and infrequent exacerbators, and the predictive relationships investigated using receiver operating characteristic curves and area under the curve (AUC) values. Results: Of the 95 participants, 89 completed the exacerbation follow-up, where 30 participants (n=22 COPD, n=8 asthma) had two or more exacerbations. At the baseline visit, expressions of IRAK2, IRAK3, PELI1, and IL1R1 were elevated in participants with frequent exacerbations of both asthma and COPD combined and separately. In the combined population, sputum gene expression of IRAK3 (AUC=75.4%; P

Netsanet A Negewo,1,2 Vanessa M McDonald,1–4 Katherine J Baines,1,2 Peter AB Wark,1–3 Jodie L Simpson,1,2 Paul W Jones,5 Peter G Gibson1–3 1Priority Research Centre for Healthy Lungs, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, Australia; 2Hunter Medical Research Institute, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, Australia; 3Department of Respiratory and Sleep Medicine, John Hunter Hospital, Newcastle, NSW, Australia; 4School of Nursing and Midwifery, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, Australia; 5Institute for Infection and Immunity, St George’s, University of London, London, UK Introduction: Sputum eosinophilia occurs in approximately one-third of stable chronic obstructive pulmonary disease (COPD) patients and can predict exacerbation risk and response to corticosteroid treatments. Sputum induction, however, requires expertise, may not always be successful, and does not provide point-of-care results. Easily applicable diagnostic markers that can predict sputum eosinophilia in stable COPD patients have the potential to progress COPD management. This study investigated the correlation and predictive relationship between peripheral blood and sputum eosinophils. It also examined the repeatability of blood eosinophil counts.Methods: Stable COPD patients (n=141) were classified as eosinophilic or noneosinophilic based on their sputum cell counts (≥3%), and a cross-sectional analysis was conducted comparing their demographics, clinical characteristics, and blood cell counts. Receiver operating characteristic curve analysis was used to assess the predictive ability of blood eosinophils for sputum eosinophilia. Intraclass correlation coefficient was used to examine the repeatability of blood eosinophil counts.Results: Blood eosinophil counts were significantly higher in patients with sputum eosinophilia (n=45) compared to those without (0.3×109/L vs 0.15×109/L; P

Mothers with asthma or atopy have a higher likelihood of having autistic children, with maternal immune activation in pregnancy implicated as a mechanism. This study aimed to determine, in a prospective cohort of mothers with asthma and their infants, whether inflammatory gene expression in pregnancy is associated with likelihood of future autism. Mothers with asthma were recruited to the Breathing for Life Trial. RNA was extracted from blood samples collected at mid-pregnancy. 300 ng total RNA was hybridized with the nCounter Human Inflammation gene expression panel (Nanostring Technologies, 249 inflammation-related genes). Parents completed the First Year Inventory (FYI) at 12-month follow-up, which assessed an infant's likelihood for autism across 2 behavioural domains: social communication and sensory regulation. A total score ≥19.2 indicated increased likelihood for future autism. Inflammatory gene expression was profiled from 24 mothers: four infants scored in the high autism likelihood range; 20 scored in the low autism likelihood range. Six inflammatory genes were differentially expressed and significantly up-regulated in the high autism likelihood group: CYSLTR2 , NOX1 , C1QA , CXCL10 , C8A , IL23R . mRNA count significantly correlated with social communication FYI score for CYSLTR2 (Pearson r = 0.46, p = 0.024) and CXCL10 (r = 0.43, p = 0.036) and with sensory regulation score for ALOX5 (r = -0.43, p = 0.038) and MAFK (r = -0.46, p = 0.022). In this proof-of-concept study, inflammatory gene expression during pregnancy in mothers with asthma was associated with an infant's likelihood of future autism as well as scores relating to social communication and sensory regulation., Competing Interests: On behalf of all listed authors of the enclosed submission, I declare that there are no known conflicts of interest, whether personal, professional, or financial., (© 2024 The Authors.)

Background: Non-neuronal cholinergic system (NNCS) contributes to various inflammatory airway diseases. However, the role of NNCS in severe asthma (SA) remains largely unexplored., Objective: To explore airway NNCS in SA., Methods: In this prospective cohort study based on the Australasian Severe Asthma Network in a real-world setting, patients with SA (n = 52) and non-SA (n = 104) underwent clinical assessment and sputum induction. The messenger RNA (mRNA) levels of NNCS components and proinflammatory cytokines in the sputum were detected using real-time quantitative polymerase chain reaction, and the concentrations of acetylcholine (Ach)-related metabolites were evaluated using liquid chromatography coupled with tandem mass spectrometry. Asthma exacerbations were prospectively investigated during the next 12 months. The association between NNCS and future asthma exacerbations was also analyzed., Results: Patients with SA were less controlled and had worse airway obstruction, a lower bronchodilator response, higher doses of inhaled corticosteroids, and more add-on treatments. The sputum mRNA levels of NNCS components, such as muscarinic receptors M1R-M5R, OCT3, VACHT, and ACHE; proinflammatory cytokines; and Ach concentration in the SA group were significantly higher than those in the non-SA group. Furthermore, most NNCS components positively correlated with non-type (T) 2 inflammatory profiles, such as sputum neutrophils, IL8, and IL1B. In addition, the mRNA levels of sputum M2R, M3R, M4R, M5R, and VACHT were independently associated with an increased risk of moderate-to-severe asthma exacerbations., Conclusion: This study indicated that the NNCS was significantly activated in SA, leading to elevated Ach and was associated with clinical features, non-T2 inflammation, and future exacerbations of asthma, highlighting the potential role of the NNCS in the pathogenesis of SA., Clinical Trial Registration: ChiCTR-OOC-16009529 (http://www.chictr.org.cn)., Competing Interests: Disclosures Dr G. Wang is on advisory boards for and has received speaker's honoraria from AstraZeneca, GlaxoSmithKline, Chiesi, and Novartis and has received research support from AstraZeneca. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Introduction: Bronchiectasis is a worldwide chronic lung disorder where exacerbations are common. It affects people of all ages, but especially Indigenous populations in high-income nations. Despite being a major contributor to chronic lung disease, there are no licensed therapies for bronchiectasis and there remain relatively few randomised controlled trials (RCTs) conducted in children and adults. Our RCT will address some of these unmet needs by evaluating whether the novel mucoactive agent, erdosteine, has a therapeutic role in children and adults with bronchiectasis.Our primary aim is to determine in children and adults aged 2-49 years with bronchiectasis whether regular erdosteine over a 12-month period reduces acute respiratory exacerbations compared with placebo. Our primary hypothesis is that people with bronchiectasis who regularly use erdosteine will have fewer exacerbations than those receiving placebo.Our secondary aims are to determine the effect of the trial medications on quality of life (QoL) and other clinical outcomes (exacerbation duration, time-to-next exacerbation, hospitalisations, lung function, adverse events). We will also assess the cost-effectiveness of the intervention., Methods and Analysis: We are undertaking an international multicentre, double-blind, placebo-RCT to evaluate whether 12 months of erdosteine is beneficial for children and adults with bronchiectasis. We will recruit 194 children and adults with bronchiectasis to a parallel, superiority RCT at eight sites across Australia, Malaysia and Philippines. Our primary endpoint is the rate of exacerbations over 12 months. Our main secondary outcomes are QoL, exacerbation duration, time-to-next exacerbation, hospitalisations and lung function., Ethics and Dissemination: The Human Research Ethics Committees (HREC) of Children's Health Queensland (for all Australian sites), University of Malaya Medical Centre (Malaysia) and St. Luke's Medical Centre (Philippines) approved the study. We will publish the results and share the outcomes with the academic and medical community, funding and relevant patient organisations., Trial Registration Number: ACTRN12621000315819., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

The gut microbiota influences anti-tumor immunity and can induce or inhibit response to immune checkpoint inhibitors (ICIs). Therefore, microbiome features are being studied as predictive/prognostic biomarkers of patient response to ICIs, and microbiome-based interventions are attractive adjuvant treatments in combination with ICIs. Specific gut-resident bacteria can influence the effectiveness of immunotherapy; however, the mechanism of action on how these bacteria affect anti-tumor immunity and response to ICIs is not fully understood. Nevertheless, early bacterial-based therapeutic strategies have demonstrated that targeting the gut microbiome through various methods can enhance the effectiveness of ICIs, resulting in improved clinical responses in patients with a diverse range of cancers. Therefore, understanding the microbiota-driven mechanisms of response to immunotherapy can augment the success of these interventions, particularly in patients with treatment-refractory cancers., Competing Interests: Declaration of interests S.M.V. is a former member of the board of directors of IMV Inc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Due to the intracellular expression of Foxp3 it is impossible to purify viable Foxp3 + cells on the basis of Foxp3 staining. Consequently CD4 + Foxp3 + regulatory T cells (Tregs) in mice have mostly been characterized using CD4 + CD25 + T cells or GFP-Foxp3 reporter T cells. However, these two populations cannot faithfully represent Tregs as the expression of CD25 and Foxp3 does not completely overlap and GFP + Foxp3 + reporter T cells have been reported to be functionally altered. The aim of this study was to characterize normal Tregs without separating Foxp3 + and Foxp3 - cells for the expression of the main functional molecules and proliferation behaviors by flow cytometry and to examine their gene expression characteristics through differential gene expression. Our data showed that the expressions of Foxp3, CD25, CTLA-4 (both intracellular and cell surface) and PD-1 was mostly confined to CD4 + T cells and the expression of Foxp3 did not completely overlap with the expression of CD25, CTLA-4 or PD-1. Despite higher levels of expression of the T cell inhibitory molecules CTLA-4 and PD-1, Tregs maintained higher levels of Ki-67 expression in the homeostatic state and had greater proliferation in vivo after allo-activation than Tconv. Differential gene expression analysis revealed that resting Tregs exhibited immune activation markers characteristic of activated Tconv. This is consistent with the flow data that the T cell activation markers CD25, CTLA-4, PD-1, and Ki-67 were much more strongly expressed by Tregs than Tconv in the homeostatic state., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Liu, Baines, Niessen, Heer, Clark, Bishop and Trevillian.)

The differences between males and females begin shortly after birth, continue throughout prenatal development, and eventually extend into childhood and adult life. Male embryos and fetuses prioritize proliferation and growth, often at the expense of the fetoplacental energy reserves. This singular focus on growth over adaptability leaves male fetuses and neonates vulnerable to adverse outcomes during pregnancy and birth and can have lasting impacts throughout life. Beyond this prioritization of growth, male placentas and fetuses also respond to infection and inflammation differently than female counterparts. Pregnancies carrying female fetuses have a more regulatory immune response, whereas pregnancies carrying male fetuses have a stronger inflammatory response. These differences can be seen as early as the innate immune response with differences in cytokine and chemokine signaling. The sexual dimorphism in immunity then continues into the adaptive immune response with differences in T-cell biology and antibody production and transfer. As it appears that these sex-specific differences are amplified in pathologic pregnancies, it stands to reason that differences in the placental, fetal, and maternal immune responses in pregnancy contribute to increased male perinatal morbidity and mortality. In this review, we will describe the genetic and hormonal contributions to the sexual dimorphism of fetal and placental immunity. We will also discuss current research efforts to describe the sex-specific differences of the maternal-fetal interface and how it impacts fetal and maternal health., (Published by Oxford University Press on behalf of Society for the Study of Reproduction 2023.)

Introduction: Uterine Natural Killer (NK) cells are the predominant immune cells within the decidua during early pregnancy. These cells are thought to regulate aspects of decidualization and placental development, but their functions remain poorly characterized, especially in species with deeply invading trophoblasts such as humans and rats. Interleukin-15 (IL-15) is a cytokine required for NK cell development and survival. IL-15 mutant ( IL15Δ/Δ ) rats lack NK cells and exhibit altered placental development with precocious trophoblast invasion. In this study, we profiled gene expression differences between wild-type and IL15Δ/Δ implantation sites to reveal candidate factors produced by uterine NK cells that may regulate placentation and trophoblast invasion. Methods: Clariom S gene expression profiling was performed using implantation sites collected from pregnant wild-type and IL15Δ/Δ rats on gestational day 9.5. Levels and localization of perforin and osteopontin in implantation sites from wild-type and IL15Δ/Δ rats were further analyzed. The effect of osteopontin on the invasive capacity of rat trophoblasts was evaluated using Matrigel-based Transwell assays. Results: There were 257 genes differentially expressed between wild-type and IL15Δ/Δ implantation sites on gestational day 9.5, including decreased expression of various NK cell markers in IL15Δ/Δ rats, as well as Spp1 , which encodes osteopontin. In wild-type rats, osteopontin was present within the decidua basalis and adjacent to the primitive placenta, and osteopontin colocalized with the NK cell marker perforin. Osteopontin was also detectable in uterine glands. Conversely, in IL15Δ/Δ rats, osteopontin and perforin were not readily detectable in the decidua despite robust osteopontin levels in uterine glands. Neutralization of osteopontin in media conditioned by cells isolated from the decidua decreased invasion of rat trophoblasts, suggesting that reduced levels of osteopontin are unlikely to account for the precocious trophoblast invasion in IL15Δ/Δ rats. Conclusion: Osteopontin is expressed by NK cells at the maternal-fetal interface in rats and may contribute to modulation of trophoblast invasion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Baines, Klausner, Patterson and Renaud.)

Remodeling of the uterine vasculature by invasive extravillous trophoblasts (EVTs) is a critical aspect of human placentation. Insufficient EVT invasion can lead to severe obstetrical complications like preeclampsia, intrauterine growth restriction, and preterm birth. Glial cells missing-1 (GCM1) is a transcription factor that is crucial for proper placentation in mice, and is highly expressed in human syncytiotrophoblast (ST) and EVTs. GCM1 is classically considered a master regulator of ST formation, but little is known about its contribution to the development and function of EVTs. Therefore, in this study we test the hypothesis that GCM1 is a critical regulator of both EVT and ST development and function. We show that GCM1 is highly expressed in human trophoblast stem (TS) cells differentiated into either ST or EVTs. Knockdown of GCM1 in TS cells hindered differentiation into both ST and EVT pathways. When placed in ST media, GCM1-knockdown cells formed small, unstable clusters; when placed in EVT media, cells had altered morphology and transcript profiles resembling cells trapped in an intermediate state between CT and EVT, and invasive capacity through matrix was reduced. RNA sequencing analysis of GCM1-deficient TS cells revealed downregulation of EVT-associated genes and enrichment in transcripts related to WNT signaling, which was linked to decreased expression of the EVT master regulator ASCL2 and the WNT antagonist NOTUM. Our findings reveal an essential role of GCM1 during ST and EVT development, and suggest that GCM1 regulates differentiation of human TS cells into EVTs by inducing expression of ASCL2 and NOTUM.

Background and Objective: Severe asthma (SA) is a heterogeneous disease. Transcriptomic analysis contributes to the understanding of pathogenesis necessary for developing new therapies. We sought to identify and validate mechanistic pathways of SA across two independent cohorts., Methods: Transcriptomic profiles from U-BIOPRED and Australian NOVocastrian Asthma cohorts were examined and grouped into SA, mild/moderate asthma (MMA) and healthy controls (HCs). Differentially expressed genes (DEGs), canonical pathways and gene sets were identified as central to SA mechanisms if they were significant across both cohorts in either endobronchial biopsies or induced sputum., Results: Thirty-six DEGs and four pathways were shared across cohorts linking to tissue remodelling/repair in biopsies of SA patients, including SUMOylation, NRF2 pathway and oxidative stress pathways. MMA presented a similar profile to HCs. Induced sputum demonstrated IL18R1 as a shared DEG in SA compared with healthy subjects. We identified enrichment of gene sets related to corticosteroid treatment; immune-related mechanisms; activation of CD4 + T cells, mast cells and IL18R1; and airway remodelling in SA., Conclusion: Our results identified differentially expressed pathways that highlight the role of CD4 + T cells, mast cells and pathways linked to ongoing airway remodelling, such as IL18R1, SUMOylation and NRF2 pathways, as likely active mechanisms in the pathogenesis of SA., (© 2022 The Authors. Respirology published by John Wiley & Sons Australia, Ltd on behalf of Asian Pacific Society of Respirology.)

Children with asthma are at risk of acute exacerbations triggered mainly by viral infections. A diet high in fruit and vegetables (F&V), a rich source of carotenoids, may improve innate immune responses in children with asthma. Children with asthma (3−11 years) with a history of exacerbations and low F&V intake (≤3 serves/d) were randomly assigned to a high F&V diet or control (usual diet) for 6 months. Outcomes included respiratory-related adverse events and in-vitro cytokine production in peripheral blood mononuclear cells (PBMCs), treated with rhinovirus-1B (RV1B), house dust mite (HDM) and lipopolysaccharide (LPS). During the trial, there were fewer subjects with ≥2 asthma exacerbations in the high F&V diet group (n = 22) compared to the control group (n = 25) (63.6% vs. 88.0%, p = 0.049). Duration and severity of exacerbations were similar between groups. LPS-induced interferon (IFN)-γ and IFN-λ production showed a small but significant increase in the high F&V group after 3 months compared to baseline (p < 0.05). Additionally, RV1B-induced IFN-λ production in PBMCs was positively associated with the change in plasma lycopene at 6 months (rs = 0.35, p = 0.015). A high F&V diet reduced asthma-related illness and modulated in vitro PBMC cytokine production in young children with asthma. Improving diet quality by increasing F&V intake could be an effective non-pharmacological strategy for preventing asthma-related illness by enhancing children’s innate immune responses.

Background: Conflicting literature exists regarding the risk factors for exacerbations among pregnant women with asthma. This systematic review and meta-analysis aimed to determine risk factors for asthma exacerbations during pregnancy., Methods: Electronic databases were searched for the following terms: (asthma or wheeze) and (pregnan* or perinat* or obstet*) and (exacerb* or flare up or morbidit* or attack*).All studies published between 2000 and 24 August 2021 were considered for inclusion if they reported at least one potential risk factor of asthma exacerbations in pregnant women with asthma. Of the 3337 references considered, 35 publications involving 429 583 pregnant women with asthma were included. Meta-analyses were conducted to determine mean difference in risk factor between exacerbation groups, or the relative risks of exacerbation with certain risk factors. Good study quality was found through the Newcastle-Ottawa Scale (median score 8, interquartile range 7-9)., Results: Increased maternal age (mean difference 0.62, 95% CI 0.11-1.13), obesity (relative risk 1.25, 95% CI 1.15-1.37), smoking (relative risk 1.35, 95% CI 1.04-1.75), black ethnicity (relative risk 1.62, 95% CI 1.52-1.73), multiparity (relative risk 1.31, 95% CI 1.01-1.68), depression/anxiety (relative risk 1.42, 95% CI 1.27-1.59), moderate-severe asthma (relative risk 3.44, 95% CI 2.03-5.83, versus mild) and severe asthma (relative risk 2.70, 95% CI 1.85-3.95, versus mild-moderate) were associated with an increased risk of asthma exacerbations during pregnancy., Conclusions: Future interventions aimed at reducing exacerbations in pregnancy could address the modifiable factors, such as smoking and depression/anxiety, and introduce more regular monitoring for those with nonmodifiable risk factors such as obesity and more severe asthma., Competing Interests: Conflict of interest: M.P. Bokern reports support for the present manuscript from Groningen Research Institute of Pharmacy, PharmacoTherapy, Epidemiology and Economics, University of Groningen, Groningen, the Netherlands and Department of Non-Communicable Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, UK. M.P. Bokern reports grants or contracts from GlaxoSmithKline (GSK PhD scholarship at London School of Hygiene and Tropical Medicine), outside the submitted work. V.E. Murphy reports support for the present manuscript from Medical Research Future Fund Investigator Grant (paid to her institution). The remaining authors have nothing to disclose., (Copyright ©The authors 2022.)

Background: Asthma is the most common medical condition to affect pregnancy. Asthma exacerbations occur in up to 45% of pregnant women and have been associated with adverse perinatal and infant outcomes. Conflicting literature exists regarding the risk factors for exacerbations, and no synthesis of the literature currently exists. Therefore, this systematic review and meta-analysis aims to determine risk factors for asthma exacerbations during pregnancy among pregnant women with asthma., Methods: This protocol has been reported according to the Preferred Reporting Items for Systematic Review and Meta-Analysis protocols checklist. A systematic search will be conducted in the electronic MEDLINE, Embase, CINAHL and Cochrane Clinical Trials Register databases (from January 2000 onwards). Eligibility of each publication will be determined based on predefined selection criteria. Prospective cohort studies, retrospective cohort studies, case-control studies and randomised controlled trials (RCTs) will be included. Quality of included studies will be determined using the Newcastle Ottawa Scale and the Cochrane Risk of Bias tool. Pooled relative risk will be computed using random-effects meta-analyses. Heterogeneity will be assessed using the chi-squared test and the I 2 parameter. Publication bias will be assessed by inspecting a funnel plot for asymmetry and with the Egger's test of analyses including ten studies or more., Discussion: The results of this systematic review and meta-analysis will discuss the potential risk factors for asthma exacerbations during pregnancy. This may aid healthcare professionals in early identification of pregnant women with asthma at risk of poor outcomes, providing the opportunity to implement early interventions in order to avoid deterioration of asthma symptoms during pregnancy., Systematic Review Registration: PROSPERO CRD42020196190., (© 2022. The Author(s).)

Chronic low-grade systemic inflammation (SI), including activation of the NLRP3 inflammasome, is a feature of obesity, associated with increased circulating saturated fatty acids, such as palmitic acid (PA), and bacterial endotoxin lipopolysaccharide (LPS). PA and LPS may contribute to SI observed in obesity, while the dietary antioxidant sulforaphane has been shown to reduce activation of the NLRP3 inflammasome. This study investigated immune cell responses from obese subjects to PA, and the effects of sulforaphane on NLRP3 activation/inflammation. Peripheral blood monocytes isolated from obese (n = 8) and non-obese (n = 8) subjects and adipose tissue macrophages (ATMs) isolated from visceral fat obtained from obese subjects (n = 10) during bariatric surgery were pre-treated with/without sulforaphane (40 µM) for 3 hours then stimulated with PA (500 µM) ± LPS (1 ng/mL monocytes; 100 ng/mL ATMs) for 15 hours. Culture supernatants were assessed for tumor necrosis factor-α and interleukin-β (IL-1β) by enzyme-linked immunosorbent assay, NLRP3 inflammasome gene expression was assessed by quantitative polymerase chain reaction, IL-1β and NLRP3 expression were higher in both unstimulated and PA treated monocytes from obese compared to nonobese subjects. In ATMs neither PA alone or combined with LPS increased cytokine production or inflammasome gene expression. Sulforaphane reduced tumor necrosis factor-α and IL-1β from monocytes in both groups, however inflammasome associated genes were only reduced in monocytes from obese subjects. Sulforaphane reduced cytokine production and inflammasome gene expression in ATMs. NLRP3 inflammasome activation by PA is higher in obesity, which maybe driven by baseline activation of the NLRP3 inflammasome. Sulforaphane modulates inflammatory responses in immune cells and may play a role in reducing SI in obesity., Competing Interests: Declaration of competing interests The author declares that there are no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Maternal infections during pregnancy are linked with an increased risk for disorders like Autism Spectrum Disorder and schizophrenia in the offspring. Although precise mechanisms are still unclear, clinical and preclinical evidence suggest a strong role for maternal immune activation (MIA) in the neurodevelopmental disruptions caused by maternal infection. Previously, studies using the Polyinosinic:Polycytidylic (Poly I:C) MIA preclinical model showed that cytokines like Interleukin 6 (Il6) are important mediators of MIA's effects. In this study, we hypothesized that Il15 may similarly act as a mediator of Poly I:C MIA, given its role in the antiviral immune response. To test this hypothesis, we induced Poly I:C MIA at gestational day 9.5 in wildtype ( WT ) and Il15 -/- rat dams and tested their offspring in adolescence and adulthood. Poly I:C MIA and Il15 knockout produced both independent and synergistic effects on offspring behaviour. Poly I:C MIA decreased startle reactivity in adult WT offspring but resulted in increased adolescent anxiety and decreased adult locomotor activity in Il15 -/- offspring. In addition, Poly I:C MIA led to genotype-independent effects on locomotor activity and prepulse inhibition. Finally, we showed that Il15 -/- offspring exhibit distinct phenotypes that were unrelated to Poly I:C MIA including altered startle reactivity, locomotion and signal transduction in the auditory brainstem. Overall, our findings indicate that the lack of Il15 can leave offspring either more or less susceptible to Poly I:C MIA, depending on the phenotype in question. Future studies should examine the contribution of fetal versus maternal Il15 in MIA to determine the precise developmental mechanisms underlying these changes., (© 2022 The Authors.)

Introduction: The significance of endoplasmic reticulum (ER) stress in asthma is unclear. Here, we demonstrate that ER stress and the unfolded protein response (UPR) are related to disease severity and inflammatory phenotype., Methods: Induced sputum (n=47), bronchial lavage (n=23) and endobronchial biopsies (n=40) were collected from participants with asthma with varying disease severity, inflammatory phenotypes and from healthy controls. Markers for ER stress and UPR were assessed. These markers were also assessed in established eosinophilic and neutrophilic murine models of asthma., Results: Our results demonstrate increased ER stress and UPR pathways in asthma and these are related to clinical severity and inflammatory phenotypes. Genes associated with ER protein chaperone ( BiP, CANX, CALR ), ER-associated protein degradation ( EDEM1, DERL1) and ER stress-induced apoptosis ( DDIT3, PPP1R15A ) were dysregulated in participants with asthma and are associated with impaired lung function (forced expiratory volume in 1 s) and active eosinophilic and neutrophilic inflammation. ER stress genes also displayed a significant correlation with classic Th2 (interleukin-4, IL-4/13) genes, Th17 (IL-17F/CXCL1) genes, proinflammatory (IL-1b, tumour necrosis factor α, IL-8) genes and inflammasome activation (NLRP3) in sputum from asthmatic participants. Mice with allergic airway disease (AAD) and severe steroid insensitive AAD also showed increased ER stress signalling in their lungs., Conclusion: Heightened ER stress is associated with severe eosinophilic and neutrophilic inflammation in asthma and may play a crucial role in the pathogenesis of asthma., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Neutrophilic asthma (NA) is a clinically important asthma phenotype, the cellular and molecular basis of which is not completely understood. Airway macrophages are long-lived immune cells that exert important homeostatic and inflammatory functions which are dysregulated in asthma. Unique transcriptomic programmes reflect varied macrophage phenotypes in vitro. We aimed to determine whether airway macrophages are transcriptomically altered in NA., Methods: We performed RNASeq analysis on flow cytometry-isolated sputum macrophages comparing NA (n = 7) and non-neutrophilic asthma (NNA, n = 13). qPCR validation of RNASeq results was performed (NA n = 13, NNA n = 23). Pathway analysis (PANTHER, STRING) of differentially expressed genes (DEGs) was performed. Gene set variation analysis (GSVA) was used to test for enrichment of NA macrophage transcriptomic signatures in whole sputum microarray (cohort 1 - controls n = 16, NA n = 29, NNA n = 37; cohort 2 U-BIOPRED - controls n = 16, NA n = 47, NNA n = 57)., Results: Flow cytometry-sorting significantly enriched sputum macrophages (99.4% post-sort, 44.9% pre-sort, p < .05). RNASeq analysis confirmed macrophage purity and identified DEGs in NA macrophages. Selected DEGs (SLAMF7, DYSF, GPR183, CSF3, PI3, CCR7, all p < .05 NA vs. NNA) were confirmed by qPCR. Pathway analysis of NA macrophage DEGs was consistent with responses to bacteria, contribution to neutrophil recruitment and increased expression of phagocytosis and efferocytosis factors. GSVA demonstrated neutrophilic macrophage gene signatures were significantly enriched in whole sputum microarray in NA vs. NNA and controls in both cohorts., Conclusions: We demonstrate a pathophysiologically relevant sputum macrophage transcriptomic programme in NA. The finding that there is transcriptional activation of inflammatory programmes in cell types other than neutrophils supports the concept of NA as a specific endotype., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Background: Obesity is a risk factor for asthma, and obese asthmatic individuals are more likely to have severe, steroid-insensitive disease. How obesity affects the pathogenesis and severity of asthma is poorly understood. Roles for increased inflammasome-mediated neutrophilic responses, type 2 immunity, and eosinophilic inflammation have been described., Objective: We investigated how obesity affects the pathogenesis and severity of asthma and identified effective therapies for obesity-associated disease., Methods: We assessed associations between body mass index and inflammasome responses with type 2 (T2) immune responses in the sputum of 25 subjects with asthma. Functional roles for NLR family, pyrin domain-containing (NLRP) 3 inflammasome and T2 cytokine responses in driving key features of disease were examined in experimental high-fat diet-induced obesity and asthma., Results: Body mass index and inflammasome responses positively correlated with increased IL-5 and IL-13 expression as well as C-C chemokine receptor type 3 expression in the sputum of subjects with asthma. High-fat diet-induced obesity resulted in steroid-insensitive airway hyperresponsiveness in both the presence and absence of experimental asthma. High-fat diet-induced obesity was also associated with increased NLRP3 inflammasome responses and eosinophilic inflammation in airway tissue, but not lumen, in experimental asthma. Inhibition of NLRP3 inflammasome responses reduced steroid-insensitive airway hyperresponsiveness but had no effect on IL-5 or IL-13 responses in experimental asthma. Depletion of IL-5 and IL-13 reduced obesity-induced NLRP3 inflammasome responses and steroid-insensitive airway hyperresponsiveness in experimental asthma., Conclusion: We found a relationship between T2 cytokine and NLRP3 inflammasome responses in obesity-associated asthma, highlighting the potential utility of T2 cytokine-targeted biologics and inflammasome inhibitors., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Type 2 inflammation is characterized by enhanced activity of interleukin (IL)-4, -5 and -13, and treatments targeting these pathways are available for treatment of severe asthma. At present, the pattern of pathway activity and the implications overlapping of pathway activity are unknown., Objective: We hypothesized that clustering of airway mRNA expression would identify distinct molecular subtypes of severe asthma and thereby uncover the prevalence and overlap of pathway activity., Methods: Sputum mRNA expression of genes related to expression of IL-5(CLC, CPA3 and DNASE1L3), IL-13(IL13Ra1, TNFSF14 and SERPINB2), T1/Th17 activity(IL1B, ALPL and CXCR2) and in vitro response to corticosteroids (FKBP512) and mepolizumab (ARAP3) was analysed in patients (n = 109) with severe asthma and healthy controls (n = 22). A cluster analysis of gene expression was performed. The response to a short course of OCS was assessed in a subset of patients (n = 29)., Results: Five molecular clusters were identified. Three had abundant T2 gene expression of which two (n = 39 and n = 9) were characterized by abundant expression of both IL-13- and IL-5-related genes. The last (n = 6) had only abundant IL-5-related gene expression. These T2-high molecular clusters could not be distinguished using T2 biomarkers. T2- and Th1/Th17-related mRNA expression were co-expressed across all clusters. OCS significantly reduced T2 gene expression (CLC, IL13Ra1, SERPINB2 and ARAP3) and significantly increase expression of Th1/Th17-related genes (ALPL and CXCR2)., Conclusions and Clinical Relevance: Clustering of airway mRNA expression identified five molecular clusters of severe asthma of which three were considered T2 high. Co-expression of IL-5- and IL-13-related genes at moderate levels was present in almost half of patients, while marked elevated expression of both was rare. In contrast to IL-5, clusters with isolated IL-13- and Th1/Th17-related gene expression were not identified., (© 2021 John Wiley & Sons Ltd.)

Iron deficiency, which occurs when iron demands chronically exceed intake, is prevalent in pregnant women. Iron deficiency during pregnancy poses major risks for the baby, including fetal growth restriction and long-term health complications. The placenta serves as the interface between a pregnant mother and her baby, and it ensures adequate nutrient provisions for the fetus. Thus, maternal iron deficiency may impact fetal growth and development by altering placental function. We used a rat model of diet-induced iron deficiency to investigate changes in placental growth and development. Pregnant Sprague-Dawley rats were fed either a low-iron or iron-replete diet starting 2 weeks before mating. Compared with controls, both maternal and fetal hemoglobin were reduced in dams fed low-iron diets. Iron deficiency decreased fetal liver and body weight, but not brain, heart, or kidney weight. Placental weight was increased in iron deficiency, due primarily to expansion of the placental junctional zone. The stimulatory effect of iron deficiency on junctional zone development was recapitulated in vitro, as exposure of rat trophoblast stem cells to the iron chelator deferoxamine increased differentiation toward junctional zone trophoblast subtypes. Gene expression analysis revealed 464 transcripts changed at least 1.5-fold (P < 0.05) in placentas from iron-deficient dams, including altered expression of genes associated with oxygen transport and lipoprotein metabolism. Expression of genes associated with iron homeostasis was unchanged despite differences in levels of their encoded proteins. Our findings reveal robust changes in placentation during maternal iron deficiency, which could contribute to the increased risk of fetal distress in these pregnancies., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Neutrophil extracellular traps (NETs) increasingly are implicated in acute and chronic conditions involving multiple organ systems., Research Question: Are NET concentrations higher in parapneumonic effusions compared with effusions of other origin and does this reflect the inflammatory nature of these effusions?, Study Design and Methods: Patients (N = 101) seeking hospital treatment for undifferentiated pleural effusion underwent pleural fluid classification based on cytologic analysis results, biochemical findings, microbiological characteristics, and clinical judgement. Concentrations of NET markers (extracellular DNA [eDNA], citrullinated histone H3 [citH3]), neutrophils (α-defensins), and inflammation (IL-1β)-related proteins were quantified by enzyme-linked immunosorbent assay. Differences between groups were analyzed using the Kruskal-Wallis one-way analysis of variance. Correlations used Spearman coefficient. Receiver operating characteristic (ROC) curves were calculated., Results: Effusions were classified into four groups: parapneumonic (n = 18), malignant (n = 35), transudative (n = 22), and unclassifiable (n = 26). Concentrations of NETs markers were significantly higher in the parapneumonic group compared with malignant, transudative, and unclassifiable groups (median eDNA, 12.8 ng/mL vs 0.77 ng/mL, 0.44 ng/mL, and 0.86 ng/mL [P < .001]; and median citH3, 127.1 ng/mL vs 0.44 ng/mL, 0.34 ng/mL, and 0.49 ng/mL [P < .001]). citH3 and eDNA were correlated highly with lactate dehydrogenase (LDH; Spearman r = 0.66 and r = 0.73, respectively; P < .001) and moderately negatively correlated with pH (r = -0.55 and r = -0.62, respectively; P < .001). α-Defensins and IL-1β were higher in the parapneumonic group than in other groups (median α-defensins, 124.4 ng/mL vs 4.7 ng/mL,7 ng/mL, and 6.9 ng/mL [P < .001]; and median IL-1β, 145 pg/mL vs 1.87 pg/mL, 1.39 pg/mL, and 2.6 pg/mL [P < .001]) and moderately correlated with LDH (r = 0.60 and r = 0.57; P < .001). ROC curves showed high sensitivity and specificity for NET markers for prediction of parapneumonic effusion., Interpretation: High levels of some NET-related mediators in parapneumonic effusions correlate with inflammation. Effusions of other causes do not show high levels of NETs. These results may have treatment implications because NETs may be an important contributor to the inflammation and viscosity of parapneumonic effusions and may help us to understand the therapeutic benefit of deoxyribonuclease in empyema., (Copyright © 2021 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Background: Severe asthma is a complex disease. Transcriptomic profiling has contributed to understanding the pathogenesis of asthma, especially type-2 inflammation. However, there is still poor understanding of non-type-2 asthma, and consequently, there are limited treatment options., Objective: The aim of this study was to identify differentially expressed genes (DEGs) and pathways in endobronchial biopsies associated with inflammatory phenotypes of severe asthma., Methods: This cross-sectional study examined endobronchial biopsies from 47 adults with severe asthma (neutrophilic asthma (NA) n = 9, eosinophilic asthma (EA) n = 22 and paucigranulocytic asthma (PGA) n = 16) and 13 healthy controls (HC). RNA was extracted and transcriptomic profiles generated (Illumina Humanref-12 V4) and analysed using GeneSpring GX14.9.1. Pathway identification using Ingenuity Pathway Analysis., Results: NA had the most distinct profile, with signature of 60 top-ranked DEGs (FC >±2) including genes associated with innate immunity response, neutrophil degranulation and IL-10 signalling. NA presented enrichment to pathways previously linked to neutrophilic inflammation; dendritic cell maturation, Th1, TREM1, inflammasome, Th17 and p38 MAPK, as well as novel links to neuroinflammation, NFAT and PKCθ signalling. EA presented similar transcriptomic profiles to PGA and HC. Despite the higher proportion of bacterial colonization in NA, no changes were observed in the transcriptomic profiles of severe asthma culture positive compared with severe asthma culture negative., Conclusions & Clinical Relevance: NA features a distinct transcriptomic profile with seven pathways enriched in NA compared to EA, PGA and HC. All those with severe asthma had significant enrichment for SUMOylation, basal cell carcinoma signalling and Wnt/β-catenin pathways compared to HC, despite high-dose inhaled corticosteroids. These findings contribute to the understanding of mechanistic pathways in endobronchial biopsies associated with NA and identify potential novel treatment targets for severe asthma., (© 2021 John Wiley & Sons Ltd.)

Background: A high fruit and vegetable (F&V) diet reduces asthma exacerbations in adults; this has not been examined in children to date., Objective: To investigate the effect of a 6-month, high F&V diet on the time to first asthma exacerbation in children with asthma, in a parallel-group, randomized, controlled trial., Methods: Children (aged 3-11 years) with asthma, history of exacerbations and usual low F&V intake (≤3 serves/day) were randomized to the intervention (high F&V diet) or control group (usual diet) for 6 months. The primary outcome was time to first exacerbation requiring medical intervention. Secondary outcomes included exacerbation rate, lung function, plasma TNF-α, CRP, and IL-6, faecal microbiota and peripheral blood mononuclear cell (PBMC) histone deacetylase (HDAC) activity and G-protein coupled receptor (GPR) 41/43 and HDAC (1-11) expression., Results: 67 children were randomized between September 2015 and July 2018. F&V intake (difference in change (∆): 3.5 serves/day, 95% CI: [2.6, 4.4] p < 0.001) and plasma total carotenoids (∆: 0.44 µg/ml [0.19, 0.70] p = 0.001) increased after 6 months (intervention vs control). Time to first exacerbation (HR: 0.81, 95% CI: [0.38, 1.69], p = 0.569; control vs. intervention) and exacerbation rate (IRR: 0.84, [0.47, 1.49], p = 0.553; control vs. intervention) were similar between groups. In per-protocol analysis, airway reactance z-scores increased in the intervention versus control group (X 5 ∆: 0.76 [0.04, 1.48] p = 0.038, X 20 ∆: 0.93 [0.23, 1.64] p = 0.009) and changes in faecal microbiota were observed though there was no difference between groups in systemic inflammation or molecular mechanisms. In the control group, CRP and HDAC enzyme activity increased, while GPR41 expression decreased. No adverse events attributable to the interventions were observed., Conclusion & Clinical Relevance: A high F&V diet did not affect asthma exacerbations over the 6-month intervention, though warrants further investigation as a strategy for improving lung function and protecting against systemic inflammation in children with asthma., (© 2021 John Wiley & Sons Ltd.)

Background: Mast cells (MCs) and basophils are important in asthma pathophysiology, however direct measurement is difficult, and clinical and inflammatory associations in severe asthma are poorly understood. Transcriptomic hallmarks of MCs/basophils may allow their measurement in sputum using gene expression., Objectives: This study sought to develop and validate a sputum MC/basophil gene signature and investigate its relationship to inflammatory and clinical characteristics of severe asthma., Methods: A total of 134 candidate MC/basophil genes (identified by the Immunological Genome Project Consortium) were screened in sputum microarray for differential expression among control subjects (n = 18), patients with eosinophilic (n = 29), and patients with noneosinophilic asthma (n = 30). Candidate genes were validated by confirming correlation of gene expression with flow cytometry-quantified sputum MCs and basophils in a separate asthma cohort (n = 20). The validated gene signature was measured in a severe asthma cohort (n = 81), and inflammatory and clinical associations were tested., Results: Through microarray screening and subsequent validation, we found quantitative PCR gene expression of 8 targets correlated with sputum MCs/basophils: TPSAB1/TPSB2, CPA3, ENO2, GATA2, KIT, GPR56, HDC, SOCS2. In severe asthma, MC/basophil genes were associated with eosinophilic airway inflammation (GATA2, TPSB2, CPA3, GPR56, HDC, SOCS2), blood eosinophils (TPSB2, CPA3, GATA2, SOCS2, FCER1A, HDC), fractional exhaled NO (GATA2, SOCS2), decreased lung function (KIT, ENO2), and moderate exacerbation history (GATA2, SOCS2)., Conclusions: Quantitative PCR-based measures reflect varying sputum MC/basophil abundance, demonstrating associations of MCs/basophils with eosinophilic inflammation, spirometry and exacerbation history in severe asthma., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Dysregulation of tumour necrosis factor-α (TNF-α) signalling is implicated in neutrophilic asthma. TNF-α signalling involves membrane-bound and soluble ligand (TNF-α) and receptors (TNFRs); however, little is known about how these proteins are altered in asthma. We hypothesised that intercompartment-, immune cell- and/or asthma inflammatory phenotype-dependent regulation could relate to TNF dysregulation in neutrophilic asthma., Methods: Measurements were made in 45 adults with asthma (36 non-neutrophilic, 9 neutrophilic) and 8 non-asthma controls. Soluble TNF-α, TNF receptor 1 (TNFR1) and TNFR2 were quantified in plasma and sputum supernatant by ELISA, and membrane-bound TNF-α/TNFR1/TNFR2 measured on eosinophils, neutrophils, monocytes, and macrophages in blood and sputum by flow cytometry. Marker expression was compared between inflammatory phenotypes and compartments, and relationship of membrane-bound and soluble TNF markers and immune cell numbers tested by correlation., Results: Soluble sputum TNFR1 and TNFR2 were increased in neutrophilic versus non-neutrophilic asthma (p=0.010 and p=0.029). Membrane-bound TNF-α expression was upregulated on sputum versus blood monocytes, while TNFR1 and TNFR2 levels were reduced on airway versus blood monocytes and neutrophils. Soluble TNFR1 and TNFR2 in sputum significantly correlated with the number of airway monocytes (p=0.016, r=0.358 and p=0.029, r=0.327)., Conclusion: Our results imply that increased sputum soluble TNF receptor levels observed in neutrophilic asthma relate to the increased recruitment of monocytes and neutrophils into the airways and their subsequent receptor shedding. Monocytes also increase TNF-α ligand expression in the airways. These results suggest an important contribution of airway monocytes to the altered inflammatory milieu in neutrophilic asthma., Competing Interests: Conflict of interests: N.M. Niessen has nothing to disclose. Conflict of interest: P.G. Gibson reports, during the conduct of this study, that he holds a Practitioner Fellowship to the National Health and Medical Research Council. Outside the submitted works, grant funding has been received from AstraZeneca and GlaxoSmithKline, and he has participated in educational symposia funded by AstraZeneca, GlaxoSmithKline and Novartis. Conflict of interest: J.L. Simpson has nothing to disclose. Conflict of interest: H.A. Scott has nothing to disclose. Conflict of interest: K.J. Baines reports project grant funds paid by NHMRC to their institution outside the submitted works. Conflict of interest: M. Fricker reports receiving grant funding from GlaxoSmithKline outside the submitted works., (Copyright ©The authors 2021.)

Background: The AMAZES randomized controlled trial demonstrated that long-term low-dose azithromycin treatment reduces exacerbations of poorly controlled asthma, but the therapeutic mechanisms remain unclear. Dysregulation of the inflammatory tumour necrosis factor (TNF) pathway is implicated in asthma and could be suppressed by azithromycin. We aimed to determine the inflammatory and clinical associations of soluble TNF signalling proteins (TNF receptors [TNFR] 1 and 2, TNF) in sputum and serum, and to test the effect of 48 weeks of azithromycin vs placebo on TNF markers., Methods: Sputum supernatant and serum TNFR1, TNFR2 (n = 142; 75 azithromycin-treated, 67 placebo-treated) and TNF (n = 48; 22 azithromycin-treated, 26 placebo-treated) were measured by ELISA in an AMAZES trial sub-population at baseline and end of treatment. Baseline levels were compared between sputum inflammatory phenotypes, severe/non-severe asthma and frequent/non-frequent exacerbators. Effect of azithromycin on markers was tested using linear mixed models., Results: Baseline sputum TNFR1 and TNFR2 were significantly increased in neutrophilic vs non-neutrophilic asthma phenotypes, while serum markers did not differ. Sputum TNFR1 and TNFR2 were increased in severe asthma and correlated with poorer lung function, worse asthma control and increasing age. Serum TNFR1 was also increased in severe asthma. Sputum and serum TNFR2 were increased in frequent exacerbators. Azithromycin treatment significantly reduced sputum TNFR2 and TNF relative to placebo, specifically in non-eosinophilic participants., Conclusions: We demonstrate dysregulation of TNF markers, particularly in the airways, that relates to clinically important phenotypes of asthma including neutrophilic and severe asthma. Suppression of dysregulated TNF signalling by azithromycin could contribute to its therapeutic mechanism., (© 2021 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: Airway and systemic eosinophilia are important treatable traits in both severe asthma and COPD. The molecular basis of eosinophilia in COPD is poorly understood but could involve type 2 cytokines (IL5, IL13) and prostaglandin D 2 (PGD 2 )., Methods: This study included non-obstructive airways disease (OAD) controls (n = 19), a COPD cohort (n = 96) and a severe asthma cohort (n = 84). Demographics, exacerbation history, disease impact (SGRQ) and spirometry were assessed. Participants were categorized as eosinophilic using either sputum eosinophil proportion (≥3%) or blood eosinophil count (≥300/μL). Sputum type 2 inflammatory measures included PGD 2 by ELISA and gene expression (qPCR) of IL5, IL13 and the haematopoietic PGD 2 synthase (HPGDS)., Results: Type 2 markers did not differ across groups except HPGDS mRNA which was highest in non-OAD controls and lowest in COPD. IL5 and IL13 mRNA and PGD 2 levels were significantly increased in eosinophilic vs non-eosinophilic severe asthma but did not differ between eosinophilic COPD and eosinophilic severe asthma or non-eosinophilic COPD. HPGDS expression was higher in eosinophilic severe asthma compared with eosinophilic COPD. Results were similar using sputum or blood eosinophil cut-offs. Sputum IL5 and IL13 were highly intercorrelated in severe asthma (r = 0.907, p < 0.001) and COPD (r = 0.824, p < 0.001), were moderately correlated with sputum eosinophils in severe asthma (IL5 r = 0.440, p < 0.001; IL13 r = 0.428, p < 0.001) and were weakly correlated in COPD (IL5 r = 0.245, p < 0.05; IL13 r = 0.317, p < 0.05)., Conclusions: Molecular markers of type 2 airway inflammation do not differ between eosinophilic asthma and eosinophilic COPD; however, the relationship between eosinophilia and type 2 airway markers appears weaker in COPD than in severe asthma., (© 2021 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: Asthma is the most frequent cause of hospitalisation among children; however, little is known regarding the effects of asthma on immune responses in children., Objective: The present study aimed to evaluate cytokine responses of peripheral blood mononuclear cells (PBMCs), PBMC composition and lung function in children with and without asthma., Methods: Using a case-control design, we compared 48 children with asthma aged 3-11 years with 14 age-matched healthy controls. PBMC composition and cytokine production including interferon (IFN)-γ, interleukin (IL)-1β, IL-5 and lL-6 following stimulation with rhinovirus-1B (RV1B), house dust mite (HDM) and lipopolysaccharide (LPS) were measured. Lung function was assessed using impulse oscillometry and nitrogen multiple breath washout., Results: The frequency of group 2 innate lymphoid cells were significantly higher in asthmatics and PBMCs from asthmatics had deficient IFN-γ production in response to both RV1B and LPS compared with controls (P<0.01). RV1B-induced IL-1β response and HDM-stimulated IL-5 production was higher in asthmatics than controls (P<0.05). In contrast, IL-1β and IL-6 were significantly reduced in response to HDM and LPS in asthmatics compared to controls (P<0.05). Children with asthma also had reduced pulmonary function, indicated by lower respiratory reactance as well as higher area of-reactance and lung clearance index values compared with controls (P<0.05)., Conclusion: Our study indicates that children with asthma have a reduced lung function in concert with impaired immune responses and altered immune cell subsets. Improving our understanding of immune responses to viral and bacterial infection in childhood asthma can help to tailor management of the disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hosseini, Berthon, Starkey, Collison, McLoughlin, Williams, Nichol, Wark, Jensen, Da Silva Sena, Baines, Mattes and Wood.)

Background and Objective: Long-term data on children with PBB has been identified as a research priority. We describe the 5-year outcomes for children with PBB to ascertain the presence of chronic respiratory disease (bronchiectasis, recurrent PBB and asthma) and identify the risk factors for these., Methods: Prospective cohort study was undertaken at the Queensland Children's Hospital, Brisbane, Australia, of 166 children with PBB and 28 controls (undergoing bronchoscopy for symptoms other than chronic wet cough). Monitoring was by monthly contact via research staff. Clinical review, spirometry and CT chest were performed as clinically indicated., Results: A total of 194 children were included in the analysis. Median duration of follow-up was 59 months (IQR: 50-71 months) post-index PBB episode, 67.5% had ongoing symptoms and 9.6% had bronchiectasis. Significant predictors of bronchiectasis were recurrent PBB in year 1 of follow-up (OR adj = 9.6, 95% CI: 1.8-50.1) and the presence of Haemophilus influenzae in the BAL (OR adj = 5.1, 95% CI: 1.4-19.1). Clinician-diagnosed asthma at final follow-up was present in 27.1% of children with PBB. A significant BDR (FEV 1 improvement >12%) was obtained in 63.5% of the children who underwent reversibility testing. Positive allergen-specific IgE (OR adj = 14.8, 95% CI: 2.2-100.8) at baseline and bronchomalacia (OR adj = 5.9, 95% CI: 1.2-29.7) were significant predictors of asthma diagnosis. Spirometry parameters were in the normal range., Conclusion: As a significant proportion of children with PBB have ongoing symptoms at 5 years, and outcomes include bronchiectasis and asthma, they should be carefully followed up clinically. Defining biomarkers, endotypes and mechanistic studies elucidating the different outcomes are now required., (© 2020 Asian Pacific Society of Respirology.)