Gender and sex hormone effects on neonatal innate immune function.

OBJECTIVES: Scientific evidence provides a widened view of differences in immune response between male and female neonates. The X-chromosome codes for several genes important in the innate immune response and neonatal innate immune cells express receptors for, and are inhibited by, maternal sex hormones. We hypothesized that sex differences in innate immune responses may be present in the neonatal population which may contribute to the increased susceptibility of premature males to sepsis. We aimed to examine the in vitro effect of pro-inflammatory stimuli and hormones in neutrophils and monocytes of male and female neonates, to examine the expression of X-linked genes involved in innate immunity and the miRNA profiles in these populations. METHODS: Preterm infants (n = 21) and term control (n = 19) infants were recruited from the Coombe Women and Infants University Hospital Dublin with ethical approval and explicit consent. The preterm neonates (eight female, 13 male) were recruited with a mean gestation at birth (mean ± SD) of 28 ± 2 weeks and corrected gestation at the time of sampling was 30 + 2.6 weeks. The mean birth weight of preterm neonates was 1084 ± 246 g. Peripheral blood samples were used to analyze immune cell phenotypes, miRNA human panel, and RNA profiles for inflammasome and inflammatory genes. RESULTS: Dividing neutrophil results by sex showed no differences in baseline CD11b between sexes among either term or preterm neonates. Examining monocyte CD11b by sex shows, that at baseline, total and classical monocytes have higher CD11b in preterm females than preterm males. Neutrophil TLR2 did not differ between sexes at baseline or following lipopolysaccharide (LPS) exposure. CD11b expression was higher in preterm male non-classical monocytes following Pam3CSK treatment when compared to females, a finding which is unique to our study. Preterm neonates had higher TLR2 expression at baseline in total monocytes, classical monocytes and non-classical monocytes than term. A sex difference was evident between preterm females and term females in TLR2 expression only. Hormone treatment showed no sex differences and there was no detectable difference between males and females in X-linked gene expression. Two miRNAs, miR-212-3p and miR-218-2-3p had significantly higher expression in preterm female than preterm male neonates. CONCLUSIONS: This study examined immune cell phenotypes and x-linked gene expression in preterm neonates and stratified according to gender. Our findings suggest that the responses of females mature with advancing gestation, whereas male term and preterm neonates have very similar responses. Female preterm neonates have improved monocyte activation than males, which likely reflects improved innate immune function as reflected clinically by their lower risk of sepsis. Dividing results by sex showed changes in preterm and term infants at baseline and following LPS stimulation, a difference which is reflected clinically by infection susceptibility. The sex difference noted is novel and may be limited to the preterm or early neonatal population as TLR2 expression on monocytes of older children does not differ between males and females. The differences shown in female and male innate immune cells likely reflect a superior innate immune defense system in females with sex differences in immune cell maturation. Existing human studies on sex differences in miRNA expression do not include preterm patients, and most frequently use either adult blood or cord blood. Our findings suggest that miRNA profiles are similar in neonates of opposite sexes at term but require further investigation in the preterm population. Our findings, while novel, provide only very limited insights into sex differences in infection susceptibility in the preterm population leaving many areas that require further study. These represent important areas for ongoing clinical and laboratory study and our findings represent an important contribution to exiting literature.

Global respiratory health priorities at the beginning of the 21st century.

Respiratory health has become a prevailing priority amid the diverse global health challenges that the 21st century brings, due to its substantial impact on individuals and communities on a global scale. Due to rapid advances in medicine, emerging knowledge gaps appear along with new challenges and ethical considerations. While breakthroughs in medical science can bring about encouraging possibilities for better treatments and interventions, they also lead to unanswered questions and areas where further research is warranted. A PubMed search on the topic "global respiratory health priorities" between the years 2000 and 2023 was conducted, which returned 236 articles. Of these, 55 were relevant and selected for inclusion in this article. The selection process took into account literature reviews, opinions from expert groups and careful analysis of existing gaps and challenges within the field; our selection encompasses specific infectious and noninfectious respiratory conditions in both adults and children. The global respiratory health priorities identified were selected on the basis that they have been recognised as critical areas of investigation and potential advancement and they span across clinical, translational, epidemiological and population health domains. Implementing these priorities will require a commitment to fostering collaboration and knowledge-sharing among experts in different fields with the ultimate aim to improve respiratory health outcomes for individuals and communities alike.

TGF-ß1 Inhibition of ACE2 Mediated by miRNA Uncovers Novel Mechanism of SARS-CoV-2 Pathogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, utilizes receptor binding domain (RBD) of spike glycoprotein to interact with angiotensin (Ang)-converting enzyme 2 (ACE2). Altering ACE2 levels may affect entry of SARS-CoV-2 and recovery from COVID-19. Decreased cell surface density of ACE2 leads to increased local levels of Ang II and may contribute to mortality resulting from acute lung injury and fibrosis during COVID-19. Studies published early during the COVID-19 pandemic reported that people with cystic fibrosis (PwCF) had milder symptoms, compared to people without CF. This finding was attributed to elevated ACE2 levels and/or treatment with the high efficiency CFTR modulators. Subsequent studies did not confirm these findings reporting variable effects of CFTR gene mutations on ACE2 levels. Transforming growth factor (TGF)-ß signaling is essential during SARS-CoV-2 infection and dominates the chronic immune response in severe COVID-19, leading to pulmonary fibrosis. TGF-ß1 is a gene modifier associated with more severe lung disease in PwCF but its effects on the COVID-19 course in PwCF is unknown. To understand whether TGF-ß1 affects ACE2 levels in the airway, we examined miRNAs and their gene targets affecting SARS-CoV-2 pathogenesis in response to TGF-ß1. Small RNAseq and micro(mi)RNA profiling identified pathways uniquely affected by TGF-ß1, including those associated with SARS-CoV-2 invasion, replication, and the host immune responses. TGF-ß1 inhibited ACE2 expression by miR-136-3p and miR-369-5p mediated mechanism in CF and non-CF bronchial epithelial cells. ACE2 levels were higher in two bronchial epithelial cell models expressing the most common CF-causing mutation in CFTR gene F508del, compared to controls without the mutation. After TGF-ß1 treatment, ACE2 protein levels were still higher in CF, compared to non-CF cells. TGF-ß1 prevented the modulator-mediated rescue of F508del-CFTR function while the modulators did not prevent the TGF-ß1 inhibition of ACE2 levels. Finally, TGF-ß1 reduced the interaction between ACE2 and the recombinant spike RBD by lowering ACE2 levels and its binding to RBD. Our data demonstrate novel mechanism whereby TGF-ß1 inhibition of ACE2 in CF and non-CF bronchial epithelial cells may modulate SARS-CoV-2 pathogenicity and COVID-19 severity. By reducing ACE2 levels, TGF-ß1 may decrease entry of SARS-CoV-2 into the host cells while hindering the recovery from COVID-19 due to loss of the anti-inflammatory and regenerative effects of ACE2. The above outcomes may be modulated by other, miRNA-mediated effects exerted by TGF-ß1 on the host immune responses, leading to a complex and yet incompletely understood circuitry.

Basic, translational and clinical aspects of bronchiectasis in adults.

Bronchiectasis is a common progressive respiratory disease with recognisable radiological abnormalities and a clinical syndrome of cough, sputum production and recurrent respiratory infections. Inflammatory cell infiltration into the lung, in particular neutrophils, is central to the pathophysiology of bronchiectasis. Herein we explore the roles and relationships between infection, inflammation and mucociliary clearance dysfunction in the establishment and progression of bronchiectasis. Microbial and host-mediated damage are important processes underpinning bronchiectasis and the relative contribution of proteases, cytokines and inflammatory mediators to the propagation of inflammation is presented. We also discuss the emerging concept of inflammatory endotypes, defined by the presence of neutrophilic and eosinophilic inflammation, and explore the role of inflammation as a treatable trait. Current treatment for bronchiectasis focuses on treatment of underlying causes, enhancing mucociliary clearance, controlling infection and preventing and treating complications. Data on airway clearance approaches via exercise and mucoactive drugs, pharmacotherapy with macrolides to decrease exacerbations and the usefulness of inhaled antibiotics and bronchodilators are discussed, finishing with a look to the future where new therapies targeting host-mediated immune dysfunction hold promise.

ERS International Congress 2022: highlights from the Basic and Translational Science Assembly.

In this review, the Basic and Translational Science Assembly of the European Respiratory Society provides an overview of the 2022 International Congress highlights. We discuss the consequences of respiratory events from birth until old age regarding climate change related alterations in air quality due to pollution caused by increased ozone, pollen, wildfires and fuel combustion as well as the increasing presence of microplastic and microfibres. Early life events such as the effect of hyperoxia in the context of bronchopulmonary dysplasia and crucial effects of the intrauterine environment in the context of pre-eclampsia were discussed. The Human Lung Cell Atlas (HLCA) was put forward as a new point of reference for healthy human lungs. The combination of single-cell RNA sequencing and spatial data in the HLCA has enabled the discovery of new cell types/states and niches, and served as a platform that facilitates further investigation of mechanistic perturbations. The role of cell death modalities in regulating the onset and progression of chronic lung diseases and its potential as a therapeutic target was also discussed. Translational studies identified novel therapeutic targets and immunoregulatory mechanisms in asthma. Lastly, it was highlighted that the choice of regenerative therapy depends on disease severity, ranging from transplantation to cell therapies and regenerative pharmacology.

Long Non-Coding RNA Expression in Alpha-1 Antitrypsin Deficient Monocytes Pre- and Post-AAT Augmentation Therapy.

Long non-coding RNAs (lncRNAs) regulate gene expression. Their expression in alpha-1 antitrypsin (AAT) deficiency has not been investigated. Treatment of AAT deficiency involves infusion of plasma-purified AAT and this augmentation therapy has previously been shown to alter microRNA expression in monocytes of AAT-deficient (ZZ) individuals. Here, we assess the effect of AAT augmentation therapy on the lncRNA expression profile in ZZ monocytes. Peripheral blood monocytes were isolated from ZZ individuals pre (Day 0)- and post (Day 2)-AAT augmentation therapy. Arraystar lncRNA microarray profiling was performed; a total of 17,761 lncRNAs were detectable across all samples. The array identified 7509 lncRNAs with differential expression post-augmentation therapy, 3084 were increased and 4425 were decreased (fold change ≥ 2). Expression of many of these lncRNAs were similarly altered in ZZ monocytes treated ex vivo with 27.5 µM AAT for 4 h. These properties may contribute to the manifold effects of AAT augmentation therapy.

Sexual Dimorphism in Interstitial Lung Disease.

Interstitial lung diseases (ILD) are a group of heterogeneous progressive pulmonary disorders, characterised by tissue remodelling and/or fibrotic scarring of the lung parenchyma. ILD patients experience lung function decline with progressive symptoms, poor response to treatment, reduced quality of life and high mortality. ILD can be idiopathic or associated with systemic or connective tissue diseases (CTD) but idiopathic pulmonary fibrosis (IPF) is the most common form. While IPF has a male predominance, women are affected more greatly by CTD and therefore associated ILDs. The mechanisms behind biological sex differences in these progressive lung diseases remain unclear. However, differences in environmental exposures, variable expression of X-chromosome related inflammatory genes and sex hormones play a role. Here, we will outline sex-related differences in the incidence, progression and mechanisms of action of these diseases and discuss existing and novel cellular and pre-clinical studies. Furthermore, we will highlight how sex-differences are not adequately considered in pre-clinical disease models, how gender bias exists in clinical diagnosis and how women are underrepresented in clinical trials. Future action on these observations will hopefully shed light on the role of biological sex in disease development, identify potential targets for intervention and increase female participant numbers in clinical trials.

Epithelial damage in the cystic fibrosis lung: the role of host and microbial factors.

INTRODUCTION: The airway epithelium is a key system within the lung. It acts as a physical barrier to inhaled factors, and can actively remove unwanted microbes and particles from the lung via the mucociliary escalator. On a physiological level, it senses the presence of pathogens and initiates innate immune responses to combat their effects. Hydration of the airways is also controlled by the epithelium. Within the cystic fibrosis (CF) lung, these properties are suboptimal and contribute to the pulmonary manifestations of CF. AREAS COVERED: In this review, we discuss how various host and microbial factors can contribute to airway epithelium dysfunction in the CF lung focusing on mechanisms relating to the mucociliary escalator and protease expression and function. We also explore how alterations in microRNA expression can impact the behavior of the airway epithelium. EXPERT OPINION: Notwithstanding the unprecedented benefits that CFTR modulator drugs now provide to the health of CF sufferers, it will be important to delve more deeply into additional mechanisms underpinning CF lung disease such as those illustrated here in an attempt to counteract these aberrant processes and further enhance quality of life for people with CF.

miR-224-5p and miR-545-5p Levels Relate to Exacerbations and Lung Function in a Pilot Study of X-Linked MicroRNA Expression in Cystic Fibrosis Monocytes.

Altered microRNA expression patterns in bronchial brushings from people with versus without cystic fibrosis (CF) relate to functional changes and disease pathophysiology. The expression of microRNAs encoded on the X chromosome is also altered in peripheral blood monocytes of p. Phe508del homozygous versus non-CF individuals. Here we investigate whether levels of the top seven X-linked microRNAs (miR-224-5p, miR-452-5p, miR-450b-5p, miR-542-3p, miR-450a-5p, miR-424-5p, and miR-545-5p) that are significantly increased over 1.5 fold in CF versus non-CF monocytes correlate with lung function. CD14+ monocytes were isolated from males and females with (n = 12) and without cystic fibrosis (n = 12) and examined for the expression of X-linked microRNAs by qRT-PCR array. MicroRNA target mRNA levels were quantified using qRT-PCR. Clinical correlations with lung function data were analysed in the CF cohort. Increasing levels of miR-545-5p correlated moderately with FEV1% predicted (r = -0.4553, p > 0.05) and strongly with exacerbation rate (r = 0.5858, p = 0.0483). miR-224-5p levels were significantly higher in the severe (FEV1 <40%) versus mild (FEV1 ≥80%, p = 0.0377) or moderate (FEV1 40-79%, p = 0.0350) groups. MiR-224-5p expression inversely correlated with lung function (FEV1%: r = -0.5944, p = 0.0457) and positively correlated with exacerbation rates (r = 0.6139, p = 0.0370). These data show that peripheral blood monocyte miR-545-5p and miR-224-5p levels correlate with exacerbation rate, whilst miR-224-5p levels also correlate with lung function in cystic fibrosis.

Research priorities to address the global burden of chronic obstructive pulmonary disease (COPD) in the next decade.

BACKGROUND: The global prevalence of chronic obstructive pulmonary disease (COPD) has increased markedly in recent decades. Given the scarcity of resources available to address global health challenges and respiratory medicine being relatively under-invested in, it is important to define research priorities for COPD globally. In this paper, we aim to identify a ranked set of COPD research priorities that need to be addressed in the next 10 years to substantially reduce the global impact of COPD. METHODS: We adapted the Child Health and Nutrition Research Initiative (CHNRI) methodology to identify global COPD research priorities. RESULTS: 62 experts contributed 230 research ideas, which were scored by 34 researchers according to six pre-defined criteria: answerability, effectiveness, feasibility, deliverability, burden reduction, and equity. The top-ranked research priority was the need for new effective strategies to support smoking cessation. Of the top 20 overall research priorities, six were focused on feasible and cost-effective pulmonary rehabilitation delivery and access, particularly in primary/community care and low-resource settings. Three of the top 10 overall priorities called for research on improved screening and accurate diagnostic methods for COPD in low-resource primary care settings. Further ideas that drew support involved a better understanding of risk factors for COPD, development of effective training programmes for health workers and physicians in low resource settings, and evaluation of novel interventions to encourage physical activity. CONCLUSIONS: The experts agreed that the most pressing feasible research questions to address in the next decade for COPD reduction were on prevention, diagnosis and rehabilitation of COPD, especially in low resource settings. The largest gains should be expected in low- and middle-income countries (LMIC) settings, as the large majority of COPD deaths occur in those settings. Research priorities identified by this systematic international process should inform and motivate policymakers, funders, and researchers to support and conduct research to reduce the global burden of COPD.

High-Throughput Identification of miRNA-Target Interactions in Melanoma Using miR-CATCHv2.0.

MicroRNAs (miRNAs) can regulate the expression of potentially every transcript in the cell, and the definition of miRNA-target interactions is crucial to understand their role in all biological processes. However, the identification of the miRNAs that target a specific mRNA remains a challenge. Here, we describe an innovative method called miR-CATCHv2.0 for the high-throughput identification of the miRNA species bound to an RNA of interest. We also describe how this method can overcome the limitations of the current computational and experimental methods available in this field.

Impaired Airway Epithelial Barrier Integrity in Response to Stenotrophomonas maltophilia Proteases, Novel Insights Using Cystic Fibrosis Bronchial Epithelial Cell Secretomics.

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can chronically colonize the lungs of people with cystic fibrosis (CF) and is associated with lethal pulmonary hemorrhage in immunocompromised patients. Its secreted virulence factors include the extracellular serine proteases StmPR1, StmPR2, and StmPR3. To explore the impact of secreted virulence determinants on pulmonary mucosal defenses in CF, we examined the secretome of human CFBE41o- bronchial epithelial cells in response to treatment with S. maltophilia K279a cell culture supernatant (CS) using a liquid-chromatography-tandem mass spectrometry (LC-MS/MS) based label-free quantitative (LFQ) shotgun proteomics approach for global profiling of the cell secretome. Secretome analysis identified upregulated pathways mainly relating to biological adhesion and epithelial cell signaling in infection, whereas no specific pathways relating to the immune response were enriched. Further exploration of the potentially harmful effects of K279a CS on CF bronchial epithelial cells, demonstrated that K279a CS caused CFBE41o- cell condensation and detachment, reversible by the serine protease inhibitor PMSF. K279a CS also decreased trans-epithelial electrical resistance in CFBE41o- cell monolayers suggestive of disruption of tight junction complexes (TJC). This finding was corroborated by an observed increase in fluorescein isothiocyanate (FITC) dextran permeability and by demonstrating PMSF-sensitive degradation of the tight junction proteins ZO-1 and occludin, but not JAM-A or claudin-1. These observations demonstrating destruction of the CFBE41o- TJC provide a novel insight regarding the virulence of S. maltophilia and may explain the possible injurious effects of this bacterium on the CF bronchial epithelium and the pathogenic mechanism leading to lethal pulmonary hemorrhage.

Alpha-1 Antitrypsin-A Target for MicroRNA-Based Therapeutic Development for Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder arising from mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Disruption to normal ion homeostasis in the airway results in impaired mucociliary clearance, leaving the lung more vulnerable to recurrent and chronic bacterial infections. The CF lung endures an excess of neutrophilic inflammation, and whilst neutrophil serine proteases are a crucial part of the innate host defence to infection, a surplus of neutrophil elastase (NE) is understood to create a net destructive effect. Alpha-1 antitrypsin (A1AT) is a key antiprotease in the control of NE protease activity but is ineffective in the CF lung due to the huge imbalance of NE levels. Therapeutic strategies to boost levels of protective antiproteases such as A1AT in the lung remain an attractive research strategy to limit the damage from excess protease activity. microRNAs are small non-coding RNA molecules that bind specific cognate sequences to inhibit expression of target mRNAs. The inhibition of miRNAs which target the SERPINA1 (A1AT-encoding gene) mRNA represents a novel therapeutic approach for CF inflammation. This could involve the delivery of antagomirs that bind and sequester the target miRNA, or target site blockers that bind miRNA recognition elements within the target mRNA to prevent miRNA interaction. Therefore, miRNA targeted therapies offer an alternative strategy to drive endogenous A1AT production and thus supplement the antiprotease shield of the CF lung.

Precise Targeting of miRNA Sites Restores CFTR Activity in CF Bronchial Epithelial Cells.

MicroRNAs that are overexpressed in cystic fibrosis (CF) bronchial epithelial cells (BEC) negatively regulate CFTR and nullify the beneficial effects of CFTR modulators. We hypothesized that it is possible to reverse microRNA-mediated inhibition of CFTR using CFTR-specific target site blockers (TSBs) and to develop a drug-device combination inhalation therapy for CF. Lead microRNA expression was quantified in a series of human CF and non-CF samples and in vitro models. A panel of CFTR 3' untranslated region (UTR)-specific locked nucleic acid antisense oligonucleotide TSBs was assessed for their ability to increase CFTR expression. Their effects on CFTR activity alone or in combination with CFTR modulators were measured in CF BEC models. TSB encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles was assessed as a proof of principle of delivery into CF BECs. TSBs targeting the CFTR 3' UTR 298-305:miR-145-5p or 166-173:miR-223-3p sites increased CFTR expression and anion channel activity and enhanced the effects of ivacaftor/lumacaftor or ivacaftor/tezacaftor in CF BECs. Biocompatible PLGA-TSB nanoparticles promoted CFTR expression in primary BECs and retained desirable biophysical characteristics following nebulization. Alone or in combination with CFTR modulators, aerosolized CFTR-targeting TSBs encapsulated in PLGA nanoparticles could represent a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung.

The Estrogen-Induced miR-19 Downregulates Secretory Leucoprotease Inhibitor Expression in Monocytes.

Compared to females, males are more susceptible to acute viral and other respiratory tract infections that display greater severity and higher mortality. In contrast, females tend to fare worse with chronic inflammatory diseases. Circulating 17ß-estradiol (E2) is a female-specific factor that may influence the progression of human lung diseases. Here we hypothesize that E2 modulates the inflammatory response of monocytes through microRNA (miRNA)-based modulation of secretory leucoprotease inhibitor (SLPI), an antiprotease with immunomodulatory effects. Monocytic cells were treated ± E2, and differentially expressed miRNAs were identified using PCR profiling. Cells were transfected with miRNA mimics or antimiRs and SLPI mRNA and protein levels were quantified. Luciferase activity assay using wildtype and ΔmiR-19a/b-SLPI3'UTR reporter constructs and chromatin immunoprecipitation on E2-treated monocytes were performed. E2 downregulated SLPI and upregulated miR-19 expression in monocytes. Transfection with premiR-19b reduced SLPI mRNA and protein levels and this effect was abrogated using antimiRs against miR-19b. miR-19b directly binds the SLPI 3'UTR. The mechanism responsible for E2-mediated upregulation of miR-19 occurs via increased MIR17HG promoter activity mediated by c-MYC. Overall E2 decreases SLPI expression in human monocytic cells, via changes in miRNA expression and highlights the potential for estrogen to modulate the innate immune system.