Multiomics links global surfactant dysregulation with airflow obstruction and emphysema in COPD

RATIONALE: Pulmonary surfactant is vital for lung homeostasis as it reduces surface tension to prevent alveolar collapse and provides essential immune-regulatory and antipathogenic functions. Previous studies demonstrated dysregulation of some individual surfactant components in COPD. We investigated relationships between COPD disease measures and dysregulation of surfactant components to gain new insights into potential disease mechanisms. METHODS: Bronchoalveolar lavage proteome and lipidome were characterised in ex-smoking mild/moderate COPD subjects (n=26) and healthy ex-smoking (n=20) and never-smoking (n=16) controls using mass spectrometry. Serum surfactant protein analysis was performed. RESULTS: Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, surfactant protein (SP)-B, SP-A and SP-D concentrations were lower in COPD versus controls (log2 fold change (log2FC) -2.0, -2.2, -1.5, -0.5, -0.7 and -0.5 (adjusted p<0.02), respectively) and correlated with lung function. Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D, napsin A and CD44 inversely correlated with computed tomography small airways disease measures (expiratory to inspiratory mean lung density) (r= -0.56, r= -0.58, r= -0.45, r= -0.36, r= -0.44, r= -0.37, r= -0.40 and r= -0.39 (adjusted p<0.05)). Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D and NAPSA inversely correlated with emphysema (% low-attenuation areas): r= -0.55, r= -0.61, r= -0.48, r= -0.51, r= -0.41, r= -0.31 and r= -0.34, respectively (adjusted p<0.05). Neutrophil elastase, known to degrade SP-A and SP-D, was elevated in COPD versus controls (log2FC 0.40, adjusted p=0.0390), and inversely correlated with SP-A and SP-D. Serum SP-D was increased in COPD versus healthy ex-smoking volunteers, and predicted COPD status (area under the curve 0.85). CONCLUSIONS: Using a multiomics approach, we demonstrate, for the first time, global surfactant dysregulation in COPD that was associated with emphysema, giving new insights into potential mechanisms underlying the cause or consequence of disease

Influence of hypoxia on the epithelial-pathogen interactions in the lung: implications for respiratory disease

Under normal physiological conditions, the lung remains an oxygen rich environment. However, prominent regions of hypoxia are a common feature of infected and inflamed tissues and many chronic inflammatory respiratory diseases are associated with mucosal and systemic hypoxia. The airway epithelium represents a key interface with the external environment and is the first line of defense against potentially harmful agents including respiratory pathogens. The protective arsenal of the airway epithelium is provided in the form of physical barriers, and the production of an array of antimicrobial host defense molecules, proinflammatory cytokines and chemokines, in response to activation by receptors. Dysregulation of the airway epithelial innate immune response is associated with a compromised immunity and chronic inflammation of the lung. An increasing body of evidence indicates a distinct role for hypoxia in the dysfunction of the airway epithelium and in the responses of both innate immunity and of respiratory pathogens. Here we review the current evidence around the role of tissue hypoxia in modulating the host-pathogen interaction at the airway epithelium. Furthermore, we highlight the work needed to delineate the role of tissue hypoxia in the pathophysiology of chronic inflammatory lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease in addition to novel respiratory diseases such as COVID-19. Elucidating the molecular mechanisms underlying the epithelial-pathogen interactions in the setting of hypoxia will enable better understanding of persistent infections and complex disease processes in chronic inflammatory lung diseases and may aid the identification of novel therapeutic targets and strategies

The role of extracellular vesicles as a shared disease mechanism contributing to multimorbidity in patients with COPD

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide. Individuals with COPD typically experience a progressive, debilitating decline in lung function as well as systemic manifestations of the disease. Multimorbidity, is common in COPD patients and increases the risk of hospitalisation and mortality. Central to the genesis of multimorbidity in COPD patients is a self-perpetuating, abnormal immune and inflammatory response driven by factors including ageing, pollutant inhalation (including smoking) and infection. As many patients with COPD have multiple concurrent chronic conditions, which require an integrative management approach, there is a need to greater understand the shared disease mechanisms contributing to multimorbidity. The intercellular transfer of extracellular vesicles (EVs) has recently been proposed as an important method of local and distal cell-to-cell communication mediating both homeostatic and pathological conditions. EVs have been identified in many biological fluids and provide a stable capsule for the transfer of cargo including proteins, lipids and nucleic acids. Of these cargo, microRNAs (miRNAs), which are short 17-24 nucleotide non-coding RNA molecules, have been amongst the most extensively studied. There is evidence to support that miRNA are selectively packaged into EVs and can regulate recipient cell gene expression including major pathways involved in inflammation, apoptosis and fibrosis. Furthermore changes in EV cargo including miRNA have been reported in many chronic diseases and in response to risk factors including respiratory infections, noxious stimuli and ageing. In this review, we discuss the potential of EVs and EV-associated miRNA to modulate shared pathological processes in chronic diseases. Further delineating these may lead to the identification of novel biomarkers and therapeutic targets for patients with COPD and multimorbidities.</p

Dysregulation of anti-viral function of CD8+T cells in the COPD lung: role of the PD1/PDL1 axis

Rationale: COPD patients are susceptible to respiratory viral infections which cause exacerbations. Mechanisms underlying susceptibility are not understood. Effectors of the adaptive immune response; CD8+ T cells which clear viral infections, are present in increased numbers in lungs of COPD patients but fail to protect against infection and may contribute to the immunopathology of the disease. Objectives: CD8+ function and signalling through the Programmed Cell Death (PD-1) exhaustion pathway was investigated as a potential key mechanism of viral exacerbation of the COPD lung. Methods: Tissue from control or COPD patients undergoing lung resection was infected with live influenza virus ex vivo. Viral infection and expression of lung cell markers was analysed using flow cytometry. Measurements and Main Results: The proportion of lung CD8+ T cells expressing PD-1 was greater in COPD(mean=16.2%) than controls(4.4%, p=0.029). Only epithelial cells and macrophages were infected with influenza and there was no difference in the proportion of infected cells between controls and COPD. Infection upregulated T cell PD-1 expression in control and COPD samples. Concurrently, influenza significantly upregulated the marker of cytotoxic degranulation (CD107a) on CD8+ T cells(p=0.03) from controls, but not from COPD patients. Virus-induced expression of the ligand PD-L1 was decreased on COPD macrophages(p=0.04) with a corresponding increase in IFN? release from infected COPD explants compared to controls(p=0.04). Conclusions: This study has established a signal of cytotoxic immune dysfunction and aberrant immune regulation in the COPD lung that may explain both the susceptibility to viral infection and the excessive, inflammation associated with exacerbations

The role of non-typeable Haemophilus influenzae biofilms in Chronic Obstructive Pulmonary Disease

Non-typeable Haemophilus influenzae (NTHi) is an ubiquitous commensal-turned-pathogen that colonises the respiratory mucosa in airways diseases including Chronic Obstructive Pulmonary Disease (COPD). COPD is a progressive inflammatory syndrome of the lungs, encompassing chronic bronchitis that is characterised by mucus hypersecretion and impaired mucociliary clearance and creates a static, protective, humid, and nutrient-rich environment, with dysregulated mucosal immunity; a favourable environment for NTHi colonisation. Several recent large COPD cohort studies have reported NTHi as a significant and recurrent aetiological pathogen in acute exacerbations of COPD. NTHi proliferation has been associated with increased hospitalisation, disease severity, morbidity and significant lung microbiome shifts. However, some cohorts with patients at different severities of COPD do not report that NTHi is a significant aetiological pathogen in their COPD patients, indicating other obligate pathogens including Moraxella catarrhalis, Streptococcus pneumoniae and Pseudomonas aeruginosa as the cause. NTHi is an ubiquitous organism across healthy non-smokers, healthy smokers and COPD patients from childhood to adulthood, but it currently remains unclear why NTHi becomes pathogenic in only some cohorts of COPD patients, and what behaviours, interactions and adaptations are driving this susceptibility. There is emerging evidence that biofilm-phase NTHi may play a significant role in COPD. NTHi displays many hallmarks of the biofilm lifestyle and expresses key biofilm formation-promoting genes. These include the autoinducer-mediated quorum sensing system, epithelial- and mucus-binding adhesins and expression of a protective, self-produced polymeric substance matrix. These NTHi biofilms exhibit extreme tolerance to antimicrobial treatments and the immune system as well as expressing synergistic interspecific interactions with other lung pathogens including S. pneumoniae and M. catarrhalis. Whilst the majority of our understanding surrounding NTHi as a biofilm arises from otitis media or in-vitro bacterial monoculture models, the role of NTHi biofilms in the COPD lung is now being studied. This review explores the evidence for the existence of NTHi biofilms and their impact in the COPD lung. Understanding the nature of chronic and recurrent NTHi infections in acute exacerbations of COPD could have important implications for clinical treatment and identification of novel bactericidal targets.</p

Viral infection of human lung macrophages increases PDL1 expression via IFNβ

Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM) and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFN? release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFN? mRNA and protein release by MDM in response to influenza infection. Knockdown of IFN? by siRNA, resulted in a 37.5% reduction in IFN? gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFN?, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFN? release in response to virus and that this expression is regulated by autologous IFN? production

Macrophage inflammatory responses to Non-typeable Haemophilus influenzae (NTHi) are strain-dependent

The airway macrophage is crucial for immune surveillance and pathogen clearance, however, this function is impaired in chronic respiratory diseases such as COPD and asthma. This may lead to colonization of the airways by Nontypeable Haemophilus influenzae (NTHi). NTHi is a heterogeneous organism and this heterogeneity could result in strain-dependent differences in macrophage responses, leading to airway persistence of certain NTHi strains. The aim of this work was to investigate macrophage responses to different clinical strains of NTHi. Strain diversity assessment of eight NTHi clinical isolates was performed using ParSNP. Subsequently, three strains (ST14, 201 and 408) were chosen to infect monocyte-derived macrophages (MDM) isolated from the blood of healthy volunteers to investigate expression of inflammatory pathways by PCR or ELISA. Measurement of NTHi hel gene expression revealed increased presence of ST14 and ST408 compared to ST201 (p&lt;0.05). MDM expression of IFN-β and RIG-I was significantly higher for ST14 compared to ST408 (p&lt;0.05), but not ST201. In contrast, expression of CXCL10, RIG-I and NFκB was significantly higher for ST14 compared to ST201 (p&lt;0.05) but not ST408. MDM expression of TLR4 and TLR7 did not differ between strains. There were also no significant differences in cytokine production between ST408 and ST14, however ST14 induced higher release of IL-1β, IL-6 and IL-8 from MDM compared to ST201 (p&lt;0.05). This work suggests ST14 elicits a differential MDM inflammatory response compared to ST201 and ST408. These differences highlight that NTHi heterogeneity must be further explored in order to comprehend the role of NTHi in chronic respiratory disease

IFN-γ influences epithelial anti-viral responses via histone methylation of the RIG-I promoter

The asthmatic lung is prone to respiratory viral infections that exacerbate the symptoms of the underlying disease. Recent work has suggested that a deficient Th1 response in early life may lead to these aberrant anti-viral responses. We investigated whether the inflammatory environment of the airway epithelium could modulate anti-viral gene expression via epigenetic mechanisms, in order to study the development of a long-term dysregulation of innate responses, which are a hallmark of asthma.We primed AALEB, a human bronchial epithelial cell line, with IFNγ and IL13 and subsequently infected cells with Respiratory Syncytial Virus (RSV) and innate anti-viral genes expression and their epigenetic markers were analysed.Priming epithelial cells with IFNγ reduced RSV viral load. Microarray analysis identified that IFNγ-priming enhanced RIG-I mRNA expression and this expression correlated with epigenetic changes at the RIG-I promoter that influenced its transcription. Using chromatin immunoprecipitation, we observed a reduction of trimethylated Histone3 Lysine9 (H3K9me3) at the RIG-I promoter. Addition of inhibitor BIX-01294 to this model indicated an involvement of lysine methyltransferase G9a in RIG-I epigenetic regulation.These data suggest that prior exposure to IFNγ may leave an epigenetic mark upon the chromatin that enhances airway cells’ ability to better resist infection possibly via epigenetic upregulation of RIG-I. These observations provide further evidence for a crucial role of IFNγ in the development of mature anti-viral responses within a model of respiratory infection. Further clinical validation is required to determine if this effect in early life leads to changes in anti-viral responses associated with asthma

Human lung fibroblasts present bacterial antigen to autologous lung T helper cells

Lung fibroblasts are key structural cells that reside in the submucosa where they are in contact with large numbers of CD4+ T helper cells. During severe viral infection and chronic inflammation the submucosa is susceptible to bacterial invasion by lung microbiota such as Nontypeable Haemophilus influenzae (NTHi). Given their proximity in tissue, we hypothesised that human lung fibroblasts play an important role in modulating T helper cell responses to NTHi. We demonstrate that fibroblasts express the critical CD4+ T cell antigen-presentation molecule HLA-DR within the human lung, and that this expression can be recapitulated in vitro in response to interferon (IFN)?. Furthermore, we observed that cultured lung fibroblasts could internalize live NTHi. While unable to express CD80 and CD86 in response to stimulation, fibroblasts expressed the co-stimulatory molecules 4-1BBL, OX-40L and CD70, all of which are related to memory T cell activation and maintenance. CD4+ T cells isolated from the lung were predominantly (mean 97.5%) CD45RO+ memory cells. Finally, cultured fibroblasts activated IFN? and IL-17A cytokine production by autologous, NTHi-specific lung CD4+ T cells, and cytokine production was inhibited by a HLA-DR blocking antibody. These results indicate a novel role for human lung fibroblasts in contributing to responses against bacterial infection through activation of bacteria-specific CD4+ T cells