The COVID-19 pandemic has led to more than 760,000 deaths worldwide (correct as of 16th August 2020). Studies suggest a hyperinflammatory response is a major cause of disease severity and death. ...Identitfying COVID-19 patients with hyperinflammation may identify subgroups who could benefit from targeted immunomodulatory treatments. Analysis of cytokine levels at the point of diagnosis of SARS-CoV-2 infection can identify patients at risk of deterioration.
We used a multiplex cytokine assay to measure serum IL-6, IL-8, TNF, IL-1β, GM-CSF, IL-10, IL-33 and IFN-γ in 100 hospitalised patients with confirmed COVID-19 at admission to University Hospital Southampton (UK). Demographic, clinical and outcome data were collected for analysis.
Age > 70 years was the strongest predictor of death (OR 28, 95% CI 5.94, 139.45). IL-6, IL-8, TNF, IL-1β and IL-33 were significantly associated with adverse outcome. Clinical parameters were predictive of poor outcome (AUROC 0.71), addition of a combined cytokine panel significantly improved the predictability (AUROC 0.85). In those ≤70 years, IL-33 and TNF were predictive of poor outcome (AUROC 0.83 and 0.84), addition of a combined cytokine panel demonstrated greater predictability of poor outcome than clinical parameters alone (AUROC 0.92 vs 0.77).
A combined cytokine panel improves the accuracy of the predictive value for adverse outcome beyond standard clinical data alone. Identification of specific cytokines may help to stratify patients towards trials of specific immunomodulatory treatments to improve outcomes in COVID-19.
Abstract Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and hospitalisation of infants in developed countries. Surfactant protein A (SP-A) is an important innate immune ...molecule, localized in pulmonary surfactant. SP-A binds to carbohydrates on the surface of pathogens in a calcium-dependent manner to enable neutralisation, agglutination and clearance of pathogens including RSV. SP-A forms trimeric units and further oligomerises through interactions between its N-terminal domains. Whilst a recombinant trimeric fragment of the closely related molecule (surfactant protein D) has been shown to retain many of the native protein’s functions, the importance of the SP-A oligomeric structure in its interaction with RSV has not been determined. The aim of this study was to produce a functional trimeric recombinant fragment of human (rfh)SP-A, which lacks the N-terminal domain (and the capacity to oligomerise) and test its ability to neutralise RSV in an in vitro model of human bronchial epithelial infection. We used a novel expression tag derived from spider silk proteins (‘NT’) to produce rfhSP-A in Escherichia coli , which we found to be trimeric and to bind to mannan in a calcium-dependent manner. Trimeric rfhSP-A reduced infection levels of human bronchial epithelial (AALEB) cells by RSV by up to a mean (±SD) of 96.4 (±1.9) % at 5 μg/ml, which was significantly more effective than dimeric rfhSP-A (34.3 (±20.5) %) (p < 0.0001). Comparatively, native human SP-A reduced RSV infection by up to 38.5 (±28.4) %. For the first time we report the development of a functional trimeric rfhSP-A molecule which is highly efficacious in neutralising RSV, despite lacking the N-terminal domain and capacity to oligomerise.
Based on evidence suggesting similarities to human embryonic stem cells, human embryonic germ (hEG) cells have been advocated as an alternative pluripotent stem cell resource but have so far received ...limited attention. To redress this imbalance, human fetal gonads were collected for the isolation and culture of primordial germ cells at 7-9 weeks postconception. We provide evidence for the derivation, culture, and differentiation of hEG cells in vitro. This evidence includes the expression of markers characteristic of pluripotent cells, the retention of normal XX or XY karyotypes, and the demonstration of pluripotency, as suggested by the expression of markers indicative of differentiation along the three germ lineages (ectoderm, mesoderm, and endoderm) and an associated loss of pluripotent markers. In assessing this differentiation, however, we also demonstrate a hitherto unacknowledged overlap in gene expression profiles between undifferentiated and differentiated cell types, highlighting the difficulty in ascribing cell lineage by gene expression analyses. Furthermore, we draw attention to the problems inherent in the management of these cells in prolonged culture, chiefly the difficulty in preventing spontaneous differentiation, which hinders the isolation of pure, undifferentiated clonal lines. While these data advocate the pursuit of pluripotent hEG cell studies with relevance to early human embryonic development, culture limitations carry implications for their potential applicability to ambitious cell replacement therapies.
Lung fibroblasts are key structural cells that reside in the submucosa where they are in contact with large numbers of CD4
Th 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 hypothesized that human lung fibroblasts play an important role in modulating Th cell responses to NTHi. We demonstrate that fibroblasts express the critical CD4
T cell Ag-presentation molecule HLA-DR within the human lung, and that this expression can be recapitulated in vitro in response to IFN-γ. Furthermore, we observed that cultured lung fibroblasts could internalize live NTHi. Although unable to express CD80 and CD86 in response to stimulation, fibroblasts expressed the costimulatory 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-γ and IL-17A cytokine production by autologous, NTHi-specific lung CD4
T cells, and cytokine production was inhibited by a HLA-DR blocking Ab. 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.
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 IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production.
Chronic obstructive pulmonary disease (COPD) patients are at increased risk of poor outcome from Coronavirus disease (COVID-19). Early data suggest elevated Severe Acute Respiratory Syndrome ...Coronavirus 2 (SARS-CoV-2) receptor angiotensin converting enzyme 2 (ACE2) expression, but relationships to disease phenotype and downstream regulators of inflammation in the Renin-Angiotensin system (RAS) are unknown. We aimed to determine the relationship between RAS gene expression relevant to SARS-CoV-2 infection in the lung with disease characteristics in COPD, and the regulation of newly identified SARS-CoV-2 receptors and spike-cleaving proteases, important for SARS-CoV-2 infection.
We quantified gene expression using RNA sequencing of epithelial brushings and bronchial biopsies from 31 COPD and 37 control subjects.
ACE2 gene expression (log2-fold change (FC)) was increased in COPD compared to ex-smoking (HV-ES) controls in epithelial brushings (0.25, p = 0.042) and bronchial biopsies (0.23, p = 0.050), and correlated with worse lung function (r = - 0.28, p = 0.0090). ACE2 was further increased in frequent exacerbators compared to infrequent exacerbators (0.51, p = 0.00045) and associated with use of ACE inhibitors (ACEi) (0.50, p = 0.0034), having cardiovascular disease (0.23, p = 0.048) or hypertension (0.34, p = 0.0089), and inhaled corticosteroid use in COPD subjects in bronchial biopsies (0.33, p = 0.049). Angiotensin II receptor type (AGTR)1 and 2 expression was decreased in COPD bronchial biopsies compared to HV-ES controls with log2FC of -0.26 (p = 0.033) and - 0.40, (p = 0.0010), respectively. However, the AGTR1:2 ratio was increased in COPD subjects compared with HV-ES controls, log2FC of 0.57 (p = 0.0051). Basigin, a newly identified potential SARS-CoV-2 receptor was also upregulated in both brushes, log2FC of 0.17 (p = 0.0040), and bronchial biopsies, (log2FC of 0.18 (p = 0.017), in COPD vs HV-ES. Transmembrane protease, serine (TMPRSS)2 was not differentially regulated between control and COPD. However, various other spike-cleaving proteases were, including TMPRSS4 and Cathepsin B, in both epithelial brushes (log2FC of 0.25 (p = 0.0012) and log2FC of 0.56 (p = 5.49E-06), respectively) and bronchial biopsies (log2FC of 0.49 (p = 0.00021) and log2FC of 0.246 (p = 0.028), respectively).
This study identifies key differences in expression of genes related to susceptibility and aetiology of COVID-19 within the COPD lung. Further studies to understand the impact on clinical course of disease are now required.
Patients with chronic obstructive pulmonary disease (COPD) are susceptible to respiratory viral infections that cause exacerbations. The mechanisms underlying this susceptibility are not understood. ...Effectors of the adaptive immune response-CD8(+) T cells that clear viral infections-are present in increased numbers in the lungs of patients with COPD, but they fail to protect against infection and may contribute to the immunopathology of the disease.
CD8(+) function and signaling through the programmed cell death protein (PD)-1 exhaustion pathway were investigated as a potential key mechanism of viral exacerbation of the COPD lung.
Tissue from control subjects and patients with COPD undergoing lung resection was infected with live influenza virus ex vivo. Viral infection and expression of lung cell markers were analyzed using flow cytometry.
The proportion of lung CD8(+) T cells expressing PD-1 was greater in COPD (mean, 16.2%) than in 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 up-regulated T-cell PD-1 expression in control and COPD samples. Concurrently, influenza significantly up-regulated the marker of cytotoxic degranulation (CD107a) on CD8(+) T cells (P = 0.03) from control subjects but not on those from patients with COPD. Virus-induced expression of the ligand PD-L1 was decreased on COPD macrophages (P = 0.04) with a corresponding increase in IFN-γ release from infected COPD explants compared with controls (P = 0.04).
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.
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.
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.
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.
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