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Background 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. Methods 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. Results 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). Conclusions 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.

The development of asthma is linked to early life environmental exposures and the occurrence of severe viral infections. Rapid maturation of adaptive immunity from a tolerant (Th2) to an anti-infective (Th1) state occurs in the neonatal period. We hypothesised that the airway inflammatory milieu, driven by the maturing immune response to environmental exposures may have important effects on the development of anti-viral innate immunity at the level of the epithelium. We studied whether the inflammatory environment of the airway epithelium modulates gene expression via epigenetic regulation of anti-viral genes as a model of the development of a long term abnormality which is a hallmark of asthma.We optimised an in-vitro model using AALEB, human immortalised bronchial epithelial-derived cells which were pre-treated for 24 h with cytokines that mimic Th1 environment (IFNy, 10 ng/ml) and Th2 (IL-13, 10 ng/ml) before being infected with RSV A, MOI=2 for 48 h. Quantitative real-time PCR with Taqman primers was used to assess expression of innate genes. Cells were collected after 48 h and stored in Trizol. Chromatin Immuno Precipitation (ChIP) with antibodies against histone modifications was used to assess epigenetic controls. In order to confirm epigenetic regulation of innate genes we used a panel of HAT, HDAC and histone demethylase inhibitors.We initially studied the impact of cytokines on a range of innate anti-viral genes. RIG1 was differentially expressed and reductions in expression associated with higher viral titres. IFNy priming induced increases in RIG1 mRNA at 48 h that correlated at the promoter with enrichment of H3K9ac and RNApolII (active-promoters) and reduction of H3K9me3 (repressive-promoters). We observed a statistically significant increase of RIG1 expression by IFN gamma when co-incubated with SAHA (HDAC I and II inhibitors) and JIB-04 (Pan-Jumanji histone demethylase inhibitor). No effects of Th2 priming were seen at the level of antiviral responses.This in-vitro study suggests the inflammatory environment of naive epithelial cells can induce epigenetic modulation of innate immune responses at the level of histone methylation and acetylation and hence potentially lead to long term impacts on anti-viral immunity. The presence of a Th1 milieu appears key to the development of effective anti-viral responses.[Figure]

Introduction and ObjectivesPatient s with chronic obstructive pulmonary disease (COPD) are susceptible to the effects of recurrent respiratory infections despite increased numbers of CD8+ T cells in the lungs. We hypothesised that the inability of CD8+ T cells to successfully combat respiratory pathogens in COPD may be due to T cell \"exhaustion\" - a phenomenon described in chronic infections. Exhausted CD8+ T cells have significantly reduced cytotoxicity and inflammatory cytokine release. Exhaustion is thought to be initiated by the binding of PD-1 on T cells to its ligand (PD-L1) which is expressed on epithelial cells and macrophages. PD-1 expression is upregulated in murine models of acute and chronic viral infection, but this has yet to be elucidated in human cells.We aimed to identify and quantify PD-1+ CD4+ and CD8+ T cells and cells expressing PD-L1 in the lungs of COPD patients and non-COPD controls.MethodsLung tissue from patients undergoing surgery was digested using collagenase to form single-cell suspensions. Lung T cells were identified as populations of CD45+CD3+ cells which were either CD4+CD8- or CD4-CD8+. T cells expressing PD-1 were quantified by multi-colour flow cytometry. Patients with a FEV1/FVC ratio <70% were defined as having COPD.ResultsThe proportion of CD8+ T cells in the COPD lung (mean expression=40.87%, SD=14.67) was significantly higher (p=0.013, students t-test) than in non-COPD (mean expression=26.74%, SD=11.12), reflecting previous findings. PD-1 expression in CD4+ T cells appeared to be lower in COPD (mean expression=39.91%, SD=13.02) than non-COPD (mean expression=50.53%, sd=13.05) but this was not significant. PD-1 expressing CD4+ cells (mean expression=2.17%, SD=1.4) and CD8+ cells (mean expression=6.02%, sd=5.73) were detected in tissue, but not in the blood of the same patients. PD-L1 was undetectable on lung epithelial cells but was expressed on macrophages (mean expression = 2.85%, SD=1.91).ConclusionElements of the exhaustion pathway are expressed in the human lung in stable COPD. Further work is needed to clarify if there is an upregulation of this pathway in COPD that may explain the susceptibility of these patients to viral exacerbation. Exhaustion of cells recognising respiratory pathogens may have a significant role in COPD outcomes and requires further elucidation.

Background & ObjectiveInfluenza infection has recently been shown to cause rapid functional impairment of CD8+ T cell responses in a murine infection model via the PD1/PDL1 pathway.1 In this mouse model, it was the induction of PDL1 that was required for this impairment of CD8+ function. A previous study suggested that the anti-inflammatory cytokine, IL-10, was the principal driver of human macrophage PDL1 expression in response to HIV infection.2 The aim of this study was to investigate how human lung macrophages regulate their PDL1 expression in response to influenza infection.MethodsAlveolar macrophages washed from resected human lung tissue and purified by plate adherence or human positively-isolated CD14+ monocyte-derived macrophages (MDMs) were cultured with H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h, after which the cultures were washed and media replaced and incubation continued for a further 22 h. Virally infected cells and expression of cell surface markers were identified using flow cytometry. Gene expression was measured using RT-PCR.ResultsNo increase in MDM infection was seen using the UVX31 but incubation with X31 resulted in an average infection rate of 9.1%. Infection with X31 significantly increased cell surface expression of HLA-DR and PDL1 (p<0.05), but not of PDL2 by MDMs as measured by flow cytometry. Using RT-PCR, we observed an increase of PDL1 mRNA after X31 infection suggesting that the expression of this protein is transcriptionally regulated. In addition, we saw an increase in type I interferon expression by MDMs in response to X31 infection, but no expression of IFN gamma . In contrast we observed a trend towards decreased expression of IL-10 mRNA. In further experiments, infection of alveolar macrophages with X31 also caused significant increases in HLA-DR and PDL1 cell surface expression.ConclusionsThese data indicate that, in contrast to HIV infection of macrophages2 influenza-induced expression of PDL1 may not be regulated by IL-10 in human macrophages.Erickson et al (2012) J Clin Invest doi: 10.1172/JCI62860.Rodriguez-Garcia, et al. (2011) J Leukoc Biol 89(4):507-15.

Background & Objective Influenza infection has recently been shown to cause rapid functional impairment of CD8+ T cell responses in a murine infection model via the PD1/PDL1 pathway.1 In this mouse model, it was the induction of PDL1 that was required for this impairment of CD8+ function. A previous study suggested that the anti-inflammatory cytokine, IL-10, was the principal driver of human macrophage PDL1 expression in response to HIV infection.2 The aim of this study was to investigate how human lung macrophages regulate their PDL1 expression in response to influenza infection. Methods Alveolar macrophages washed from resected human lung tissue and purified by plate adherence or human positively-isolated CD14+ monocyte-derived macrophages (MDMs) were cultured with H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h, after which the cultures were washed and media replaced and incubation continued for a further 22 h. Virally infected cells and expression of cell surface markers were identified using flow cytometry. Gene expression was measured using RT-PCR. Results No increase in MDM infection was seen using the UVX31 but incubation with X31 resulted in an average infection rate of 9.1%. Infection with X31 significantly increased cell surface expression of HLA-DR and PDL1 (p<0.05), but not of PDL2 by MDMs as measured by flow cytometry. Using RT-PCR, we observed an increase of PDL1 mRNA after X31 infection suggesting that the expression of this protein is transcriptionally regulated. In addition, we saw an increase in type I interferon expression by MDMs in response to X31 infection, but no expression of IFNγ. In contrast we observed a trend towards decreased expression of IL-10 mRNA. In further experiments, infection of alveolar macrophages with X31 also caused significant increases in HLA-DR and PDL1 cell surface expression. Conclusions These data indicate that, in contrast to HIV infection of macrophages2 influenza-induced expression of PDL1 may not be regulated by IL-10 in human macrophages. Erickson et al (2012) J Clin Invest doi: 10.1172/JCI62860. Rodriguez-Garcia, et al. (2011) J Leukoc Biol 89(4):507–15.

Introduction and Objectives Patient s with chronic obstructive pulmonary disease (COPD) are susceptible to the effects of recurrent respiratory infections despite increased numbers of CD8+ T cells in the lungs. We hypothesised that the inability of CD8+ T cells to successfully combat respiratory pathogens in COPD may be due to T cell “exhaustion” - a phenomenon described in chronic infections. Exhausted CD8+ T cells have significantly reduced cytotoxicity and inflammatory cytokine release. Exhaustion is thought to be initiated by the binding of PD-1 on T cells to its ligand (PD-L1) which is expressed on epithelial cells and macrophages. PD-1 expression is upregulated in murine models of acute and chronic viral infection, but this has yet to be elucidated in human cells. We aimed to identify and quantify PD-1+ CD4+ and CD8+ T cells and cells expressing PD-L1 in the lungs of COPD patients and non-COPD controls. Methods Lung tissue from patients undergoing surgery was digested using collagenase to form single-cell suspensions. Lung T cells were identified as populations of CD45+CD3+ cells which were either CD4+CD8- or CD4-CD8+. T cells expressing PD-1 were quantified by multi-colour flow cytometry. Patients with a FEV1/FVC ratio <70% were defined as having COPD. Results The proportion of CD8+ T cells in the COPD lung (mean expression=40.87%, SD=14.67) was significantly higher (p=0.013, students t-test) than in non-COPD (mean expression=26.74%, SD=11.12), reflecting previous findings. PD-1 expression in CD4+ T cells appeared to be lower in COPD (mean expression=39.91%, SD=13.02) than non-COPD (mean expression=50.53%, sd=13.05) but this was not significant. PD-1 expressing CD4+ cells (mean expression=2.17%, SD=1.4) and CD8+ cells (mean expression=6.02%, sd=5.73) were detected in tissue, but not in the blood of the same patients. PD-L1 was undetectable on lung epithelial cells but was expressed on macrophages (mean expression = 2.85%, SD=1.91). Conclusion Elements of the exhaustion pathway are expressed in the human lung in stable COPD. Further work is needed to clarify if there is an upregulation of this pathway in COPD that may explain the susceptibility of these patients to viral exacerbation. Exhaustion of cells recognising respiratory pathogens may have a significant role in COPD outcomes and requires further elucidation.

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.

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.

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.