Rationale: 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 1 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. Objectives: CD81 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.Methods: 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. Measurements and Main Results:The proportion of lung CD8 1 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 CD81 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-g release from infected COPD explants compared with 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.

Viral infections are major drivers of exacerbations and clinical burden in patients with asthma and COPD. IFN-β is a key component of the innate immune response to viral infection. To date studies of inhaled IFN-β treatment have not demonstrated a significant effect on asthma exacerbations. OBJECTIVES: The dynamics of exogenous IFN-β activity were investigated to inform on future clinical indications for this potential anti-viral therapy. METHODS: Monocyte-derived macrophages (MDMs), alveolar macrophages (AMs) and primary bronchial epithelial cells (PBECs) were isolated from healthy controls and COPD patients and infected with influenza virus either prior to or after IFN-β stimulation. Infection levels were measured by % nucleoprotein 1 positive (NP1+) cells using flow cytometry. Viral RNA shedding and interferon stimulated gene expression were measured by qPCR. Production of inflammatory cytokines was measured using MSD. MEASUREMENTS AND MAIN RESULTS: Adding IFN-β to MDMs, AMs and PBECs prior to, but not after, infection reduced %NP1 + cells by 85%, 56% and 66%, respectively (p<0.05). Inhibition of infection lasted for 24h following removal of IFN-β and was maintained albeit reduced up to 1 week in MDMs and 72h in PBECs; 2 this was similar between health and COPD. IFN-β did not induce inflammatory cytokine production by MDMs or PBECs but reduced influenza-induced IL-1β production by PBECs. CONCLUSIONS: In vitro modelling of IFN-β dynamics highlights the potential for intermittent prophylactic doses of exogenous IFN-β to modulate viral infection. This provides important insights to aid the future design of clinical trials of IFN-β in asthma and COPD.

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.