Abstract
BackgroundInfluenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Application of high GM-CSF levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established.MethodsMice were infected intranasally with influenza A virus (PR8 strain). Supra-physiologic levels of GM-CSF were induced in the airways using the double transgenic GM-CSF (DTGM) or littermate control mice starting on 3 days post-infection (dpi). Assessment of respiratory mechanical parameters was performed using the flexiVent rodent ventilator. RNA sequence analysis was performed on FACS-sorted airway macrophage subsets at 8 dpi.ResultsSupra-physiologic levels of GM-CSF conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (BAL) fluid to near baseline levels. Transcriptome analysis, and subsequent validation ELISA assays, revealed that excess GM-CSF re-directs macrophages from an “M1-like” to a more “M2-like” activation state as revealed by alterations in the ratios of CXCL9 and CCL17 in BAL fluid, respectively. Ingenuity pathway analysis predicted that GM-CSF surplus during IAV infection elicits expression of anti-inflammatory mediators and moderates M1 macrophage pro-inflammatory signaling by Type II interferon (IFN-γ).ConclusionsOur data indicate that application of high levels of GM-CSF in the lung after influenza A virus infection alters pathogenic “M1-like” macrophage inflammation. These results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury.
Highlights
Influenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract
Doxycycline-inducible airway GM-CSF over-expression confers protection against severe influenza A virus (IAV) infection To characterize the pathogenicity of our H1N1 Puerto Rico/8/34 (PR8) IAV preparation virus, wild-type C57BL/6 J mice (The Jackson Laboratory, MA) were purchased and we determined the lethal dose 50% (LD50) of our PR8 IAV preparation
In the absence of infection, BAL fluid levels of GM-CSF in double transgenic GM-CSF (DTGM) mice are near the limit of detection, similar to littermate controls (Additional file 4: Figure S2a), and their alveolar macrophages appear identical by multiparameter flow cytometry
Summary
Influenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Halstead et al Respiratory Research (2018) 19:3 response leading to collateral injury [2] This combination can lead to an ARDS-like syndrome characterized by increased capillary leak, oxygen diffusion difficulty and ventilation/perfusion mismatch [1]. The effect of local elevation of GM-CSF on IAV infection in the lung has been investigated in transgenic models with expression of GM-CSF under the control of constitutive or doxycycline-inducible promoters in lungs of alveolar or small airway epithelial cells of GM-CSF knockout (csf2−/−) mice [3, 4]. Differential effects on morbidity and mortality from IAV infection in these studies was associated with increased alveolar macrophage (AM) numbers in the constitutive GM-CSF expression models [3, 5] and AM differentiation in the GM-CSF-inducible model [4]. Differential results on morbidity and survival were obtained after prolonged or brief administration of supra-physiological levels of GM-CSF before or at the onset of IAV infection [6]
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