The online version of this article (https://doi.org/10.1186/s12931-017-0708-5) contains supplementary material, which is available to authorized users.
Influenza 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.
Mice 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.
Supra-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).
Our 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.
Additional file 1: Table S1. All protein concentration measurements were made as described in the manuscript text using the reagents and kits listed. (TIFF 3075 kb)12931_2017_708_MOESM1_ESM.tiff
Additional file 2: Table S2. Multi-parameter flow cytometry was utilized to characterize the alveolar and exudative macrophages as shown in Fig. 4. All monoclonal antibodies were purchased from either AbD Serotec, BD Bioscience or eBioscience, respectively. (TIFF 3075 kb)
Additional file 3: Figures S1A-D. Lethal dose 50% (LD50) determination of influenza virus strain A/Puerto Rico/8/1934 (PR8) in female (A, B) and male (C, D) mice, demonstrating an LD50 of 728 vs. 3728 fluorescent focus units (FFU) in female and males, respectively. (ZIP 87 kb)12931_2017_708_MOESM3_ESM.zip
Additional file 4: Figure S2A and B. Characterization of the Double Transgenic GM-csf (DTGM) mouse model. In the absence of influenza A virus infection, GM-CSF levels (A) in bronchoalveolar lavage (BAL) fluid was low, near the limit of detection in littermate (LM) and DTGM mice. Upon influenza A virus infection DTGM mice without doxycycline-induction (DTGM noDox) demonstrate "leakiness" that corresponds to the peak of type II interferon levels at days 7-8 post-infection. DTGM +Dox mice demonstrate supra-physiologic levels of GM-CSF in BAL fluid at all time points after induction. DTGM mice were less susceptible to IAV infection (B) even in the absence of doxycycline induction, whereas doxycycline administration to LM mice had no effect. (ZIP 55 kb)12931_2017_708_MOESM4_ESM.zip
Additional file 5: Figure S3A and B. Measurement of serum proteins in BAL fluid. Elevated levels of GM-CSF neither affected the quantity of mouse albumin (A) nor IgM (B) in BAL fluid at 10 and 14 days post-infection. (ZIP 39 kb)12931_2017_708_MOESM5_ESM.zip
Additional file 6: Figures S4A-D. Characterization of the kinetics of BAL cytokines. Type I interferon (A), type II interferon (B), type III interferon (C), and IL-10 (D), were measured in BAL fluid from wild-type (WT, gray bars) or DTGM +Dox (red bars) mice by multiplex analysis (Luminex, https://www.luminexcorp.com) at the indicated time points. (ZIP 74 kb)
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- GM-CSF overexpression after influenza a virus infection prevents mortality and moderates M1-like airway monocyte/macrophage polarization
E. Scott Halstead
Todd M. Umstead
Michael L. Davies
Yuka Imamura Kawasawa
Eranda Kurundu Hewage
Zissis C. Chroneos
- BioMed Central
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