GM-CSF overexpression after influenza a virus infection prevents mortality and moderates M1-like airway monocyte/macrophage polarization

Abstract

Background: 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.

Methods: 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.

Results: 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).

Conclusions: 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.

Keywords: Alveolar; Exudative; GM-CSF; Influenza; Interferon; Macrophage; Pneumonia; RNA-seq.

Fig. 1

Therapeutic model of GM-CSF during IAV infection using an inducible airway GM-CSF over-expression transgenic mouse model, and effects on survival and body mass during IAV infection. To simulate a therapeutic model of GM-CSF administration doxycycline was administered to both DTGM and LM control mice starting 3 days after i.n. infection with PR8 IAV. Doxycycline-containing water was protected from light and changed every three days (a). DTGM (n = 23, red circles/lines) and LM control (n = 15, black squares/lines) mice were administered approximately 2 LD₅₀ of IAV PR8 virus i.n. and administered doxycycline in water starting on +3 dpi, and the effects on survival and body weight are shown. Mice were euthanized if they lost >30% body weight and were moribund. GM-CSF over-expression (DTGM mice) conferred a significant survival benefit (b) but not a significant effect on weight loss/recovery (c) as compared to wild-type levels (LM mice). Results shown represent three independent experiments (**p < 0.005)

Fig. 2

Effects of supra-physiologic levels of GM-CSF on lung mechanical properties during IAV infection. Pressure-volume (PV) curves showing the mean±SEM of lung volume (mL) of each group, LM (black) and DTGM (red) at each indicated preprogramed pressure (cmH₂O) at 7 dpi (a) and at 10 dpi (b)(n = 5-9 mice per group per time point). The PV curves of uninfected mice (gray) are shown on each graph for comparison. Shown are the lung mechanical parameters of static compliance (Cst, mL*cmH₂O⁻¹)(c), total respiratory system resistance (Rrs, cmH₂O*s⁻¹*mL⁻¹)(d), tissue damping or peripheral airway resistance (G, cmH₂O*s⁻¹*mL⁻¹)(e),Newtonian or central airway resistance (Rn, cmH₂O*s⁻¹*mL⁻¹)(f), and the curvature of the deflation limb of the PV curve (K, cmH₂O⁻¹)(g). Results shown represent three independent experiments, n = 4-10 mice per group per time point (*p < 0.05)

Fig. 3

Effects of supra-physiologic levels of GM-CSF on bronchoalveolar lavage fluid content and influenza A virus load. BAL fluid was recovered from mice at indicated time points after IAV infection and total protein (a) was measured by BCA assay, while BAL concentrations of alpha-2-macroglobulin (b) and amphiregulin (c) levels were quantitated by ELISA. The number of influenza A virus matrix protein (M1) transcripts was quantitated from whole lung by RT-PCR (d). Results shown represent three independent experiments (*p < 0.05, ***p < 0.0005)

Fig. 4

Flow cytometric discrimination of alveolar and exudative macrophages by surface marker expression. Representative FACS plots from an IAV-infected LM mouse at 10 dpi, which detail our 12-color flow cytometry gating strategy of single cell suspensions from BAL and enzyme-digested lung (a). Alveolar macrophages (AM) were designated as F4/80⁺ SiglecF⁺ CD11b^neg/dim, whereas exudative macrophages (EM) were designated as F4/80⁺ SiglecF^neg/dim CD11b+. Supra-physiologic GM-CSF levels during IAV infection had no effect on the absolute number of either airway (BAL-recovered) AM or EM cell numbers at 10 dpi (b)

Fig. 5

Characterization of the changes in transcriptome patterns of airway macrophages during IAV infection. BAL airway macrophages were sorted using the gating strategy described in Fig. 4a and next generation RNA-sequencing was used to profile the complete transcriptome data of AMs (a, orange bars) and EMs (b, blue bars) at 8 dpi, the time point at which the survival curves diverge (n = 5 mice per group). The effect of supraphysiologic GM-CSF levels on each of the 43,628 sequenced macrophage genes was examined: differential gene expression was determined using with transcripts having a q-value <0.2 being included. The relative expression of each transcript was calculated using the equation, Log2 Expression Ratio (DTGM:LM) = Log₂ $(\overline{X}$ transcript^DTGM) - Log₂ ($\overline{X}$ transcript^LM), and the differential expression of transcripts is shown. To investigate the impact of GM-CSF on M1/M2 macrophage polarization, the Log2 Expression Ratios were plotted against known M1 and M2 macrophage-associated transcripts from AMs (c, d) and EMs (e, f)

Fig. 6

Effect of GM-CSF overexpression on airway levels of CCL17, CXCL9 and MMP12. Mouse CCL17 (a), CXCL9^# (b), and MMP12^# (c) were measured by ELISA in BAL fluid from doxycycline-treated LM (black) and DTGM (red) uninfected and IAV-infected (10 dpi) mice. Furthermore, the ratio of CXCL9:CCL17^# in each BAL sample was determined to examine the relative effect of supraphysiologic GM-CSF levels on macrophage chemokine polarization (d). Results from three independent experiments. (^#Please note the log₁₀ scale, *p < 0.05, **p < 0.005)