Activation of A1-adenosine receptors promotes leukocyte recruitment to the lung and attenuates acute lung injury in mice infected with influenza A/WSN/33 (H1N1) virus

Abstract

We have shown that bronchoalveolar epithelial A1-adenosine receptors (A1-AdoR) are activated in influenza A virus-infected mice. Alveolar macrophages and neutrophils also express A1-AdoRs, and we hypothesized that activation of A1-AdoRs on these cells will promote macrophage and neutrophil chemotaxis and activation and thereby play a role in the pathogenesis of influenza virus-induced acute lung injury. Wild-type (WT) C57BL/6 mice, congenic A1-AdoR knockout (A1-KO) mice, and mice that had undergone reciprocal bone marrow transfer were inoculated intranasally with 10,000 PFU/mouse influenza A/WSN/33 (H1N1) virus. Alternatively, WT mice underwent daily treatment with the A1-AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) from 1 day prior to inoculation. Infection increased bronchoalveolar lining fluid (BALF) adenosine comparably in WT and A1-KO mice. Infection of WT mice resulted in reduced carotid arterial O2 saturation (hypoxemia), lung pathology, pulmonary edema, reduced lung compliance, increased basal airway resistance, and hyperresponsiveness to methacholine. These effects were absent or significantly attenuated in A1-KO mice. Levels of BALF leukocytes, gamma interferon (IFN-γ), and interleukin 10 (IL-10) were significantly reduced in infected A1-KO mice, but levels of KC, IP-10, and MCP-1 were increased. Reciprocal bone marrow transfer resulted in WT-like lung injury severity, but BALF leukocyte levels increased only in WT and A1-KO mice with WT bone barrow. Hypoxemia, pulmonary edema, and levels of BALF alveolar macrophages, neutrophils, IFN-γ, and IL-10 were reduced in DPCPX-treated WT mice. Levels of viral replication did not differ between mouse strains or treatment groups. These findings indicate that adenosine activation of leukocyte A1-AdoRs plays a significant role in their recruitment to the infected lung and contributes to influenza pathogenesis. A1-AdoR inhibitor therapy may therefore be beneficial in patients with influenza virus-induced lung injury.

Importance: Because antiviral drugs are of limited efficacy in patients hospitalized for influenza virus-induced respiratory failure, there is an urgent need for new therapeutics that can limit the progression of lung injury and reduce influenza death rates. We show that influenza A virus infection results in increased production of the nucleoside adenosine in the mouse lung and that activation of A1-subtype adenosine receptors by adenosine contributes significantly to both recruitment of innate immune cells to the lung and development of acute lung injury following influenza virus infection. We also show that treatment with an A1-adenosine receptor antagonist reduces the severity of lung injury in influenza virus-infected mice. Our findings indicate that adenosine plays an important and previously unrecognized role in the innate immune response to influenza virus infection and suggest that drugs which can inhibit either generation of adenosine or activation of A1-adenosine receptors may be beneficial in treating influenza patients hospitalized for respiratory failure.

FIG 1

Hypoxemia, but not viral replication, was significantly attenuated in influenza A virus-infected A₁-KO mice. Effects of intranasal infection of wild-type C57BL/6 (WT) and A₁-adenosine receptor-knockout (A₁-KO) mice with 10,000 PFU/mouse of influenza A/WSN/33 (H1N1) virus on body weight (BWT [percent change from day 0]; n > 25 per group) (A), carotid arterial oxygen saturation (percent S_aO₂; n > 10 per group) (B), and lung homogenate viral titers (log PFU/g; n = 5 to 10 per group) (C). #, P < 0.001 versus value for uninfected WT mice; ‡, P < 0.005 versus value for WT mice at the same time point. Data are presented as means ± SEM.

FIG 2

Intranasal inoculation of WT or A₁-KO mice with 10,000 PFU/mouse influenza A/WSN/33 (H1N1) virus does not result in viral replication in the brain. (A) Western blot for H1N1 influenza nucleoprotein (NP) in brain and lung homogenates from 3 A₁-KO mice and 3 WT mice at 6 days p.i. Probing for GAPDH and β-actin (which appear to be differentially expressed between lung and brain) demonstrated that protein loadings were comparable between groups. (B) Relative expression of NP in lung and brain homogenates from WT and A₁-KO mice (normalized to GAPDH). Data are presented as means ± SEM.

FIG 3

Influenza A virus infection induced an adenosine response in the lung. Effect of influenza A/WSN/33 (H1N1) virus infection of WT and A₁-KO mice on bronchoalveolar lavage fluid (BALF) adenosine content (μM) (n = 5 per group). #, P < 0.001 versus the value for uninfected WT mice. Data are presented as means ± SEM.

FIG 4

Pulmonary leukocyte infiltration in response to influenza A virus infection was decreased in A₁-KO mice. Effects of influenza A/WSN/33 (H1N1) virus infection of WT and A₁-KO mice on BALF alveolar macrophages (AMs) (A), BALF neutrophils (PMNs) (B), BALF lymphocytes (Lymphs) (C), and lung homogenate myeloperoxidase (MPO) activity (D) (n = 6 to 10 per group). *, P < 0.05; **, P < 0.005; #, P < 0.001 versus values for uninfected WT mice. †, P < 0.05; §, P < 0.001 versus values for WT mice at the same time point. Data are presented as means ± SEM.

FIG 5

Severity of lung pathology was reduced in influenza A virus-infected A₁-KO mice. Representative photomicrographs of lung histology in an uninfected WT mouse (A), a WT mouse at 2 days p.i. (B), a WT mouse at 6 days p.i. (C), a WT mouse (same as in panel C) at 6 days p.i. (D), an uninfected A₁-KO mouse (E), an A₁-KO mouse at 2 days p.i. (F), an A₁-KO mouse at 6 days p.i. (G), and an A₁-KO mouse (same as in panel G) at 6 days p.i. (H). Magnifications, ×10 (A to C and E to G) and ×40 (D and H). Bars, 100 μm (A) and 50 μm (D). All sections were prepared from formalin-fixed, paraffin-embedded tissues and stained with hematoxylin and eosin by standard methods.

FIG 6

Influenza A virus-infected A₁-KO mice were partially protected from pulmonary edema and bronchoalveolar epithelial injury. Effects of influenza A/WSN/33 (H1N1) virus infection of WT and A₁-KO mice on lung water content (wet weight/dry weight ratio; n = 7 to 12 per group) (A) and BALF protein content (μg/ml; n = 5 to 10 per group) (B). #, P < 0.001 versus values for uninfected WT mice. †, P < 0.05; ‡, P < 0.005 versus values for WT mice at the same time point. Data are presented as means ± SEM.

FIG 7

Lung compliance and airway resistance were not altered by influenza A virus infection in A₁-KO mice. Effects of influenza A/WSN/33 (H1N1) virus infection of WT and A₁-KO mice on static lung compliance (C_ST; ml/cm H₂O; n = 6 to 14 per group) (A), dynamic lung compliance (C_DYN; ml/cm H₂O; n = 6 to 14 per group) (B), baseline total lung resistance (R_BASAL, cm H₂O · s/ml; n = 6 to 14 per group) (C), and maximal lung resistance following nebulization of 50 mg/ml methacholine (R_MAX, cm H₂O · s/ml; n = 5 to 9 per group) (D). #, P < 0.001 versus values for uninfected WT mice; §, P < 0.001 versus values for WT mice at the same time point. Data are presented as means ± SEM.

FIG 8

Reciprocal bone marrow transfer altered the outcome of influenza A virus infection. Effects of infection with influenza A/WSN/33 (H1N1) virus for 6 days following reciprocal bone marrow transfer on body weight (BWT; percent change from day 0) (A), carotid arterial oxygen saturation (percent S_aO₂) (B), baseline total lung resistance (R_BASAL, cm H₂O · s/ml) (C), static lung compliance (C_ST; ml/cm H₂O) (D), BALF alveolar macrophages (AMs) (E), BALF neutrophils (PMNs) (F), BALF KC content (G), and lung homogenate viral titers (H). n, >5 per group. **, P < 0.005; #, P < 0.001 versus values for WT mice. Data are presented as means ± SEM.

FIG 9

Treatment with the A₁-AdoR antagonist DPCPX attenuated influenza-induced ALI in WT mice. Effects of daily i.p. administration of 100 μl saline vehicle (SALINE-Tx) or 1 mg/kg 8-cyclopentyl-1,3-dipropylxanthine (DPCPX-Tx) in 100 μl saline to WT mice infected with influenza A/WSN/33 virus on body weight (BWT; percent change from day 0; n > 10 per treatment group) (A), carotid arterial oxygen saturation (percent S_aO₂; n > 10 per treatment group) (B), BALF alveolar macrophages (AMs; n = 5 per treatment group) and neutrophils (PMNs; n = 5 per treatment group) (C), and lung water content (wet weight/dry weight ratio; n = 5 per treatment group) (D). **, P < 0.005; #, P < 0.001 versus values for uninfected WT mice. †, P < 0.05; §, P < 0.001 versus values for untreated WT mice at the same time point. Data are presented as means ± SEM.