Attenuation of interferon regulatory factor 7 activity in local infectious sites of trachea and lung for preventing the development of acute lung injury caused by influenza A virus

Abstract

The excessive activation of interferon regulatory factor 7 (IRF7) promotes the development of acute lung injury (ALI) caused by influenza A virus (IAV). However, the deficiency of IRF7 increases the susceptibility to deadly IAV infection in both humans and mice. To test whether the attenuation rather than the abolishment of IRF7 activity in local infectious sites could alleviate IAV-induced ALI, we established IAV-infected mouse model and trachea/lung-tissue culture systems, and designed two IRF7-interfering oligodeoxynucleotides, IRF7-rODN M1 and IRF7-rODN A1, based on the mouse and human consensus sequences of IRF7-binding sites of Ifna/IFNA genes, respectively. In the model mice, we found a close relationship between the IAV-induced ALI and the level/activity of IRF7 in local infectious sites, and also found that the reduced IRF7 level or activity in the lungs of mice treated with IRF7-rODN M1 led to decreased mRNA levels of Ifna genes, reduced neutrophil infiltration in the lungs and prolonged survival of mice. Furthermore, we found that the effects of IRF7-rODN M1 on alleviating IAV-induced ALI could be correlated to the reduced translocation of IRF7, caused by the IRF7-rODN M1, from cytosol to nucleus in IAV-infected cells. These data suggest that the proper attenuation of IRF7 activity in local infectious sites could be a novel approach for treating IAV-induced ALI.

Keywords: acute lung injury; influenza A virus; interferon regulatory factor 7; oligodeoxynucleotide.

Figure 1

Inflammatory responses induced by influenza virus in mice. Female BALB/c mice (n = 6 mice/group) were intranasally infected with 10 LD ₅₀ H1N1 influenza virus or an equivalent dilution of non‐infectious allantoic fluid (AF), followed by recording the survivals (a) and weights (b) of the mice. The lungs were isolated and sectioned for hematoxylin & eosin staining. The stained sections were observed under the microscope (magnification × 400, Scale bars = 20 μm) and scored (c) on days 1, 3, 5 and 7 post‐infection (p.i.). The cells of bronchoalveolar lavage fluid (BALF) collected from the H1N1 influenza virus‐infected mice were harvested, labeled with Ly6G and analysed the ratio of Ly6G⁺ cells (d). Ly6G⁺ cells represent the neutrophils. FSC, forward scatter; Isotype, Isotype control; SSC, side scatter. Data are represented as mean ± SD (n = 3 mice/group).

Figure 2

Influenza A virus (IAV) induced local immune responses in the cultured tracheas and lungs. The cultured trachea and lung tissues isolated from the naive mice were infected with H1N1 influenza virus for different times followed by detecting the inflammatory response molecules by qRT‐PCR and Western blotting. (a–f) The mRNA levels of Tlr7, Irf7, Irf3, Ifna, Ifnb and Cxcl10 in cultured trachea tissues infected with H1N1 influenza virus. The mRNA levels were normalized with the mRNA levels of Actb. (g, h) The protein levels of interferon regulatory factor 7 (IRF7) in cultured trachea and lung tissues from the naive mice. (i, j) The protein levels of IRF7 and p‐IRF7 in cultured trachea and lung tissues infected with H1N1 influenza virus by Western blotting. n.s. denotes the insignificance. Data are represented as mean ± SD (n = 3).

Figure 3

The mRNA levels of IRF7,IFNA and CXCL10 in epithelial cells of upper respiratory tract from child patients with or without influenza A virus (IAV) infection. (a) Immunofluorescence staining sections for detecting IAV infection. The epithelial cells of upper respiratory tract from child patients with or without IAV infection were coated on the glasses for detection of IAV by direct fluorescent antigen (DFA) assays. The cells were stained for red colour, whereas the IAV were stained for green colour. (b) The mRNA levels of IRF7,IFNA and CXCL10 in the epithelial cells of the upper respiratory tract of the patients were analysed by qRT‐PCR. The mRNA levels were normalized with the mRNA levels of GAPDH. Each point represents one patient. Data are represented as mean ± SD.

Figure 4

The interfering role of interferon regulatory factor 7–oligodeoxynucleotides (IRF7‐rODNs) on expression and activation of interferon regulatory factor 7 (IRF7) in influenza A virus (IAV) ‐infected RAW264.7 cells. The IRF7 expression and translocation were detected in RAW264.7 cells infected with H1N1 influenza virus. (a) The mRNA levels of Irf7 in H1N1 influenza virus infected RAW264.7 cells. The mRNA levels were normalized with the mRNA levels of Actb. (b) The effect of IRF7‐rODNs on the Irf7 mRNA expression induced by H1N1 influenza virus in vitro. (c) The IRF7 protein expression in H1N1 influenza virus‐infected RAW264.7 cells treated with or without IRF7‐rODNs. (d) The impact of IRF7‐rODNs on cytoplasm IRF7 level in H1N1 influenza virus‐infected RAW264.7 cells. (e) The inhibitory function of IRF7‐rODNs on the IRF7 nuclear translocation. n.s. denotes the insignificance. Data are represented as mean ± SD (n = 3).

Figure 5

Effects of interferon regulatory factor 7–oligodeoxynucleotides (IRF7‐rODNs) in H1N1 influenza virus‐infected mice. The H1N1 influenza virus‐infected mice were treated with IRF7‐rODN M1, IRF7‐rODN A1 or phosphate‐buffered saline (PBS) by intraperitoneal injection on day 2 and day 4 post‐infection (p.i.) and their survivals (a) and body weights (b) were recorded. On day 5 p.i., the lungs of the infected mice were isolated for gross observation (c) and sectioned for haematoxylin & eosin staining. The stained sections were observed under microscope (magnification ×40, Scale bars = 200 μm) and scored (d). The cells of the bronchoalveolar lavage fluid were harvested on day 5 p.i. and analysed the ratio of the Ly6G⁺ cells (e) by flow cytometry. Ly6G⁺ cells represent the neutrophils. n.s. denotes the insignificance. Data are represented as mean ± SD (n = 3 mice/group).

Figure 6

Effects of interferon regulatory factor 7–oligodeoxynucleotides (IRF7‐rODNs) on the viral load in lungs of the mice infected with H1N1 influenza virus. The H1N1 influenza virus‐infected mice were treated with IRF7‐rODN M1, IRF7‐rODN A1 or phosphate‐buffered saline (PBS) on day 2 and day 4 post‐infection (p.i.), respectively. On day 5 p.i., the viral load of the lung tissues collected from the mice was analysed by cytopatic effect assay (a) for TCID ₅₀ and haemagglutination assay (b) for HA unit. Each symbol represents the viral load in the lungs from one mouse, and the horizontal line represents the mean value ± SD for each group (n = 6). n.s. denotes the insignificance. Results shown are representative of three independent experiments.

Figure 7

Binding assay of interferon regulatory factor 7–oligodeoxynucleotides (IRF7‐rODNs) and interferon regulatory factor 7 (IRF7) in vitro and effects of IRF7‐rODNs in influenza A virus (IAV) ‐infected mice. The H1N1 influenza virus‐infected mice were treated with IRF7‐rODNs or phosphate‐buffered saline (PBS) by intraperitoneal injection on day 2 and day 4 post‐infection (p.i.). On day 5 p.i., the lungs were removed from the mice and homogenized for analysing the ODN binding affinity to IRF7 by pull‐down assay, mRNA levels of Irf7, Ifna genes and Cxcl10 by qRT‐PCR and the protein expression of IRF7 by Western blotting. (a) The binding of IRF7‐rODNs to IRF7 in lysates from homogenized lungs of the mice. Input denotes the total protein in lung homogenate. Pull‐down denotes the IRF7 bound with the IRF7‐rODNs. (b) The mRNA levels of Irf7 in lungs of the mice. The mRNA levels were normalized with the mRNA levels of Actb. (c) The protein expression of IRF7 in lungs of the mice. (d, e) The mRNA expression of Ifna genes and Cxcl10 in lungs of the mice. n.s. denotes the insignificance. Data are represented as mean ± SD (n = 3).