Receptor for advanced glycation end products is detrimental during influenza A virus pneumonia

Abstract

Pneumonia caused by influenza A virus (IAV) can have devastating effects, resulting in respiratory failure and death. The idea that a new influenza pandemic might occur in the near future has triggered renewed interests in IAV infection. The receptor for advanced glycation end products (RAGE) is expressed on different cell types and plays a key role in diverse inflammatory processes. We here investigated the role of RAGE in the host response to IAV pneumonia using wild-type (wt) and RAGE deficient ((-/-)) mice. Whereas strong RAGE was constitutively expressed in the lungs of uninfected wt mice, in particular on endothelium, IAV pneumonia was associated with enhanced expression on endothelium and de novo expression on bronchial epithelium. Additionally, the high-affinity RAGE ligand high mobility group box 1 was upregulated during IAV pneumonia. RAGE(-/-) mice were relatively protected from IAV induced mortality and showed an improved viral clearance and enhanced cellular T cell response and activation of neutrophils. These data suggest that RAGE is detrimental during IAV pneumonia.

Fig. 1

Expression of pulmonary receptor for advanced glycation end products (RAGE) during influenza A virus (IAV) pneumonia. Shown are representative RAGE stainings (original magnification, × 10) of lung tissue (A–D). Normal, uninfected wild-type (wt) mouse (A) displaying extensive RAGE expression in the interalveolar septae with an endothelial pattern. Arrow indicates bronchial epithelium being negative for RAGE staining. Absence of RAGE positivity in the lung of a RAGE^−/− mouse (B). Lungs form a wt mouse 8 days after the inoculation of IAV (C and D). Arrow in C indicates bronchial epithelium being positive for RAGE staining (compared with A) and arrows in D indicate inflammatory cells being positive for RAGE staining. Soluble (s)RAGE levels in bronchoalveolar lavage fluid (BALF) from normal, uninfected wt mice (open bar) and from mice 8 days after IAV inoculation (E) (filled bar, n=5–6 mice per group). ***P<0.005, vs. healthy, uninfected mice (Mann–Whitney U test).

Fig. 2

High mobility group box (HMGB)1 levels in bronchoalveolar lavage fluid (BALF) from normal, uninfected wt mice (open bar) and from mice 4 and 8 days after influenza A virus inoculation (filled bars, n=4–6 mice per group). **P<0.01, vs. healthy, uninfected mice (Mann–Whitney U test).

Fig. 3

(A) Receptor for advanced glycation end product deficient (RAGE^−/−) mice have a prolonged survival during influenza A virus (IAV) pneumonia. Wild-type (wt; open circles) and RAGE^−/− (filled circles) mice (n=12–14 mice/genotype) received 200 TCID₅₀ influenza virus intranasally on day 0. Mice were monitored at least twice a day after inoculation. (B) Receptor for advanced glycation end product (RAGE) deficiency diminishes viral load in the lungs. Viral load in lungs in wild-type (wt; open bars) and RAGE deficient (RAGE^−/−; filled bars) mice at 4 and 8 days after 40 TCID₅₀ intranasal oculation with influenza A virus (IAV). Data are means ± SEs (n=6–8 mice/genotype). *P<0.05, vs. wt mice (Kaplan–Meier analysis by log rank test).

Fig. 4

Lung histopathology. Shown are representative hematoxylin–eosin stainings (original magnification, × 10) of lung tissue of wt (A) and receptor for advanced glycation end product deficient (RAGE^−/−; B) mice obtained 8 days after inoculation with influenza A virus (IAV). Total histology scores of the lungs were determined in wt and RAGE^−/− mice 8 days after influenza virus inoculation (C) as described in the Materials and methods section. Data are means ± SEs (n=8–9 mice/genotype). *P<0.05, vs. wt mice (Mann–Whitney U test).

Fig. 5

Receptor for advanced glycation end product deficient (RAGE^−/−) mice show enhanced activation of pulmonary T lymphocytes and neutrophils. Wild-type (wt) and RAGE^−/− mice were intranasally inoculated with influenza A virus (IAV). After 8 days, lung cell suspensions were collected and flow cytometry was performed as described in the Materials and methods section. Results are represented as percentage of CD4+ (A), CD8+ (C) cells and neutrophils (E) in the lungs and as the mean fluorescence intensity of CD69 surface expression within the CD4+ (B) and CD8+ (D) population and of CD11b surface expression within the Gr1+ population (F). Data are means ± SEs of 8–9 mice/genotype. *P<0.05, vs. wt mice. **P<0.01, vs. wt mice. ***P<0.005, vs. wt mice (Mann–Whitney U test).

Fig. 6

Pulmonary and plasma cytokine concentrations. Cytokine levels in lungs (left panels) and plasma (right panels) from wild-type (wt; open bars) and receptor for advanced glycation end product deficient (RAGE^−/−; filled bars) mice 4 and 8 days after inoculation with influenza A virus (IAV). Data are means ± SEs (n=6–8 mice/genotype). *P<0.05, vs. wt mice.