Sirolimus alters lung pathology and viral load following influenza A virus infection

Abstract

Background: Inhibitors of mTOR, such as sirolimus, have been shown to induce thymus involution and inflammatory lung disease in mice. The latter effect supports the role of this serine/threonine kinase in ameliorating lung inflammation. Other studies have shown sirolimus reduces/delays lung disease associated with various strains of influenza A virus (IAV). Thus, the effects of mTOR inhibitors on influenza infection deserve further studies.

Methods: Here, we examined the changes in lung viral copies, pathology and pulmonary function associated with IAV (A/PR/8/34) infection in mice treated with sirolimus.

Results: Body weight loss peaked between days 6-11 post-infection and was more severe in IAV-infected mice that were administered sirolimus as compared to mice that received IAV alone (p = 0.030). Natural log viral gene copies, mean ± SD per mg lung tissue, in IAV-infected mice that were administered sirolimus were 17.31 ± 1.27 on day 4, 19.31 ± 7.46 on day 10, and 0 on day 25. The corresponding number of copies in mice that received IAV alone were 18.56 ± 0.95 on day 4 (p = 0.132), 1.52 ± 1.39 on day 10 (p = 0.008), and 0 on day 25. Lung pathology was evident on days 4, 10, and 25 post infection, with mean ± SD inflammatory score of 9.0 ± 4.5 in IAV-infected mice that were administered sirolimus, as compared to 11.5 ± 4.5 (p = 0.335) in mice received IAV alone (maximum score, 26.0). Impaired lung function was evident in IAV-infected mice on days 4 and 10, as demonstrated by increased airway resistance and decreased compliance.

Conclusions: In this model, the effects of sirolimus on influenza infection included severe weight loss and modified viral replication, respiratory function and lung inflammation. The adverse events associated with sirolimus treatment are consistent with its potent immunosuppressive activity and, thus, preclude its use in IAV infection.

Keywords: Inflammatory score; Influenza A virus; Lung function; Rapamycin; Viral replication.

Fig. 1

Weight changes in infected and uninfected mice. Values are mean ± SD (in percentages) of the daily weight divided by the starting weight for each mouse. Four separate experiments (9 to 20 mice per group, 56 mice) were performed. One mouse in the IAV group died on day 18, and one mouse in the sirolimus + IAV group died on day 9. The body weight loss peaked between days 6 and 11 post-infection, and was more pronounced in IAV-infected mice that were administered sirolimus than that in mice received IAV alone

Fig. 2

Detection of IAV in infected lungs. RNA was extracted from infected and uninfected lungs on days 4, 10, and 25, and subjected to RT-PCR using primers specific for the IAV matrix gene. Data represent three separate experiments (2–6 mice per group per day, 26 mice). On day 4 post-infection, the natural log viral gene copies in IAV-infected mice that were administered sirolimus were similar to those in mice received IAV alone. On day 10 post-infection, the viral gene copies were significantly higher in IAV-infected mice that were administered sirolimus than that in mice received IAV alone. Viral gene copies were not detected on day 25 in both groups

Fig. 3

Lung histology in infected and uninfected mice. Representative H&E stained lung sections (20×) on days 4, 10 and 25 from 1 of 3 mice per condition are shown (3 separate experiments, see Table 1). Arrows point to areas of patchy interstitial inflammatory infiltrate with alveolar wall thickening. Numbers in parentheses represent score of inflammation. Lung histology in mice treated with sirolimus alone or IAV alone showed a slightly thickened interstitium on day 4 post-infection and peribronchial inflammation (small arrows) on day 10 post-infection. Peribronchial inflammation (large arrow) on day 10 post-infection was more prominent in IAV-infected mice that were administered sirolimus

Fig. 4

Functional respiratory assessment on days 4 and 10 post-infection. Upper Panels: Baseline airway reactivity expressed as thoracic resistance (Rrs), thoracic compliance (Crs), and large airway resistance (Rn). Lower Panels: Methacholine responsiveness shown as ‘area under the curve’ (AUC) of Rrs, Crs and Rn against methacholine concentration. The values are mean ± SD (3 to 5 separate experiments, 3 to 7 mice per condition, 33 mice). Asterisks designate p < 0.05 compared with DMSO. Increased thoracic and large airway resistances and decreased thoracic compliance were evident in IAV-infected mice on days 4 and 10 (upper panels). These changes (at both baseline and after methacholine) were less pronounced on day 4 and more severe on day 10 in IAV-infected mice that were administered sirolimus (lower panels)