Melatonin alleviates lung injury in H1N1-infected mice by mast cell inactivation and cytokine storm suppression

Abstract

Influenza A virus (IAV) H1N1 infection is a constant threat to human health and it remains so due to the lack of an effective treatment. Since melatonin is a potent antioxidant and anti-inflammatory molecule with anti-viral action, in the present study we used melatonin to protect against H1N1 infection under in vitro and in vivo conditions. The death rate of the H1N1-infected mice was negatively associated with the nose and lung tissue local melatonin levels but not with serum melatonin concentrations. The H1N1-infected AANAT-/- melatonin-deficient mice had a significantly higher death rate than that of the WT mice and melatonin administration significantly reduced the death rate. All evidence confirmed the protective effects of melatonin against H1N1 infection. Further study identified that the mast cells were the primary targets of melatonin action, i.e., melatonin suppresses the mast cell activation caused by H1N1 infection. The molecular mechanisms involved melatonin down-regulation of gene expression for the HIF-1 pathway and inhibition of proinflammatory cytokine release from mast cells; this resulted in a reduction in the migration and activation of the macrophages and neutrophils in the lung tissue. This pathway was mediated by melatonin receptor 2 (MT2) since the MT2 specific antagonist 4P-PDOT significantly blocked the effects of melatonin on mast cell activation. Via targeting mast cells, melatonin suppressed apoptosis of alveolar epithelial cells and the lung injury caused by H1N1 infection. The findings provide a novel mechanism to protect against the H1N1-induced pulmonary injury, which may better facilitate the progress of new strategies to fight H1N1 infection or other IAV viral infections.

Fig 1. The blood and tissue melatonin circadian rhythms and their associations with the susceptibilities to the H1N1 infections in WT and AANAT^-/- CD1 mice.

(A) Concentrations of melatonin in the serum, nose and lung of CD1 mice at different times of a day. Data are from three independent replicates. (B) Survival rates of CD1 mice after exposure to H1N1 virus at 0:00 AM and 12:00 noon, respectively (n = 10). (C) Survival rates of WT and AANAT^-/- CD1 mice after exposure to H1N1 virus, respectively (n = 7). (D) Lung pathology at day 9 of post-infection with H&E staining and scored by an examiner blinded to the study. Black arrows indicate lymphocyte infiltration. Hollow arrows indicate hemorrhage and hyperemia. Hollow triangles indicate desquamation of epithelial cells. (E) The expression of viral NP at day 9 of post-infection with IHC staining and scored by an examiner blinded to the study. Black arrows indicate positive signals. (F) The NS1 copy numbers in lung of mice at day 9 and 11 of post-infection with RT-qPCR. Values were means ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001) vs its respective group, determined by two-way ANOVA followed by Bonferroni statistical tests.

Fig 2. Effects of exogenous melatonin treatment on H1N1 infected BALB/c mice.

(A) The schematic of experimental design with the time points of melatonin treatment (3, 10 and 30 mg/kg, respectively), H1N1 virus inoculation, melatonin detection and samples collection. (B) The levels of melatonin in the serum, nose and lung after intranasal melatonin administration detected by HPLC (n = 5). (C) Survival rates of H1N1-infected BALB/c mice treated with different doses of melatonin (n = 15). (D) Lung pathology at day 6 post-infection with H&E staining and scored by an examiner blinded to the study. Black arrows indicate lymphocyte infiltration. Hollow arrows indicate hemorrhage and hyperemia. (E) The expression of viral NP at day 6 of post-infection with IHC staining and scored by an examiner blinded to the study (n = 4). Black arrows indicate positive signals. (F) The NS1 copy numbers in lung of mice at day 3, 6 and 9 of post-infections, respectively with RT-qPCR (n = 5). (G) The expressions of TNF-α and IL-1β in lung of mice at day 3, 6 and 9 of post-infections, respectively, determined using ELISA (n = 3). (H) Apoptosis in lung of mice at day 3, 6 and 9 of post-infection, respectively, using the TUNEL assay. Green showed positive TUNEL signals. MT: melatonin. Values were means ± SEM. (*P < 0.05, **P < 0.01) vs its respective group, determined by two-way ANOVA followed by Bonferroni statistical tests.

Fig 3. Effects of melatonin on mast cell activation and lung injury in H1N1 virus infected mice.

(A-B) Number of mast cells and expression of tryptase in the nose with toluidine blue staining and IHC staining after melatonin (10 mg/kg) treatment and/or H1N1 virus inoculation (n = 4). (C) Expression of histamine and tryptase in the blood with ELISA (n = 3). (D) Number of mast cells in mucosa, lamina propria and submucosa of WT and AANAT^-/- CD1 mice, respectively, with toluidine blue staining. MT: melatonin. Values were means ± SEM. (*P < 0.05, **P < 0.01) vs its respective group, determined by two-way ANOVA followed by Bonferroni statistical tests.

Fig 4. Effects of melatonin on the gene expressions in the mast cells.

(A) The schematic of experimental design with the time points of melatonin (10⁻⁵ mol/L) treatment, H1N1 virus infection, and sample collection from P815 mast cells. (B) The numbers of DEGs between different groups with RNA-seq. Venn diagrams showing the overlap of DEGs in each comparison group. (C) The genes related to mast cell activation among groups. MT: melatonin. (D) The levels of histamine and tryptase with ELISA (n = 3). (E) KEGG pathway enrichment analysis between groups, and the heatmap of genes about HIF-1 pathway enriched from the DEGs among groups. (F) The heatmap of genes about cytokine release enriched from the DEGs among groups, and the pro-inflammatory cytokines of TNF-α, IL-1β and IL-6 as measured using ELISA (n = 3). Values were means ± SEM. (*P < 0.05, **P < 0.01) vs its respective group, determined by two-way ANOVA followed by Bonferroni statistical tests.

Fig 5. Effects of MT2 on mast cell activation.

(A) The relative gene expression of MT1 and MT2 in P815 mast cells with RT-qPCR (n = 3). (B-C) The effects of melatonin (10⁻⁵ mol/L), and melatonin (10⁻⁵ mol/L) /4P-PDOT (10⁻⁷ M) on expression of HIF-1α and p300 measured by western blot, and the levels of histamine and tryptase measured by ELISA (n = 3) in mast cells. Values were means ± SEM. (*P < 0.05) vs its respective group, determined by two-way ANOVA followed by Bonferroni statistical tests.

Fig 6. Effects of the supernatant from the melatonin treated P815 mast cells on A549 alveolar epithelial cells.

(A) The schematic of experimental design with the time points—melatonin treatment (10⁻⁵ mol/L), H1N1 virus infection, and samples collection from P815 mast cells and A549 alveolar epithelial cells. (B) The heatmap of pro-apoptotic genes enriched from the DEGs among groups. (C-D) Apoptosis and expression of caspase 3 in A549 cells at 12 h after post-infection with flow cytometric analysis and western blot, respectively (four independent replicates). MT: melatonin. Values were means ± SEM. (*P < 0.05) vs its respective group, determined by two-way ANOVA followed by Bonferroni statistical tests.

Fig 7. The summary illustration of potential mechanisms of melatonin in protecting mice against a H1N1 infection.