| Viral infection caused by influenza is a serious public health problem. Studies found that stress could increase the morbidity and severity of diseases, but the underlying mechanisms whereby stress triggers influenza susceptibility is not yet fully understood. Stress could activate the hypothalamic-pituitary-adrenal(HPA) axis and result in increase of corticosterone(CORT) level. CORT, the end product of HPA axis, play an important role in regulating the material metabolism, homeostasis and immune system. The adjustment of CORT concentration has significant impact on health. The present study sought to examine the role of CORT in mediating influenza virus susceptibility in mice.Early research found that 3 days after restraint stress 18 h, the immunity of mice was remarkably decreased, and the susceptibility of influenza infection was increased, with significant increase in morbidity and decrease in survival rate. Based on this model, the effects of restraint stress on viral load and lung injury were studied. Experimental results showed that the susceptibility of influenza infection was increased, with remarkable elevation in the viral load in lung tissue and significant lung injury compared to control mice.In innate immune system, the production of Interferon-I(IFN-I) by the RIG-I/MAVS pathways, together with AVP(antiviral protein, AVP) could inhibit viral infection synergistically. Recent studies have found that dexamethasone, a kind of glucocorticoid(GC), can competitively inhibit the production of IFN-I and IFN-stimulated genes(ISGs). This could result in blockage of the antiviral function of IFN-I and increase of viral susceptibility. Therefore, this study will investigate whether the increased influenza viral susceptibility was associated with restraint stress induced CORT fluctuation. The level of CORT in plasma of restraint stressed mice after different recovery times was detected by HPLC. Results showed that CORT level in plasma elevated significantly on the 3rd day after restraint stress. Therefore, we subcutaneously injected CORT-L(1, 2 mg/kg) for continuous 3 d to simulate restraint stress response. The CORT level of CORT-L(1 mg/kg) group was similar to the CORT level in restraint stressed mice. Based on this result KM mice were randomly divided into 5 groups with 10 mice each group, including vehicle control group, virus control group, restraint + virus group, CORT + virus group and RU486(GR antagonist) group. Mice were infected with influenza virus on the 3rd day after restraint stress. CORT and RU486 were injected intraperitoneally(i.p.) to mice after stress for 3 consecutive days. All mice were sacrificed 4 days after virus infection. Susceptibility markers(MAVS, p-IRF3, NF-κB, IFN-β and IFITM 3) and viral loads were determined. Compared to virus control group, the viral loads of restraint + virus group and CORT + virus group were increased. Meanwhile, the expression of MAVS was downregulated and the phosphorylation of IRF3 was decreased. As a result, the productions of IFN-β and IFITM3 were decreased, while the expression of NF-κB was increased. These alterations promoted the production of pro-inflammatory factors, and resulted inaggravation of secondary pneumonia. However, RU486 lowered the viral loads in the lung tissue of mice, and effectively restored the expression levels of susceptible factors(MAVS, p-IRF3, IFN-β, IFITM3). Moreover, the current study utilized A549 cell model to explore the mechanism of CORT in influenza virus susceptibility. In vitro, different treatment was conducted, including control, virus, CORT, CORT+virus, RU486+CORT and RU486+CORT+virus. A549 cells were pretreated with RU486 for 1 h, and then exposed to CORT(100 μM) for 48 h, and finally infected with H1N1. Eight hour after infection, cells were analyzed by RT-PCR for the gene expression of NP, and by western blot for the expression of susceptible factors MAVS, p-IRF3, IFN-β and IFITM3. It was showed that the viral load of CORT+virus group was higher than that of control group. Meanwhile, the downregulated MAVS and decreased IRF3 phosphorylation decreased the production of IFN-β and IFITM3, whereas the expression of NF-κB was increased, which promoted the release of pro-inflammatory factors. However, pretreatment with RU486(10 μM, 1 h) effectively lowered the viral load in A549 cells, and restored the expression levels of susceptible factors(MAVS, p- IRF3, IFN-β, IFITM3). These experimental results indicated that restraint stress increased CORT level, which couldbind to GR, alternate the expressions of susceptible factors and ultimately increase influenza viral susceptibility.Recent study found that Mfn2 could inhibit RIG-I mediated MAVS antiviral signaling pathway and reduce the production of IFN-β, implying a relation between Mfn2 and viral susceptibility. Moreover, the level of GC could affect the migration and distribution of GR, regulate the expression of Mfn2, and consequently affect the structure and function of mitochondria. Therefore, this study will examine the potential role of Mfn2 in CORT-increased viral susceptibility. The expression of Mfn2 in animal and cell models was determined by western blot respectively. Furthermore, Mfn2 was silenced by RNA interference to investigate viral load and expressions of IFN-β and IFITM3. Results showed that the levels of Mfn2 were increased after restraint stress in mice, as well as treatment with CORT(100 μM, 48 h) in A549 cells. Silencing Mfn2 by RNA interference decreased the viral load in A549 cells, whereas upregulated the expressions of IFN-β and IFITM3. These might suggest that CORT increased viral susceptibility through upregulating the level of Mfn2, which inhibited the production of IFN-β and IFITM3.In summary, our study indicated that restraint stress caused increases in corticosterone and enhanced Mfn2 expression. These alterations caused inhibition of the MAVS antiviral signaling pathway and increased viral susceptibility. |
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