ZIKV infection causes testicular damage and testosterone decline in mice
ZIKV has obvious organization preferences, and the previous studies have shown that the brain and testicles are the main target organs of the virus in male mice [12, 23]. To study the effect of ZIKV on testicular injury, the male A129 mice were pathologically analyzed at 8 days after ZIKV infection. Compared with the control group, the vas deferens and testicles were obviously congested (Fig. 1A-B). The seminiferous tubules of ZIKV infected mice were lost the original structure, and spermatogenic cells arranged loose and mussily (Fig. 1C). Testosterone is mainly produced by the testes. To test whether testicular damage caused by ZIKV infection could affect testosterone secretion, blood samples were taken from the tail of mice on day 8 post ZIKV infection to detect serum testosterone concentration, and testicles were ground to detect testosterone concentration in testicles. The results showed that ZIKV infection reduced the levels of testosterone in serum and testis (Fig. 1D-E).
Testosterone supplementation decreases ZIKV-induced mouse lethality
Previous studies have shown that ZIKV can enter the brain of mice and cause severe inflammation[25] while testosterone has an immunosuppressive effect. To investigate whether testosterone plays a protective role during ZIKV infection, ZIKV-infected A129 mice were injected with testosterone or the same dose of sesame oil at the second day and third day after ZIKV infection (Fig. 2A). On day 8 post infection, blood samples were collected to detect the testosterone concentration. As shown in the results, testosterone was maintained at a higher level in the ZIKV-infected and testosterone-treated group compared with the ZIKV-infected and sesame oil-treated group (Fig. 2B). All mice in the testosterone or sesame oil-treated group survived during the observation, while mice in the ZIKV-infected and sesame oil treated or testosterone-treated group started to display morbidity and mortality on day 9 or 10 post-infection. After ZIKV infection, a high mortality rate (83%) of mice was observed in the sesame oil group, while the mortality rate of mice in testosterone treated group was 50% (Fig. 2C). In addition, treatment with testosterone caused fewer declines in body weight (Fig. 2D) and improved behavioral signs in ZIKV-infected mice (Fig. 2E). These results suggest that testosterone treatment after ZIKV infection can alleviate the symptoms and increase the survival rate of mice, revealing that testosterone plays a protective role on mice infected with ZIKV.
Testosterone treatment reduces ZIKV-induced inflammatory response in mouse brain tissues
Brain is one of the main target organs for ZIKV, which can invade the brain of both fetuses and adults and cause encephalitis, myelitis and meningoencephalitis [26, 27]. Given the role of testosterone in reducing the ZIKV-induced mouse lethality, we investigated whether testosterone can eliminate the neuroinflammation caused by ZIKV infection. A129 mice were injected with testosterone or sesame oil following ZIKV infection. H&E staining was performed for histopathological changes analysis of the brain sections on the eighth day after virus infection. Results showed the signatures of perivascular cuffing in ZIKV-infected mice at 8 days post infection, whereas these indicators of encephalitis were reduced in ZIKV-infected mice receiving testosterone treatment (Fig. 3A). In addition, testosterone treatment can reduce meningitis (Fig. 3B) and haemorrhage in the hippocampus (Fig. 3C) caused by ZIKV infection. Furthermore, RT-qPCR results showed that testosterone treatment reduced the expression of proinflammatory factors IL-6 and TNF-α induced by ZIKV infection (Fig. 3D-E). Taken together, these results demonstrate that testosterone treatment reduced inflammation and pathological changes caused by ZIKV infection in brains of mice.
Testosterone treatment does not affect the viral replication and humoral immunity in ZIKV infected mice
To investigate whether testosterone protects mice and alleviate the neuroinflammation by interfering with viral replication, A549 cells and TM4 cells were used to detect the in vitro replication of ZIKV. The cytotoxicity of testosterone on A549 cells and TM4 cells was examined by cell viability assay. Viable cells were determined on the basis of ATP quantification of cells, which indicates the presence of metabolically active cells. Different concentrations of testosterone were added on A549 cells and TM4 cells, and the results revealed that 10µM testosterone exhibits no cytotoxic effect, while a 100µM concentration was significantly toxic to cells (Fig. 4A). A concentration of 10µM was then used to test the effect of testosterone on viral replication. A549 cells and TM4 cells were treated with 10µM testosterone followed by ZIKV infection. At 24, 36, and 48 hours post infection, cells were collected to detect the RNA level of and viral titer of ZIKV. The results showed that testosterone did not inhibit the replication of ZIKV (Fig. 4B-C).
Since testosterone does not affect the replication of the virus, we detected the antibody level in the serum of ZIKV infected mice to explore whether testosterone protected the mice by increasing the antibody level. Serum samples were collected on 8 days after ZIKV infection, and the antibody against ZIKV E protein was detected by ELISA. No significant change of antibody level was observed upon testosterone treatment in mice (Fig. 4D). These results suggest that testosterone treatment does not play protective role on ZIKV infection by interfering with the viral replication and humoral immunity.
Testosterone inhibits T cell activation caused by ZIKV infection
To further investigate how testosterone participate the pathogenic process of ZIKV infection, we analyzed the proportion of CD4+ T cells and CD8+ T cells in spleen of mice by flow cytometry. The results showed that ZIKV infection led to a significant increase in the proportion of CD3+ T cells in spleen, while testosterone injection could reduce the increased CD3+ T cells ratio caused by ZIKV infection (Fig. 5A-B). Although testosterone treatment did not significantly change the proportion of CD8+ T cells in CD3+ T cells, it significantly reduced the proportion of CD8+ T cells in spleen cells (Fig. 5A, C-D). However, infection with ZIKV did not change the proportion of CD4+ T cells in the spleen (Fig. 5A, E-F).
Testosterone treatment reduces the infiltration of CD8+ T cells in brain of mice infected with ZIKV
ZIKV infection can cause CD8+ T cells to enter the brain and produce an inflammatory response[28]. Here to determine the effect of ZIKV infection and testosterone supplementation on infiltration of CD8+ T cells in mouse brain, the immunohistochemical assay was performed to analyze the CD8+ T cell numbers. More CD8+ T cells in the ZIKV infected mouse brain than those in the uninfected mouse brain was observed, while treatment of testosterone reduced the CD8+ T cell infiltration caused by ZIKV infection (Fig. 6A). To further verify the result, the brain cells in mice were isolated for flow cytometry assay (Fig. 6B). Consistently, the results showed that CD8+ T cells increased significantly after ZIKV infection in mice, while testosterone supplementation reduced the CD8+ T cell number (Fig. 6C-D). These results suggest that testosterone treatment could reduce the infiltration of CD8+ T cells in the brain of ZIKV infected mice.
Testosterone treatment reduces IFN-γ and chemokine expression in brain of ZIKV infected mice
IFN-γ is mainly expressed by T cells, and previous studies have reported that testosterone can inhibit IFN-γ secretion signaling pathway[29], so we detected the expression of IFN-γ in mouse serum and brain. Mice were sacrificed and blood was collected on day 8 after infection with ZIKV, and their brains were subjected to grind and centrifugation to separate the supernatant. IFN-γ levels in brain and serum of mice were detected by ELISA kit. The results showed that the level of IFN-γ in the brain was significantly increased after ZIKV infection, and the increase was inhibited by testosterone treatment (Fig. 7A). However, the level of IFN-γ in serum was not significantly changed after ZIKV infection (Fig. 7B). Given that chemokines play important role in directing T cell trafficking, the mRNA levels of chemokines, such as CCL2 and CCL5, in mouse brain were analyzed by real-time RT-PCR. It was found that testosterone inhibited the upregulation of chemokines caused by ZIKV infection (Fig. 7C-D).