CD8+ T cells promote ZIKV clearance and mitigate testicular damage in mice

Abstract

Zika virus (ZIKV) causes human testicular inflammation and alterations in sperm parameters and causes testicular damage in mouse models. The involvement of individual immune cells in testicular damage is not fully understood. We detected virus in the testes of the interferon (IFN) α/β receptor^-/- A129 mice three weeks post-infection and found elevated chemokines in the testes, suggesting chronic inflammation and long-term infection play a role in testicular damage. In the testes, myeloid cells and CD4⁺ T cells were absent at 7 dpi but were present at 23 days post-infection (dpi), and CD8⁺ T cell infiltration started at 7 dpi. CD8^-/- mice with an antibody-depleted IFN response had a significant reduction in spermatogenesis, indicating that CD8⁺ T cells are essential to prevent testicular damage during long-term ZIKV infections. Our findings on the dynamics of testicular immune cells and the importance of CD8⁺ T cells function as a framework to understand mechanisms underlying observed inflammation and sperm alterations in humans.

Fig. 1. Human primary Sertoli cells support long-term infection of ZIKV with minimal cell death, but widespread damage is observed in testes of A129 mice.

A ZIKV PRVABC59 growth curve on human primary Sertoli (hpSertoli) cells or mouse Sertoli (15P-1) cell line at an MOI of 0.1, the supernatant was collected at each time point and titrated. B Sertoli cells show minimal cell death when MTT assay is used as a proxy for cell viability. For A, B, bars show mean and error bars represent standard deviation. Statistical significance was assessed with one-way ANOVA with Sidak’s multiple comparison correction. C Viremia in A129 mice at 2 dpi after mice were infected intraperitoneally with a target dose of 3log₁₀ PFU. D A129 mice testicular weight 60 dpi with ZIKV. Statistical significance was assessed using a two-tailed t-test. Lines represent the mean. E Histopathological damage observed in mice testes 60 dpi with ZIKV. In the experiment related to figures C–E, 5 mice were used per condition. *p < 0.05; ****p < 0.0001.

Fig. 2. ZIKV replication in testes of A129 mice after clearance of systemic infection.

A Weights of A129 mice infected with ZIKV PRVABC59. Mice were infected intraperitoneally with a target dose of 3log₁₀ PFU. Statistical significance was assessed with two-way ANOVA with Tukey’s multiple comparison correction. Error bars represent standard error of the mean. B Virus detection in the plasma of A129 mice, uninfected, or at 2 dpi or 3 wpi. Lines represent the mean. C Viral titer in the testes at 3 wpi. D Testicular weight of the mice at 3 wpi. Bars show mean. In the experiment related to this figure, 5 mice were used in the PBS group, and 6 in the infection group. Horizontal dotted lines represent the limit of detection. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 3. Long-term ZIKV infection of A129 mice induces local activation of cytokines and chemokines.

A Log₂ of the fold-change of cytokines and chemokines measured in the plasma of ZIKV-infected mice. B Log₂ fold-change induction of cytokines and chemokines in the testes of ZIKV-infected mice. The data shown in this figure are from the same mouse groups as in Fig. 2. Statistical significance was assessed with one-way ANOVA with Sidak’s multiple comparison correction in between PBS control groups (N = 5) and the infected groups (N = 6), for each cytokine and chemokine. Bars show mean and error bars represent standard error of the mean. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 4. Macrophages, neutrophils and DCs infiltrate testes at 23 dpi.

Cell count and myeloid cells for A the spleen, B the inguinal lymph nodes, C the mesenteric lymph nodes and D testes. Mice were infected intraperitoneally with a target dose of 3log₁₀ PFU. In the experiment related to this figure, 5 mice were used for the PBS group, 5 mice for the 7 dpi group, and 6 mice for the 23 dpi group. Red bars show ZIKV-infected mice, and black bars denote uninfected baseline controls. Statistical significance was assessed with one-way ANOVA with Sidak’s multiple comparison correction. Comparisons were made between each of the groups and only significant results are shown. Bars show mean and error bars represent standard error of the mean. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 5. CD4⁺ T cells accumulate in testes at 23 dpi.

CD4⁺ T cells for A the spleen, B the inguinal lymph nodes, C the mesenteric lymph nodes and D testes. Red bars show ZIKV-infected mice, black bars denote uninfected baseline controls. The data shown in this figure are from the same mouse groups as in Fig. 4. Statistical significance was assessed with one-way ANOVA with Sidak’s multiple comparison correction. Comparisons were made in between each of the groups and only significant results are shown. Bars show the mean and error bars represent the standard error of the mean. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 6. CD8⁺ T cells accumulate in testes at 7 and 23 dpi and in lymph nodes at 7 dpi.

CD8⁺ T cells for A the spleen, B the inguinal lymph nodes, C the mesenteric lymph nodes, D testes. Red bars show ZIKV-infected mice, black bars denote uninfected baseline controls. The data shown in this figure are from the same mouse groups as in Fig. 4. Statistical significance was assessed with one-way ANOVA with Sidak’s multiple comparison correction. Comparisons were made in between each of the groups and only significant results are shown. Bars show mean and error bars represent standard error of the mean. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 7. CD8⁺ T cells are important to prevent significant testicular damage upon long-term ZIKV infection in C57BL/6 J mice transiently treated with anti-IFN antibodies.

A Histology of WT C57BL/6 J mice or CD8^−/− mice testes. Mice were infected intraperitoneally with a target dose of 5log₁₀ PFU/mouse. In the experiment related to this figure, 3 mice were used for each of the PBS groups, and 10 mice were used for each of the CD8^−/− groups. B Testicular weight. Statistical significance was assessed with one-way ANOVA with Sidak’s multiple comparison correction in between WT and CD8^−/− conditions, either uninfected or infected. Bars show mean and error bars represent standard deviation. C Percentage of spermatogenesis quantified from the histology by a pathologist blinded to the samples. Statistical significance was assessed using one-way ANOVA with Sidak’s multiple comparison correction. Groups using WT mice were compared against groups using CD8^−/− mice only within the same treatment, and groups of mice injected with PBS were compared to groups of mice injected with ZIKV only for the same mouse type. Lines represent the mean. D, E Immunofluorescence of testicular samples showing cell nuclei (blue), ZIKV envelope protein (red), and DDX-4 (green). The brightness and contrast of these images were adjusted for optimal visibility. The white arrows show cell detachment from the wall of the ST. *p < 0.05.