T cell cytokine synthesis at the single-cell level in BALB/c and C57BL/6 mice infected with ectromelia virus.

BACKGROUND: The purpose of the study was to evaluate synthesis of IFN-γ, IL-2, TNF-α (Th1/Tc1) and IL-4 (Th2/Tc2) at CD4+ T and CD8+ T cell level in BALB/c and C57BL/6 mice in the course of infection with ectromelia virus Moscow strain (ECTV-MOS). MATERIAL/METHODS: Synthesis of IFN-γ, IL-2, TNF-α and IL-4 in CD4+ T and CD8+ T cells in draining lymph nodes (DLNs) and spleens of BALB/c and C57BL/6 mice was detected by intracellular staining and flow cytometry analysis. RESULTS: Our results showed an increase in percentage of IFN-γ -synthesizing CD8+ T cells only in DLNs and spleens of C57BL/6 mice at the early stages of infection. Moreover, synthesis of IL-2 by CD4+ and CD8+ T cells occurred earlier and was stronger in C57BL/6 mice compared to BALB/c mice. The increase in TNF-α synthesis by CD4+ T and CD8+ T cells was detected mainly in DLNs of infected animals. We did not observe any changes in the percentage of IL-4-synthesizing T cells (Th2 and Tc2) during ECTV-MOS infection in both strains of mice. CONCLUSIONS: Results presented in this study confirmed that during the early phase of infection, C57BL/6 mice mounted a strong Th1 and Tc1 immune response against ECTV-MOS. BALB/c mice that survived the acute stage of mousepox, were able to mount an adequate cellular response to ECTV-MOS, however successful elimination of the virus in susceptible mice may occur more slowly compared to resistant strains of mice. Intracellular detection of IL-4 by flow cytometry was not sensitive enough to distinguish the differences in IL-4-synthesizing Th2 and Tc2 cells between susceptible and resistant strains of mice during ECTV-MOS infection.

Beclin 1 is involved in regulation of apoptosis and autophagy during replication of ectromelia virus in permissive L929 cells.

Several reports have brought to light new and interesting findings on the involvement of autophagy and apoptosis in pathogenesis of viral and bacterial diseases, as well as presentation of foreign antigens. Our model studies focused on the involvement of apoptosis during replication of highly virulent Moscow strain of ectromelia virus (ECTV-MOS). Here, we show evidence that autophagy is induced during mousepox replication in a cell line. Fluorescence microscopy revealed increase of LC3 (microtubule-associated protein 1 light chain 3) aggregation in infected as opposed to non-infected control L929 cells. Furthermore, Western blot analysis showed that replication of ECTV-MOS in L929 cells led to the increase in LC3-II (marker of autophagic activity) expression. Beclin 1 strongly colocalized with extranuclear viral replication centers in infected cells, whereas expression of Bcl-2 decreased in those centers as shown by fluorescence microscopy. Loss of Beclin 1-Bcl-2 interaction may lead to autophagy in virus-infected L929 cells. To assess if Beclin 1 has a role in regulation of apoptosis during ECTV-MOS infection, we used small interfering RNA directed against beclin 1 following infection. Early and late apoptotic cells were analyzed by flow cytometry after AnnexinV and propidium iodide staining. Silencing of beclin 1 resulted in decreased percentage of early and late apoptotic cells in the late stage of ECTV-MOS infection in L929 cells. We conclude that Beclin 1 plays an important role in regulation of both, autophagy and apoptosis, during ECTV-MOS replication in L929 permissive cells.