Table 1.
The sequence of the primers for RT-qPCR.
Fig 1.
Identification of C57BL/6-Tg (HCMV-UL122) mice.
(A) Illumina NovaSeq6000 sequencing platform to sequence the whole genome of the F0 generation transgenic mice. (B) Detection of transgenic fragments. The fragment’s detection is indicated by intervals with sequencing depth. (C) The expression of Fam47c and Cfap47 mRNA level was detected by RT-qPCR. Date are mean ± SEM. (D) Identification of the IE2 positive and negative mice by PCR. (Lanes 1-2, 4-7, 10) positive mice; (Lanes 3, 8) the negative mice; (Lanes 10, 11) the positive and water control. PCR product size: 470 bp. (E) IE2 expressed in macrophages were confirmed by western-blot technology.
Fig 2.
IE2 inhibits the activation of macrophages in vivo.
(A) All mice were immunized with LPS via intraperitoneal injection, after 12h, spleen lymphocytes were isolated, and the surface markers CD80, CD86, MHC I, and MHC II in CD11b+ macrophages were detected by flow cytometry. (B) The percentages of CD80, CD86, MHC I and MHC II expression in CD11b+ macrophages. WT + T means WT mice injected intraperitoneally with LPS. IE2 + T means IE2 mice injected intraperitoneally with LPS. (C) Assessment of TFN-α, IL-1β, IL-6 and IL-12p70 were detected in primary peritoneal macrophages after LPS-treated 48h in vitro. Statistically significant differences as determined by t-test from ANOVA. Date are mean ± SEM. n = 5. Significance levels were defined as *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 3.
The effect of IE2 on function of primary macrophages.
(A) M1 phenotype macrophages (F4/80+ iNOS+) were detected in the spleen by flow cytometry (right). The percentage of M1 phenotype macrophages (F4/80+ iNOS+) (left). (B) M2 phenotype macrophages (F4/80+ CD206+) were detected in the spleen by flow cytometry (right). The percentage of M2 phenotype macrophages (F4/80+CD206+) (left). WT + T means WT mice injected intraperitoneally with LPS. IE2 + T means IE2 mice injected intraperitoneally with LPS. (C) Primary macrophages were obtained and used fluorescence microscope to detect phagocytic function. (D)The percentages of phagocytic in primary macrophages. (E) Primary macrophages recruitment by MCP-1 (20 ng/mL) for 20 min was measured using transwell assay. (F) Summarized data of migration in primary macrophages. Statistically significant differences as determined by t-test from ANOVA. Date are mean ± SEM. All experiments were performed independently at least three times, and significance levels were defined as *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 4.
The effect of IE2 on macrophage stimulated T cell differentiation.
(A) The secretion of IL-4 and IFN-γ in CD4+ T cells were detected by flow cytometry. (B) The percentages of IL-4 and IFN-γ in CD4+ T cells. (C) The secretion of IFN-γ and TNF-α in CD8+ T cells were detected by flow cytometry. (D) The percentages of TNF-α and IFN-γ in CD8+ T cells. Statistically significant differences as determined by t-test from ANOVA. Date are mean ± SEM. n = 5. Significance levels were defined as *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 5.
IE2 up-regulated IL10/STAT3 signaling pathway.
(A) Primary macrophages were cocultured with LPS for 48h. The expression of antigen presentation related molecules mRNA level was detected by RT-qPCR. MHC I antigen presentation pathway: β-2m and H2-D1; MHC II antigen presentation pathway: CD74. (B) The expression of IL-12b and IL-10 mRNA level were detected by RT-qPCR. (C) Whole cell proteins were extracte from the mouse spleen lymphocytes with cultured macrophages, and the STAT3 and p-STAT3 levels were measured was detected by western blot. (D) Summarized data of panel C. Statistically significant differences as determined by t-test from ANOVA. Date are mean ± SEM. All experiments were performed independently at least three times, and significance levels were defined as *P < 0.05 and ***P < 0.001.