Anti-TGEV Miller Strain Infection Effect of Lactobacillus plantarum Supernatant Based on the JAK-STAT1 Signaling Pathway

Wang, Kai; Ran, Ling; Yan, Tao; Niu, Zheng; Kan, Zifei; Zhang, Yiling; Yang, Yang; Xie, Luyi; Huang, Shilei; Yu, Qiuhan; Wu, Di; Song, Zhenhui

Although there was no significant difference in the levels of p-STAT1 between the Lp-1s group and the TGEV group at 12 hpi, the level of p-STAT1 in the Lp-1s group was significantly higher than that in the TGEV group at the later stages (24 and 48 h). The level of p-STAT1 in the Lp-1s group correlated positively with time from 12-48 h. The results showed that IPEC-J2 cells treated with Lp-1s could effectively increase STAT1 phosphorylation in the late stage of virus infection. At the same time, the level of p-STAT1 changed slightly from 12-48 h after infection with TGEV. The results were quite different from those reported in previous studies on the infection of ST cells by TGEV. In addition, the level of p-STAT1 in IPEC-J2 cells infected with TGEV from 24-48 h was significantly lower than that in ST cells infected with TGEV from 24-48 h, which might reflect the difference between the cell lines and viruses used. Further experiments showed that the level p-STAT1 nuclear translocation in the Lp-1s group was significantly higher than that in the TGEV group at the same time points, and p-STAT1 nuclear accumulation correlated positively with time. In TGEVinfected cells, p-STAT1 at the late stage of infection (24-48 h) accumulated in large amounts near the cell membrane and only a few nuclear translocations occurred. We speculated that the reason might be that the intracellular STAT1 protein is activated by IFN-β after IPEC-J2 cells are directly infected with TGEV to form a homologous or heterodimer, and the virus interacts with its receptor IRF9. The interaction resulted in the inability of activated STAT1 to undergo nuclear translocation through receptor-induced endocytosis, and could only dissociate around IFNAR, which reduced the JAK-STAT signaling pathway cascade response and antagonized the antiviral effect of IFN-β. The fluorescence value of activated STAT1 was significantly higher than that of blank control group. This indicated that TGEV could not completely escape the immune mechanism of IFN-β FIGURE 9 | Proposed mechanism of the anti-TGEV effect of Lp-1s. Lp-1s increases IFN-β expression, and the binding of IFN-β to its receptor IFNAR leads to activation of the Janus family kinase (JAK) and subsequent activation of signal transduction and transcriptional activator 1 (STAT1) signaling cascades. These signaling pathways upregulate downstream interferon-stimulated genes (ISGs), including MX1, MX2, PKR, OAS, ISG15, and ZAP), which produce the corresponding antiviral proteins, e.g., ZAP and PKR, ultimately activating IFN-β to exert an antiviral effect. production in IPEC-J2 cells. At the same time, the fluorescence intensity of p-STAT in the nucleus of the cells in the Lp-1s treatment group correlated positively with time, and the intensity of FITC-labeled TGEV N protein was significantly lower than that in the TGEV treatment group at the same time point. These results showed that IPEC-J2 cells treated with Lp-1s could indeed activate the JAK-STAT1 signaling pathway when infected with TGEV, and the intensity of JAK-STAT1 signaling pathway was significantly higher in the Lp-1s-treated cells than in the cells directly infected with TGEV. As the signaling pathway cascade response increased, the replication level of TGEV in IPEC-J2 cells was further inhibited.