Transmissible Gastroenteritis Virus Infection Up-Regulates FcRn Expression via Nucleocapsid Protein and Secretion of TGF-ß in Porcine Intestinal Epithelial Cells

Qian, Shaoju; Gao, Zitong; Cao, Rui; Yang, Kang; Cui, Yijie; Li, Shaowen; Meng, Xianrong; He, Qigai; Li, Zili

To determine whether TLRs are involved in mediating TGEVinduced FcRn activation, specific siRNAs were synthesized to target binding molecules in the TLR2, TLR3, TLR4, TLR8, TLR9, TRIF, MyD88, and RIG-I pathways. Transient transfection and RT-qPCR assays proved the low knockout efficiency of each siRNA, and the silencing efficiencies of the siRNAs to these receptors were over 50% (Figure 2A) . The cells were transfected with each siRNA and then infected with TGEV; then, the changes in FcRn expression were detected by RT-qPCR and Western blot. The results showed that, compared with the cells transfected with NC siRNA, siRNA knockdown of TLR3, TLR9, RIG-I, TRIF, and MyD88 effectively reduced the TGEVinduced up-regulation of FcRn, while FcRn mRNA expression was not inhibited in cells transfected with siRNA targeting TLR2, TLR4, or TLR8 ( Figure 2B) . Next, IPEC-J2 cells were transfected with TLR-specific siRNA or NC siRNA. After 12 h, the cells were infected with 1 MOI TGEV for an additional 36 h. Compared with NC siRNA, TLR3, TLR9, RIG-I, TRIF, and MyD88-specific siRNAs inhibited TGEV-induced FcRn expression, while no significant change was detected in s TLR2, TLR4 and TLR8-specific siRNAs (Figures 2C,D) . These data suggest that TGEV infection activates FcRn via TLR3, TLR9, TRIF, MyD88, and RIG-I. IPEC-J2 were co-transfected with pRL-TK and FcRn-Luc, followed by alive TGEV or UV-inactivated TGEV (MOI 1). Cells were harvested at 12, 24, 36 or 48 hpi, and the lysates were analyzed by dual-luciferase assay. (B) IPEC-J2 cells were infected with alive TGEV or UV-inactivated TGEV at a MOI of 1. Cells were harvested at 12, 24, 36 or 48 hpi, and the lysates were analyzed by RT-qPCR. (C) IPEC-J2 were infected with alive TGEV at a MOI of 1. Cells were harvested at 12, 24, 36 or 48 hpi, and the lysates were analyzed by Western blot. * p < 0.05, * * p < 0.01.