Studies On FcRn-Mediated Transcytosis Of IgG And TGEV Infection Regulates FcRn Expression Via Activation Of NF-κB Signalling

Most pathogens invade organisms through mucosal surfaces. Thus, the mucosal immunity is gaining more attention recent years. It has been well characterized that the major Ig species of mucosal secretions is secretory IgA(SIgA), which is transported across the polarized epithelium by polymeric immunoglobulin receptor(pIgR). However, previous studies have found that a certain level of IgG is also showed on the mucosal secretions and played an important role in the defense of pathogens invasion. Recently, most of the studies about FcRn were focused on the function of human and mice FcRn as well as the regulation mechanism of human and mice FcRn expressions, however, a handful of studies were reported regarding the function of porcine FcRn. Meanwhile, the regulation mechanism of FcRn during the infection of pathogens remains unclear. In this study, we constructed the transwell system in vitro with porcine intestinal epithelial cells(IPEC-J2) and investigated the role of porcine FcRn that mediated bidirectional IgG transport across polarized IPEC-J2 cells. After that, we found that transmissible gastroenteritis virus(TGEV) infection regulating FcRn expression via activation of NF-κB signaling. More details are as follows: 1. The distribution and expression of porcine FcRn on intestinal epithelial cellsWe designed the specific primers based on the sequences of porcine FcRn from the Genbank. Then, we extracted the total RNA from the IPEC-J2 cells and amplified FcRn segment by RT-PCR. Meanwhile, we confirmed the expression of FcRn protein in IPEC-J2 cells by Western blot. Furthermore, we confirmed that FcRn was mainly distributed on the apical side of IPEC-J2 cells and piglets jejunal villi epithelial cells by indirect immunofluorescence. 2. The mechanism of FcRn-mediated bidirectional transcytosis of IgGWe used the IPEC-J2 cells to construct the transwell system, porcine biotin-IgG was added into the transwell system and then detected the level of biotin-IgG in opposite chamber by Western blot. Our results showed that biotin-Ig G could be bidirectional transported in polarized IPEC-J2 cells under 37°C but not 4°C. These results suggested that Ig G was bidirectional transported across the epithelial barrier by transcellular pathway. In order to further detect whether transcytosis of IgG was receptor-mediated and specific for FcRn. We added Protein A or unlabeled IgG as inhibitors during the transcytosis of biotin-IgG. The results showed that the transcytosis of biotin-IgG was inhibited after adding protein A or unlabeled IgG. The Laser Scanning Confocal Microscope results also suggested that FcRn bound the IgG within the cells and medicated the transcytosis of IgG. Furthermore, the co-immunoprecipitation showed FcRn binds to IgG at pH<6.5 but releases them at pH>7.0 which indicated that the binding of FcRn and IgG is pH dependent. The TCID50 assay results showed that FcRn-transcytosed viral-specific IgG reduced TGEV yield from the lumenal directiondue to the transcytosis of IgG by FcRnOur results indicated that Fc Rn-dependent bidirectional IgG transport across the intestinal epithelium which plays a critical role in the acquisition of humoral immunity in early life and in host defense at mucosal surfaces. 3. TGEV infection regulates FcRn expression via activation of NF-κB signallingNF-κB is one of the extensive expressed transcription factors in cells, which is mainly related to the immune response, inflammatory response and cell differentiation related gene transcription. Our studies found that NF-κB pathway was activated by TGEV infection. Firstly, we detected the luciferase activity of NF-κB luciferase report plasmid by TGEV infection. The results showed the NF-κB luciferase activity increased after the TGEV infection. We further transfected pEGFP-p65 plasmid into the IPEC-J2 cells after TGEV infection. Our results showed that p65 rapidly translocated to the nucleus by the Laser Scanning Confocal Microscope. We infected IPEC-J2 cells with TGEV and then detected the total mRNA and protein expression level of FcRn by real time RT-PCR and Western blot, respectively. The results indicated that the expression of FcRn was up-regulated by TGEV infection. We analyzed the transcriptional binding sites of FcRn promoter regions with online softwares and found both NF-κB and MAPK transcription factor binding sites. In order to determine which signaling pathway plays the important role in the regulation of FcRn expression, we treated cells with the NF-κB inhibitor or MAPK inhibitors and then detected the expression level of Fc Rn. The results show that FcRn expression was inhibited by NF-κB inhibitor, which further supported the conclusion that the up-regulation of FcRn expression by TGEV infection is related to NF-κB pathway. Meanwhile, according to the transcriptional binding sites of FcRn promoter regions, we constructed 9 luciferase reporting plasmids of FcRn promoter. The luciferase reporting results showed that the NF-κB sensitive region is located in the sequence between-1381 and-208 of the FcRn promoter. Furthermore, we confirmed the binding sites of transcription factor p65 for promoter of FcRn by Chromatin Immunoprecipitation(CHIP) and Electrophoretic Mobility Shift Assay(EMSA).