Persistent Transmissible Gastroenteritis Virus Infection Enhances Enterotoxigenic Escherichia coli K88 Adhesion by Promoting Epithelial-Mesenchymal Transition in Intestinal Epithelial Cells

Abstract

Transmissible gastroenteritis virus (TGEV) is a coronavirus characterized by diarrhea and high morbidity rates, and the mortality rate is 100% in piglets less than 2 weeks old. Pigs infected with TGEV often suffer secondary infection by other pathogens, which aggravates the severity of diarrhea, but the mechanisms remain unknown. Here, we hypothesized that persistent TGEV infection stimulates the epithelial-mesenchymal transition (EMT), and thus enterotoxigenic (ETEC) can more easily adhere to generating cells. Intestinal epithelial cells are the primary targets of TGEV and ETEC infections. We found that TGEV can persistently infect porcine intestinal columnar epithelial cells (IPEC-J2) and cause EMT, consistent with multiple changes in key cell characteristics. Infected cells display fibroblast-like shapes; exhibit increases in levels of mesenchymal markers with a corresponding loss of epithelial markers; have enhanced expression levels of interleukin-1β (IL-1β), IL-6, IL-8, transforming growth factor β (TGF-β), and tumor necrosis factor alpha (TNF-α) mRNAs; and demonstrate increases in migratory and invasive behaviors. Additional experiments showed that the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways via TGF-β is critical for the TGEV-mediated EMT process. Cellular uptake is also modified in cells that have undergone EMT. TGEV-infected cells have higher levels of integrin α5 and fibronectin and exhibit enhanced ETEC K88 adhesion. Reversal of EMT reduces ETEC K88 adhesion and inhibits the expression of integrin α5 and fibronectin. Overall, these results suggest that TGEV infection induces EMT in IPEC-J2 cells, increasing the adhesion of ETEC K88 in the intestine and facilitating dual infection. Transmissible gastroenteritis virus (TGEV) causes pig diarrhea and is often followed by secondary infection by other pathogens. In this study, we showed that persistent TGEV infection induces an EMT in porcine intestinal columnar epithelial cells (IPEC-J2) and enhances the adhesion of the secondary pathogen ETEC K88. Additional experiments suggest that integrin α5 and fibronectin play an important role in TGEV-enhanced ETEC K88 adhesion. Reversal of EMT reduces the expression of integrin α5 and fibronectin and also reduces ETEC K88 adhesion. We conclude that TGEV infection triggers EMT and facilitates dual infection. Our results provide new insights into secondary infection and suggest that targeted anti-EMT therapy may have implications for the prevention and treatment of secondary infection.

Porcine transmissible gastroenteritis virus nonstructural protein 2 contributes to inflammation via NF-κB activation

“…Considering that the small intestine was the main target organ attacked by TGEV and that IPEC-J2 cells, a spontaneously immortalized cell line from the porcine intestine, are likely much more representative of porcine physiology and the true state of virus infection in animals [25,26], IPEC-J2 cells were used in current study in addition to ST cells. Previous studies showed that IPEC-J2 cells infected with TGEV displayed increased expression levels of proinflammatory cytokines, including IL-1β, IL-6, IL-8, TGF-β, and TNF-α [27,28]. The current study further demonstrated that TGEV could activate NF-κB in IPEC-J2 cells, by using a luciferase reporter gene assay and IFA.…”

“…Even with the uncertainty regarding the effect of the ZnF, our analyzes, which were performed using truncated mutants allowed us to map the key domain responsible for activating NF-κB to the 120 amino acids of the N-terminal end of the Nsp2 protein. Although the above experiments were conducted in vitro, IPEC-J2 cells are morphologically differentiated cells derived from the small intestine of young piglets, which can be used as a well-defined in vitro model with comparable intestinal physiology [25][26][27][28]. Based on these results, we predicted that Nsp2 could promote intestinal inflammation in TGEV-infected piglets, and the Nsp2-induced inflammation may cause damage of intestine barrier which would benefit the spread of TGEV to deeper intestine tissue.…”

Susceptibility of porcine IPEC-J2 intestinal epithelial cells to infection with porcine deltacoronavirus (PDCoV) and serum cytokine responses of gnotobiotic pigs to acute infection with IPEC-J2 cell culture-passaged PDCoV

“…Based on our data, IPEC-J2 cells are susceptible to infection with PDCoV, like for the other enteric CoVs, PEDV and TGEV (Guo et al, 2016;Lin et al, 2017;Shi et al, 2017;Xia et al, 2017;Zhao et al, 2014). Compared with LLC-PK or ST cells, our previous study revealed no or less susceptibility of IPEC-J2 cells to PDCoV infection using feces or intestinal contents or tissues of PDCoV-infected pigs (Hu et al, 2015).…”

“…Because of the significant physiologic and morphologic similarities to enterocytes in vivo, this primary cell line has been used increasingly to characterize the interactions of intestinal epithelial cells with enteric bacteria and viruses in vitro. IPEC-J2 cells have been infected with porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) (Lin et al, 2017;Shi et al, 2017;Xia et al, 2017;Zhao et al, 2014). Compared with PEDV-infected Vero (African green monkey kidney) cells (Guo et al, 2016), the proteomic data from PEDV-infected IPEC-J2 cells appeared to better coincide with the physiologic and pathologic outcomes in PEDV-infected enterocytes in vivo (Lin et al, 2017).…”

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

“…TNF-α and IL-1β can activate NF-κB to up-regulate the expression of human FcRn, which enhances FcRn-mediated IgG transport (Liu et al, 2007). TGEV infection was also found to induce EMT via TGFβ in IPEC-J2 cells (Xia et al, 2017). The aim of this study was to identify the TGEV-encoded proteins involved in inducing FcRn production.…”