A Preliminary Study On The Function Of Intraepithelial Lymphocytes In Porcine Small Intestine Against PEDV Infection

The intestinal mucosal immune system constitutes the first barrier against intestinal pathogen infection in the body,which exerts an important role in maintaining the integrity of the structure and function of the porcine small intestinal mucosa.Intraepithelial lymphocytes(IELs)are located in and at the base of intestinal epithelial cells(IECs).They are the most preferentially immune cells in the small intestinal mucosal immune system that contact with intestinal microbes and foreign antigens,which are vital in maintain the integrity of the intestinal epithelial barrier and resistance to pathogenic infection.Porcine epidemic diarrhea virus(PEDV)is one of the important pathogens causing diarrhea in neonatal piglets,and the main target organ for infection and pathogenicity is the small intestinal epithelium.Considering the pathogenic characteristics of PEDV,the function of IELs located between IECs requires an in-depth exploration against PEDV.In this study,we first performed challenging test in neonatal piglets to examine the influence of PEDV infection on the distribution and migration of IELs.A co-culture model of porcine IELs and IECs was further generated,aiming to investigate the specific mechanism underlying the transepithelial migration of IELs after PEDV infection.Finally,in vitro and in vivo experiments were conducted to validate the protective role of IELs in PEDV infection,and preliminarily clarified the anti-viral mechanism of IELs.The main contents of this study were divided into the following three sections.1.The effect of PEDV infection on the distribution of IELs in pigletsSince T lymphocytes account for over 90%of the immune cells in IELs of piglets,immunohistochemical staining of the anti-pig CD3 monoclonal antibody was performed in this study to reveal the distribution of IELs in the intestinal mucosa of newborn piglets at day 0 and piglets at 1 month old.It is shown that the number of IELs in the small intestinal mucosa of piglets at 1 month old was significantly higher than that of newborn piglets(p<0.01),and IELs were mainly distributed in the jejunum and ileum of piglets.Subsequently,oral infection of PEDV was performed in piglets at 1 month old,and the distribution of IELs in each intestinal segment at 48 h was examined.Immunohistochemical results showed that the number of IELs distributed in the small intestine after PEDV infection significantly increased(p<0.01).Finally,the in-situ perfusion model in intestines of piglets was generated to explore the characteristics of PEDV-induced migration of IELs.It is shown that PEDV significantly triggered the migration of IELs into the intestinal epithelial cells after 1 h of PEDV injection,and the number of IELs entering the interepithelial cells significantly increased after 3 hours of perfusion(p<0.01).Notably,some IELs could pass across the intestinal epithelial cells and enter the intestinal lumen.Immunohistochemical staining of PCNA was performed to identify cell proliferation,and it is found that IELs that migrated across the epithelium had stronger proliferative activity.The above results indicated that PEDV could recruit IELs and stimulate them to migrate to the infected site after PEDV infection.The specific mechanism of regulating IELs migration requires further explorations.2.The specific mechanism of PEDV infection in regulating the migration of IELsThe co-culture model of IELs and intestinal epithelial cells(IECs)was first generated.The results of transepithelial electrical resistance(TEER)and immunofluorescence staining of the tight junction protein showed that co-culture of IEL/IEC did not affect the barrier structure of IECs.Furthermore,in the co-culture model of IEL/IEC,IECs infected with PEDV could induce the transepithelial migration of IELs.Finally,transcription profiles of three host cell lines infected with PEDV(Vero E6 and IPEC-J2)and small intestinal mucosa of piglets were analyzed.The chemokine CCL2 was identified,and its function in triggering PEDV-induced migration of IELs was validated.It is shown that both in vitro and in vivo infection of PEDV could significantly promote the secretion of the chemokine CCL2 by IECs(p<0.01).CCL2 treatment in the co-culture model of IEL/IEC consistently induced transepithelial migration of IELs.As expected,the inductive role of PEDV infection in IELs was significantly inhibited by the preconditioning of the inhibitor of CCR2,which was the ligand of CCL2.It is concluded that PEDV-infected IECs induced migration of IELs to the infected cells by secreting the chemokine CCL2.However,whether IELs exerted anti-infection function after they were migrated surrounding infected cells,and the underlying mechanism remain unclear.3.The mechanism of IELs against PEDV infectionTo examine the potential function of IELs in influencing PEDV-infected IECs,IELs were co-cultured with PEDV-infected IECs.It is shown that IELs did not significantly influence the infection level of PEDV.However,activated IELs by 1-h stimulation of inactivated PEDV in advance significantly inhibited PEDV infection in IECs(p<0.01).Further results showed that either activated IELs were co-cultured with PEDV-infected IECs or not could inhibit PEDV infection in host cells,with the similar inhibitory rate.Therefore,we speculated that activated IELs may exert resistance through their secretion products.Since the proportion of CD8~+T lymphocytes in isolated and purified IELs in vitro was nearly 60%,the cytotoxic effect of activated IELs was examined.It is shown that IELs could secrete perforin,which contributed to kill PEDV-infected IECs,induce apoptosis of infected cells and release abundant lactate dehydrogenase.Besides the cytotoxic effect,IELs could also upregulate multiple interferon-stimulating genes in PEDV-infected IECs by secreting interferon-γ(IFN-γ),thus exerting the anti-viral effect.Briefly,IFN-γsecreted by IELs was significantly upregulated in IELs co-cultured with PEDV-infected IECs(p<0.01).After the induction of the antibody to antagonize IFN-γin the co-culture model,the anti-viral ability of IELs was remarkably inhibited.Moreover,the inhibited interferon pathway in PEDV-infected IECs was significantly activated.The above data demonstrated that IELs recognized and inhibited PEDV infection in IECs only after being activated by the inactivated PEDV.Activated IELs not only directly killed PEDV-infected IECs through cell cytotoxicity effect,but also exerted the anti-infection effect through activating the innate immune response of IEPCs by secreting IFN-γ.In the present study,we explored the role of IELs in piglets in resisting PEDV infection by generating several in vivo and in vitro models.Our findings not only proved the communication mechanism between epithelial cells and IELs after PEDV infection,but also preliminarily clarified the anti-viral activity of IELs in piglets and the underlying mechanism.The results of this study contribute to further reveal the role and function of IELs in the porcine intestinal mucosal immunity,and provide theoretical references for the effective prevention and control of the infection of porcine intestinal diarrhea pathogens and the disease onset.