Immortalization Of Swine Intestinal Epithelial Cells And Effects On The Cells Infected With Porcine Rotavirus

Porcine rotavirus (PRV) are the leading etiologic agent of viral gastroenteritis in pigletand commonly characterized of diarrhea. Rotavirus can bind to a variety of cell lines butproductive infection is restricted to only a sub of renal or intestinal epithelial cell origin. Thestudies of pathogenesis on porcine rotavirus are limited by the lack of proper porcineintestinal epithelil cell lines. In vitro, cells will stop dividing after a finite number ofpopulation doublings and enter replicative senescence. Primary swine intestinal epithelialcells (pSIECs) as well as other primary cells derived from normal tissue will stop dividingafter8-13passages. In the present study, we address that newly established SIEC02cell linepresented normal epithelial cell morphological and functional characteristics as primarySIECs. This cell line can effectively response to E.coli, Salmonella typhimurium and FBS andparticipate in the cell immune response. The SIEC02cell line is the first to exhibit any signsof CPE following PRV infection. Indexes related with apoptosis were used to determine cellapoptosis. Bax was activated by NSP4protein and NSP4viroporins activity; NSP4and NSP4viroporins-induced apoptosis were partly depended on caspase activity. The results were asfollows:1. The estalishment of immortalized swine intestinal epithelial cell line(1) Eukaryotic expression plasmid pCI-neo-hTERT was transfected into primary porcineintestinal epithelial cells to immortalize the cells by lipofection.The immortalized swineintestinal epithelial cell line was named SIEC02.Newly established epithelial cells (SIEC02)were detected significantly higher protein expression of hTERT and activity of telomerase ascompare to primary cells, suggesting the lifespan of pSIECs was successfully extended.(2) SIEC02cell line presented normal epithelial cell morphology and ultrastructure asprimary SIECs. Cells were stained with antibodies that recognized marker proteins fordifferent cell phenotypes, including cytokeratin, tight junction and intermediate filamentsproteins. The result showed that SIEC02cells were positively stained for cytokeratin18,pan-cytokeratin, occludin and vimentin. This result suggests that the SIEC02cell line has anepithelial phenotype, but co-expresses vimentin. Western blot analysis confirmed theexpression of epithelial characteristic markers in cultured cells, the protein of cytokeratin18, E-cadherin, ZO-1, occludin, sucrase-isomaltase and villin were stably expressed in SIEC02cells. the cell line presented a greater proliferation activity and a lower apoptosis rate than thatof primary swine intestinal epithelial cells. The optical concetration of FBS was5%, and ITScould partially substitute for the mitogenic effects of FBS and was capable of sustainingproliferation of SIEC02cells.We observed a normal complement of chromosomes in SIEC02swith a modal chromosome number of38. The newly established SIEC02cell line wasanchorage-independent, serum dependent, monolayer, with the characteristics of contactinhibition and was not tumorigenic in nude mice, indicating that this cell line can be used inaddition to pSIECs. Thus, the immortalization via the telomerase activity activated by hTERTis closer to the physiological process.2. The immunological function of immortalized swine intestinal epithelial cells(1) The results of adhesion and invasion assays suggest that E.coli k88and C83903couldadhere to SIEC02cells effectively. Invasion efficiency of Salmonella typhimurium in theSIEC02cell line and pSIECs were24.3%and30.55%, respectively, suggesting that bothpSIECs and SIEC02cell can engulf Salmonella.(2) Salmonella typhimurium infection caused an upregulated mRNA expression of IL-8and TNF-α. Interestingly, cells that stimulated with LPS demonstrated a significant reductionin IL-8and TNF-α mRNA expression compared to controls, but TLR4and TLR6wereupregulated in LPS stimulated SIEC02s. In order to determine if challenged SIEC02cellsexhibit differential protein expression during an inflammatory response, IL-8were evaluatedby ELISA. Exposure to S.T. resulted in an initial trend toward an increase in IL8secretion.However, the lack of response of swine intestinal epithelial cell in vitro to stimulatewith LPS was found in terms of IL8production.(3) The results of the FBS (with or without) effect on SIEC02cells showd that the cellswere capable of expressing the genes encoding CCL20, IL-8, IL-1α, IL-15, IL-18, MyD88,NF-κB, NOD2, pPGRP-L2, TLR4, TLR7, TNF-α and the epithelial group were CD47,CLDN3, CLDN4, KRT8, MUC1, OCLN, VILLN, CD58. The result revealed that severalselected immune genes up-regulated in the medium serum-free (TLR2,TLR3, TLR4, TLR6,TLR9, NOD2, OCLN, PGRP-L1, TGFβ1,IL-7, CCL2,GM-CSF, MyD88, NF-Кb, TNF-α,IL-8,IL-1β) and TLR1,TLR5, TLR7, TLR8, TLR10, NOD1, IL-18, IL-2, Caspase1, IL-15,PGRP-L2, CCL20, FCGR3β, IL-10, IL-12p40were down-regulated in the same cell medium.Epithelial cell marker gene were also enhanced in the medium with FBS (MUC1, CLDN3,CD47, ICAM1,Keratin8). No significant changes were observed for IL-6, TLR4, TLR6,IL-15, Villin, IL-2, IL-1a between the two condition. 3. Immortalized porcine intestinal epithelial cell cultures susceptible to porcinerotavirus infection(1) Light microscope observed the cytopathic changes in cultured porcine intestinalepithelial cells during virus invasion. Cellular degradation and viral-induced cytolysis werevary between different porcine rotavirus strains.(2) Following infection with porcine rotavirus, the cell cultures contained viral protein at16h post-infection as detected by direct immunofluorescence. Rotavirus infection does notlead to a time-dependent apoptosis but is MOI-dependent.4. The subcellular localization of porcine rotavirus NSP4and the effect to apoptosisof SIEC02cellsNSP4and NSP4viroporin were localized in the endoplasmic reticulum (ER). Westernblot anysis showed that rotavirus induced apoptosis may contribute to Bax activation viaNSP4and viroporins. NSP4viroporin-induced apoptosis at least partly depended on thecaspase activation.In conclusion, the lifespan of pSIECs was successfully extended and immortalized swinrintestinal epithelial cells remain the normal biological characteristics and immunologicalfunction, suggesting that the immortalization via the telomerase activity activated by hTERTis closer to the physiological process. Cytopathic effect was found in SIEC02cells followinginfection with porcine rotavirus, NSP4and NSP4viroporins localized in ER.NSP4viroporin-induced apoptosis at least partly depended on the caspase activation.