Limits...
Characteristic and functional analysis of a newly established porcine small intestinal epithelial cell line.

Wang J, Hu G, Lin Z, He L, Xu L, Zhang Y - PLoS ONE (2014)

Bottom Line: TLR4, TLR6 and IL-6 mRNA expression were upregulated following stimulation with LPS, ZYM-SIEC02 cells were hyporeponsive to LPS with respect to IL-8 mRNA expression and secretion.TNFα mRNA levels were significantly decreased after LPS stimulation and TNF-α secretion were not detected challenged with S.Typhimurium neither nor LPS.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.

ABSTRACT
The mucosal surface of intestine is continuously exposed to both potential pathogens and beneficial commensal microorganisms. Recent findings suggest that intestinal epithelial cells, which once considered as a simple physical barrier, are a crucial cell lineage necessary for maintaining intestinal immune homeostasis. Therefore, establishing a stable and reliable intestinal epithelial cell line for future research on the mucosal immune system is necessary. In the present study, we established a porcine intestinal epithelial cell line (ZYM-SIEC02) by introducing the human telomerase reverse transcriptase (hTERT) gene into small intestinal epithelial cells derived from a neonatal, unsuckled piglet. Morphological analysis revealed a homogeneous cobblestone-like morphology of the epithelial cell sheets. Ultrastructural indicated the presence of microvilli, tight junctions, and a glandular configuration typical of the small intestine. Furthermore, ZYM-SIEC02 cells expressed epithelial cell-specific markers including cytokeratin 18, pan-cytokeratin, sucrase-isomaltase, E-cadherin and ZO-1. Immortalized ZYM-SIEC02 cells remained diploid and were not transformed. In addition, we also examined the host cell response to Salmonella and LPS and verified the enhanced expression of mRNAs encoding IL-8 and TNF-α by infection with Salmonella enterica serovars Typhimurium (S. Typhimurium). Results showed that IL-8 protein expression were upregulated following Salmonella invasion. TLR4, TLR6 and IL-6 mRNA expression were upregulated following stimulation with LPS, ZYM-SIEC02 cells were hyporeponsive to LPS with respect to IL-8 mRNA expression and secretion. TNFα mRNA levels were significantly decreased after LPS stimulation and TNF-α secretion were not detected challenged with S. Typhimurium neither nor LPS. Taken together, these findings demonstrate that ZYM-SIEC02 cells retained the morphological and functional characteristics typical of primary swine intestinal epithelial cells and thus provide a relevant in vitro model system for future studies on porcine small intestinal pathogen-host cell interactions.

Show MeSH

Related in: MedlinePlus

Inhibition of LPS-mediated response.ZYM-SIEC02 cells were stimulated with the indicated dose of E.coli 055:B55 LPS in the presence or absence of polymyxin B sulfate(Sigma) for 3 hours. A. Polymyxin B increased TNF-α mRNA expression when the concentration of LPS was lower than 250 ng/ml. B. Polymyxin B prevented the increase in IL-6 mRNA expression. The data shown are means±SEM of 3 independent experiments. * P<0.05;** P<0.01; *** P<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4206455&req=5

pone-0110916-g009: Inhibition of LPS-mediated response.ZYM-SIEC02 cells were stimulated with the indicated dose of E.coli 055:B55 LPS in the presence or absence of polymyxin B sulfate(Sigma) for 3 hours. A. Polymyxin B increased TNF-α mRNA expression when the concentration of LPS was lower than 250 ng/ml. B. Polymyxin B prevented the increase in IL-6 mRNA expression. The data shown are means±SEM of 3 independent experiments. * P<0.05;** P<0.01; *** P<0.001.

Mentions: Interestingly, antagonism of TLR4 signaling with polymyxin B prevented the increase in IL-6 mRNA expression but increased TNF-α mRNA expression when the concentration of LPS was lower than 250 ng/ml (Fig. 9A). As expected, ZYM-SIEC02 cells responded to LPS in a dose-dependent manner and polymyxin B blocked LPS recognition by host cells inhibited this response (Fig. 9B).


Characteristic and functional analysis of a newly established porcine small intestinal epithelial cell line.

Wang J, Hu G, Lin Z, He L, Xu L, Zhang Y - PLoS ONE (2014)

Inhibition of LPS-mediated response.ZYM-SIEC02 cells were stimulated with the indicated dose of E.coli 055:B55 LPS in the presence or absence of polymyxin B sulfate(Sigma) for 3 hours. A. Polymyxin B increased TNF-α mRNA expression when the concentration of LPS was lower than 250 ng/ml. B. Polymyxin B prevented the increase in IL-6 mRNA expression. The data shown are means±SEM of 3 independent experiments. * P<0.05;** P<0.01; *** P<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206455&req=5

pone-0110916-g009: Inhibition of LPS-mediated response.ZYM-SIEC02 cells were stimulated with the indicated dose of E.coli 055:B55 LPS in the presence or absence of polymyxin B sulfate(Sigma) for 3 hours. A. Polymyxin B increased TNF-α mRNA expression when the concentration of LPS was lower than 250 ng/ml. B. Polymyxin B prevented the increase in IL-6 mRNA expression. The data shown are means±SEM of 3 independent experiments. * P<0.05;** P<0.01; *** P<0.001.
Mentions: Interestingly, antagonism of TLR4 signaling with polymyxin B prevented the increase in IL-6 mRNA expression but increased TNF-α mRNA expression when the concentration of LPS was lower than 250 ng/ml (Fig. 9A). As expected, ZYM-SIEC02 cells responded to LPS in a dose-dependent manner and polymyxin B blocked LPS recognition by host cells inhibited this response (Fig. 9B).

Bottom Line: TLR4, TLR6 and IL-6 mRNA expression were upregulated following stimulation with LPS, ZYM-SIEC02 cells were hyporeponsive to LPS with respect to IL-8 mRNA expression and secretion.TNFα mRNA levels were significantly decreased after LPS stimulation and TNF-α secretion were not detected challenged with S.Typhimurium neither nor LPS.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.

ABSTRACT
The mucosal surface of intestine is continuously exposed to both potential pathogens and beneficial commensal microorganisms. Recent findings suggest that intestinal epithelial cells, which once considered as a simple physical barrier, are a crucial cell lineage necessary for maintaining intestinal immune homeostasis. Therefore, establishing a stable and reliable intestinal epithelial cell line for future research on the mucosal immune system is necessary. In the present study, we established a porcine intestinal epithelial cell line (ZYM-SIEC02) by introducing the human telomerase reverse transcriptase (hTERT) gene into small intestinal epithelial cells derived from a neonatal, unsuckled piglet. Morphological analysis revealed a homogeneous cobblestone-like morphology of the epithelial cell sheets. Ultrastructural indicated the presence of microvilli, tight junctions, and a glandular configuration typical of the small intestine. Furthermore, ZYM-SIEC02 cells expressed epithelial cell-specific markers including cytokeratin 18, pan-cytokeratin, sucrase-isomaltase, E-cadherin and ZO-1. Immortalized ZYM-SIEC02 cells remained diploid and were not transformed. In addition, we also examined the host cell response to Salmonella and LPS and verified the enhanced expression of mRNAs encoding IL-8 and TNF-α by infection with Salmonella enterica serovars Typhimurium (S. Typhimurium). Results showed that IL-8 protein expression were upregulated following Salmonella invasion. TLR4, TLR6 and IL-6 mRNA expression were upregulated following stimulation with LPS, ZYM-SIEC02 cells were hyporeponsive to LPS with respect to IL-8 mRNA expression and secretion. TNFα mRNA levels were significantly decreased after LPS stimulation and TNF-α secretion were not detected challenged with S. Typhimurium neither nor LPS. Taken together, these findings demonstrate that ZYM-SIEC02 cells retained the morphological and functional characteristics typical of primary swine intestinal epithelial cells and thus provide a relevant in vitro model system for future studies on porcine small intestinal pathogen-host cell interactions.

Show MeSH
Related in: MedlinePlus