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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.

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Cytokine expression in ZYM-SIEC02 cells infected with Salmonella typhimurium and LPS.Confluent ZYM-SIEC02 cell monolayers were infected for 4 h with S. Typhimurium or LPS. A. Relative mRNA expression of IL-8 in ZYM-SIEC02 cells were upregulated approximately 2.5-fold within 4 h of S. Typhimurium challenge relative to control, while the cells that stimulated with LPS demonstrated a significant reduction; B. TNF-α mRNA expression was a modest increased by S. Typhimurium stimulation, while the cells that stimulated with LPS demonstrated a significant reduction; C. TLR4 and TLR6 mRNA expression were upregulated following stimulation with LPS. D. IL-8 protein expression in ZYM-SIEC02 cells infected with Salmonella typhimurium and LPS. ZYM-SIEC02 cells were incubated with S. Typhimurium at a MOI of 100∶1 followed by an incubation in media containing gentamicin, and then cell culture supernatants were harvested after 4 h. ZYM-SIEC02 cells were stimulated with LPS, and cells were harvested after 4 h. IL-8 concentrations were determined by ELISA. The data shown are means±SEM of 3 independent experiments. * P<0.05;** P<0.01; *** P<0.001; ns, not significant.
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pone-0110916-g008: Cytokine expression in ZYM-SIEC02 cells infected with Salmonella typhimurium and LPS.Confluent ZYM-SIEC02 cell monolayers were infected for 4 h with S. Typhimurium or LPS. A. Relative mRNA expression of IL-8 in ZYM-SIEC02 cells were upregulated approximately 2.5-fold within 4 h of S. Typhimurium challenge relative to control, while the cells that stimulated with LPS demonstrated a significant reduction; B. TNF-α mRNA expression was a modest increased by S. Typhimurium stimulation, while the cells that stimulated with LPS demonstrated a significant reduction; C. TLR4 and TLR6 mRNA expression were upregulated following stimulation with LPS. D. IL-8 protein expression in ZYM-SIEC02 cells infected with Salmonella typhimurium and LPS. ZYM-SIEC02 cells were incubated with S. Typhimurium at a MOI of 100∶1 followed by an incubation in media containing gentamicin, and then cell culture supernatants were harvested after 4 h. ZYM-SIEC02 cells were stimulated with LPS, and cells were harvested after 4 h. IL-8 concentrations were determined by ELISA. The data shown are means±SEM of 3 independent experiments. * P<0.05;** P<0.01; *** P<0.001; ns, not significant.

Mentions: To further verify the immunomodulatory responses of the ZYM-SIEC02 cell line, we evaluated the expression of the inflammatory factors and chemokines expressed in intestinal epithelial cells that are known to be involved in the immune response to pathogenic infection. ZYM-SIEC02 cells were used to evaluate the effect of S. Typhimurium and LPS on IL8 and TNF-α mRNA expression. As shown in Fig. 8, the expression of IL-8, TNF-α was upregulated following S. Typhimurium challenge and IL8 expression was upregulated approximately 2.5-fold within 4 h of S. Typhimurium challenge relative to control, while TNF-α mRNA expression was a modest increased by S. Typhimurium stimulation. Interestingly, cells that stimulated with LPS demonstrated a significant reduction in IL-8 and TNF-α mRNA expression compared to controls (Fig. 8A, B). To determine if this functional immune response were via TLR4 recognition on intestinal epithelial cells, we choose to detect TLR4 and TLR6 mRNA expression following stimulation with LPS. The result showed that TLR4 and TLR6 were upregulated (Fig. 8C).


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)

Cytokine expression in ZYM-SIEC02 cells infected with Salmonella typhimurium and LPS.Confluent ZYM-SIEC02 cell monolayers were infected for 4 h with S. Typhimurium or LPS. A. Relative mRNA expression of IL-8 in ZYM-SIEC02 cells were upregulated approximately 2.5-fold within 4 h of S. Typhimurium challenge relative to control, while the cells that stimulated with LPS demonstrated a significant reduction; B. TNF-α mRNA expression was a modest increased by S. Typhimurium stimulation, while the cells that stimulated with LPS demonstrated a significant reduction; C. TLR4 and TLR6 mRNA expression were upregulated following stimulation with LPS. D. IL-8 protein expression in ZYM-SIEC02 cells infected with Salmonella typhimurium and LPS. ZYM-SIEC02 cells were incubated with S. Typhimurium at a MOI of 100∶1 followed by an incubation in media containing gentamicin, and then cell culture supernatants were harvested after 4 h. ZYM-SIEC02 cells were stimulated with LPS, and cells were harvested after 4 h. IL-8 concentrations were determined by ELISA. The data shown are means±SEM of 3 independent experiments. * P<0.05;** P<0.01; *** P<0.001; ns, not significant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4206455&req=5

pone-0110916-g008: Cytokine expression in ZYM-SIEC02 cells infected with Salmonella typhimurium and LPS.Confluent ZYM-SIEC02 cell monolayers were infected for 4 h with S. Typhimurium or LPS. A. Relative mRNA expression of IL-8 in ZYM-SIEC02 cells were upregulated approximately 2.5-fold within 4 h of S. Typhimurium challenge relative to control, while the cells that stimulated with LPS demonstrated a significant reduction; B. TNF-α mRNA expression was a modest increased by S. Typhimurium stimulation, while the cells that stimulated with LPS demonstrated a significant reduction; C. TLR4 and TLR6 mRNA expression were upregulated following stimulation with LPS. D. IL-8 protein expression in ZYM-SIEC02 cells infected with Salmonella typhimurium and LPS. ZYM-SIEC02 cells were incubated with S. Typhimurium at a MOI of 100∶1 followed by an incubation in media containing gentamicin, and then cell culture supernatants were harvested after 4 h. ZYM-SIEC02 cells were stimulated with LPS, and cells were harvested after 4 h. IL-8 concentrations were determined by ELISA. The data shown are means±SEM of 3 independent experiments. * P<0.05;** P<0.01; *** P<0.001; ns, not significant.
Mentions: To further verify the immunomodulatory responses of the ZYM-SIEC02 cell line, we evaluated the expression of the inflammatory factors and chemokines expressed in intestinal epithelial cells that are known to be involved in the immune response to pathogenic infection. ZYM-SIEC02 cells were used to evaluate the effect of S. Typhimurium and LPS on IL8 and TNF-α mRNA expression. As shown in Fig. 8, the expression of IL-8, TNF-α was upregulated following S. Typhimurium challenge and IL8 expression was upregulated approximately 2.5-fold within 4 h of S. Typhimurium challenge relative to control, while TNF-α mRNA expression was a modest increased by S. Typhimurium stimulation. Interestingly, cells that stimulated with LPS demonstrated a significant reduction in IL-8 and TNF-α mRNA expression compared to controls (Fig. 8A, B). To determine if this functional immune response were via TLR4 recognition on intestinal epithelial cells, we choose to detect TLR4 and TLR6 mRNA expression following stimulation with LPS. The result showed that TLR4 and TLR6 were upregulated (Fig. 8C).

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