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Nucleotide Binding Oligomerization Domain 1 Is an Essential Signal Transducer in Human Epithelial Cells Infected with Helicobacter pylori That Induces the Transepithelial Migration of Neutrophils.

Kim BJ, Kim JY, Hwang ES, Kim JG - Gut Liver (2015)

Bottom Line: Cytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls.NF-κB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain.NF-κB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Chung-Ang University College of Medicine, Seoul, Korea.

ABSTRACT

Background/aims: The cytosolic host protein nucleotide binding oligomerization domain 1 (Nod1) has emerged as a key pathogen recognition molecule for innate immune responses in epithelial cells. The purpose of the study was to elucidate the mechanism by which Helicobacter pylori infection leads to transepithelial neutrophil migration in a Nod1-mediated manner.

Methods: Human epithelial cell lines AGS and Caco-2 were grown and infected with H. pylori. Interleukin (IL)-8 mRNA expression and IL-8 secretion were assessed, and nuclear factor κB (NF-κB) activation was determined. Stable transfections of AGS and Caco-2 cells with dominant negative Nod1 were generated. Neutrophil migration across the monolayer was quantified.

Results: Cytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls. NF-κB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain. NF-κB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls. The transepithelial migration of neutrophils in DN-Nod1 stably transfected cells was reduced compared with that in controls.

Conclusions: Signaling through Nod1 plays an essential role in neutrophil migration induced by the upregulated NF-κB activation and IL-8 expression in H. pylori-infected human epithelial cells.

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Related in: MedlinePlus

Interleukin (IL)-8 mRNA expression and IL-8 production in AGS cells infected with cytotoxin-associated gene pathogenicity island (cagPAI) (+) Helicobacter pylori (HP99). (A) As assayed by qualitative reverse transcription-polymerase chain reaction (RT-PCR), the incubation of AGS cells with HP99 resulted in increased IL-8 mRNA expression compared with that in controls by 24 hours after infection. (B) IL-8 mRNA expression in response to infection with HP99 was significantly increased at 4 hours after infection, as determined by real-time RT-PCR. (C) As determined by enzyme-linked immunosorbent assay, infected cells produced IL-8 at a mean level in excess of 500 pg/mL. The error bars indicate the standard error of the mean of triplicate samples, which were representative of three independent experiments.
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f2-gnl-09-358: Interleukin (IL)-8 mRNA expression and IL-8 production in AGS cells infected with cytotoxin-associated gene pathogenicity island (cagPAI) (+) Helicobacter pylori (HP99). (A) As assayed by qualitative reverse transcription-polymerase chain reaction (RT-PCR), the incubation of AGS cells with HP99 resulted in increased IL-8 mRNA expression compared with that in controls by 24 hours after infection. (B) IL-8 mRNA expression in response to infection with HP99 was significantly increased at 4 hours after infection, as determined by real-time RT-PCR. (C) As determined by enzyme-linked immunosorbent assay, infected cells produced IL-8 at a mean level in excess of 500 pg/mL. The error bars indicate the standard error of the mean of triplicate samples, which were representative of three independent experiments.

Mentions: As shown in Fig. 2, incubation of AGS cells with HP99 resulted in the increased expression of IL-8 mRNA compared with those in controls by 4 hours after infection. IL-8 mRNA expression and secretion of IL-8 protein into the culture medium were confirmed by the real-time RT-PCR and by ELISA. As assayed by real-time PCR, IL-8 mRNA expression in AGS cells in response to infection with HP99 was significantly increased within the first 4 hours after infection (Fig. 2B). In addition, infected cells responded to produce a mean level in excess of 500 pg/mL of IL-8 by ELISA (Fig. 2C).


Nucleotide Binding Oligomerization Domain 1 Is an Essential Signal Transducer in Human Epithelial Cells Infected with Helicobacter pylori That Induces the Transepithelial Migration of Neutrophils.

Kim BJ, Kim JY, Hwang ES, Kim JG - Gut Liver (2015)

Interleukin (IL)-8 mRNA expression and IL-8 production in AGS cells infected with cytotoxin-associated gene pathogenicity island (cagPAI) (+) Helicobacter pylori (HP99). (A) As assayed by qualitative reverse transcription-polymerase chain reaction (RT-PCR), the incubation of AGS cells with HP99 resulted in increased IL-8 mRNA expression compared with that in controls by 24 hours after infection. (B) IL-8 mRNA expression in response to infection with HP99 was significantly increased at 4 hours after infection, as determined by real-time RT-PCR. (C) As determined by enzyme-linked immunosorbent assay, infected cells produced IL-8 at a mean level in excess of 500 pg/mL. The error bars indicate the standard error of the mean of triplicate samples, which were representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4413970&req=5

f2-gnl-09-358: Interleukin (IL)-8 mRNA expression and IL-8 production in AGS cells infected with cytotoxin-associated gene pathogenicity island (cagPAI) (+) Helicobacter pylori (HP99). (A) As assayed by qualitative reverse transcription-polymerase chain reaction (RT-PCR), the incubation of AGS cells with HP99 resulted in increased IL-8 mRNA expression compared with that in controls by 24 hours after infection. (B) IL-8 mRNA expression in response to infection with HP99 was significantly increased at 4 hours after infection, as determined by real-time RT-PCR. (C) As determined by enzyme-linked immunosorbent assay, infected cells produced IL-8 at a mean level in excess of 500 pg/mL. The error bars indicate the standard error of the mean of triplicate samples, which were representative of three independent experiments.
Mentions: As shown in Fig. 2, incubation of AGS cells with HP99 resulted in the increased expression of IL-8 mRNA compared with those in controls by 4 hours after infection. IL-8 mRNA expression and secretion of IL-8 protein into the culture medium were confirmed by the real-time RT-PCR and by ELISA. As assayed by real-time PCR, IL-8 mRNA expression in AGS cells in response to infection with HP99 was significantly increased within the first 4 hours after infection (Fig. 2B). In addition, infected cells responded to produce a mean level in excess of 500 pg/mL of IL-8 by ELISA (Fig. 2C).

Bottom Line: Cytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls.NF-κB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain.NF-κB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Chung-Ang University College of Medicine, Seoul, Korea.

ABSTRACT

Background/aims: The cytosolic host protein nucleotide binding oligomerization domain 1 (Nod1) has emerged as a key pathogen recognition molecule for innate immune responses in epithelial cells. The purpose of the study was to elucidate the mechanism by which Helicobacter pylori infection leads to transepithelial neutrophil migration in a Nod1-mediated manner.

Methods: Human epithelial cell lines AGS and Caco-2 were grown and infected with H. pylori. Interleukin (IL)-8 mRNA expression and IL-8 secretion were assessed, and nuclear factor κB (NF-κB) activation was determined. Stable transfections of AGS and Caco-2 cells with dominant negative Nod1 were generated. Neutrophil migration across the monolayer was quantified.

Results: Cytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls. NF-κB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain. NF-κB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls. The transepithelial migration of neutrophils in DN-Nod1 stably transfected cells was reduced compared with that in controls.

Conclusions: Signaling through Nod1 plays an essential role in neutrophil migration induced by the upregulated NF-κB activation and IL-8 expression in H. pylori-infected human epithelial cells.

Show MeSH
Related in: MedlinePlus