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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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Interleukin (IL)-8 mRNA expression and IL-8 production in dominant-negative nucleotide binding oligomerization domain 1 (DN-Nod1) stably transfected AGS cells infected with HP99. (A) IL-8 mRNA expression in DN-Nod1 stably transfected AGS cells was reduced compared with that in the controls. (B) At 4 hours after infection, IL-8 mRNA expression in DN-Nod1 stably transfected AGS cells was reduced compared with the control vector-transfected cells, as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). (C) IL-8 secretion by the DN-Nod1 stably transfected AGS cells was reduced compared with the control vector-transfected cells. (D) IκB-α degradation and IκB-α phosphorylation were reduced in the DN-Nod1 stably transfected Caco-2 cells compared with the controls. (E) Nuclear factor κB (NF-κB) binding in the DN-Nod1 stably transfected Caco-2 cells was reduced compared with that in the controls. The error bars indicate the standard error of the mean of triplicate samples, which were representative of three independent experiments. Control vector, pcDNA3.
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f5-gnl-09-358: Interleukin (IL)-8 mRNA expression and IL-8 production in dominant-negative nucleotide binding oligomerization domain 1 (DN-Nod1) stably transfected AGS cells infected with HP99. (A) IL-8 mRNA expression in DN-Nod1 stably transfected AGS cells was reduced compared with that in the controls. (B) At 4 hours after infection, IL-8 mRNA expression in DN-Nod1 stably transfected AGS cells was reduced compared with the control vector-transfected cells, as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). (C) IL-8 secretion by the DN-Nod1 stably transfected AGS cells was reduced compared with the control vector-transfected cells. (D) IκB-α degradation and IκB-α phosphorylation were reduced in the DN-Nod1 stably transfected Caco-2 cells compared with the controls. (E) Nuclear factor κB (NF-κB) binding in the DN-Nod1 stably transfected Caco-2 cells was reduced compared with that in the controls. The error bars indicate the standard error of the mean of triplicate samples, which were representative of three independent experiments. Control vector, pcDNA3.

Mentions: To investigate whether Nod1 might be involved in cagPAI-dependent recognition of H. pylori, we established Nod1-deficient cell lines by stable transfection of DN-Nod1 construct into AGS cells. To assay IL-8 mRNA expression in DN-Nod1 stably transfected AGS cells, IL-8 mRNA levels were determined by qualitative PCR. As a result, IL-8 mRNA expression in these cells were reduced compared with those in controls (Fig. 5A). As shown in Fig. 5B, decreased expression of IL-8 mRNA in DN-Nod1 stably trasfected AGS cells was observed in real-time RT-PCR assay. In order to determine whether Nod1 regulates IL-8 secretion, DN-Nod1 stably transfected AGS cells along with the controls were stimulated with HP99, and cell culture supernatants were collected. At 4 hours after infection, we observed approximately 4-fold induction of IL-8 in DN-Nod1 stably transfected AGS cells, compared with approximately 18-fold in the control vector transfected cells (Fig. 5C). Western blotting showed that IκB-α degradation and IκB-α phosphorylation were reduced in DN-Nod1 stably transfected AGS cells compared with those in controls (Fig. 5D). To evaluate Nod1-dependent NF-κB activation, we performed EMSA. As shown in Fig. 5E, NF-κB binding in DN-Nod1 stably transfected AGS cells infected with HP99 was reduced compared with those in controls.


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 dominant-negative nucleotide binding oligomerization domain 1 (DN-Nod1) stably transfected AGS cells infected with HP99. (A) IL-8 mRNA expression in DN-Nod1 stably transfected AGS cells was reduced compared with that in the controls. (B) At 4 hours after infection, IL-8 mRNA expression in DN-Nod1 stably transfected AGS cells was reduced compared with the control vector-transfected cells, as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). (C) IL-8 secretion by the DN-Nod1 stably transfected AGS cells was reduced compared with the control vector-transfected cells. (D) IκB-α degradation and IκB-α phosphorylation were reduced in the DN-Nod1 stably transfected Caco-2 cells compared with the controls. (E) Nuclear factor κB (NF-κB) binding in the DN-Nod1 stably transfected Caco-2 cells was reduced compared with that in the controls. The error bars indicate the standard error of the mean of triplicate samples, which were representative of three independent experiments. Control vector, pcDNA3.
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f5-gnl-09-358: Interleukin (IL)-8 mRNA expression and IL-8 production in dominant-negative nucleotide binding oligomerization domain 1 (DN-Nod1) stably transfected AGS cells infected with HP99. (A) IL-8 mRNA expression in DN-Nod1 stably transfected AGS cells was reduced compared with that in the controls. (B) At 4 hours after infection, IL-8 mRNA expression in DN-Nod1 stably transfected AGS cells was reduced compared with the control vector-transfected cells, as determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). (C) IL-8 secretion by the DN-Nod1 stably transfected AGS cells was reduced compared with the control vector-transfected cells. (D) IκB-α degradation and IκB-α phosphorylation were reduced in the DN-Nod1 stably transfected Caco-2 cells compared with the controls. (E) Nuclear factor κB (NF-κB) binding in the DN-Nod1 stably transfected Caco-2 cells was reduced compared with that in the controls. The error bars indicate the standard error of the mean of triplicate samples, which were representative of three independent experiments. Control vector, pcDNA3.
Mentions: To investigate whether Nod1 might be involved in cagPAI-dependent recognition of H. pylori, we established Nod1-deficient cell lines by stable transfection of DN-Nod1 construct into AGS cells. To assay IL-8 mRNA expression in DN-Nod1 stably transfected AGS cells, IL-8 mRNA levels were determined by qualitative PCR. As a result, IL-8 mRNA expression in these cells were reduced compared with those in controls (Fig. 5A). As shown in Fig. 5B, decreased expression of IL-8 mRNA in DN-Nod1 stably trasfected AGS cells was observed in real-time RT-PCR assay. In order to determine whether Nod1 regulates IL-8 secretion, DN-Nod1 stably transfected AGS cells along with the controls were stimulated with HP99, and cell culture supernatants were collected. At 4 hours after infection, we observed approximately 4-fold induction of IL-8 in DN-Nod1 stably transfected AGS cells, compared with approximately 18-fold in the control vector transfected cells (Fig. 5C). Western blotting showed that IκB-α degradation and IκB-α phosphorylation were reduced in DN-Nod1 stably transfected AGS cells compared with those in controls (Fig. 5D). To evaluate Nod1-dependent NF-κB activation, we performed EMSA. As shown in Fig. 5E, NF-κB binding in DN-Nod1 stably transfected AGS cells infected with HP99 was reduced compared with those in controls.

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