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Helicobacter pylori protein JHP0290 binds to multiple cell types and induces macrophage apoptosis via tumor necrosis factor (TNF)-dependent and independent pathways.

Pathak SK, Tavares R, de Klerk N, Spetz AL, Jonsson AB - PLoS ONE (2013)

Bottom Line: Furthermore, rJHP0290-induced TNF release was partly dependent on activation of nuclear transcription factor-κB (NF-κB).These results provide mechanistic insight into the potential role of the protein JHP0290 during H. pylori-associated disease development.By virtue of its ability to induce TNF, an acid suppressive proinflammatory cytokine and induction of macrophage apoptosis, JHP0290 possibly helps in persistent survival of the bacterium inside the stomach.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
Activated macrophages at the sub-mucosal space play a major role in generating innate immune responses during H. pylori infection. Final disease outcome largely depends on how H. pylori and bacterium-derived products modulate macrophage responses. Here, we report that JHP0290, a functionally unknown protein from H. pylori, regulates macrophage functions. Recombinant purified JHP0290 (rJHP0290) had the ability to bind to several cell types including macrophages, human gastric epithelial cell lines, human monocyte-derived dendritic cells (MoDC) and human neutrophils. Exposure to rJHP0290 induced apoptosis in macrophages concurrent with release of proinflammatory cytokine tumor necrosis factor (TNF). A mutant strain of H. pylori disrupted in the jhp0290 gene was significantly impaired in its ability to induce apoptosis and TNF in macrophages confirming the role of endogenous protein in regulating macrophage responses. Intracellular signaling involving Src family of tyrosine kinases (SFKs) and ERK MAPK were required for rJHP0290-induced TNF release and apoptosis in macrophages. Furthermore, rJHP0290-induced TNF release was partly dependent on activation of nuclear transcription factor-κB (NF-κB). Neutralizing antibodies against TNF partially blocked rJHP0290-induced macrophage apoptosis indicating TNF-independent pathways were also involved. These results provide mechanistic insight into the potential role of the protein JHP0290 during H. pylori-associated disease development. By virtue of its ability to induce TNF, an acid suppressive proinflammatory cytokine and induction of macrophage apoptosis, JHP0290 possibly helps in persistent survival of the bacterium inside the stomach.

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The role of endogenous JHP0290 on macrophage apoptosis and TNF release.(A) Equal amount of cell lysates from H. pylori wild-type strain (J99 Wt) or mutant strains (J99 Δjhp0290) were immunoblotted with anti-JHP0290 antibody. Blots were reprobed with antibody raised against another H. pylori protein, alkyl hydroperoxide-reductase (AhpC), to confirm equal loading. Blot shown is representative of results obtained in three independent experiments. MDM and RAW264.7 cells were infected either with H. pylori wild-type strain J99 (Wt) or mutant strain (Δjhp0290) at MOI100. Percentage of apoptotic cells (B) or TNF release in culture supernatant (C) was determined. (D) RAW264.7 cells were infected either with J99 Wt or J99 Δjhp0290 mutant strain at MOI100 for 6 h. RT-PCR was performed to assess the fold changes (RQ) in mRNA level of TNF. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by *(p<0.05).
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pone-0077872-g004: The role of endogenous JHP0290 on macrophage apoptosis and TNF release.(A) Equal amount of cell lysates from H. pylori wild-type strain (J99 Wt) or mutant strains (J99 Δjhp0290) were immunoblotted with anti-JHP0290 antibody. Blots were reprobed with antibody raised against another H. pylori protein, alkyl hydroperoxide-reductase (AhpC), to confirm equal loading. Blot shown is representative of results obtained in three independent experiments. MDM and RAW264.7 cells were infected either with H. pylori wild-type strain J99 (Wt) or mutant strain (Δjhp0290) at MOI100. Percentage of apoptotic cells (B) or TNF release in culture supernatant (C) was determined. (D) RAW264.7 cells were infected either with J99 Wt or J99 Δjhp0290 mutant strain at MOI100 for 6 h. RT-PCR was performed to assess the fold changes (RQ) in mRNA level of TNF. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by *(p<0.05).

Mentions: In order to study the apoptosis and TNF-inducing ability of endogenous JHP0290, an isogenic mutant strain of H. pylori J99 disrupted in the jhp0290 gene was constructed. Inactivation of the jhp0290 gene in mutant strain was verified by PCR and immunoblotting with anti-JHP0290 antibody (Fig. 4A). Inactivation of the jhp0290 gene led to a significant reduction in the induction of apoptosis (Fig. 4B) and TNF release (Fig. 4C) compared with the wild-type parent strain both in RAW264.7 and primary MDM. The impaired TNF inducing ability of the mutant strain was also observed at mRNA level.(Fig. 4D). However, the apoptosis and TNF-inducing ability was not totally abolished in the mutant strain. This was expected because several other apoptosis and TNF-inducing factors from H. pylori also contribute to apoptosis and TNF release from macrophages [16], [17], [22]. Taken together, these results suggested the involvement of JHP0290 in the induction of apoptosis and TNF in macrophages.


Helicobacter pylori protein JHP0290 binds to multiple cell types and induces macrophage apoptosis via tumor necrosis factor (TNF)-dependent and independent pathways.

Pathak SK, Tavares R, de Klerk N, Spetz AL, Jonsson AB - PLoS ONE (2013)

The role of endogenous JHP0290 on macrophage apoptosis and TNF release.(A) Equal amount of cell lysates from H. pylori wild-type strain (J99 Wt) or mutant strains (J99 Δjhp0290) were immunoblotted with anti-JHP0290 antibody. Blots were reprobed with antibody raised against another H. pylori protein, alkyl hydroperoxide-reductase (AhpC), to confirm equal loading. Blot shown is representative of results obtained in three independent experiments. MDM and RAW264.7 cells were infected either with H. pylori wild-type strain J99 (Wt) or mutant strain (Δjhp0290) at MOI100. Percentage of apoptotic cells (B) or TNF release in culture supernatant (C) was determined. (D) RAW264.7 cells were infected either with J99 Wt or J99 Δjhp0290 mutant strain at MOI100 for 6 h. RT-PCR was performed to assess the fold changes (RQ) in mRNA level of TNF. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by *(p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815203&req=5

pone-0077872-g004: The role of endogenous JHP0290 on macrophage apoptosis and TNF release.(A) Equal amount of cell lysates from H. pylori wild-type strain (J99 Wt) or mutant strains (J99 Δjhp0290) were immunoblotted with anti-JHP0290 antibody. Blots were reprobed with antibody raised against another H. pylori protein, alkyl hydroperoxide-reductase (AhpC), to confirm equal loading. Blot shown is representative of results obtained in three independent experiments. MDM and RAW264.7 cells were infected either with H. pylori wild-type strain J99 (Wt) or mutant strain (Δjhp0290) at MOI100. Percentage of apoptotic cells (B) or TNF release in culture supernatant (C) was determined. (D) RAW264.7 cells were infected either with J99 Wt or J99 Δjhp0290 mutant strain at MOI100 for 6 h. RT-PCR was performed to assess the fold changes (RQ) in mRNA level of TNF. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by *(p<0.05).
Mentions: In order to study the apoptosis and TNF-inducing ability of endogenous JHP0290, an isogenic mutant strain of H. pylori J99 disrupted in the jhp0290 gene was constructed. Inactivation of the jhp0290 gene in mutant strain was verified by PCR and immunoblotting with anti-JHP0290 antibody (Fig. 4A). Inactivation of the jhp0290 gene led to a significant reduction in the induction of apoptosis (Fig. 4B) and TNF release (Fig. 4C) compared with the wild-type parent strain both in RAW264.7 and primary MDM. The impaired TNF inducing ability of the mutant strain was also observed at mRNA level.(Fig. 4D). However, the apoptosis and TNF-inducing ability was not totally abolished in the mutant strain. This was expected because several other apoptosis and TNF-inducing factors from H. pylori also contribute to apoptosis and TNF release from macrophages [16], [17], [22]. Taken together, these results suggested the involvement of JHP0290 in the induction of apoptosis and TNF in macrophages.

Bottom Line: Furthermore, rJHP0290-induced TNF release was partly dependent on activation of nuclear transcription factor-κB (NF-κB).These results provide mechanistic insight into the potential role of the protein JHP0290 during H. pylori-associated disease development.By virtue of its ability to induce TNF, an acid suppressive proinflammatory cytokine and induction of macrophage apoptosis, JHP0290 possibly helps in persistent survival of the bacterium inside the stomach.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
Activated macrophages at the sub-mucosal space play a major role in generating innate immune responses during H. pylori infection. Final disease outcome largely depends on how H. pylori and bacterium-derived products modulate macrophage responses. Here, we report that JHP0290, a functionally unknown protein from H. pylori, regulates macrophage functions. Recombinant purified JHP0290 (rJHP0290) had the ability to bind to several cell types including macrophages, human gastric epithelial cell lines, human monocyte-derived dendritic cells (MoDC) and human neutrophils. Exposure to rJHP0290 induced apoptosis in macrophages concurrent with release of proinflammatory cytokine tumor necrosis factor (TNF). A mutant strain of H. pylori disrupted in the jhp0290 gene was significantly impaired in its ability to induce apoptosis and TNF in macrophages confirming the role of endogenous protein in regulating macrophage responses. Intracellular signaling involving Src family of tyrosine kinases (SFKs) and ERK MAPK were required for rJHP0290-induced TNF release and apoptosis in macrophages. Furthermore, rJHP0290-induced TNF release was partly dependent on activation of nuclear transcription factor-κB (NF-κB). Neutralizing antibodies against TNF partially blocked rJHP0290-induced macrophage apoptosis indicating TNF-independent pathways were also involved. These results provide mechanistic insight into the potential role of the protein JHP0290 during H. pylori-associated disease development. By virtue of its ability to induce TNF, an acid suppressive proinflammatory cytokine and induction of macrophage apoptosis, JHP0290 possibly helps in persistent survival of the bacterium inside the stomach.

Show MeSH
Related in: MedlinePlus