Helicobacter pylori tumor necrosis factor-α inducing protein promotes cytokine expression via nuclear factor-κB

Abstract

AIM: To study the effects of Helicobacter pylori (H. pylori) tumor necrosis factor-α (TNF) inducing protein (Tip-α) on cytokine expression and its mechanism.

METHODS: We cloned Tip-α from the H. pylori strain 26695, transformed Escherichia coli with an expression plasmid, and then confirmed the expression product by Western blotting. Using different concentrations of Tip-α that affected SGC7901 and GES-1 cells at different times, we assessed cytokine levels using enzyme-linked immunosorbent assay. We blocked SGC7901 cells with pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of nuclear factor κB (NF-κB). We then detected interleukin (IL)-1β and TNF-α levels in SGC7901 cells.

RESULTS: Western blot analysis using an anti-Tip-α antibody revealed a 23-kDa protein, which indicated that recombinant Tip-α protein was recombined successfully. The levels of IL-1β, IL-8 and TNF-α were significantly higher following Tip-α interference, whether GES-1 cells or SGC-7901 cells were used (P < 0.05). However, the levels of cytokines (including IL-1β, IL-8 and TNF-α) secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-α at the same concentration and for the same duration (P < 0.05). After blocking NF-κB with PDTC, the cells (GES-1 cells and SGC-7901 cells) underwent interference with Tip-α. We found that IL-1β and TNF-α levels were significantly decreased compared to cells that only underwent Tip-α interference (P < 0.05).

CONCLUSION: Tip-α plays an important role in cytokine expression through NF-κB.

Key Words: Helicobacter pylori, Tumor necrosis factor-α inducing protein, Interleukin-1β, Interleukin-8, Tumor necrosis factor-α, Nuclear factor-κB

INTRODUCTION

Infection with Helicobacter pylori (H. pylori) leads to chronic gastritis, peptic ulcer, and gastric lymphoma[1-3]. H. pylori has also been associated with gastric cancer[4], is classified as a class I carcinogen by the International Agency for Research on Cancer[5], and H. pylori exerts its pathogenesis by secreting toxins, including hemolysin, lipopolysaccharides, CagA and VacA[6-9]. CagA and VacA are the major virulence factors. Persistent infection by H. pylori enables these toxins to stimulate gastric epithelial cells to produce a large number of cytokines such as tumor necrosis factor (TNF-α) and interleukin 1, 6 and 8 (IL-1, IL-6 and IL-8), thus generating an inflammatory reaction[10-14]. Tumor necrosis factor-α inducing protein (Tip-α) is a new toxin discovered recently, and likely accelerates the inflammation and cancers caused by H. pylori[15]. However, its function and the mechanism underlying these effects remain unclear. The present work was conducted to determine the effects of recombinant Tip-α (rTip-α) on human gastric epithelial cells and gastric cancer cytokine expression, as well as explore the mechanisms involved.

MATERIALS AND METHODS

Materials

H. pylori strain 26695 was obtained from the Shanghai Institute of Digestive Disease. The following reagents were used in this study: Dual Promoter TA Cloning^® Kit pCR^®II and pET28a vectors (Invitrogen); monoclonal rabbit anti-Tip-α antibody (Beijing Aviva Systems Biology); BamHI, Xho I and Prestained Protein Molecular Weight Markers (Fermentas); DNA and gel extraction kit from Tiangen Biotech (Beijing) Co. Ltd.; DNA marker (TaKaRa); His Trap^TMH. pylori affinity chromatography column (GE Healthcare); and enhanced chemiluminescence kit (Pierce Protein Biology Products). The polymerase chain reaction primer sequences were 5’-TTGGATCCATGCTGCAGGCTTG-3’, which contained an Xho I restriction site, and 5’-GGCTCGAGCTACATGGCTATAG-3’, which contained a BamH I restriction site. The primers were synthesized by Invitrogen. The human gastric epithelial cell line GES-1 and gastric cancer SGC7901 cells were purchased from the Shanghai Cancer Institute. Enzyme-linked immunosorbent assay (ELISA) kits were obtained from MultiSciences Biotech (Shanghai) Co., Ltd., while pyrrolidine dithiocarbamate (PDTC) was purchased from Jingmei Biotech Co., Ltd.

Methods

Expression, purification, and identification of Tip-α: We cloned Tip-α from the genome of H. pylori strain 26695. The Tip-α gene and pET28a vector (His tag) were digested with BamH I and Xho I, purified, and then ligated together to generate the ET28a-Tip-α plasmid expressing recombinant Tip-α. This plasmid was transformed into Escherichia coli and the resultant protein was purified by Ni-NTA affinity chromatography and verified by Western blotting.

Cell recovery, culture, and passage: Cryopreserved GES-1 and SGC-7901 cells were centrifuged at 1000 rpm for 5 min. After removal of the supernatant, these cells were cultured in 60 mm × 60 mm dishes containing Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum.

IL-1β, IL-8 and TNF-αlevels at different times following interference by 12.5μg/mL rTip-αin GES-1 and SGC7901 cells: GES-1 and SGC7901 cells during their logarithmic growth phase underwent interference with 12.5 μg/mL rTip-α after starvation in serum-free medium for 24 h. The levels of IL-1β, IL-8 and TNF-α cytokines were then assessed at 0, 1, 2, 4 and 8 h post-interference using ELISA.

Levels of IL-1β, IL-8 and TNF-αin GES-1 and SGC7901 cells following incubation with different concentrations of rTip-α: We incubated GES-1 and SGC7901 cells with the following concentrations of rTip-α: 0, 12.5, 25 and 50 μg/mL. After 2 h, we examined the levels of IL-1β, IL-8 and TNF-α using ELISA.

Effects of rTip-αon IL-1βand TNF-αexpression after PDTC-mediated inhibition of NF-κB: Four groups consisting of the same number of GES-1 and SGC7901 cells were starved in serum-free medium for 24 h before undergoing different treatments. Group A was treated with 12.5 μg/mL rTip-α for 2 h. Group B was treated similarly after PDTC blocking of NF-κB for 4 h. Groups C and D were incubated with serum-free medium and dimethyl sulfoxide (the vehicle with which PDTC was diluted), respectively. ELISA was performed to detect the levels of IL-1β and TNF-α in each group.

Statistical analysis

Data are presented as the mean ± SD and analyzed using SPSS 17.0. The Student’s t test was used to compare two groups, while one-way analysis of variance was used to compare among several groups. A P value < 0.05 was considered statistically significant.

RESULTS

Identification by Western blotting after rTip-α expression and purification

Western blotting analysis demonstrated that the Tip-α recombinant protein and anti-human Tip-α monoclonal antibody could be specifically bound; specific bands were found (Figure 1). Western blotting analysis by non-denaturing gel electrophoresis showed active dimer bands (46 kDa).

Figure 1 Western blotting identification of recombinant tumor necrosis factor-α inducing protein.

IL-1β, IL-8 and TNF-α levels at different times following interference by 12.5 μg/mL rTip-α in GES-1 and SGC7901 cells

The levels of IL-1β, IL-8 and TNF-α were significantly higher after GES-1 and SGC7901 cells underwent interference by 12.5 μg/mL rTip-α for 1, 2, 4 and 8 h than those at 0 h. Cytokine secretion by GES-1 and SGC7901 cells peaked after rTip-α interference for 2 h, indicating no obvious dependence on time (Tables 1 and 2). However, after interference by rTip-α for 2 h, the levels of IL-8 (2.53 ± 0.50) and TNF-α (1.41 ± 0.10) in SGC7901 cells were significantly higher than those in GES-1 cells (0.84 ± 0.11 for IL-8 and 0.72 ± 0.08 for TNF-α). As shown in Table 1, the levels of IL-1β in GES-1 and SGC7901 cells (2.07 ± 0.10 and 2.07 ± 0.30, respectively) were not statistically different after rTip-α interference for 2 h.

Table 1 Cytokine levels at different times after interference of GES-1 and SGC7901 cells with 12.5 μg/mL recombinant tumor necrosis factor-α inducing protein.

Groups	0 h	1 h	2 h	4 h	8 h
GES-1 (IL-1β)	0.34 + 0.04	0.88 + 0.09a	2.07 + 0.30a	1.35 + 0.20a	1.41 + 0.15a
SGC-7901 (IL-1β)	0.22 + 0.04	0.35 + 0.05	2.07 + 0.10a	1.11 + 0.04a	1.14 + 0.04a
GES-1 (IL-8)	0.35 + 0.05	0.60 + 0.12a	0.84 + 0.11a	0.64 + 0.06a	0.50 + 0.07a
SGC-7901 (IL-8)	0.70 + 0.02	0.78 + 0.19	2.53 + 0.50a	2.26 + 0.24a	2.14 + 0.68a
GES-1 (TNF-α)	0.39 + 0.06	0.39 + 0.07	0.72 + 0.08a	0.53 + 0.03a	0.51 + 0.14a
SGC-7901 (TNF-α)	0.33 + 0.09	1.02 + 0.09a	1.41 + 0.10a	0.86 + 0.07a	0.47 + 0.03a

^aP < 0.05 vs 0 h. IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α.

Table 2 Cytokine levels in GES-1 and SGC7901 cells after interference with different concentrations of recombinant tumor necrosis factor-α inducing protein for 2 h.

Groups	0	12.5μg/mL	25μg/mL	50μg/mL
GES-1 (IL-1β)	0.59 + 0.11	2.07 + 0.30a	2.20 + 0.09a	1.23 + 0.13a
SGC-7901 (IL-1β)	0.36 + 0.01	2.07 + 0.10a	1.22 + 0.03a	1.02 + 0.04a
GES-1 (IL-8)	0.39 + 0.08	0.84 + 0.11a	0.75 + 0.09a	0.61 + 0.15a
SGC-7901 (IL-8)	0.78 + 0.09	2.53 + 0.50a	1.50 + 0.16a	1.41 + 0.14a
GES-1 (TNF-α)	0.30 + 0.06	0.72 + 0.08a	0.54 + 0.13a	0.63 + 0.10a
SGC-7901 (TNF-α)	0.26 + 0.18	1.41 + 0.10a	0.62 + 0.07a	0.62 + 0.02a

^aP < 0.05 vs group 0. IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α.

Levels of IL-1β, IL-8 and TNF-α in GES-1 and SGC7901 cells following incubation with different concentrations of rTip-α

The levels of IL-1β, IL-8 and TNF-α were significantly higher than those in the blank control in GES-1 and SGC7901 cells after rTip-α interference for 2 h. Cytokine secretion of GES-1 and SGC7901 cells peaked at 12.5 μg/mL, suggesting that this effect was not concentration-dependent (Table 2).

Effects of rTip-α on IL-1β and TNF-α expression after PDTC-mediated inhibition of NF-κB

The levels of IL-1β and TNF-α in SGC7901 cells in Group B (treated with PDTC + rTip-α) were higher than those in Groups C and D (no rTip-α and PDTC), but markedly lower than those in Group A (only treated with rTip-α). As shown in Table 3, these differences were statistically significant (P < 0.05, F = 40.15).

Table 3 Effects of recombinant tumor necrosis factor-α inducing protein on the levels of interleukin-1β and tumor necrosis factor-α in SGC7901 cells under different conditions.

Groups	A	B	C	D
SGC-7901 (IL-1β)	2.32 + 0.25	1.05 + 0.75a	084 + 0.08	0.57 + 0.09
SGC-7901 (TNF-α)	1.51 + 0.64	0.72 + 0.20a	0.43 + 0.07	0.71 + 0.23
GES-1 (IL-1β)	2.07 + 0.30	0.98 + 0.34a	0.69 + 0.06	0.63 + 0.06
GES-1 (TNF-α)	0.81 + 0.08	0.36 + 0.03a	0.27 + 0.03	0.29 + 0.04

^aP < 0.05 vs group A. IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α.

DISCUSSION

Tip-α is a novel gene that was discovered recently in H. pylori. Located in the H. pylori 0596 open reading frame of the H. pylori 26695 strain, Tip-α is also called H. pylori 0596 protein. Its open reading frame is 519 bp in length and constitutes 173 amino acids. Tip-α has a molecular weight of 19 kDa and can form active homodimers with a molecular weight of 38 kDa through disulfide bonding[15]. Recent studies have found that Tip-α is associated with the adsorption and colonization of H. pylori in gastric mucosa[16]. Some studies have shown that the structure of Tip-α is different from penicillin binding proteins. Tip-α is composed of three closely linked domains that interact with other proteins and nucleic acids. In particular, the homodimer formed by cysteine ​​C25 and C27 is essential for Tip-α to serve its role in the gastric mucosal acidic environment[17]. Detected by gene chip technology, expression of the chemokines Cc 12 Cc17, Cc120, Cx11 and Cx15 was enhanced after Tip-α treatment in gastric cancer cells and gastric epithelial cells[18]. These chemokines can induce chemotaxis of immune cells to local sites of infection, resulting in immune regulation and pathology, and ultimately the inflammatory response[19].

Because our pET28a-Tip-α vector also expresses a 3.74 kDa His tag, the recombinant Tip-α protein we produced possesses a molecular weight of about 23 kDa and can form active homodimers with a molecular weight of 46 kDa through disulfide bonding. Our work indicates that after affecting gastric epithelial and cancer cells, rTip-α can promote the expression of IL-1β, IL-8 and TNF-α. These cytokines are important in promoting the inflammatory response, thus linking Tip-α to inflammation and the occurrence of H. pylori-related gastrointestinal diseases. After incubating the cells for 2 h with rTip-α, the levels of cytokine expression peaked at 12.5 μg/mL, which was the best concentration for interference. The levels of cytokine expression induced by rTip-α were not time- or concentration-dependent. These results suggest that Tip-α affects the host by inducing toxin secretion into the exterior environment of the bacteria through the type II secretion system[20]. The toxins then enter host cells via receptor-mediated endocytosis instead of through injection via the IV secretion system[21-23]. Studies have shown that Tip-α possesses DNA binding activity. In particular, DNA affecting gastric mucosal cells can combine with some transcription factors to promote IL-1β, IL-8 and TNF-α expression, leading to the occurrence and development of H. pylori-related gastrointestinal diseases[24].

PDTC is a specific NF-κB inhibitor that works by blocking degradation of the NF-κB p65 subunit or IκB, thereby reducing NF-κB nuclear translocation[25]. Our data demonstrated no significant increase in IL-1β and TNF-α levels after pretreatment of SGC7901 cells with PDTC followed by rTip-α interference. This finding suggests that the promotion of cytokine expression by Tip-α may be regulated by NF-κB. However, further study is required to determine the underlying mechanism.

In addition, we found that when gastric epithelial and cancer cells were treated with the same concentration of Tip-α for the same duration, the level of cytokine expression in gastric cancer SGC7901 cells was significantly higher than that in gastric epithelial GES-1 cells. This difference may be due to differential effects of rTip-α on NF-κB expression in the cell types or may be related to variations in the DNA binding activity of Tip-α in the cells. Some studies suggest that the increased cytokine expression promoted by Tip-α may hasten the invasion and metastasis of gastric cancer[26]. Overall, Tip-α can activate cytokine expression in an NF-κB-dependent manner. Tip-α plays a major role in the pathogenesis of H. pylori, however, its mechanism requires further investigation.

COMMENTS

Background

Helicobacter pylori (H. pylori) exerts its pathogenesis by secreting toxins. Tumor necrosis factor-α (TNF-α) inducing protein (Tip-α) is a new toxin discovered recently, however, its function and mechanism of pathogenesis remain unclear.

Research frontiers

The pathogenesis of H. pylori is partially clear, as H. pylori may secrete many types of toxins. With the exception of CagA and VacA, new H. pylori toxins have been discovered, such as Tip-α, mammalian, prokaryotic high-temperature requirement A, and the duodenal ulcer-promoting gene. Only the function of the toxins and their mechanism of pathogenesis are clear, as the mechanism of H. pylori pathogenesis is known.

Innovations and breakthroughs

Other studies discovered that Tip-α promoted the expression of cytokines, however, this article first showed the difference in the promotion of the expression of cytokines between gastric epithelial cells and cancer cells, and that Tip-α activates cytokine expression in a nuclear factor κB (NF-κB)-dependent manner.

Applications

Tip-α may become a marker of H. pylori virulence. The virulence of H. pylori may be determined by detecting Tip-α.

Terminology

Tip-α is the abbreviation for tumor necrosis factor-α inducing protein. It was first discovered that this new H. pylori toxin can promote the expression of TNF-α, therefore, it was called Tip-α.

Peer review

The authors certified that Tip-α-a new toxin of H. pylori, promoted the expression of cytokine, discovered the difference of this function between gastric epithelial cells and cancer cells, and in an NF-κB-dependent manner, further interpreted the mechanism of pathogenesis of H. pylori.