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Introduction

H. pylori (H. pylori) is a gram-negative, microaerophilic bacterium that can survive in the highly acid environment of the human stomach. Most infected individuals are asymptomatic; however, for a significant number of individuals, infection with H. pylori causes the development of gastritis, gastric-duodenal ulcers, and even cancer. For this reason, H. pylori is listed as a Class I carcinogen (1). The first virulence factor of H. pylori to be identified cagA, is an oncoprotein encoded by the cagA gene localized on the Cag pathogenicity island (2). cagA protein is delivered into host cells by a type-4 secretory system (T4SS) and then induces cellular alterations that can lead to pathological changes, via activation of signaling pathways leading to gene expression (3). The presence of cagA+ H. pylori is associated with the infiltration of neutrophils and peripheral mononuclear cells (PBMCs) with the secretion of pro-inflammatory cytokines such as IL-1b, IL-8, IL-6, and TNF-a in the gastric mucosa (4). cagA+ H. pylori induces gastric epithelial cells to secrete IL-8, which attracts neutrophils and causes mucosal tissue damage (5). Therefore, cagA+ H. pylori strains are associated with strong inflammatory responses and severe clinical outcomes (6).

Neutrophils are the most abundant circulating immune cells. They play a crucial role in innate immune responses through the secretion of toxic molecules, such as reactive oxygen species (ROS) to kill invading bacteria. To generate ROS, the nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase becomes activated and provides electrons to oxygen (O2) to generate superoxide (O2-). Then superoxide dismutase catalyzes O2- to hydrogen peroxide (H2O2) which is a substrate for myeloperoxidase to generate hypohalous acids (7). Although ROS is generated as part of the mechanisms used to kill invading pathogens, if unregulated, bystander effects of ROS can cause tissue injury including cellular DNA damage to host tissues (8,9). Hence, neutrophil functions are usually highly regulated by serum/plasma factors such as complement proteins and immunoglobulins (10).

The majority of studies on neutrophil H. pylori interactions involve experiments using live bacteria and purified neutrophils (11,12). While these experiments can shed light on live pathogen: immune cell interactions, they may fail to give insights into the full range of effects of pathogenicity factors as the immune cells may not be exposed to intra-bacterial molecules. In the present study, novel activation of neutrophils by H. pylori extracts that was only observed when neutrophils were co-incubated with plasma was identified. When PBMCs were co-incubated with neutrophils and extracts, this activity was not seen. It was also shown that extracts containing cagA generated significantly higher levels of ROS compared to extracts devoid of this protein. These novel data identify a new and pathologically important process whereby cagA can stimulate adverse immune processes that contribute to tissue damage.

Materials and methods

Study workflow

Neutrophils, PBMCs, and plasma were separated from whole blood using the Ficoll density gradient separation method (Fig. 1A). Stocks of cagA+ and cagA- H. pylori strains were grown on Brucella agar plates for 3 days before being expanded into broth media for 7 days. Bacterial cells were disrupted using an ultra-sonicator and centrifuged to collect total protein extract (Fig. 1B). As a positive control for ROS production, purified neutrophils were activated by PMA. Experiment 1 was designed to investigate whether H. pylori extract could stimulate ROS of neutrophils in the absence of plasma. Experiment 2 was designed to investigate whether cell-to-cell contact between purified neutrophils and PBMCs could induce ROS production by extracts in the absence of plasma. In this experiment, PBMCs were added to purified neutrophils in ratios of 1:1 and 5:1. These mixtures were then co-cultured with extracts from cagA+ and cagA- H. pylori. Experiment 3 was designed to determine whether H. pylori extract could trigger neutrophils to produce ROS in the presence of plasma. In this experiment, neutrophils were mixed with autologous plasma (1:1 v/v) and co-incubated with H. pylori extract at 37˚C for 60 min. All experiments were performed in technical duplicates, n=3 donors.

Figure 1 Schematic overview of the experimental design. (A) Isolation of purified neutrophils. (B) Preparation of H. pylori extracts. (C) ROS measurement by flow cytometry ROS, reactive oxygen species; PBMC, peripheral blood monocytes; PMA, Phorbol myristate acetate; DHR123, dihydrodichlorofluorescein.

Participants

Blood was provided by healthy blood donors from the Blood Bank of Srinagarind University Hospital. The present study was approved by the Ethics Committee of Khon Kaen University, Faculty of Medicine, Khon Kaen, Thailand (approval no. HE651442). The 6 donors were 30-65 years old, with a male: female ratio of 1:1. Patients did not disclose any underlying infections or inflammatory conditions.

H. pylori strains and extract preparation

cagA + and cagA- H. pylori strains were provided by the Tropical Disease Research Center, Khon Kaen University. CagA+ and CagA- H. pylori isogenic strains of P12(13) were generously provided by Professor R. Haas (Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany) and grown on Brucella solid agar plates containing H. pylori selective supplement (Dent) (cat. no. SR0147, Oxoid Limited), at 37˚C in microaerophilic conditions for 7 days. Colonies were then inoculated into Dent-supplemented Brucella broth-culture flasks and incubated at 37˚C, 10% CO2.

From broth media, bacterial cells were pelleted and washed twice by centrifugation at 800 x g, 18˚C, for 5 min, then re-suspended in 1 ml PBS. The bacterial cells were fragmented by an ultrasonic processor at 22 kHz for 3 min on ice. The sonicated suspension was centrifuged at 7,000 x g, 4˚C, for 10 min. The supernatant containing total protein extract was removed and the protein concentration was measured using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Inc.; OD A280-A310). The extracts were stored at -20˚C until required.

Neutrophil isolation

Neutrophils were isolated from heparinized whole blood using the density gradient separation method. HetaSep™ (cat. no. 7906; Stem Cell Technologies, Inc.) was added to whole blood at a ratio of 1:5. The mixture was incubated at 37˚C for 30 min until the RBC interface was 50% of the total volume. The leucocyte-rich plasma was removed and overlaid gently onto Ficoll-Hypaque (cat. no. 17144002; Cytiva), at a ratio of 1:1, and centrifuged at 500 x g for 30 min at 25˚C. The upper layer (containing platelets and plasma) and the second interface (containing PBMCs) were collected and retained, while the Ficoll suspension above the cell pellet was discarded. To the pellet, 1 ml RPMI 1640 (cat. no. SH303555.02; Cytiva) was added, and gently re-suspended before adding 9 ml ammonium chloride lysis buffer (13.4 mM KHCO3, 155 mM NH4Cl, 96.7 µM EDTA) and then incubated for 3 min at 25˚C to disrupt the red blood cells. This mixture was centrifuged at 500 x g, 25˚C for 3 min. The supernatant was discarded, and neutrophils were resuspended in 2 ml RPMI 1640. An aliquot of the purified neutrophils was stained with Trypan Blue (0.4%, w/v) for 1 min at 25˚C (cat. no. 15250061, Thermo Fisher Scientific, Inc.) and counted on a hemocytometer slide before adjusting the neutrophil concentration to 2x106 cells/ml with RPMI 1640 medium. Purity was determined using Wright's staining and morphological staining (14) and was routinely >95% neutrophils.

ROS measurement

A total of 250 µl plasma and/or 250 µl RPMI 1640 were added sequentially to 250 µl purified neutrophils containing 5x105 cells, then co-incubated with 200 µg/ml cagA+ and cagA- H. pylori extracts for 1 h at 37˚C. A total of 2 µg/ml dihydrodichlorofluorescein (cat. no. 309825, MilliporeSigma), used to detect H2O2, was added and incubated for a further 15 min at 37˚C. Phorbol myristate acetate (PMA, final concentration 0.1 µg/ml) (cat. no. P1585-1MG; MilliporeSigma) was used as a positive control. ROS production was detected using flow cytometry on a BD FACSCanto™ II flow cytometer (BD Biosciences). The flow cytometry results were analyzed using FlowJo™ v10.8 Software (BD Biosciences).

Gating strategy

The gating strategy for ROS detection is shown in Fig. 2. Neutrophils were gated by forward and side scatter (Fig. 2A) and then single neutrophils were analyzed by forward scatter area and forward scatter high (Fig. 2B). The cut-off value for a positive signal was identified based on comparisons of negative control values (Fig. 2C) vs. positive control values with PMA stimulation (Fig. 2D).

Figure 2 Gating strategy. (A) Neutrophil population. (B) Single neutrophils. (C) Negative control (neutrophils only, without stimulus). (D) Positive control (PMA-treated neutrophils). (E) ROS production in the negative control (neutrophil), and PMA-treated neutrophils (neutrophils + PMA). n=3. **P<0.01. ROS, reactive oxygen species; PMA, Phorbol myristate acetate; NEU, neutrophils; FSC, forward scatter; SSC, side scatter.

Statistical analysis

All data are presented as the mean ± SEM. Statistical comparisons were performed using a Mann-Whitney U test between groups. P<0.05 was considered to indicate a statistically significant difference. All data were analyzed using GraphPad Prism version 8.0 (GraphPad Software, Inc.).

Results

cagA+ and cagA- H. pylori extracts do not directly trigger ROS production in purified neutrophils

To identify whether H. pylori extracts can trigger ROS production directly, purified neutrophils were co-cultured with cagA+ and cagA- H. pylori extracts. Very few neutrophils produced ROS in the untreated control samples (<5%, Fig. 3A), whereas PMA effectively stimulated the majority of the neutrophils to release ROS. Therefore, the control system was reliable.

Figure 3 H. pylori extract does not induce ROS production by human neutrophils. (A) Neutrophils only (no extract)-untreated controls. (B) ROS production by neutrophils treated with cagA+ H. pylori extract. (C) ROS production by neutrophils treated with cagA- H. pylori extract. (D) Summary of ROS production induced by cagA+ and cagA- H. pylori extracts compared to untreated neutrophils. All experiments were performed in technical duplicates. n=3 donors. ns, not significant; ROS, reactive oxygen species; NEU, neutrophils; FSC, forward scatter.

Neither cagA+ (P=0.200) nor cagA- (P=0.3429) H. pylori extract increased the number of ROS-producing cells (Fig. 3B and C, respectively) when compared to the untreated control (Fig. 3D). Thus, it was concluded that these extracts could not induce ROS production directly, or otherwise, neutrophils require other factors to prime them, such as cytokines from PBMCs before encountering the antigens.

cagA+ and cagA- H. pylori extracts do not trigger the production of ROS by neutrophils in the presence of PBMCs

To determine whether cell-to-cell contact with PBMCs induced neutrophil ROS production, purified PBMCs were added to the neutrophils before incubation with cagA+ and cagA- H. pylori extracts, and the ROS levels were measured. There were no notable levels of ROS detected in the control group (only neutrophils and PBMCs) (Fig. 4A) nor in the cagA+ (Fig. 4B) or in the cagA- H. pylori-treated neutrophils (Fig. 4C). we increased the ratio of PBMCs to neutrophil was increased to 5:1, there was still no measurable ROS production detected (Fig. 4E-G).

Figure 4 Neutrophil-PBMC cell-to-cell contact did not induce ROS production by neutrophils stimulated with H. pylori extracts. (A) Neutrophils mixed with PBMCs at a ratio=1:1. (B) Neutrophils and PBMCs (1:1) co-cultured with cagA+ H. pylori extract. (C) Neutrophils and PBMCs (1:1) co-cultured with cagA- H. pylori extract. (D) Summary of ROS production of neutrophil co-incubation with PBMCs (ratio 1:1) and cagA+ and cagA- H. pylori extracts. (E) Neutrophils mixed with PBMCs at a ratio=1:5. (F) Neutrophils mixed with PBMCs (1:5) co-cultured with cagA+ H. pylori extract. (G) Neutrophils mixed with PBMCs (1:5) co-cultured with cagA- H. pylori extract. (H) Summary of ROS production by neutrophils co-incubated with PBMCs (1:5) and cagA+ and cagA- H. pylori extracts. All experiments were performed in technical duplicates. n=3 donors. ROS, reactive oxygen species; PBMC, peripheral blood monocytes; ns, not significant; NEU, neutrophils; FSC, forward scatter.

cagA+ and cagA- H. pylori extracts induce ROS production by neutrophils in the presence of human plasma

To investigate whether human plasma affects ROS production by neutrophils in response to cagA+ and cagA- H. pylori extracts, autologous plasma was used to pre-treat neutrophils, with or without co-culture with cagA+ and cagA- H. pylori extracts, and measured ROS production after 1 h. There was no ROS production in the negative controls (plasma-treated neutrophils without H. pylori extracts; Fig. 5A). However, in the presence of plasma, both cagA+ (Fig. 5B; P=0.0286) and cagA- H. pylori extracts stimulated neutrophils to produce significantly more ROS than the negative controls (Fig. 5C; P=0.0286). Additionally, cagA+ H. pylori extract induced the production of significantly more ROS by neutrophils than cagA- H. pylori extract in the presence of plasma (Fig. 5D).

Figure 5 Plasma primes neutrophils for generation of ROS induced by H. pylori extracts. (A) Neutrophils were cultured in the presence of plasma (no H. pylori extract). (B) ROS production by neutrophils in the presence of plasma after addition of cagA+ H. pylori extract. (C) ROS production by neutrophils in the presence of plasma after addition of cagA- H. pylori extract. (D) Summary data of ROS production in the absence or presence of cagA+ and cagA- H. pylori extract. All experiments were performed in technical duplicates. n=3 donors. *P<0.05. ROS, reactive oxygen species; NEU, neutrophils; FSC, forward scatter; SSC, side scatter.

Discussion

This study shows, for the first time, that complex host-pathogen inflammatory processes regulate the activation of human neutrophils using H. pylori extracts. It was found that these extracts could only activate ROS production by human neutrophils in the presence of plasma, and that this activation could not be replicated by the addition of PBMCs. It was also shown that H. pylori extracts containing the cagA protein generated significantly higher levels of ROS than extracts devoid of this protein. This is an important observation in view of the fact that infection with cagA+ H. pylori strains usually results in more adverse pathological outcomes, such as an increased risk of gastric cancer, than those that do not express this pathogenicity factor (15). The results of the present study indicated that serum factors are necessary to prime neutrophils to generate ROS after incubation with these extracts, but also showed that extracellular CagA activated neutrophils in the presence of these plasma factors.

Several components in plasma may be involved in the process of ROS activation of neutrophils. Firstly, following interaction with immune cells, activated platelets can release chemokines (to attract neutrophils) and proinflammatory cytokines such as CD40L and IL-1β (16), which may prime neutrophil functions. Second, immunoglobulins in plasma may elicit neutrophil ROS production, either alone or via the formation of immune complexes after interactions with their cognate antigen. It has been shown that immunoglobulins for intravenous use, named Gamimune N, Sandoglobuin, and Intraglobin F can enhance neutrophil respiratory-burst activity in-vitro (17). Moreover, these immunoglobulins also promote killing activity towards multi-drug-resistant bacteria and autophagy of neutrophils in immunocompromised patients (18). However, whether the plasma of the volunteers contained anti-Helicobacter antibodies was not determined, although this is now being explored in the follow-up studies. Third, complement proteins such as C3a, C5a, and the surface-bound opsonins, C3b and C4b, can enhance the ability of neutrophils to phagocytose opsonized particles, release proinflammatory cytokines, generate ROS, and form neutrophil extracellular traps (NETs) (19). It is postulated that these components in plasma can prime neutrophils, and once these primed neutrophils encounter specific antigens in H. pylori extracts, they become activated. It is also possible that components in plasma interact with H. pylori proteins, subsequently activating neutrophils. Nevertheless, these results are the first to demonstrate that neutrophil ROS production can be stimulated by H. pylori extracts even without phagocytosis of intact live bacteria; however, factors present within plasma are required for this ROS production to occur.

It is hypothesized that cell-to-cell contact with PBMCs may facilitate neutrophil ROS production in the presence of H. pylori extracts. However, this was shown not to be the case. The results of the present study suggest that cell-to-cell contact with, or cytokines from, PBMCs do not contribute to neutrophil ROS production at least after incubation with cagA+ and cagA- H. pylori extract under the experimental conditions employed in the present study.

H. pylori has been studied largely given its role in gastric cancer development and several in vitro experiments support this property. For example, co-culture of H. pylori extract with gastric epithelial cell lines leads to elevated cell proliferation as well as inhibition of cell apoptosis and autophagy (20). In addition, H. pylori extract induces mRNA expression of gastric cancer biomarkers such as chloride channel-3 and slingshot protein phosphatase 1, which suggests that H. pylori extract may contribute to gastric cancer progression (20). H. pylori extract also causes extra-gastric disorders. For example, an animal-model study indicated that this extract promoted the expression of chemokines and elevated the levels of TGF-β1/NF-κB-mediated inflammation in a rat hepatic stellate cell line (21). For these reasons, H. pylori extract has been used for vaccine development. Flagella are important for H. pylori motility and colonization, and flagella-sheath proteins or total protein lysate have been used to vaccinate mice (22). These immunized mice were then infected with H. pylori orally and both forms of vaccination led to an equally significant decrease in H. pylori burden relative to non-vaccinated controls (22).

A major finding of the present study was that cagA+ H. pylori extract induced significantly higher ROS levels than cagA-extracts, which suggests a novel immunopathogenic pathway induced by cagA+ H. pylori. As the two strains that were used in the present study were isogenic (that is, the cagA-strain was identical to the wild-type strain except that it was specifically depleted of cagA), the only difference in protein composition of the two extracts used was the absence or presence of CagA. Thus, this pathogenicity factor induced high levels of ROS production in plasma-treated neutrophils, and it is hypothesized that this mechanism plays a role in tissue damage associated with this organism in human diseases.

cagA is normally inserted into host cells via the type-4 secretory system and once within the cytoplasm, it becomes phosphorylated and then interacts with and activates intracellular signaling pathways leading to altered gene expression and oncogenesis (3). Intact H. pylori are phagocytosed by human neutrophils and can survive intracellularly and delay neutrophil apoptosis (23). It is noteworthy that in the present study, extracellular cagA activated human neutrophils (in the presence of serum) to generate ROS. Previously, it has been reported that in addition to their role in gastric diseases, Helicobacter spp are responsible for a number of hepatobiliary pathologies including several types of liver cancer (24-27). In addition, cagA+ H. pylori is detected at considerably higher levels in individuals infected with the liver fluke, Opisthorchis viverrini (compared to uninfected controls) and at even higher levels in those with advanced periductal fibrosis, which is an outcome of liver fluke infection (28). O. viverrini is a reservoir for H. pylori and hence carries this bacterial pathogen to the bile ducts during liver fluke infection (29). At a 2-year follow-up, >40% of those initially diagnosed with liver fibrosis were now parasite free but had persistent or worsening fibrosis (30) and these individuals had significantly higher levels of cagA + H. pylori (31).

The present study highlights the importance of cagA as an important pathogenicity factor that can activate neutrophils to generate molecules that may damage host tissues, as ROS may induce oxidative stress, resulting in DNA damage. Identification of the molecular mechanisms responsible for the receptor/intracellular signaling processes mediated by cagA is important in order, not only to define this mechanism, but to identify ways by which this pathway can be experimentally blocked. Further work will also include identifying the factor(s) within plasma that can regulate this process and establish the full range of neutrophil functions (including secreted proteases and cytokines/chemokines) that are activated by this novel mechanism.

In conclusion, total extracts from cagA+ and cagA- H. pylori had little effect on neutrophil ROS production in the absence of plasma. However, the addition of plasma significantly primed human neutrophils to generate ROS in response to the extracts. cagA+ H. pylori extract-treated neutrophils produced significantly more ROS than did neutrophils treated with cagA- H. pylori extracts. These data show that H. pylori proteins, perhaps actively secreted by the bacteria or after release from dead bacteria, can, in the presence of plasma factors, activate ROS by neutrophils. CagA+ H. pylori extract significantly induced higher ROS than cagA-extract, which suggests a novel immunopathogenic pathway of cagA+ H. pylori.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by a grant from the Faculty of Medicine, Khon Kaen University, Thailand (grant no. IN66011) and partly supported by a Royal Society International Collaboration Award (grant no. ICA\R1\201299).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

TDT, SWE and KS conceived and designed the study. TDT, CC, DW, and KS performed the experiments. TDT, KF, BS, SWE and KS analysed and interpreted the data. TDT and KS wrote the first draft of the manuscript. TDT, KF, BS, SE and KS edited and finalized the manuscript. All authors have read and approved the final manuscript. TDT, SWE and KS confirm the authenticity of all the raw data.

Ethics approval and consent to participant

The present study was approved by the Ethics Committee of Khon Kaen University, Faculty of Medicine (Khon Kaen, Thailand; approval no. HE651442) and written informed consent was obtained from each participant.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References