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. 2024 Jul 26;19(13):1145–1156. doi: 10.1080/17460913.2024.2360798

Helicobacter pylori infection delays neutrophil apoptosis and exacerbates inflammatory response

Yu Song a,b, Peng Liu b, Xi Qi b,c, Xiao-Lin Shi b,c, Yu-Shan Wang b,c, Dong Guo b, Hong Luo c, Zong-Jun Du a, Ming-Yi Wang a,b,*
PMCID: PMC11529197  PMID: 39056165

Abstract

Aim: Understanding molecular mechanisms of Helicobacter pylori (H. pylori)-induced inflammation is important for developing new therapeutic strategies for gastrointestinal diseases.

Materials & methods: We designed an H. pylori-neutrophil infection model and explored the effects of H. pylori infection on neutrophils.

Results: H. pylori infected neutrophils showed a low level of apoptosis. H. pylori stimulation activated the NACHT/LRR/PYD domain-containing protein 3 (NLRP3)-gasdermin-D (GSDMD) pathway for interleukin (IL)-1β secretion. However, IL-1β secretion was not completely dependent on GSDMD, as inhibition of autophagy significantly reduced IL-1β release, and autophagy-related molecules were significantly upregulated in H. pylori-infected neutrophils.

Conclusion: Therefore, H. pylori infection inhibits neutrophils apoptosis and induces IL-1β secretion through autophagy. These findings may be utilized to formulate therapeutic strategies against H. pylori mediated chronic gastritis.

Keywords: : Apoptosis, autophagy, H. pylori, IL-1β, neutrophil

Plain language summary

Article highlights.

Background

  • Helicobacter pylori (H. pylori) is a common opportunistic morbigenous bacterium worldwide and associated with gastrointestinal diseases.

Methods

  • H. pylori strain ATCC 11637 was used in this study.

  • We designed a H. pylori neutrophils infection model in vitro utilizing in vivo harvested neutrophils.

  • Pathological tissue analysis were carried out by multiplexed immunohistochemistry.

Results

  • The H. pylori infected neutrophils showed a lower frequency of annexin V-labeling and upregulation of CXCR4.

  • H. pylori stimulation promote the activation of the NLRP3-GSDMD-IL1β pathway.

  • Autophagy inhibitor significantly reduced IL-1β release and autophagy related molecules were upregulated significantly in H. pylori infected neutrophils.

Conclusion

  • H. pylori inhibited neutrophil apoptosis and prolonged neutrophil survival.

  • The inhibition of neutrophil apoptosis by H. pylori was mediated by inhibition of caspase-3 activation.

  • H. pylori infection induced IL-1β secretion by neutrophils via an autophagy-dependent mechanism.

1. Background

Helicobacter pylori (H. pylori), discovered in 1982, is a common opportunistic morbigenous bacterium worldwide and associated with gastrointestinal diseases [1]. It has been identified as a major causative factor of gastric adenocarcinoma (GA), with almost 90% of new cases of GA attributed to this bacterium [2]. The prevalence of H. pylori infection varies with geographic regions, socioeconomic status, living environment, smoking status, high-salt diet and occupation [3]. Although the average prevalence of H. pylori infection worldwide is more than 50%, most H. pylori positive individuals remain asymptomatic throughout lifetime [4,5]. Approximately 10% carriers of H. pylori develop peptic ulcer, and 1–3% cases progress to gastric cancer [6]. Typical virulence factors of H. pylori include cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA), which are encoded by the cag pathogenicity island (PAI) and translocated to host cells via the Type IV secretion system (T4SS) [7]. When injected into targeted cells, CagA activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induces the production of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-6 [8]. The variable response to H. pylori infection is partly affected by the high expression levels of inflammatory cytokines [9]. Therefore, understanding the secretory mechanisms of H. pylori induced inflammatory cytokines is important for developing new strategies against GA.

Polymorphisms in inflammatory cytokine genes, such as IL-6 and IL-1β, are related to various cancers [9,10]. Polymorphisms in IL-1β significantly increase the risk of certain tumors [11,12], particularly GA [9,13]. The works by Shuiping Tu, et al. showed direct evidence that IL-1β combined with H. pylori contribute to the pathogenesis of GA, and both are indispensable [14]. IL-1β, synthesized in the cytoplasm as a precursor, is proteolytically processed to mature peptide with biological activity. Enzymatic processing of IL-1β in humans relies on caspases-1/4/5 and neutral proteases such as elastase and proteinase-3 [15,16]. Mature IL-1β without a leader signal peptide cannot be secreted outside the cell through the conventional secretory pathway, the endoplasmic reticulum-Golgi unit.

Several unconventional pathways have been reported to explain IL-1β secretion in macrophages or other cells; however, the definitions of these pathways remain controversial [17]. The pore-forming protein gasdermin-D (GSDMD)-dependent IL-1β release accompanied by pyroptosis has been primarily evidenced within monocytes/macrophages [18–20]. Neutrophils are the most abundant leukocytes in peripheral human blood. H. pylori can induce neutrophils infiltration into the gastric mucosal layers in patients with chronic gastritis [10,14]. Chen K and Karmakar M, et al. reported that neutrophils can secrete IL-1β in the absence of pyroptosis along with activation of NACHT/LRR/PYD domain-containing protein 3 (NLRP3) inflammasome, a mechanism different from that in macrophages [21–23]. Studies by Heilig, et al. and Monteleone, et al. showed that IL-1β secretion in neutrophils is reduced in GSDMD knockout mice, similar to that in macrophages [24,25]. Furthermore, the active fragment of GSDMD, GSDMD-NH2 segment protein (N-GSDMD), do not polymerize into the pores of the neutrophil plasma membrane; rather it is located on the membranes of the primary granules and autophagosomes in neutrophils; however, the reason behind this phenomenon and underlying mechanism remains unclear [26]. Moreover, autophagy related proteins are necessary for IL-1β secretion in neutrophils [26].

While most studies have focused on monocytes and macrophages as the main source of IL-1β, neutrophils are also important producer of IL-1β because they could be recruited in large numbers to the infection sites [27]. Traditionally, neutrophils are considered to have only antimicrobial potential, such as phagocytosis, and killing microbes by producing reactive oxygen species and granular proteases [28,29]. However, highly purified neutrophils exhibit considerable phenotypic and functional plasticity as they produce cytokines, show nuclear hypersegmentation, and change surface receptors [30,31]. Neutrophils have disadvantage that they are shortlived, with an average circulatory lifespan of 8 h to 5 days [32,33]. Whether the survival time of neutrophils is altered by H. pylori infection in vivo and the resulting effects remain unknown.

Therefore, the present study assessed the effect of H. pylori infection on neutrophil apoptosis and IL-1β secretory pathways.

2. Materials & methods

2.1. Isolation of human neutrophils

Whole blood was collected from healthy donors aged of 20–50 years in accordance with the Declaration of Helsinki guidelines and the Institutional Review Board of Weihai Municipal Hospital. Written informed consent was obtained from all donors. Neutrophils were then isolated using a human peripheral blood neutrophil isolation kit (TBD Science, Cat #LZS11131, TSN, CHN) by density gradient centrifugation, which yields >90% purity, as assessed by flow cytometry and Wright–Giemsa staining. A human neutrophil separation kit (Miltenyi Biotec, Cat #130-104-434, BB, DEU) was used to isolate highly purified neutrophils. APC antihuman CD184 (CXCR4) antibody (BioLegend®, Cat #306510, CA, USA) was used to detect CXCR4 by flow cytometry (BD Biosciences, Canto II, CA, USA).

2.2. Transwell migration assay

Transwell migration assay [34] was performed in a 24-well Transwell plate, 5 μm pore size (Jet Bio-Filtration, Cat #TCS004024, CAN, CHN). Approximately 2 × 105 neutrophils were seeded in the upper chambers with 200 μl Dulbecco’s modified eagle medium (DMEM) (Yeasen Biotechnology, Cat #41401ES76, SHA, CHN) containing 10% fetal bovine serum (Yeasen Biotechnology, Cat #40130ES76, SHA, CHN), and 1 mL of DMEM was added to the lower chambers. Then 10 μl phosphate-buffered saline containing 2 × 106 H. pylori was added to each well. The plates was then incubated at 37°C for 15–120 min. Neutrophils that migrated through the pores into the lower chambers were counted, and cells on the lower side of the filter were fixed with 4% paraformaldehyde, stained with Wright–Giemsa stain, and counted (Figure 1A). Four duplicate wells were set up in each group, and the experiments were repeated four-times.

Figure 1.

Figure 1.

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Transwell assay using neutrophils and Helicobacter pylori. (A) Simplified diagram of the Transwell assay chamber. (B) Number of neutrophils migrated to the lower chamber. (C) Number of neutrophils migrated to the membrane filter. (D) Wright–Giemsa staining of membrane filters. The experiment was repeated four-times.

2.3. Patient recruitment

Patients from the Physical Examination Department, who underwent the urea breath test between June 2020 and July 2022 were recruited in this study. All samples were obtained with written informed consent from the patients prior to their inclusion, in accordance with the Helsinki Declaration. H. pylori status was assessed using a Urea 13C Breath Test kit (Huagen Anbang Technology, Cat #211013, PEK, CHN) and a Typing Detection Kit for antibodies against H. pylori (Shenzhen Blot Biotech, Cat #202107039, SZX, CHN). The patients were considered positive for H. pylori infection when both examinations yielded positive results.

2.4. Multiplexed immunohistochemistry (mIHC)

mIHC was performed by staining 4 μm thick formalin-fixed paraffin-embedded whole tissue sections using primary antibodies and a tyramide signal amplification 7-color kit (Absinbio, Cat #abs50015, PEK, CHN) [35]. Briefly, formalin-fixed paraffin-embedded slides were deparaffinized with xylene and hydrated using an ethanol series (100, 95 and 70%) and distilled water. Deparaffinized slides were incubated with antimyeloperoxidase (MPO) rabbit monoclonal antibody (Beyotime Biotechnology, Cat #AG2657, SHA, CHN) for 30 min, followed by incubation with an antirabbit horseradish peroxidase-conjugated secondary antibody for 10 min. Labeling was then developed with strict observation for 10 min using tyramide signal amplification 520 per manufacturer’s instructions. Slides were washed with Tris-buffered saline with Tween-20 and transferred to a preheated citrate solution (90°C) before being heat-treated using a microwave oven at 20% maximum power for 15 min. The slides were then cooled to room temperature using the same solution. This process was repeated when the slides were incubated with anti-CXCR4 monoclonal antibody (Proteintech Group, Cat #60042-1-Ig, IL, USA). Each slide was then incubated with two drops of 4′,6-diamidino-2-phenylindole (DAPI), washed in distilled water and mounted using a coverslip. The slides were air-dried and photographed using a Pannoramic MIDI Scanner (3DHISTECH, BP, HUN). Images were analyzed using Indica Halo software.

2.5. Western blot analysis

Proteins in neutrophils lysates were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. Membranes were incubated with primary monoclonal antibodies against IL-1β (Proteintech Group, Cat #66737-1-Ig, IL, USA), gasdermin D(GSDMD) (SIGMA-ALDRICH, Cat #G7422, MO, USA), LC3B (Abmart, Cat #T55992M, SHA, CHN), ULK1 (Proteintech Group, Cat #68445-1-Ig, IL, USA), ATG5 (Beyotime Biotechnology, Cat #AG4459, SHA, CHN), ATG16L (Proteintech Group, Cat #67943-1-Ig, IL, USA), NLRP3 (Beyotime Biotechnology, Cat #AF2155, SHA, CHN), caspase-1 (Cell Signal Technology, Cat #2225S, MA, USA), caspase-3 (Cell Signal Technology, Cat #14220S, MA, USA), Akt (Cell Signal Technology, Cat #9272S, MA, USA), Bax (Cell Signal Technology, Cat #2774S, MA, USA), Bcl-x (Cell Signal Technology, Cat #2764S, MA, USA), GAPDH (Cell Signal Technology, Cat #2118S, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signal Technology, Cat #7074S or 7076S, MA, USA). Protein bands were visualized using a Chemidoctoc XRS Molecular Imager (Bio-Rad, CA, USA). Protein expression was normalized with respect to GAPDH expression.

2.6. Cytokine assay

Enzyme-linked immunosorbent assay (ELISA) was used to quantify the levels of IL-1β in media. Cells were treated with 10 μM disulfiram (DSF), an inhibitor of GSDMD pore formation, 20 μM MCC950, an inhibitor of NLRP3, or 50 μM chloroquine (CQ), an autophagy inhibitor, 1 h before the addition of H. pylori. After treatment, cells were centrifuged at 1500 rpm for 5 min, and the supernatant was used for ELISA. A LEGEND MAX™ Human IL-1β ELISA Kit (BioLegend®, Cat #437007, CA, USA) was used, and optical density at 450 nm was determined using a Varioskan Lux plate reader (Thermo scientific, MA, USA).

2.7. Analysis of neutrophil apoptosis using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)

Neutrophils apoptosis was measured by flow cytometry using an annexin V-FITC/PI apoptosis assay kit (BD Biosciences, Cat #556547, CA, USA) according to the manufacturer’s instructions. Briefly, neutrophils were washed twice with ice-cold phosphate-buffered saline and resuspended in 100 μl binding buffer. Annexin V-FITC and PI were added and incubate away from light for 15 min, then 400 μl binding buffer was added and neutrophils were analyzed by flow cytometry within 1 h. Viable neutrophils were defined as those negative for annexin V-FITC and PI staining; apoptotic neutrophils were defined as those positive for annexin V-FITC but negative for PI staining; and necrotic cells were defined as those positive for both annexin V-FITC and PI staining.

2.8. Statistical analysis

Statistical analysis was performed using Student’s t test (two-tailed) to compare the results between the control and H. pylori-treated groups, using GraphPad Prism. Error bars indicate mean ± standard error, and a p-values < 0.05 was considered significant.

3. Results

3.1. H. pylori mildly attracted neutrophils

H. pylori infection is often histologically characterized by leukocyte infiltration, including neutrophils recruitment into the gastric mucosal layers [36,37]. The migration of highly purified neutrophils to H. pylori (ATCC 11637) was analyzed using a Transwell assay in vitro (Figure 1A & Supplementary Figure S1). The number of migrating neutrophils in the lower chamber did not differ between the control and H. pylori treated groups (Figure 1B). The number of neutrophils that migrated through the membrane filter was slightly higher in the H. pylori group than in the control group; however, the difference was not significant (Figures 1C & D). This suggests that H. pylori is not a strong attractor of neutrophils. Therefore, we investigated whether factors other than attraction that contribute to neutrophils accumulation in gastric foci during H. pylori infection.

3.2. H. pylori infection delayed neutrophil apoptosis

When cultured in vitro, rapid and spontaneous apoptosis of neutrophils resulted in a decrease in cell count over time, whereas H. pylori infection significantly inhibited the decrease in neutrophil counts (Figures 2A & B). More neutrophils survived in the H. pylori infection group than in the control group, indicating that H. pylori infection prolonged the survival time of neutrophils. H. pylori infected neutrophils showed a relatively low frequency of Annexin V+ cells, but a relatively high abundance of PI and Annexin V double-negative cells, indicating the presence of live neutrophils even after 24–48 h (Figure 2C–F & Supplementary Figure S2). Owing to the delay in apoptosis, relatively high number of apoptotic neutrophils (Annexin V+) accumulated in the H. pylori infected group, resulting in an increase in the absolute count of Annexin V-positive cells (Figure 2G). These data demonstrate that neutrophils infected by H. pylori display a slower rate of apoptosis than do the uninfected cells.

Figure 2.

Figure 2.

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Helicobacter pylori infection delayed neutrophil apoptosis. (A) Morphology of neutrophils 24 h post H. pylori infection (right). (B) Number of neutrophils. (C) Analysis of neutrophil apoptosis by flow cytometry using propidium iodide (PI)/annexin V staining. (D) Frequency of live (PI-Annexin V-) neutrophils. (E) Frequency of apoptotic (Annexin V+) neutrophils. (F) Absolute number of live (PI-Annexin V-) neutrophils. (G) Absolute number of apoptotic (Annexin V+) neutrophils. (H) C-X-C motif chemokine receptor 4 (CXCR4) analysis in neutrophils by flow cytometry. (I) Frequency of CXCR4hi/CXCR4low neutrophils. (J) Transcriptional levels of SEC14 like lipid binding 1 (SEC14L1) and CXCR4 detected by qRT-PCR. The experiment was repeated five-times.

*p < 0.05; **p < 0.01.

qRT-PCR: Quantitative real-time polymerase chain reaction.

Neutrophils with prolonged survival times would exhibit senescence characteristics. Studies have shown that senescent neutrophils exhibit an increase in markers such as CXCR4 and SEC14 like lipid binding 1 (SEC14L1) [38–40]. H. pylori infected neutrophils showed upregulation in mRNA and protein expression of CXCR4, which was significantly different from that in the control group (Figure 2H–J).

3.3. H. pylori infection increased CXCR4+ neutrophils in vivo

Next, we investigated whether H. pylori infection delayed neutrophils apoptosis in vivo. We used CXCR4 as a marker of senescent neutrophils. Histopathological sections from three healthy controls without H. pylori infection (HC) and six patients with chronic atrophic gastritis owing to H. pylori infection (H. pylori+) were analyzed using mIHC, which showed that during H. pylori infection, neutrophils (with multilobed nuclei) showed upregulation of CXCR4 expression in vivo, which was significantly different from that in healthy controls (Figure 3). These data indicate that H. pylori infection delays neutrophil apoptosis in vivo.

Figure 3.

Figure 3.

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Helicobacter pylori infection upregulated CXCR4+ neutrophils in vivo. Representative immunofluorescent staining of cell nuclei and CXCR4. HC: Healthy controls. H.p+: patients of chronic atrophic gastritis with H. pylori infection.

3.4. H. pylori infection delayed neutrophil apoptosis through multiple pathways

H. pylori infection inhibited neutrophils apoptosis, which led to the accumulation of senescent neutrophils; therefore, we aimed to decipher the underlying mechanism. Protein chip array showed that the expression of anti-apoptotic proteins, including B cell lymphoma 2 (Bcl-2), B-cell lymphoma extra (Bcl-x), survivin and X-linked inhibitor of apoptosis (XIAP), were upregulated, whereas the expression of apoptosis-promoting proteins, such as active caspase-3, was downregulated in H. pylori infected neutrophils (Figure 4A & Supplementary Figure S3). However, apoptosis-promoting proteins, such as Bcl-2-associated death promoter (Bad), Bcl-2-associated X protein (Bax), cytochrome c and tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor (TRAILR), were slightly upregulated (Figure 4A & Supplementary Figure S3).

Figure 4.

Figure 4.

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Helicobacter pylori infection delayed neutrophil apoptosis. (A) Analysis of apoptosis-related proteins by protein chip Human Apoptosis Array Kit (A) and western blotting (B). (C) Densitometric analysis of western blots. (D) Analysis of apoptosis in CAGA knockout H. pylori (H.p-ΔcagA)-infected neutrophils by flow cytometry. (E) Frequency of live (PI-annexin V-) neutrophils as determined by flow cytometry. (F) Frequency of apoptotic (Annexin V+) neutrophils as determined by flow cytometry. The protein chip assay was repeated twice, and the other experiments were repeated thrice.

*p < 0.05.

NS: Not significantly.

Western blot analysis showed a slight increase in caspase-3 expression and a significant decrease in the expression of active caspase-3 (Figures 4B, C & Supplementary Figure S4), which was consistent with the protein chip results. However, we did not observe any obvious upregulation of Bcl-2, Bcl-x, survivin and XIAP in H. pylori-infected neutrophils, as indicated by the protein chip array results. WB results showed a decrease in Bax expression, which was inconsistent with the protein chip array result. We also detected an obvious increase in the expression of protein kinase B (Akt) (Figure 4B & C), an important mediator of the delay in neutrophil apoptosis caused by inflammation. As neutrophil apoptosis involves complex regulatory networks, these limited data demonstrated that H. pylori infection may activate the anti-apoptotic proteins Bcl-2 and Bcl-x in neutrophils and then restrain the activation of caspase-3, extending the lifespan of neutrophils.

Cytotoxin associated gene A (CagA) is a virulence factor of H. pylori. However, the CAGA-knockout strain of H. pylori (H.p-cagA) did not show a reduced anti-apoptotic effect compared with that of the wild-type controls (H.p-wt) (Figure 4D–F). These results indicate that Cag A does not play an anti-apoptotic role in H. pylori infected neutrophils.

3.5. H. pylori infection promote neutrophils secrete IL-1β

The NLRP3 inflammasome is usually activated in response to several physical and chemical stimuli, such as extracellular adenosine triphosphate, uric acid crystals, aluminum salts and several bacterial pathogens [41–43]. We found that neutrophils infected with H. pylori showed increased transcription (Figure 5A–E) and protein expression (Figure 5F & G) of intracellular NLRP3 inflammasome, whereas other types of inflammasome receptors, including absent in melanoma 2 (AIM2), NOD-like receptor family pyrin domain-containing 1 (NLRP1) and NOD-like receptor C4 (NLRC4), showed no significant changes at the transcriptional level (Figure 5B–D). The expression and activation of caspase-1, an enzyme downstream of NLRP3 and an activator of pro-IL-1β, and mature form IL-1β increased (Figures 5F & G). The expression of GSDMD, the executor of IL-1β secretion, did not increase at the transcriptional level (Figure 5E), but increased at the protein level (Figures 5F & G). NLRP3 inflammasome inhibitor (MCC950) inhibited the release of IL-1β (Figure 5H) without affecting IL-6 and TNFα secretion (Figures 5I & J). These results suggest that H. pylori stimulation activates NLRP3 inflammasome signaling pathway in neutrophils and promotes the expression and secretion of IL-1β through this pathway.

Figure 5.

Figure 5.

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Helicobacter pylori infection induced IL-1β secretion through the NACHT/LRR/PYD domain-containing protein 3 (NLRP3) inflammasome in neutrophils.

qRT-PCR analysis of mRNA levels of NLRP3 (A), AIM2 (B), NLRP1 (C), NLRC4 (D) and GSDMD (E). (F) Western blot analysis of NLRP3 inflammasome-related proteins. (G) Densitometric measurements of the western blots. (H–J) Analysis of the levels of inflammatory cytokines by ELISA. The experiment was repeated four-times.

*p < 0.05.

qRT-PCR: Quantitative real-time polymerase chain reaction.

3.6. H. pylori infection promote IL-1β release through autophagy-related pathways

GSDMD specific inhibitor (disulfiram, DSF) treatment did not decrease IL-1β secretion (Figure 6A); However, the NLRP3/Caspase-1 pathway inhibitor (MCC950) significantly blocked IL-1β secretion (Figure 6A), suggesting that the expression and/or secretion of IL-1β in neutrophils mainly depend on the NLRP3/caspase-1 pathway, but not the GSDMD-pore.

Figure 6.

Figure 6.

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Helicobacter pylori infection promoted IL-1β release through autophagy-related pathways in neutrophils. (A) ELISA of IL-1β. (B) qRT-PCR analysis of mRNA expression of autophagy-related genes. (C) western blot analysis of autophagy-related proteins. (D) Densitometric measurements of western blots. (E) Analysis of lysosome activity by flow cytometry with Lyso–Dye staining. (F) Frequency of Lyso-Dye labeled neutrophils tested by flow cytometry. (G) Representative immunofluorescent images of co-staining of autophagy related proteins in neutrophils. The experiments were repeated four-times.

*p < 0.05; **p < 0.01.

CQ: Chloroquine; DSF: Disulfiram; qRT-PCR: Quantitative real-time polymerase chain reaction.

The autophagy inhibitor (chloroquine, CQ) treatment significantly reduced IL-1β secretion (Figure 6A). Simultaneously, increased expression of autophagy related molecules, including microtubule associated protein light chain 3B (LC3B I/II), autophagy-related gene 5 (ATG5), UNC-51 like autophagy activating kinase 1 (ULK1) at the transcriptional (Figure 6B) and translational levels (Figures 6C, D & G) was noticed. Autophagy in phagocytes involves the fusion of phagosomes and lysosomes and formation of autophagosomes. The occurrence of this process can be reflected by detecting pH changes in lysosomes using specific probes (Lyso–dye). The activity of lysosomes in neutrophils significantly increased after infection with H. pylori (Figures 6E & F), confirming that H. pylori infection promotes the formation of autophagosomes in neutrophils.

4. Discussion

We established an in vitro infection model using H. pylori and neutrophils and found that neutrophil apoptosis significantly decreased by H. pylori infection. Neutrophils with prolonged survival times exhibit an increase in cell senescence markers such as CXCR4 and SEC14L1 [38–40]. SEC14L1 and CXCR4 are signature genes of the neutrophil cluster hG5c that displays the highest maturation and apoptosis scores [40]. As H. pylori infection delayed neutrophil apoptosis, we selected these two molecules as reference markers for neutrophil aging and observed elevated mRNA levels of SEC14L1 and CXCR4 (Figure 2J). This indicated that H. pylori infection prolongs neutrophil survival. CXCR4/CXCL12 signaling also plays an important role in neutrophil retention at inflammatory sites and in the bone marrow [39]. Although we cannot rule this out, the two explanations do not contradict each other.

During infection, the molecular mechanisms underlying neutrophil apoptosis are characterized by multiple pathways that have not yet been fully elucidated. Previously, we found that H. pylori can abnormally activate the NF-κB signaling pathway in neutrophils through the virulence factor CagA and then upregulate anti-apoptotic proteins, such as Bcl-2 and Bcl-x, and activate or downregulate proapoptotic proteins, such as Bax and caspase-3, thereby hindering neutrophil apoptosis. A low Bax/Bcl-2 ratio is associated with impaired neutrophil apoptosis and weak caspase-3 activity [44]. In H. pylori infected neutrophils, increasing of Bcl-2 and unaltered or decreased Bax expression resulted in a relatively low Bax/Bcl-2 ratio. Akt, also known as Protein Kinase B, is an important signaling molecule, which mediates the delay in neutrophil apoptosis caused by inflammation [45]. We also detected increased Akt levels in H. pylori infected neutrophils. These data show that after H. pylori infection, neutrophil apoptosis can be regulated by multiple signaling axes, resulting in a prolonged life cycle and aggravation of the inflammatory response.

IL-1β is a leaderless cytosolic protein, the secretion of which does not follow the classical endoplasmic reticulum-Golgi pathway, and a canonical mechanism of secretion remains to be established. Notably, IL-1β is an important inflammatory factor associated with GA, and its synthesis and secretion are closely related to pyroptosis [46,47]. Pyroptosis is a recently discovered form of programmed cell death. In contrast to apoptosis, pyroptosis-related caspase molecules can cleave the downstream target protein GSDMD, after activation. Active N-GSDMD produced by cleavage can directly bind to phospholipid molecules and form a pore structure on the cell membrane, resulting in the loss of membrane integrity [46]. This process releases several inflammatory factors, such as IL-1β, in large quantity.

After H. pylori infection, NLRP3 expression increase in immune cells; caspase-1 activity is enhanced; and IL-1β secretion is increased [41,43]. We also found that after H. pylori infection, the expression of NLRP3 inflammasome, caspase-1, and GSDMD in neutrophils increased, and IL-1β secretion was significantly induced. Consistent results have been obtained in previous studies [41–43]. Inhibition of the NLRP3/caspase-1 pathway downregulated the downstream proteins, and IL-1β secretion was significantly reduced, while GSDMD inhibition failed to significantly inhibit IL-1β secretion. These results suggest that the NLRP3/caspase-1 pathway plays an important role in IL-1β expression, whereas activated N-GSDMD fails to mediate the transmembrane release of IL-1β.

IL-1β release involves two mechanisms: directly penetrating the cell membrane to form a transport pathway through perforated proteins, and relying on intracellular vesicle transport [48,49]. Neutrophils do not release IL-1β through the GSDMD pores under lipopolysaccharide stimulation, but GSDMD mediates the release of azurophilic protease [31]. Autophagy is a new pathway for unconventional secretion of cellular contents [49]. The NLRP3 inflammasome and caspase-1 are closely associated with autophagy [50]. During this process, the amount of reactive oxygen species, and activities of phosphatidylinositol 3-kinase (PI3K), and/or AMP-activated protein kinase (AMPK) increase in neutrophils, causing the activation of beclin1 or the inhibition of mechanistic target of rapamycin (mTOR), thereby inducing the formation of autophagosomes through downstream ATG5/ATG12. Additionally, LC3BII mediates the fusion and maturation of autophagosomes and phagosomes. Mature autophagosomes/phagosomes contain IL-1β and other substances that fuse with the cell membrane, resulting in IL-1β secretion [51]. We found that after H. pylori infection, the autophagosome formation-related proteins ATG5 and ATG16L were significantly upregulated in neutrophils, and the expression of ULK1, an essential protein for autophagosome formation in the mTOR pathway, significantly increased. LC3BII protein expression also increased. The autophagy inhibitor, CQ significantly reduced IL-1β secretion. These results indicate that H. pylori infection stimulates the initiation of autophagic signaling in neutrophils, which may play a leading role in IL-1β secretion.

This study suffers from some limitations. Multilobed nuclei, CD15 staining or MPO single staining did not adequately characterize neutrophils in the gastric tissue. Therefore, identifying gastric neutrophils using polychromatic co-staining is necessary. VacA and CagA are the two most important determinants of virulence of H. pylori. However, we did not perform VacA deficiency related experiments. Experiments with knockout neutrophil cell lines could not be performed for validating the necessity of molecules, such as caspase 3, Bcl-xL and XIAP, in IL-1β secretion. Neutrophils derived from the gene-knockout mice should be used to verify the function of a certain protein.

5. Conclusion

In conclusion, this study contributes to our understanding of the mechanism of IL-1β secretion in H. pylori stimulated human neutrophils. During the chronic phase of H. pylori infection, complex interactions occur between the pathogenic microorganisms and immune cells. H. pylori inhibits neutrophil apoptosis, prolongs survival time and increases the secretion of inflammatory factors, which are not conducive to curing chronic inflammation. These findings may contribute to identifying potential therapeutic targets for controlling H. pylori mediated chronic gastritis.

Supplementary Material

Supplementary Figures S1-S4
IFMB_A_2360798_SM0001.zip (1.3MB, zip)

Funding Statement

This work was supported by grants from the Natural Science Foundation of Shandong Province (No. ZR2020MH296 and ZR2021MH409) and the National Natural Science Foundation of China (No. 82102398).

Supplemental material

Supplementary data for this article can be accessed at https://doi.org/10.1080/17460913.2024.2360798

Author contributions

Y Song, P Liu, X Qi, XL Shi and YS Wang designed and performed the experiments and analyzed the data. D Guo, H Luo and ZJ Du helped with designing the experiments, analyzing the data and evaluating the manuscript. MY Wang designed the experiments, analyzed the data and wrote the paper. All authors reviewed the manuscript and approved the submitted version.

Financial disclosure

This work was supported by grants from the Natural Science Foundation of Shandong Province (No. ZR2020MH296 and ZR2021MH409) and the National Natural Science Foundation of China (No. 82102398). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

This study was approved by the Ethics Committee of Weihai Municipal Hospital (No. 2021020). All samples were obtained with written informed consent from the patients prior to their inclusion, in accordance with the Helsinki Declaration.

References

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