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Occurrences and phenotypes of RIPK3-positive gastric cells in Helicobacter pylori infected gastritis and atrophic lesions

  • Guanglin Cui
    Correspondence
    Corresponding author at: Faculty of Health Science, Nord University, Levanger Campus, Norway or Research Group of Gastrointestinal Diseases, the Second Affiliated Hospital of Zhengzhou University, China.
    Affiliations
    Research Group of Gastrointestinal Diseases, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China

    Faculty of Health Science, Nord University, Campus Levanger, Norway
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  • Aping Yuan
    Affiliations
    Research Group of Gastrointestinal Diseases, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
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  • Zhenfeng Li
    Affiliations
    Research Group of Gastrointestinal Diseases, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
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      Abstract

      Background

      Research evidences suggest that diverse forms of programmed cell death (PCD) are involved in the helicobacter pylori (H. pylori)-induced gastric inflammation and disorders.

      Aims

      To characterize occurrences and phenotypes of necroptosis in gastric cells in H. pylori infected gastritis and atrophic specimens.

      Methods

      Occurrences and phenotypes of necroptosis in gastric cells were immunohistochemically characterized with receptor-interacting protein kinase 3 (RIPK3) antibody in both human H. pylori infected gastric gastritis, atrophic specimens, and transgenic mice.

      Results

      Increased populations of RIPK3-positive cells were observed in both gastric glands and lamina propria in H. pylori infected human oxyntic gastritis and atrophic specimens. Phenotypic analysis revealed that many RIPK3-positive cells were H + K+ ATPase-positive parietal cells in the gastric glands and were predominantly CD3-positive T lymphocytes, CD68-positive macrophages, and SMA-alpha-positive stromal cells in the lamina propria. Furthermore, we found an increased expression of RIPK3-positive gastric glandular cells along with the histological process of hyperplasia-atrophy-dysplasia progression in hypergastrinemic INS-GAS mice.

      Conclusions

      An increased population of RIPK3-positive cells was observed in several types of gastric cells, future studies that define the effects and mechanisms of PCD implicated in the development of H. pylori induced gastric disorders are needed.

      Keywords

      1. Introduction

      Although the incidence of gastric cancer is decreased in western countries, it remains as one of the leading causes of cancer mortality worldwide, particularly in Asian countries [
      • Fock K.M.
      • Ang T.L.
      Epidemiology of Helicobacter pylori infection and gastric cancer in Asia.
      ]. It is now well recognized that infection with Helicobacter pylori (H. pylori) is a major cause of gastric cancer [
      • Sitarz R.
      • Skierucha M.
      • Mielko J.
      • Offerhaus G.J.A.
      • Maciejewski R.
      • Polkowski W.P.
      Gastric cancer: epidemiology, prevention, classification, and treatment.
      ]. According to the Correa pathway, the development of gastric cancer is through a multistep histopathologic cascade that now has been labelled as the atrophy-metaplasia-dysplasia-intestinal-type gastric cancer sequence [
      • Moss S.F.
      The Clinical Evidence Linking Helicobacter pylori to Gastric Cancer.
      ]. In which, the induction of parietal cell loss has been known as the key premalignant step of gastric atrophy development [
      • Moss S.F.
      The Clinical Evidence Linking Helicobacter pylori to Gastric Cancer.
      ]. However, the mechanisms by which H. pylori leads to gastric parietal cell loss (atrophy) remain as an unresolved issue and becomes a question of great interest currently.
      Physiologically, gastric cellular homeostasis is kept by the balance between cell death and renewal. Once this balance is disturbed, it may cause significant morphological and functional damages. For example, resistance to cell death can lead to hyperplasia and dysplasia as seen in the process of gastric carcinogenesis. On the other hand, excessive cell death will result in gastric cell loss as seen in gastric atrophy or ulceration [
      • Jones M.K.
      • Tomikawa M.
      • Mohajer B.
      • Tarnawski A.S.
      Gastrointestinal mucosal regeneration: role of growth factors.
      ].
      Considerable amount of in vitro and in vivo evidence suggest that H. pylori is a major pathogen that causes impaired balance of gastric cellular turnover and result in excessive process of programmed cell death (PCD) and atrophy in the gastric mucosa, which accelerates H. pylori-induced gastric carcinogenesis [
      • Yahiro K.
      • Satoh M.
      • Nakano M.
      • et al.
      Low-density lipoprotein receptor-related protein-1 (LRP1) mediates autophagy and apoptosis caused by Helicobacter pylori VacA.
      ,
      • Cui G.
      • Takaishi S.
      • Ai W.
      • et al.
      Gastrin-induced apoptosis contributes to carcinogenesis in the stomach.
      ,
      • Shirin H.
      • Moss S.F.
      Helicobacter pylori induced apoptosis.
      ,
      • Wagner S.
      • Beil W.
      • Westermann J.
      • et al.
      Regulation of gastric epithelial cell growth by Helicobacter pylori: offdence for a major role of apoptosis.
      ,
      • Rosania R.
      • Varbanova M.
      • Wex T.
      • et al.
      Regulation of apoptosis is impaired in atrophic gastritis associated with gastric cancer.
      ,
      • Alzahrani S.
      • Lina T.T.
      • Gonzalez J.
      • Pinchuk I.V.
      • Beswick E.J.
      • Reyes V.E.
      Effect of Helicobacter pylori on gastric epithelial cells.
      ]. Studies have revealed that H. pylori infection induced gastric parietal cell loss may be related to an increased rate of PCD [
      • Cui G.
      • Takaishi S.
      • Ai W.
      • et al.
      Gastrin-induced apoptosis contributes to carcinogenesis in the stomach.
      ,
      • Rosania R.
      • Varbanova M.
      • Wex T.
      • et al.
      Regulation of apoptosis is impaired in atrophic gastritis associated with gastric cancer.
      ,
      • Cai Y.
      • Cong X.
      • Fei R.
      • Wang J.T.
      [Effects of Helicobacter pylori, omeprazole and gastrin on the proliferation and apoptosis of gastric epithelial cell].
      . Several PCD forms such as apoptosis, autophagy and pyroptosis have been shown to be involved in the physiology of gastric mucosal homeostasis [
      • Cui G.
      • Yuan Y.
      • Wang Y.
      • Li Z.
      The expression of RIPK3 is associated with cell turnover of gastric mucosa in the mouse and human stomach.
      ] and associated with H. pylori-induced gastric inflammation and disorders [
      • Cui G.
      • Takaishi S.
      • Ai W.
      • et al.
      Gastrin-induced apoptosis contributes to carcinogenesis in the stomach.
      ,
      • Pachathundikandi S.K.
      • Blaser N.
      • Bruns H.
      • Backert S.
      Helicobacter pylori Avoids the Critical Activation of NLRP3 Inflammasome-Mediated Production of Oncogenic Mature IL-1beta in Human Immune Cells.
      ,
      • Semper R.P.
      • Mejias-Luque R.
      • Gross C.
      • et al.
      Helicobacter pylori-induced IL-1beta secretion in innate immune cells is regulated by the NLRP3 inflammasome and requires the cag pathogenicity island.
      . Recently, a novel PCD form, necroptosis, was identified. Which can be realized as a programmed necrosis that occurs in an orderly manner [
      • Christofferson D.E.
      • Yuan J.
      Necroptosis as an alternative form of programmed cell death.
      ,
      • Gunther C.
      • Neumann H.
      • Neurath M.F.
      • Becker C.
      Apoptosis, necrosis and necroptosis: cell death regulation in the intestinal epithelium.
      . In terms of morphology, unlike cell membrane integrity during apoptosis, the occurrence of necroptosis will destruct the integrity of cell membrane and release a large number of intracellular substances e.g., cytokines and enzymes, these factors could result in a strong immune response and pathological inflammation [
      • Dagenais M.
      • Douglas T.
      • Saleh M.
      Role of programmed necrosis and cell death in intestinal inflammation.
      ,
      • Pasparakis M.
      • Vandenabeele P.
      Necroptosis and its role in inflammation.
      . Therefore, excessive increases in necroptosis are frequently associated with the development of inflammatory diseases [
      • Dagenais M.
      • Douglas T.
      • Saleh M.
      Role of programmed necrosis and cell death in intestinal inflammation.
      ,
      • Pasparakis M.
      • Vandenabeele P.
      Necroptosis and its role in inflammation.
      ,
      • Ofengeim D.
      • Ito Y.
      • Najafov A.
      • et al.
      Activation of necroptosis in multiple sclerosis.
      ,
      • Pierdomenico M.
      • Negroni A.
      • Stronati L.
      • et al.
      Necroptosis is active in children with inflammatory bowel disease and contributes to heighten intestinal inflammation.
      ,
      • Zhou W.
      • Yuan J.
      Necroptosis in health and diseases.
      ,
      • Lee S.H.
      • Kwon J.Y.
      • Moon J.
      • et al.
      Inhibition of RIPK3 Pathway Attenuates Intestinal Inflammation and Cell Death of Inflammatory Bowel Disease and Suppresses Necroptosis in Peripheral Mononuclear Cells of Ulcerative Colitis Patients.
      ,
      • Zhang J.
      • Lei H.
      • Hu X.
      • Dong W.
      Hesperetin ameliorates DSS-induced colitis by maintaining the epithelial barrier via blocking RIPK3/MLKL necroptosis signaling.
      ]. For example, recent interest has been focused on the pathological role of necroptosis in intestinal inflammation [
      • Gunther C.
      • Neumann H.
      • Neurath M.F.
      • Becker C.
      Apoptosis, necrosis and necroptosis: cell death regulation in the intestinal epithelium.
      ,
      • Dagenais M.
      • Douglas T.
      • Saleh M.
      Role of programmed necrosis and cell death in intestinal inflammation.
      ,
      • Takahashi N.
      • Vereecke L.
      • Bertrand M.J.
      • et al.
      RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis.
      ,
      • Negroni A.
      • Cucchiara S.
      • Stronati L.
      Apoptosis, Necrosis, and Necroptosis in the Gut and Intestinal Homeostasis.
      ,
      • Jeon M.K.
      • Kaemmerer E.
      • Schneider U.
      • et al.
      Notch inhibition counteracts Paneth cell death in absence of caspase-8.
      . Accumulative results suggested that increase in the rate of necroptosis could unbalance epithelial cell turnover and is associated with the development of inflammation in the intestine [
      • Gunther C.
      • Neumann H.
      • Neurath M.F.
      • Becker C.
      Apoptosis, necrosis and necroptosis: cell death regulation in the intestinal epithelium.
      ,
      • Dagenais M.
      • Douglas T.
      • Saleh M.
      Role of programmed necrosis and cell death in intestinal inflammation.
      ,
      • Takahashi N.
      • Vereecke L.
      • Bertrand M.J.
      • et al.
      RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis.
      ,
      • Negroni A.
      • Cucchiara S.
      • Stronati L.
      Apoptosis, Necrosis, and Necroptosis in the Gut and Intestinal Homeostasis.
      . Interestingly, previous studies have revealed that chronic infection of H. pylori could induce a significantly elevation of tumor necrosis factor (TNF)-α level in both human and mice [
      • Moss S.F.
      • Legon S.
      • Davies J.
      • Calam J.
      Cytokine gene expression in Helicobacter pylori associated antral gastritis.
      ,
      • Goll R.
      • Gruber F.
      • Olsen T.
      • et al.
      Helicobacter pylori stimulates a mixed adaptive immune response with a strong T-regulatory component in human gastric mucosa.
      ,
      • Goll R.
      • Cui G.
      • Olsen T.
      • et al.
      Alterations in antral cytokine gene expression in peptic ulcer patients during ulcer healing and after Helicobacter pylori eradication.
      ,
      • Neu B.
      • Puschmann A.J.
      • Mayerhofer A.
      • et al.
      TNF-alpha induces apoptosis of parietal cells.
      ], which has been shown to be a factor to promote H. pylori induced inflammation and PCD in parietal cell [
      • Neu B.
      • Puschmann A.J.
      • Mayerhofer A.
      • et al.
      TNF-alpha induces apoptosis of parietal cells.
      ]. H. pylori vacuolating cytotoxin A (VacA) has been shown to promote progressive vacuolation and induce gastric mucosal injury. More recently, the induction of VacA on necroptosis in gastric epithelial cells has been reported in vitro [
      • Radin J.N.
      • Gonzalez-Rivera C.
      • Ivie S.E.
      • McClain M.S.
      • Cover T.L.
      Helicobacter pylori VacA induces programmed necrosis in gastric epithelial cells.
      ]. The findings suggested that H. pylori might induce necroptosis and change the PCD rate of gastric cells [
      • Radin J.N.
      • Gonzalez-Rivera C.
      • Ivie S.E.
      • McClain M.S.
      • Cover T.L.
      Helicobacter pylori VacA induces programmed necrosis in gastric epithelial cells.
      ]. This leads to us to hypothesize that necroptosis might be involved in the H. pylori infection induced gastric atrophy. On the other hand, hypergastrinemia is a consequence of chronic H. pylori infection and involved in the development of hyperplasia-atrophy-dysplasia in the stomach [
      • Cui G.
      • Takaishi S.
      • Ai W.
      • et al.
      Gastrin-induced apoptosis contributes to carcinogenesis in the stomach.
      ,
      • Neu B.
      • Puschmann A.J.
      • Mayerhofer A.
      • et al.
      TNF-alpha induces apoptosis of parietal cells.
      ,
      • Howlett M.
      • Chalinor H.V.
      • Buzzelli J.N.
      • et al.
      IL-11 is a parietal cell cytokine that induces atrophic gastritis.
      . However, the change of necroptosis during the hypergastrinemia-induced hyperplasia-atrophy-dysplasia process in the stomach in vivo remains unclear.
      Here, we immunohistochemically characterized the occurrence and phenotypes of RIPK3, a key element of necroptosis, positive gastric cells in H. pylori infected human gastric lesions (gastritis and atrophy). In addition, we also evaluated the dynamic of RIPK3-positive cells in fundic glands in the pathogenesis of gastric carcinogenesis in a hypergastrinemic transgenic gastric cancer mouse model.

      2. Materials and methods

      2.1 Human gastric specimens

      A total of 40 gastric paraffin blocks of H. pylori negative controls (n = 10), H. pylori infected chronic gastritis (n = 18), H. pylori infected gastric atrophy (n = 12) taken by gastric endoscopy from human oxyntic mucosa (Table 1) retrieved from tissue bank at Department of Pathology, the Second Affiliated Hospital of Zhengzhou University were included in this study. H. pylori infection was diagnosed by C13 breath test (Boran Pharmacy, Beijing, China). Human study protocol was approved by the institutional medical ethic review board of the Second Affiliated Hospital, Zhengzhou University,
      Table 1Human gastric oxyntic biopsies.
      NMale/FemaleAverage age (range)Lesion positionC13 breath test
      Control101/950.8 (31∼67)oxynticnegative
      Gastritis1812/657.89 (34∼84)oxynticpositive
      Atrophy127/557 (36∼85)oxynticpositive

      2.2 Transgenic gastric carcinogenesis mouse model

      Paraffin blocks of wild type (WT) FVB/N male mice (Charles River Lab., Wilmington, MA) at ages of 3, 6 and 12 months (each group mice N = 5), gastric carcinogenesis model of INS-GAS transgenic mice [
      • Wang T.C.
      • Dangler C.A.
      • Chen D.
      • et al.
      Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.
      ] were obtained from Drs. Theodore Koh and Timothy C. Wang (University of Columbia, New York, USA). Animals were euthanized by cervical dislocation under a anesthetized condition with pentobarbital sodium intraperitoneal injection at a dose of 50 mg/kg. Pathological features of INS-GAS mice have been described in previous publications [
      • Cui G.
      • Takaishi S.
      • Ai W.
      • et al.
      Gastrin-induced apoptosis contributes to carcinogenesis in the stomach.
      ,
      • Wang T.C.
      • Dangler C.A.
      • Chen D.
      • et al.
      Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.
      . Animal study protocol was approved by the local medical ethic committee of Second Affiliated Hospital of Zhengzhou University.

      2.3 Stomach immunohistochemistry (IHC) in mice and human specimens

      Male WT mice and INS-GAS transgenic mice at the ages of 3-, 6- and 12-month, and human gastric tissues of control, H. pylori positive gastritis and H. pylori positive atrophy were examined in this study. Midline strips along the lesser curvature of the stomach were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin. Sections were cut at 4 μm, and then stained with hematoxylin and eosin. IHC was performed with avidin-biotin-peroxidase complex (ABC) Elicit kits (Vector Laboratories, Burlingame, CA, USA) according to manufacturer's instructions and our previous published method [
      • Cui G.
      • Li C.
      • Xu G.
      • et al.
      Tumor-Associated Fibroblasts and Microvessels Contribute to the Expression of Immunosuppressive Factor Indoleamine 2, 3-Dioxygenase in Human Esophageal Cancers.
      ,
      • Cui G.
      • Ren J.
      • Xu G.
      • Li Z.
      • Zheng W.
      • Yuan A.
      Cellular and clinicopathological features of the IL-33/ST2 axis in human esophageal squamous cell carcinomas.
      ,
      • Cui G.
      • Xu G.
      • Zhu L.
      • et al.
      Temporal and spatial changes of cells positive for stem-like markers in different compartments and stages of human colorectal adenoma-carcinoma sequence.
      ,
      • Yuan A.
      • Yang H.
      • Qi H.
      • et al.
      IL-9 antibody injection suppresses the inflammation in colitis mice.
      ]. The following primary antibodies were used: against RIPK3 to stain necroptosis cells (working dilution 1:500, rabbit anti-human, mouse and rat, Thermo Fisher Scientific, Oslo, Norway) and H + K+-ATPase β subunit to parietal cells (1:2000, mouse anti-hog, Affinity Bioreagents, Golden, CO, USA). Primary antibodies were incubated at 4 °C overnight in humidified chamber. 3-Amino-9-ethylcarbazole (AEC; Vector Laboratories, Burlingame, CA, USA) was used as chromogen and slides were counterstained with Mayer's hematoxylin.

      2.4 Double immunofluorescence (DIF) staining in human gastric sections

      We have observed the positive cells for RIPK3 in human stomach in both gastric glands and lamina propria. To show RIP3 in the gastric parietal cells, double DIFs with RIPK3/HK-ATPase antibodies were done according to the protocol described in our previous publication [
      • Cui G.
      • Li C.
      • Xu G.
      • et al.
      Tumor-Associated Fibroblasts and Microvessels Contribute to the Expression of Immunosuppressive Factor Indoleamine 2, 3-Dioxygenase in Human Esophageal Cancers.
      ,
      • Cui G.
      • Yuan A.
      • Zhu L.
      • Florholmen J.
      • Goll R.
      Increased expression of interleukin-21 along colorectal adenoma-carcinoma sequence and its predicating significance in patients with sporadic colorectal cancer.
      ,
      • Cui G.
      • Yuan A.
      • Sun Z.
      • Zheng W.
      • Pang Z.
      IL-1beta/IL-6 network in the tumor microenvironment of human colorectal cancer.
      ,
      • Cui G.
      • Yuan A.
      • Li Z.
      • Goll R.
      • Florholmen J.
      ST2 and regulatory T cells in the colorectal adenoma/carcinoma microenvironment: implications for diseases progression and prognosis.
      in human oxyntic mucosa sections. To analyze phenotypes of RIPK3 positive cells in the gastric lamina propria, double DIFs with RIPK3/H + K+-ATPase (to show RIPK3 in parietal cells), RIPK3/CD3 (to show RIPK3 in T lymphocytes, mouse anti-CD3 monoclonal antibody, DAKO, Carpinteria, CA, USA), RIPK3/CD68 (to show RIPK3 in macrophages, mouse anti-CD68 monoclonal antibody, DAKO, Carpinteria, CA, USA) and RIPK3/SMA-alpha (to show RIPK3 in stromal myofibroblasts, mouse anti-SMA-α monoclonal antibody, DAKO, Carpinteria, CA, USA) in human H. pylori infected gastric lesions including chronic fundic gastritis, atrophy sections. After gastric fundic sections incubated with primary antibodies at 4 °C overnight, RIP3-immunoreactivity (IR) was developed with Texas red-conjugated secondary antibody (Jackson ImmunoRearch Lab., West Grove, PA, USA) and H + K+-ATPase -IR, CD3, CD68 and SMA-α were developed with FITC-conjugated secondary antibody (Jackson ImmunoRearch Lab.).
      Then, DIF sections were observed and photographed under a confocal microscopy (LSM-700, Carl Zeiss, Jena, Germany) under 200 × magnification.

      2.5 Morphological evaluation

      For mice sections, only RIPK3-labeling glandular cells with appropriate morphology and location in well-oriented sections were counted in at least 10 selected glands with abundant distribution under × 400 magnification (Zeiss, Germany). Since human gastric biopsies are taken by endoscopy and very small, the orientation is therefore difficult to be managed in a well-oriented position as seen in mice gastric biopsies. We have to counter oxyntic glandular cells in 10 selected glands with abundant distribution under × 400 magnification. Densities of RIPK3-positive glandular cells of oxyntic mucosa were expressed as number per gland. The average values of positive cells per glands were used for the statistical analysis.

      2.6 Statistical analysis

      Data were present as mean ± SEM (standard error of the mean) unless otherwise stated. P values were evaluated by the Mann–Whitney test. P values < 0.05 were considered statistically significant.

      3. Results

      3.1 Occurrence of RIPK3-positive glandular cells in human H. pylori positive gastric oxyntic mucosa

      Compared with human control gastric oxyntic mucosa (Fig. 1A). The population of RIPK3-positive glandular cells was increased in H. pylori infected gastritis (Fig. 1B) and even higher in H. pylori infected atrophy (Fig. 1C). Increased populations of RIPK3 in other types of gastric cells such as surface mucosal cells (Fig. 1B&C) and lamina propria cells (Fig. 1B&C) in gastritis and atrophic tissues were also observed. Further quantitative results of RIPK3-positive glandular cells showed significantly increased densities from control to gastritis and then to atrophy (Fig. 1D).
      Fig 1
      Fig. 1Immunohistochemical examination for RIPK3-positive cells in the fundic glands of H. pylori infected human gastric gastritis and atrophy. As compared with the H. pylori negative controls (A), RIPK3-positive cells in the oxyntic glands were shown in an increasing trend in H. pylori infected gastritis (B) and atrophic gastritis (C). The counting data showed that as compared with the controls (white bar in D), the number of RIPK3-positive cells in fundic glands was significantly increased in H. pylori infected gastritis (gray bar in D). It was slightly decreased in H. pylori infected atrophy (black bar in D), but still higher than that in the controls. (A-F, IHC images, counterstained with Hematoxylin, original magnification 400 ×).

      3.2 Phenotypic analysis of RIPK3-positive cells in H. pylori positive human gastric lesions

      Phenotypic analysis revealed that RIPK3-positive cells in the fundic glands were mostly H + K+-ATPase positive parietal cells (Fig. 2A-C). RIPK3-positive cells in the lamina propria were CD3-positive T lymphocytes (Fig. 2D-F) and CD68-positive macrophages (Fig. 2G-I) and occasionally SMA-α-positive stromal cells (Fig. 2J-L). This observation suggested that RIPK3 could be expressed in both gastric glandular cells and many types of cells in the lamina propria.
      Fig 2
      Fig. 2Double immunofluorescence examination of RIPK3-positive cell phenotypes in human H. pylori infected gastric lesions. Phenotypic analysis revealed that some RIPK3-positive cells (A) in oxyntic glands of human H. pylori infected gastric lesions were H+, K+, ATPase (B) positive parietal cells (merged image, C). RIPK3-positive cells (D, G, J) in oxyntic glands of human H. pylori infected gastric lesions were CD3-positive (E) lymphocytes (merged image, F), CD68-positive (H) macrophages (merged image, I) and SMA-α-positive (K) stromal cells (merged image, L). (A-L, double immunofluorescence-stained confocal images, original magnification 200 ×).

      3.3 Dynamic of RIPK3-positive cells in fundic glandular cells during the process of gastric atrophy - dysplasia sequence in hypergastrinemic transgenic gastric cancer mouse model

      Since hypergastrinemic INS-GAS transgenic gastric cancer mouse model mimics the process of hyperplasia-atrophy (parietal cell loss)-dysplasia sequence in the fundic mucosa of stomach [
      • Wang T.C.
      • Dangler C.A.
      • Chen D.
      • et al.
      Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.
      ], we have therefore examined dynamic of RIPK3-positive cells in fundic glands in this transgenic mouse model at different ages.
      As seen in Fig. 2. RIPK3-positive cells were found in both glandular parietal cells and surface mucous cells. Following the age increasing from 3-month, 6-month to 12-month, RIPK3 positive cells in fundic glands of WT FVB/N male mice were shown in a slightly increasing trend (Fig. 3A-C). The intensity of RIPK3-immunoreactivty was stronger in glandular cells in the upper part of fundic glands than those in lower part of glands. The RIPK3-positive cells in hypergastrinemic INS-GAS mice (Fig. 3D-F) were significantly changed as compared with the controls (Fig. 3A-C). The intensity of RIPK3-immunoreactivty was evenly distributed in fundic glandular cells. Counting data showed that densities of RIPK3-positive glandular cells in INS-GAS mice at ages of 3- and 6-month were higher than that in control FVB mice at the same ages (Fig. 3G). However, at the age of 12-month it was lower in the atrophy and dysplasia developed area of INS-GAS mice than control mice (Fig. 3G). But the density of RIPK3-positive cells in the fundic glands adjacent to atrophy and dysplasia (inserted image in Fig. 3F and gray bar in Fig. 3G) was still higher than that in control mice.
      Fig 3
      Fig. 3Immunohistochemical examination for RIPK3-positive fundic glandular cells in INS-GAS mice along the process of hyperplasia-atrophy-dysplasia sequence. RIPK3-psotive cells were shown in control wide-type (WT) FVB mice at different ages (A-C), some of them were located in the upper part of glands. RIPK3 positive cells in the fundic glands of INS-GAS mice were evenly distributed in the fundic glands and showed a gradual increasing trend from 3- (D) to 6-month ages (E) but decreased in atrophic region and remained in a high peak in adjacent region with hyper-parietal cells in 12-month age (F, insert image shows PRIPK3-positive glandular cells in normal region). Throughout this period a hyperplasia-atrophy-dysplasia sequence was established. Counting data showed that density changes of RIPK3-positive cells/gland (G) in the fundic mucosa were increased from 3- to 6- and 12-month along the process of hyperplasia-atrophy-dysplasia sequence. (A-F, IHC images, counterstained with Hematoxylin, original magnification 400 ×).

      4. Discussion

      It has been previously reported that chronic infection of H. pylori results in a significantly increased rate of PCD in gastric cells e.g., surface epithelial cells and parietal cells in the stomach [
      • Cui G.
      • Takaishi S.
      • Ai W.
      • et al.
      Gastrin-induced apoptosis contributes to carcinogenesis in the stomach.
      ,
      • Shirin H.
      • Moss S.F.
      Helicobacter pylori induced apoptosis.
      ,
      • Wang T.C.
      • Dangler C.A.
      • Chen D.
      • et al.
      Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.
      ,
      • Konturek P.C.
      • Kania J.
      • Konturek J.W.
      • Nikiforuk A.
      • Konturek S.J.
      • Hahn E.G.
      H. pylori infection, atrophic gastritis, cytokines, gastrin, COX-2, PPAR gamma and impaired apoptosis in gastric carcinogenesis.
      ,
      • Waldum H.L.
      • Sagatun L.
      • Mjones P.
      Gastrin and Gastric Cancer.
      ,
      • Yu P.
      • Zhang X.
      • Liu N.
      • Tang L.
      • Peng C.
      • Chen X.
      Pyroptosis: mechanisms and diseases.
      . In this study, we immunohistochemically characterized the occurrence and phenotypes of RIPK3-positive cells in the gastric glandular cells in Helicobacter infection related human specimens and the dynamic of RIPK3-positive cells in hypergastrinemia-induced atrophic and dysplastic lesions, respectively. Our results showed that increased expression of RIPK3-positive cells was demonstrated in diverse types of gastric cells in human specimens. Particularly, many of RIPK3-positive glandular cells were identified as H + K+-ATPase-positive parietal cells, which may suggest an potential role of necroptosis in the parietal cell loss and development of atrophy under H. Pylori infection condition.
      It has been previously demonstrated that PCD plays a fundamental role in tissue homeostasis and disrupted process of PCD is associated with human diseases [
      • Dhuriya Y.K.
      • Sharma D.
      Necroptosis: a regulated inflammatory mode of cell death.
      ,
      • Fuchs Y.
      • Steller H.
      Programmed cell death in animal development and disease.
      ,
      • Meng L.
      • Jin W.
      • Wang X
      RIP3-mediated necrotic cell death accelerates systematic inflammation and mortality.
      ]. Numerous studies have shown that chronic Helicobacter infection could induce an increased rate of PCD in gastric epithelial and granular cells that is associated with the development of gastric carcinogenesis [
      • Rosania R.
      • Varbanova M.
      • Wex T.
      • et al.
      Regulation of apoptosis is impaired in atrophic gastritis associated with gastric cancer.
      ,
      • Alzahrani S.
      • Lina T.T.
      • Gonzalez J.
      • Pinchuk I.V.
      • Beswick E.J.
      • Reyes V.E.
      Effect of Helicobacter pylori on gastric epithelial cells.
      ,
      • Neu B.
      • Puschmann A.J.
      • Mayerhofer A.
      • et al.
      TNF-alpha induces apoptosis of parietal cells.
      ,
      • Przemeck S.M.
      • Varro A.
      • Berry D.
      • et al.
      Hypergastrinemia increases gastric epithelial susceptibility to apoptosis.
      ,
      • Panella C.
      • Ierardi E.
      • Polimeno L.
      • et al.
      Proliferative activity of gastric epithelium in progressive stages of Helicobacter pylori infection.
      ,
      • Xia H.H.
      • Zhang G.S.
      • Talley N.J.
      • et al.
      Topographic association of gastric epithelial expression of Ki-67, Bax, and Bcl-2 with antralization in the gastric incisura, body, and fundus.
      . Rosania et al. previously [
      • Rosania R.
      • Varbanova M.
      • Wex T.
      • et al.
      Regulation of apoptosis is impaired in atrophic gastritis associated with gastric cancer.
      ] reported that the number of apoptotic epithelial cells was increased from 2% in normal mucosa to 8–17% in H. pylori related gastritis, which suggested a contributing role of PCD to the pathogenesis of H. pylori induced gastric lesions. TNF-α has been evident to be a potent inducer of necroptosis through activations of RIPK1 and then mixed lineage kinase domain-like (MLKL) that regulates cell necrosis downstream of RIPK3 [
      • Moriwaki K.
      • Chan F.K.
      Necrosis-dependent and independent signaling of the RIP kinases in inflammation.
      ,
      • Silke J.
      • Rickard J.A.
      • Gerlic M.
      The diverse role of RIP kinases in necroptosis and inflammation.
      ,
      • Yu X.
      • Deng Q.
      • Li W.
      • et al.
      Neoalbaconol induces cell death through necroptosis by regulating RIPK-dependent autocrine TNFalpha and ROS production.
      ,
      • Sun L.
      • Wang H.
      • Wang Z.
      • et al.
      Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.
      ,
      • Zhang D.W.
      • Shao J.
      • Lin J.
      • et al.
      RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis.
      ]. Interestingly, previous studies have provided massive evidences to suggest that chronic H. pylori infection can induce an remarkably increased expression of TNF-α in the gastric mucosa [
      • Goll R.
      • Gruber F.
      • Olsen T.
      • et al.
      Helicobacter pylori stimulates a mixed adaptive immune response with a strong T-regulatory component in human gastric mucosa.
      ,
      • Milic L.
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      • Popadic D.
      • et al.
      Altered cytokine expression in Helicobacter pylori infected patients with bleeding duodenal ulcer.
      ,
      • Moradipour A.
      • Khosravi A.
      • Piri F.
      Fecal Helicobacter pylori glmM and 16S rRNA genes correlate with serum TNF-alpha and IL-1beta cytokine fluctuations.
      , which in turn increases the risk of gastroduodenal mucosa injury [
      • Tourani M.
      • Habibzadeh M.
      • Karkhah A.
      • Shokri-Shirvani J.
      • Barari L.
      • Nouri H.R.
      Association of TNF-alpha but not IL-1beta levels with the presence of Helicobacter pylori infection increased the risk of peptic ulcer development.
      ]. Therefore, upregulated expression of TNF-α by Helicobacter infection might contribute to the increase in the rate of necroptosis in the gastric mucosal cells. Indeed, a recent in vitro study has demonstrated that H. pylori toxic factor VacA can significantly induce necroptosis and increase the death rate of gastric cells [
      • Radin J.N.
      • Gonzalez-Rivera C.
      • Ivie S.E.
      • McClain M.S.
      • Cover T.L.
      Helicobacter pylori VacA induces programmed necrosis in gastric epithelial cells.
      ], and suggested a potential role of necroptosis in H. pylori induced gastric disorders. RIPK3 is a critical regulator of necroptosis and facilitates the process of necroptosis and cytokine production and then contributes to the process of inflammation [
      • Dagenais M.
      • Douglas T.
      • Saleh M.
      Role of programmed necrosis and cell death in intestinal inflammation.
      ,
      • Pasparakis M.
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      ,
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      A RIPK3-caspase 8 complex mediates atypical pro-IL-1beta processing.
      . Therefore, the activation of RIPK3 is tightly associated with the induction of inflammation. In this study, we were able to demonstrate increased populations of RIPK3-positive cells in diverse gastric cells including glandular cells in H. pylori infected human gastritis and atrophic specimens. Particular interest finding was the identification of RIPK3-positive parietal cells, which are the main acid-producing glandular cells in oxyntic mucosa. This finding may imply that over process of necroptosis in the parietal cells could induce excessive loss of parietal cells and finally result in atrophy in oxyntic glands. Furthermore, we have found that RIPK3-immunoreactivity was observed in lamina propria cells. Phenotypic analysis showed that these positive cells were mostly identified as CD3-positive lymphocytes and some were CD68-positive macrophages and SMA-α-positive myofibroblasts. Since studies have demonstrated that necroptosis can destroy the integrity of cell membrane and then the release of a large number of inflammatory cytokines and contribute to the development of inflammation [
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      ,
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      , these findings might suggest a potential involvement of necroptosis occurred in the lamina propria immune and stromal cells in the development of gastric inflammation induced by H. pylori infection.
      Hypergastrinemia induced by chronic Helicobacter infection has been considered as a risk factor for the development of premalignant lesion atrophy and gastric cancer [
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      ,
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      ,
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      . The mechanisms underlying the carcinogenesis potential of hypergastrinemia are still not fully understood. Previously, an association between gastric cell PCD (apoptosis) and hypergastrinemia has been reported in rodents (Mastomys) [
      • Kidd M.
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      ] and INS-GAS transgenic mice [
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      • Ai W.
      • et al.
      Gastrin-induced apoptosis contributes to carcinogenesis in the stomach.
      ]. We have also showed that Helicobacter infection for 6 months resulted in an increased rate of apoptosis and accelerated development of gastric atrophy in INS-GAS mice, both was strongly inhibited by the treatment of CCK-2 receptor antagonist YF 476 and H-2 receptor antagonist loxtidine or both [
      • Cui G.
      • Takaishi S.
      • Ai W.
      • et al.
      Gastrin-induced apoptosis contributes to carcinogenesis in the stomach.
      ]. Thus, apoptosis as an PCD manner might contribute to the process of atrophy in the stomach [
      • Cui G.
      • Takaishi S.
      • Ai W.
      • et al.
      Gastrin-induced apoptosis contributes to carcinogenesis in the stomach.
      ]. To evaluate the dynamic of RIPK3-positive gastric glandular cells during the development of gastric carcinogenesis, we examined the occurrence and population changes of RIPK3-positive gastric glandular cells along with hyperplasia-atrophy-dysplasia histological stages in hypergastrinemic transgenic mice. We also found step-up increasingly densities of RIPK3-positive mucosal cells during the process of atrophy-dysplasia in transgenic mice as compared with WT mice, implying an occurrence of necroptosis in this region. RIPK3-immunoreactivity in fundic glands in younger (3-month) INS-GAS mice is located in an even distribution pattern, the density of RIPK3-positive glandular cells was slightly increased and greatly increased at the age of 6-month. After this time point, prolong exposure of mice to hypergastrinemia induced a significant atrophic and dysplastic lesion in the fundic mucosa and the rate of RIPK3-positive glandular cells in fundic atrophy region has decreased after parietal cell lost, but it still remained in a high level in the adjacent region with hyper-parietal cells. Such dynamic of RIPK3-positive glandular cell change along the process of hyperplasia-atrophy-dysplasia sequence in INS-GAS mice may provide a new insight into the role of necroptosis during the development of gastric carcinogenesis.
      Despite the limitation by a relatively small size of H. pylori infected gastric lesions, we have at first time demonstrated in this study an increased population of RIPK3-positive parietal cells in both human and mice. Our data provide a new insight into the role of PCD in the initiation of H. pylori infected-related gastric carcinogenesis. However, to precise the effects and mechanisms of necroptosis in the parietal cell loss and atrophy, there is still a lot of work to do. For example, several cytokines such as interleukin (IL)−11 and IL-17 have been shown to be elevated in patients with H. pylori infection and associated with the disease activity and symptoms [
      • Sabry D.
      • Abdelaleem O.O.
      • Hefzy E.M.
      • et al.
      Interplay Between Helicobacter pylori Infection, Interleukin-11, and Leukemia Inhibitory Factor in Gastric Cancer Among Egyptian Patients.
      ,
      • Kabir S.
      The role of interleukin-17 in the Helicobacter pylori induced infection and immunity.
      ,
      • Adamsson J.
      • Ottsjo L.S.
      • Lundin S.B.
      • Svennerholm A.M.
      • Raghavan S.
      Gastric expression of IL-17A and IFNgamma in Helicobacter pylori infected individuals is related to symptoms.
      ,
      • Arachchi P.S.
      • Fernando N.
      • Weerasekera M.M.
      • Senevirathna B.
      • Weerasekera D.D.
      • Gunasekara C.P.
      Proinflammatory Cytokine IL-17 Shows a Significant Association with Helicobacter pylori Infection and Disease Severity.
      ] and recently been demonstrated to participate in the process of parietal cell loss and atrophy through apoptosis in the mouse stomach [
      • Howlett M.
      • Chalinor H.V.
      • Buzzelli J.N.
      • et al.
      IL-11 is a parietal cell cytokine that induces atrophic gastritis.
      ,
      • Bockerstett K.A.
      • Osaki L.H.
      • Petersen C.P.
      • et al.
      Interleukin-17A Promotes Parietal Cell Atrophy by Inducing Apoptosis.
      . Therefore, we made a schematic summary of possible effects of necroptosis on the development of gastritis and atrophy under the H. pylori infection condition (ref. to Fig. 4). Future studies that focus on the effects and mechanisms of inflammatory mediators on necroptosis process in parietal cells remain to be conducted.
      Fig 4
      Fig. 4Schematic summary of possible effects of necroptosis on the development of gastritis and atrophy under the H. pylori infection condition incorporating integrated analysis of current and literature data. Chronic H. pylori infection resulted in the recruitment of diverse immune cells into the infected site and H. pylori toxicity factors, such as vacuolating cytotoxin A (VacA), induced necroptosis in immune cells and release high amounts of inflammatory mediators e.g., tumor necrosis factor (TNF) that triggered inflammatory cascade and inflammation in gastric mucosa. Persisted production of inflammatory mediators further induced increased necroptosis in parietal cells and parietal cell lost, and finally atrophy is developed.

      Conflict of interest

      None declared.

      Acknowledgments

      We deeply acknowledge Drs. Timothy C. Wang and Ted Koh for their help in providing paraffin blocks for INS-GAS gastric specimens and Dr. Jingli Ren for her help in providing paraffin blocks for human gastric specimens.

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