Introduction

Endoplasmic reticulum stress-induced mitochondria-dependent mucosal apoptosis and impaired mucosal defense barriers have been shown to be fundamental pathological features of portal hypertensive gastropathy (PHG) (Yi et al., Biomedicine & Pharmacotherapy 2021). Slc26a9 is a member of Slc26a family with highly expression in the stomach, which is a key regulator to maintain gastric mucosal homeostasis. Slc26a9 deletion in mice impaired gastric mucosal barrier (Liu et al., Cellular oncology 2022; Liu et al., DDW 2019, 2020 and 2022). However, the role of Slc26a9 in PHG is never investigated.

Aims & Methods

To identify the exact role of Slc26a9 in the PHG, histopathology and immunohistochemistry analysis, as well as western-blot assay were performed in PHG animal models by using parietal-specific Slc26a9 knockout (Slc26a9-KO) and Slc26a9 wild-type (Slc26a9-WT) mice. Slc26a9 mRNA and protein expression was detected in PHG patients.

Results

Compared with sham-operated mice, the mucosa of portal vein ligated (PVL) mice displayed obvious gastric mucosal damage, including inflammatory cell infiltration, edema and vasodilation. Slc26a9 KO-PVL mice displayed severe gastric mucosal injury than that in Slc26a9 WT-PVL mice, followed with significantly reduction of Slc26a9 expression both in Slc26a9 WT-PVL and Slc26a9 KO-PVL mice. Furthermore, excessive gastric epithelial cells apoptosis was detected by Tunel staining in Slc26a9 KO-PVL mice, accompanied with upregulation of endoplasmic reticulum (ER) stress markers, including Caspase12, eIFα-1, xBP-1, p53 upregulated apoptosis regulator (PUMA), as well as mitochondrial apoptosis markers, such as Bax, Bak, Cyt-c, Caspase 9 and Caspase 3, when compared with Slc26a9 WT-PVL mice. Of note, recombinant adeno-associated virus with Slc26a9 (rAAV Slc26a9) was overexpressed in Slc26a9 KO-PVL mice showed significant reversed gastric mucosal damage, including parietal and chief cells restore, reversal of cystic dilatation, and reduction of inflammatory cells, accompanied with significant reduction of gastric epithelial cell apoptosis, as well as ER stress mediated mitochondria-dependent apoptotic signalling, when compared with the control group. These results indicate that Slc26a9 reversed the PHG induced mucosal injury and functioned as the protector for the gastric mucosal defense during PHG. Moreover, consistent with animal experiments, human PHG showed significantly reduced both Slc26a9 mRNA and protein expression when compared to healthy controls.

Conclusion

Slc26a9 protects the mucosal defense barrier against PHG by attenuating ER stress mediated mitochondria-dependent apoptotic signaling. Slc26a9 may be a novel therapeutic target for PHG.

Introduction

Autoimmune gastritis (AIG) is the basis of gastric cancer (GC). Loss of parietal cells (PCs) causes the development of spasmolytic polypeptide-expressing metaplasia (SPEM), which is considered as important precancerous lesion. Previously, we demonstrated that parietal cell-specific Slc26a9 deficiency caused spontaneous SPEM to GC in mice (Liu et al., Cellular oncology; 2022; Liu et al., DDW2023, 2024). The present study was undertaken to study a potential role of Slc26a9 downregulation in the development of AIG and how to progress to SPEM.

Aims & Methods

The present study was undertaken to study a potential role of Slc26a9 downregulation in the development of AIG and how to progress to SPEM.
Slc26a9^fl/fl mice were crossed with H⁺-K⁺-Atp4b-Cre to produce the PCs-specific Slc26a9 knockout in Slc26a9^fl/fl/Atp4b-Cre mice in a mixed genomic background. Electron microscope, flow cytometry analysis, histopathological and immunohistochemical (IHC) analysis with specific markers were performed in Slc26a9^fl/fl and Slc26a9^fl/fl/Atp4b-Cre mice.

Results

Compared with Slc26a9^fl/fl mice, Slc26a9^fl/fl/Atp4b-Cre mice exhibited strongly loss of parietal cells, oxyntic atrophy followed with lymphocyte infiltration,
the mitochondria of PCs were significantly swollen, the cell membrane structure was blurred and damaged, and the cristae were broken and disappeared which was observed by electron microscope. Furthermore, Slc26a9 deletion showed an accumulation of multiple pyroptosis factors, including of NRLP3, Caspase-1, ASC, Gasdermin D, IL-1β and IL-18, and activated Gasdermin D binded to mitochondrial cardiolipin and led to the release of mitochondrial mediators, including ACO2, Cyto C, indicating that mitochondrial damage related pyroptosis of PCs. Moreover, Flow cytometry analysis showed that CD4+ Tcells that infiltrated the Slc26a9^fl/fl/Atp4b-Cre stomach expressed the TCR (TCRVα2/TCRVβ2) specific for the H⁺/K⁺-ATPase peptide, followed with upregulation of CD4+ T mediated cytokines, including IFN-γ, IL-2, IL-11 and IL-17. Taken together, PCs selective Slc26a9 deletion in mice caused Gasdermin D mediates mitochondrial damage induced PCs initial loss, resulted in the development of AIG. Furthermore, Slc26a9^fl/fl/Atp4b-Cre mice showed that hypergastrinemia induced Tuft cell migration and mediated IL-25 release, followed with upregulation of ILC2 cell marker GATA3 and M2 macrophage polarized markers F4/80 and CD163 in the base of gastric mucosal of Slc26a9^fl/fl /Atp4b-Cre mice, indicating that PCs selective Slc26a9 deletion in mice caused PCs pyroptosis and followed with gastric regional immunity remodeling, resulted in the development of SPEM based on AIG.

Conclusion

PCs-selective Slc26a9 deletion in mice caused Gasdermin D mediates mitochondrial damage induced PC cells initial loss, and followed with activation of IL-25-ILC2-M2 macrophage axis, which is the key event to induce spontaneous AIG and progress to SPEM.

Introduction

Loss of parietal cells causes the development of spasmolytic polypeptide-expressing metaplasia (SPEM) through transdifferentiation of chief cells. Slc26a9 is a member of Slc26a family with highly expression in the parietal cells to maintain gastric mucosal homeostasis (Liu et al., DDW 2019, 2020 and 2022). Our previous study showed that SLC26A9 deficiency causes gastric intraepithelial neoplasia in mice. (Liu et al., Cellular oncology 2022). However, the role of Slc26a9 in SPEM is never investigated.

Aims & Methods

Explore the role of Slc26a9 in SPEM. Slc26a9^fl/fl mice were crossed with Atp4b-Cre to generate parietal cell-specific Slc26a9 knockouts in Slc26a9^fl/fl/Atp4b-Cre mice in a mixed genomic background. Histopathology, immunohistochemistry (IHC), immunofluorescence (IF), Flow cytometry analysis, qPCR and single-cell sequencing were performed in Slc26a9^fl/fl mice and Slc26a9^fl/fl/Atp4b-Cre mice. Macrophage polarization markers and cytokine-related markers were detected by qPCR and Western-blot. Slc26a9 was overexpressed by adeno-associated virus and chronic SPEM model was established by using IL-33.

Results

Compared with Slc26a9^fl/fl mice, Slc26a9^fl/fl/Atp4b-Cre mice exhibited autoimmune gastritis (strongly loss of parietal cells, oxyntic atrophy followed with lymphocyte infiltration) at 1month, accompanied with H⁺/K⁺-ATPaseb and intrinsic factor lost. Flow cytometry analysis showed that CD4⁺T cells that infiltrated the Slc26a9^fl/fl/Atp4b-Cre stomach expressed the TCR (TCRVa2/TCRVb2) specific for the H⁺/K⁺-ATPase peptide. With advanced of age, Slc26a9^fl/fl/Atp4b-Cre mice displayed SPEM and hypergastrinemia at 6 months, followed with gastric alarmin IL-33 released and upregulation of SPEM markers, TFF2 and MUC6. Identification of gastric epithelial cell lineages using scRNA-seq and unbiased clustering showed that a variety of immune cell types. qPCR revealed significantly upregulation of macrophage molecular markers (F4/80, CD86, CD163), followed with the significantly upregulation of polarization markers of M2 macrophages and related chemokines in Slc26a9^fl/fl/Atp4b-Cre mice, including Arg1, Klf4, Fizz1, Ym1, Mrc1, CCL24 and CCL22 when compared with Slc26a9^fl/fl mice. Moreover, double immunofluorescence confirmed that M2 polarized markers F4/80⁺CD163⁺ cells were significantly accumulated in Slc26a9^fl/fl/Atp4b-Cre mice. At the same time, there was a corresponding increase in M2 macrophages capable of secreting IL-33. Notably, overexpression of Slc26a9 in IL-33-induced chronic SPEM model reduced SPEM formation and suppressed IL-33 downstream immune signaling as well as decreased M2 macrophages polarization. These results suggested that parietal cell-selective Slc26a9 deletion in mice caused SPEM development by promoting polarization of M2 macrophages via IL-33 upregulation.

Conclusion

Parietal cell-selective Slc26a9 deficiency in mice lead to the development of SPEM via IL-33 signaling. We speculated that Slc26a9 plays an important protective role in stabilizing gastric mucosal immunity and gastric epithelial cell homeostasis.

Introduction

Gastric injury induces metaplasia is based on parietal cells loss and reprogramming chief cells into proliferative, mucin-rich spasmolytic polypeptide-expressing metaplasia (SPEM) cells. Previously, we demonstrated that parietal cell-selective Slc26a9 deficiency in mice promotes the development of SPEM and progress to gastric intraepithelial neoplasia in mice (Liu et al., Cellular oncology; 2022; Deng et al., DDW2023). However, the exact role of Slc26a9 in injury-induced metaplasia is need further investigated.

Aims & Methods

We used immunostaining to characterize the expression pattern of Slc26a9 during murine gastric development, homeostasis, and injury in homeostasis, after conditionial genetic deletion of Slc26a9. Furthermore, to reveal the functional role of Slc26a9, Injury-induced metaplasia was established in Slc26a9^fl/fl mice and Slc26a9^fl/fl/Atp4b-Cre mice with high-dose tamoxifen.

Results

Mice with parietal cell selective Slc26a9 deficiency first developed spontaneous parietal cell damage, which subsequently led to SPEM. Parietal cell injury was associated with the release of typical pyroptosis factors, including NRLP3, Caspase-1, ASC, GasderminD, IL-1β and IL-18. At the same time, SLC26A9 colocalized with caspase1, NLRP3, GSDMD, and NLRP3 and GSDMD, and produced a large number of reactive oxygen species (ROS), markers of mitochondrial damage, thereby expanding membrane perforation of GSDMD-N fragments and amplifying inflammasome-mediated parietic cell pyrodeath. At the same time, electron microscopy showed that after the knockout of the Slc26a9 gene, the mitochondria of the murine wall cells were obviously swollen, the cell membrane structure was blurred and damaged, the matrix was more dissolved, the ridge was broken and disappeared, and there were many bubble protrusions. This suggests that parietal cell specific SLC26A9 gene deletion may lead to parietal cell scorch death through mitochondrial damage mediated by GSDMD. However, with a large number of parietal cell damage and loss, gastric hypergastrinemia stimulates Tuft cell proliferation and migration to the gastric mucosa for colonization, and secretes a series of alarm hormones such as IL-25, IL-33, HMGB1. Moreover, ILC2 cell secretory factors such as IL-17RA and IL-17RB, GATA3, CD90, CD25 were up-regulated. M2 macrophage polarization-related markers such as Arg1, Fizz1, Ym1, Klf4 and secreted chemokines such as CCL24 and CCL22 were significantly up-regulated. The immunofluorescence staining of F4/80+ and CD163+ was significantly increased. This suggests that the spontaneous injury of parietal cells caused by the deletion of SLC26A9 gene in gastric parietal cells may lead to gastric dysfunction (hypoacid and hypergastrinemia) and mucosal epithelial injury, which may promote the occurrence of SPEM through IL-25 mediating the activation of ILC2 cells to regulate the cellular immune mechanism of macrophage M2 polarization.

Conclusion

Slc26a9 target deletion in parietal cells leads to spontaneous damage and scorch death of parietal cells, which is the core target and cell origin of initiating SPEM. After parietal cell specific Slc26a9 gene deletion leads to spontaneous injury of parietal cells, gastric dysfunction and mucosal injury result in the release of alarm hormone IL-25, which promotes the development of SPEM by activating IL-25-ILC2-macrophage M2 polarization axis. Slc26a9 plays an important protective role in stabilizing gastric epithelial cell differentiation and gastric mucosal immune homeostasis.

Disclosure

No conflict of interest

Introduction

Loss of parietal cells causes the development of spasmolytic polypeptide-expressing metaplasia (SPEM) through transdifferentiation of chief cells. However, whether initial chief cell loss cause SPEM needs to be further investigated. Acidic mammalian chitinase (CHIA) belongs to the 18 glycosidase family and is expressed in epithelial cells in various organ, which can degrade chitin-containing pathogens, participate in Th2-mediated inflammation. CHIA is highly expressed in the chief cells, and our previous study showed that loss of CHIA promotes gastric cancer cell proliferation and progression (Hu et al., KDDW 2021), indicating that it is a tumor suppressor in gastric carcinogenesis. However, the molecular and cellular basis of CHIA function remains to be fully understood.

Aims & Methods

Histopathological and immunohistochemical (IHC) analysis, as well as In Situ Hybridization (ISH) with specific markers were performed in CHIA wild type mice (WM) and knockout mice (KM) at different age. CHIA mRNA and protein expression were measured by qPCR and Western-blot in human normal gastric epithelium, SPEM and gastric cancer tissues.

Results

With advanced of age, CHIA deficient mice exhibited severe gastric pre neoplastic phenotype, including chief cell loss followed with chronic inflammation, SPEM, and high-grade intraepithelial neoplasia (HGIN). However, slight parietal cell loss was only found in the HGIN stage. Furthermore, deletion of CHIA in the chief cells resulted in upregulation of the specifical markers of SPEM, including TFF2 and MUC6. ISH and IHC analysis showed that loss of CHIA not only significantly altered multiple lineage markers of gastric epithelial cell differentiation, but also progressively and significantly reduction of gastric stem cell (GSC) markers, including Lrg5 and Mist1 from 8 days to 15 months after birth. However, CHIA deletion in mice resulted in mild decrease of GSC maker for parietal cells (PCs) differentiation- Lrig1, as well as PCs labeling markers, including H⁺-K⁺-ATPase, Slc26a9 and SHH, which was only observed at HGIN stage, not in SPEM phase. Additionally, spontaneous inflammation was observed by upregulation of IL-6, IL33, IL25, IL-13 and IL17 in CHIA KM. Moreover, absence of CHIA caused epithelial cell hyperplasia with increased proliferation, which was detected by Ki67, but suppressed apoptosis, including BCL-2, BCL-xl, CyclinD1 via activation of IL6/STAT3 signaling pathway, when compared with wildtype mice. Consistent with animal experiment, human gastric adenocarcinoma tissues displayed significantly decreased of CHIA mRNA and protein expression when compared with healthy control. Moreover, IHC analysis of human normal gastric epithelial (n=80), SPEM (n=78), and gastric cancer tissue (n=93) indicated progressive decrease in CHIA expression, demonstrating that loss of CHIA expression in the chief cells is first step of normal gastric epithelial progressing to SPEM and malignant pathological process.

Conclusion

CHIA deficiency in the chief cells is efficient to induce SPEM via IL-6/STAT3 singling pathway, which is independent of parietal cell loss. CHIA functions as a protective role in safeguarding gastric epithelial cells against aberrant growth factor signaling and the resultant cellular plasticity and stemness.

Introduction

Loss of parietal cells causes the development of spasmolytic polypeptide-expressing metaplasia (SPEM) through transdifferentiation of chief cells. However, whether initial chief cell loss cause SPEM needs to be further investigated. Acidic mammalian chitinase (CHIA) belongs to the 18 glycosidase family and is expressed in epithelial cells in various organ, which can degrade chitin-containing pathogens, participate in Th2-mediated inflammation. CHIA is highly expressed in the chief cells, and our previous study showed that CHIA deficiency in the chief cells is the key event to induces SPEM and progress to gastric cancer, which is independent of parietal cell loss (Zhao et al., DDW 2023; Hu et al., KDDW 2021). However, the molecular role and mechanism of CHIA in the development of SPEM is need further investigated.

Aims & Methods

Aims: Explore new cellular origins and specific molecular mechanisms of SPEM occurrence.
Methods: PCR and single-cell sequencing (scRNA-seq) technology, histopathological and immunofluorescence (IF) with specific markers were performed in CHIA wild type (WT) mice and knockout (KO) mice. Multiple endoplasmic reticulum stress markers, macrophage polarization markers and cytokine-related markers were detected by qPCR in CHIA WT and KO.

Results

PCR and scRNA-seq technology data showed that increased M2 macrophage infiltration and SPEM formation, but remaining of parietal cells in CHIA KO mice, when compared to CHIA WT mice. Furthermore, deletion of CHIA in mice resulted in release of endoplasmic reticulum stress-triggered, including upregulation of endoplasmic reticulum stress markers (PERK, XBP-1, eIF2α/P- eIF2α, and GRP78), followed with upregulation of pyroptosis-related marker, including NLRP3, ACS, GSDMD, NEK7, Caspase1, Caspase11, IL-1β, and IL-18, as well as significantly increasement of IL33/IL-13 release. Moreover, Double immunofluorescence staining showed that upregulation and colocalization of NLRP3 and IL-33 induced M2 macrophage polarization markers, including F4/80 and CD163in the gastric mucosal of CHIA KO mice. Notably, IF showed that CHIA deficiency caused the mucous neck cell marker SOX9 shifted from the cervical isthmus into the SPEM cells, and co-localized with the SPEM markers CD44v, TFF2 and MUC6, suggesting that the origin of SPEM cells may transdifferentiate from mucous neck cell after CHIA gene deletion.^​

Conclusion

CHIA deficiency in the chief cells promotes SPEM through the pyroptosis mediated M2 macrophage polarization. The origin of CHIA deficiency promotes injury may origin from the transdifferentiation of mucous neck mucus cells into SPEM cells.

Disclosure

The authors declare that they have no conflicts of interest.