Most of studies related to H. pylori to date, are focused in the gastric epithelium injury produced by this bacterium, nevertheless, its extragastric presence is gaining relevance recently, due to the relationship with several diseases in distinct organs and tissues (Gravina et al. 2020; Kunovsky et al. 2021). Particularly, in pancreas, H. pylori infection has been associated to pancreatitis, diabetes and cancer (Rabelo-Gonçalves et al. 2015). Nevertheless, the mechanisms to achieve this organ and the effect in the pancreatic epithelium have not been elucidated yet. Therefore, in this study, we used an infection model in gerbils to probe the presence of H. pylori in the pancreas. There, the bacterial infection altered proteins of the intercellular junctions and the localization of some hormones, and increased levels of IL-8 in serum.

H. pylori infects humans, but several animal models are currently used to elucidate the mechanisms of infection. However, in experimentation models it is difficult to obtain efficient rates of infection, due to the complex gastric epithelium response that, just in human, could take up to decades to develop symptoms and gastric diseases. Recently, gerbils have been used as models for the study of this infection, since they accurately resemble gastric inflammation and carcinogenesis produced by H. pylori in humans; besides, it is a competent, strong, and low-cost rodent model (Ansari and Yamaoka 2022). In Mongolian gerbils, H. pylori colonizes the gastric mucosa, producing inflammatory infiltrates in the lamina propria with the presence of neutrophils and mononuclear leukocytes (Di et al. 2020); however, alterations in other organs have not been studied yet. In pancreas of cats, H. pylori has been detected by PCR (Shojaee Tabrizi et al. 2015) and in human, this bacterium has been related with pancreatitis and pancreatic cancer (Manes et al. 2003; Rieder et al. 2007). The pathogenesis and evolution of idiopathic forms of pancreatitis, is associated with changes in the secretion of the exocrine pancreas due to H. pylori infection. Furthermore, the altered epithelial gastric barrier allows the colonization of other pathogens, promoting that pancreatitis becomes chronic, and eventually, contributing to cancer development (Nilsson et al. 2006; Rieder et al. 2007; Dore et al. 2008; Bulajic et al. 2014; Rabelo-Gonçalves et al. 2015; Kunovsky et al. 2021).

Other species of Helicobacter, such as Helicobacter hepaticus, cause chronic hepatitis and hepatocellular carcinoma (HCC) in mice; and Helicobacter spp. have been identified in the liver of patients with cholestatic diseases and in HCC derived from non-cirrhotic liver (Rocha et al. 2005). According to some theories, Helicobacter spp. could go from the stomach to the liver via the duodenum and biliary tract or could enter the liver from the bloodstream via the hepatic portal vein (Pellicano et al. 2008).

In this work, we generated a gerbil model for H. pylori infection during 12 months; in addition, to promote the bacterial access to the organism, ulcers were produced by EtOH-treatment. This treatment has already been employed in other rodent models as rats, causing ulcers successfully, as observed by H&E staining (Ahmed et al. 2013). The presence of H. pylori in stomach and pancreas was demonstrated here by urease test, bacterial cultures derived from these tissues and PCR assays. Hp-inoculated animals presented more urease activity than control and EtOH-treated gerbils. In stomach, the enzymatic activity present in the latter groups could be due to the enzymes expressed by proteobacteria of the genus Proteus spp. or others (Heimesaat et al. 2014). Whilst, in pancreas, the positivity of the test is partially produced by the components of this tissue, such as digestive enzymes and sodium bicarbonate, which eventually protects the duodenum by neutralizing the acid that comes from the stomach (Pandol 2017). These components make the urea-agar mildly alkaline, hence, giving a positive result. Therefore, the urease test is not adequate to determine with certainty the H. pylori infection. Then, to specifically determine the presence of H. pylori at pancreas, bacterial cultures were performed by using tissues from stomach and pancreas, and employing antibiotics (vancomycin, trimethoprim and amphotericin B) (Mendoza-Elizalde et al. 2016) for a selective H. pylori growth. Only colonies with similar morphology to initially inoculated strains were selected and came from stomach (3 and 4 animals) and pancreas (1 and 2 gerbils) of groups 3 and 4, respectively. From these colonies, bacterial DNA was isolated, and the glmM and cagA genes were PCR-amplified. Hence, the rate of infection was 33 and 44% for groups 3 (Hp-inoculated) and 4 (Hp-inoculated + EtOH-treatment), respectively. Albeit the EtOH-treatment favoured the bacterial infection, the percentage of infection obtained was still lower than those obtained in previous studies (Fig. 1) (Velazquez-Guadarrama et al. 2007; Cortés-Márquez et al. 2018). The pronounced influence of genetic diversity, particularly, regarding to the immune system in the infection models, could explain the differential response to the bacterial infection. Therefore, it seems necessary to increase the time of infection until 18 months and the number of animals per group.

In order to corroborate if H. pylori or some of its virulence factors reach the pancreas, we used specific antibodies against H. pylori, CagA and OMP's. By immunofluorescence experiments, signals for H. pylori, CagA and OMPs were only detected in infected groups (3 and 4); while in groups 1 and 2, no fluorescence was distinguished. These findings suggest that H. pylori and some bacterial proteins are achieving the pancreas, displaying a pattern localization similar to that described in an infected stomach (Fig. 4). Alternatively, these proteins could arrive to the pancreas through outer membrane vesicles (OMVs) derived from bacteria or by exosomes produced from infected gastric cells (Chen et al. 2018; Jarzab et al. 2020).

Morphologically, infected animals showed lesions in the gastric epithelium, such as regeneration, lymphoid accumulation, and superficial gastritis, that could be attributed to the presence of H. pylori. In other studies, during prolonged infections, severe inflammation results in the loss of parietal and chief cells, usually accompanied by hyperplasia of the mucous cells of the neck (Ohkusa et al. 2003; Ansari and Yamaoka 2022). Nevertheless, in our model, no apparent morphological injuries were observed in the pancreas of infected gerbils. In all groups, empty acini and plugging in some ducts were detected, as reported in the pancreas of normal and diabetic gerbils (Fig. 2) (Li et al. 2016).

Taking into account that histologically there were no changes in the pancreatic tissue, we reviewed the epithelial structure by analysing the localization of proteins from the intercellular junctions. Proteins from the tight junction (claudin-1, claudin-4, occludin and ZO-1), adherens junction (E-cadherin and β-catenin) and desmosomes (desmoglein-2 and desmoplakin I/II) were delocalized from the cellular borders towards the cytoplasm, mainly in the groups 3 and 4, with respect to group 1. These localization changes correlated with a reduction in the amount of these proteins, as revealed by fluorescence quantification, suggesting that the expression of intercellular molecules is affected by H. pylori infection, as occurs in the stomach (Costa et al. 2013) (Fig. 6).

In the gastric mucosa the OMPs expression helps H. pylori to attach to gastric epithelial cells at the primary stage of infection and rises the virulence of this bacterium. OMPs also cooperate with other virulence factors such as CagA and VacA to increase the release of inflammatory factors, neutrophil infiltration, and helping to the colonization, persistent infection, and severe clinical consequences (Xu et al. 2020). Besides, H. pylori internalizes CagA through its T4SS system, which can be also secreted together with other virulence factors. In the epithelial cells, this oncoprotein induces the delocalization of intercellular junction proteins such as ZO-1, E-cadherin, and β-catenin, which leads to the epithelial barrier impairment (Alzahrani et al. 2014). In epithelial cells monolayers like MDCK, CagA causes polarity defects characterized by alterations of ZO-1 and E-cadherin, in a PAR1b-depending manner (Takahashi-Kanemitsu et al. 2020). Moreover, CagA has the ability to physically interact with the cytoplasmic domain of E-cadherin. The CagA/E-cadherin interaction interferes with and destabilizes the formation of the E-cadherin/ β-catenin complex and abnormally re-localizes the membrane-bound portion of β-catenin to the nucleus, where it activates Wnt-target genes in a β-catenin/TCF-dependent manner (Takahashi-Kanemitsu et al. 2020). Otherwise, the H. pylori serine protease HtrA, opens cell-to-cell junctions through cleavage of occludin, claudin-8, and E-cadherin, thus inducing the disintegration of their epithelial barrier functions (Tegtmeyer et al. 2017). In gastric epithelial cells, such as NCI-N87 and MKN28, HtrA also cuts the desmosomal cadherin, desmoglein-2. Hence, tight junctions, adherent junctions, and desmosomes are targets of this serine protease (Bernegger et al. 2021). Thus, the HtrA activity is necessary for paracellular transmigration of H. pylori across polarized cell monolayers to reach basolateral membranes and the CagA translocation across ɑ5β1 integrin (Tegtmeyer et al. 2017).

In this context, it is possible that H. pylori pathogenicity factors such as urease, OMPs, CagA, VacA and HtrA, could induce host cell signalling involved in altering cell-to-cell permeability, to impair the gastric epithelial barrier and then, the bacteria or these factors can be internalized and reach deeper tissues. In pancreas, the effect of these virulence factors over junctions is similar to that described in stomach, as we observed in this work. The breakdown of the pancreatic ductal barrier is known to contribute to the pathophysiology of pancreatitis and the development of pancreatic cancer because tight junctions in the pancreas are crucial regulators of physiologic secretion (Rieder et al. 2007; Kojima et al. 2013). Various inflammatory mediators and carcinogens can trigger tight junction disassembly and disruption of the pancreatic barrier, however, signalling events involved remain poorly understood. Furthermore, in pancreas, the adhesion molecules are crucial for proliferation, cell migration, and signal transduction, as well as in the development and tissue repair. When the cell–cell adhesion between endothelium and/or pancreatic acinar cells weakens, the accompanying interstitial oedema encourages the migration of inflammatory cells and disturbs the integrity of the tissue. In pancreatitis, occludin, claudin-1 and ZO-1 are decreased, but no changes in claudin-4 have been reported; whereas E-cadherin and β-catenin are dissociated from the plasma membrane and condensed in the cytosol of acinar cells (Sato et al. 2019). Moreover, E-cadherin is important for maintaining the architecture and homeostasis of the exocrine part and its absence contributes in the development of pathogenic conditions, such as pancreatitis or pancreatic cancer (Serrill et al. 2018). In the pancreas of the mouse model, the loss of desmoplakin expression resulted in the disruption of desmosomal adhesions, that can promote increased local tumour invasion, independently of the adherens junction status (Chun and Hanahan 2010). Altogether, these findings could explain why in this work we observed that H. pylori infection produced changes in all these proteins, which were re-localized from the plasma membrane towards the cytoplasm, and also significantly diminished (Fig. 6).

Epithelial structure is also maintained by the cytoskeleton and dynamic rearrangements of actin; but in H. pylori infected gastric epithelial cells, important changes in actin lead to the development of aberrant morphological changes, cell migration and invasive growth (Wessler et al. 2011). Translocated CagA during H. pylori infection alters SHP-2 (SH2-containing tyrosine phosphatase 2), Crk (C-terminal Src kinase), Grb2 (growth factor receptor-bound protein 2), and MARK2 (microtubule affinity-regulating kinase 2), which dysregulate key cellular biochemical pathways, apoptosis, and rearrangements of the host actin-cytoskeleton (Tohidpour et al. 2017). Similarly, it has been described actin-rearrangement in the pancreas, during both, pancreatitis and pancreatic cancer (Morris and Machesky 2015). In this work, in the stomach of infected gerbils, we also observed important rearrangements in the cytoskeleton, as well as a reduction in the amount of filamentous actin, which should be confirmed by western blot experiments in the future. Similar results were obtained in the pancreatic tissue, where reorganization of the actin-cytoskeleton was observed in infected gerbils (Figs. 5 and 6). These changes might represent the highly variable actin dynamics, trying to maintain the homeostasis of both, acini and beta cells.

In order to regulate the release of hormones in the Langerhans islet, the endocrine cells interact with one another either through homotypic or heterotypic cell–cell adhesion or in a paracrine manner (Jain and Lammert 2009). Pancreatitis can lead to diabetes mellitus, where loss of functional or structural β cells and the altered insulin secretion produced by harmed junctional proteins have been described (Singh et al. 2022). Furthermore, pancreatic inflammation leads to the decrease of islet cell mass leading to the loss of glucagon, insulin, and pancreatic polypeptide, which difficult the control of diabetes with large variations in blood glucose (Singh et al. 2022). In this work, we observed that the H. pylori infection produced alterations in the localization pattern of insulin and glucagon, and an apparent reduction in their levels at pancreas (Fig. 7). This effect could be a consequence of the modified intercellular junction proteins. For example, claudin 4 is involved in regulating the functional state of islet, and may act as a biomarker of β‐cell maturation (Li et al. 2020). Furthermore, E-cadherin plays an important role in islet formation, glucose-stimulated insulin secretion and gap junction communication (Jain and Lammert 2009). However, more experiments should be carried out in order to demonstrate the participation of junctional proteins in the endocrine altered functions of the pancreas during H. pylori infection.

During host infection, IL-8 increases in response to H. pylori, and it is a key chemokine in accumulating neutrophils. Expression of this cytokine is often regulated by NF-κB (transcription factor complex nuclear factor-κB), through κB-binding elements in the enhancer/promoter regions of their genes (Eftang et al. 2012). Here, we confirmed that infected animals presented increased levels of IL-8 in serum (Fig. 8). This finding supports the idea that IL-8 appears paramount in the inflammatory response to H. pylori infection, affecting the whole organism and other organs besides the stomach, where the induced inflammation importantly contributes to the damage in pancreas.