Helicobacter pylori-induced gastric pathology: Insights from in vivo and ex vivo models

Abstract

Gastric colonization with Helicobacter pylori induces diverse human pathological conditions, including superficial gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma and its precursors. The treatment of these conditions often relies on the eradication of H. pylori, an intervention that is increasingly difficult to achieve and that does not prevent disease progression in some contexts. There is, therefore, a pressing need to develop new experimental models of H. pylori-associated gastric pathology to support novel drug development in this field. Here, we review the current status of in vivo and ex vivo models of gastric H. pylori colonization, and of Helicobacter-induced gastric pathology, focusing on models of gastric pathology induced by H. pylori, Helicobacter felis and Helicobacter suis in rodents and large animals. We also discuss the more recent development of gastric organoid cultures from murine and human gastric tissue, as well as from human pluripotent stem cells, and the outcomes of H. pylori infection in these systems.

Full text

REVIEW
Helicobacter pylori-induced gastric pathology: insights from in vivo
and ex vivo models
Michael D. Burkitt
1
, Carrie A. Duckworth
1
, Jonathan M. Williams
2
and D. Mark Pritchard
1,
*
ABSTRACT
Gastric colonization with Helicobacter pylori induces diverse human
pathological conditions, including superficial gastritis, peptic ulcer
disease, mucosa-associated lymphoid tissue (MALT) lymphoma, and
gastric adenocarcinoma and its precursors. The treatment of these
conditions often relies on the eradication of H. pylori, an intervention
that is increasingly difficult to achieve and that does not prevent
disease progression in some contexts. There is, therefore, a pressing
need to develop new experimental models of H. pylori-associated
gastric pathology to support novel drug development in this field.
Here, we review the current status of in vivo and ex vivo models of
gastric H. pylori colonization, and of Helicobacter-induced gastric
pathology, focusing on models of gastric pathology induced by
H. pylori,Helicobacter felis and Helicobacter suis in rodents and large
animals. We also discuss the more recent development of gastric
organoid cultures from murine and human gastric tissue, as well as
from human pluripotent stem cells, and the outcomes of H. pylori
infection in these systems.
KEY WORDS: Helicobacter, Gastric cancer, Peptic ulcer disease,
MALT lymphoma, Organoid, Gastroid
Introduction
Helicobacter pylori is a bacterium that grows in close association
with the lining of the stomach and is associated with various human
gastric diseases; it causes significant morbidity and mortality
worldwide. Globally, there are wide variations in the reported
prevalence of H. pylori (Fig. 1), with particularly high levels
observed in South America, sub-Saharan Africa and the Middle
East (Asfeldt et al., 2008; Ben Mansour et al., 2016; Laszewicz
et al., 2014; Luzza et al., 2014; McDonald et al., 2015; Peleteiro
et al., 2014; Saltanova, 2001; Sanchez Ceballos et al., 2007; van
Blankenstein et al., 2013).
Many publications have promoted the concept of ‘the African
Enigma’(Holcombe, 1992), owing to the reporting of fewer cases of
peptic ulceration than expected in this continent. However, recent
studies suggest that gastric pathology is endemic where H. pylori is
endemic (Agha and Graham, 2005). They also suggest that the
geographical distribution of gastric disease and its predominant
form relates more to other co-factors, such as H. pylori virulence
properties, food preservation methods, diet and host genetics
(Graham et al., 2009; Kodaman et al., 2014).
One of the principal confounders of studies examining the impact
of H. pylori infection on gastric health in diverse populations is the
inequality of access to healthcare systems globally. Diagnosing
Helicobacter infection relies on one of four tests: endoscopy with
biopsy;
13
Carbon-hydrogen breath test; faecal antigen testing; or
serological detection of an anti-H. pylori antibody. These tests are
relatively expensive and their availability is limited, particularly in
developing countries where the highest H. pylori prevalence has
been reported.
For many H. pylori-associated conditions, the most effective
clinical intervention is to eradicate the infection using a combination
of acid-suppressing medication and antibiotics. However, this
strategy is becoming increasingly difficult to sustain because of the
emergence of antibiotic-resistant H. pylori strains. Moreover, in
some clinical circumstances, H. pylori eradication is ineffective at
preventing disease progression. There is, therefore, an unmet need
to develop new drugs, both to eradicate H. pylori more effectively
and to offer alternative strategies where eradication of infection does
not prevent the progression of gastric pathology.
To achieve this, we need to improve our understanding of the
molecular events that lead to H. pylori-induced gastric pathology,
and this requires experimental models. Here, we review the
currently available in vivo and ex vivo models of Helicobacter-
induced pathology, and describe the spectrum of pathology induced
by infection with H. pylori,Helicobacter felis and Helicobacter
suis. The in vivo models discussed here span rodent and larger
animal models, including cat, dog, pig and non-human primate
models, whilst the ex vivo models derive from mouse and human
gastric mucosa and from pluripotent stem cells. It is particularly
timely to review these ex vivo models because of the recent
development of long-lived ex vivo cultures of untransformed
gastric epithelium (Barker et al., 2010). These offer an important
adjunct to the more established animal models of gastric
carcinogenesis, and make it likely that future mechanistic studies
of gastric disease will incorporate elements of both in vivo and
ex vivo experimentation.
Helicobacter pylori: an overview
The gastric microenvironment (Fig. 2) is hostile to commensal
bacteria because of its low partial oxygen pressure, and the presence
of high concentrations of gastric acid and digestive enzymes.
H. pylori is a Gram-negative, spiral rod-shaped bacterium that has
evolved to survive in this environment. Its adaptations to these
conditions include an ability to tolerate a microaerophilic
environment (see Glossary, Box 1), the expression of a urease
enzyme that modulates the bacterial microenvironment by raising
pH, and flagellae that provide motility, allowing H. pylori to access
the deep mucous layer of the stomach wall, thereby utilizing the host
mucosal defences to develop a survivable niche.
1
Gastroenterology Research Unit, Department of Cellular and Molecular
Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool
L69 3GE, UK.
2
Pathology and Pathogen Biology, Royal Veterinary College, North
Mymms AL9 7TA, UK.
*Author for correspondence (mark.pritchard@liverpool.ac.uk)
M.D.B., 0000-0002-5055-6408; C.A.D., 0000-0001-9992-7540; D.M.P., 0000-
0001-7971-3561
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
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© 2017. Published by The Company of Biologists Ltd
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The transmission of H. pylori infection is considered to occur
through an oro-oral or faeco-oral route. Data from families indicates
that vertical transmission from parent to child is a common
transmission route. A recent phylogenetic study in an Iranian
population examined transmission by DNA fingerprinting of
H. pylori 16S ribosomal subunit DNA obtained from faecal
samples. This assay detected H. pylori DNA in 26 of 30 cases,
and demonstrated the vertical transmission of H. pylori in 46.1% of
families, with 38.4% of cases being colonized with an H. pylori
strain phylogenetically identical to their mother’s strain, and 7.7%
with a strain identical to that of their father (Mamishi et al., 2016).
These findings agree with similar studies performed in other
populations (Konno et al., 2005, 2008; McMillan et al., 2011;
Roma-Giannikou et al., 2003), and are supported by animal studies.
For example, one study reported that in an H. pylori-infected cat
colony, kittens were passively colonized by H. pylori over the first
14 weeks of life (Straubinger et al., 2003).
The global prevalence of H. pylori infection in humans is
estimated to be 50%. The association of H. pylori with humans is
longstanding, with phylogenetic studies suggesting that H. pylori
strains have co-evolved with human populations since before the
migration of early humans from Africa 58,000 years ago (Falush
et al., 2003; Linz et al., 2007). Below, we discuss the consequences
of H. pylori infection for human health, to establish the types of
pathology that need to be modelled in the laboratory.
Helicobacter-induced gastric pathology in humans
Chronic infection with H. pylori is strongly associated with gastric
pathology, including chronic active gastritis (see Glossary, Box 1),
peptic ulcer disease, gastric adenocarcinoma and gastric extranodal
marginal zone lymphoma of mucosa-associated lymphoid tissue
type (MALT lymphoma). Of these outcomes, the most significant
in terms of mortality is gastric adenocarcinoma. Recent meta-
analyses suggest that the relative risk of developing gastric cancer
is 2- to 3-times higher for people infected with H. pylori than
for those without infection (Danesh, 1999; Helicobacter and
Cancer Collaborative Group, 2001). Understanding these different
pathological conditions is important for understanding how
faithfully the available models recapitulate the clinical features of
H. pylori pathology.
Superficial gastritis
The commonest outcome of H. pylori infection is gastritis. Acute
gastritis has rarely been described in humans, but has been reported
in the context of experimentalists being exposed to H. pylori either
accidentally (Sobala et al., 1991) or in a deliberate attempt to induce
gastric pathology (Marshall et al., 1985; Morris and Nicholson,
1987; Morris et al., 1991). In these cases, the infected individuals
reported symptoms and underwent endoscopic assessment with
biopsy of the inflamed gastric mucosa. The early stages of disease
are marked by the presence of a polymorphonuclear leukocyte
infiltrate (see Glossary, Box 1) in the gastric mucosa and a transient
reduction in gastric acid output.
In the cases of Marshall et al. (1985) and Morris and Nicholson
(1987), H. pylori eradication therapy was prescribed. This was
effective in eradicating H. pylori from the gastric mucosa, and led to
the complete resolution of symptoms and of gastric histological
abnormalities. In the case of Sobala et al. (1991) symptoms and
signs resolved spontaneously, and repeat endoscopy demonstrated
low levels of Helicobacter colonizing the gastric antrum, together
with an increase in lymphocytes within the gastric mucosa. These
histological changes correlated with IgM and IgG seroconversion
>90%
Reported prevalence of H. pylori infection
20-30%
<20%
50-60%
80-90%
70-80%
40-50%
60-70%
30-40%
Unknown
Fig. 1. Worldwide prevalence of Helicobacter pylori infection. The map shows the prevalence of H. pylori infection in different parts of the world. Note, the
particularly high prevalence in sub-Saharan Africa, Latin America and the Middle East. Australasia, Switzerland, and more generally North America and Western
Europe have the lowest incidence of H. pylori infection. Data derived from Asfeldt et al., 2008; Ben Mansour et al., 2016; Laszewicz et al., 2014; Luzza et al., 2014;
McDonald et al., 2015; Peleteiro et al., 2014; Saltanova, 2001; Sanchez Ceballos et al., 2007; van Blankenstein et al., 2013.
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for H. pylori, which are typical for chronic, superficial H. pylori
gastritis. This is the most prevalent H. pylori-induced gastric
pathology worldwide (Campbell et al., 2001; Filipe et al., 1995;
Potet et al., 1993).
Peptic ulcer disease
Individuals colonized with H. pylori have a 6.8-fold [95%
confidence interval (CI), 2.9-16.1] higher risk of developing
peptic ulcer disease (PUD) than those not exposed to this
infection (Li et al., 2010). In line with this, the reduced incidence
of H. pylori infection worldwide has coincided with a reduction in
PUD (Groenen et al., 2009). In contrast to the 1980s, when the
association of H. pylori and PUD was first established (Graham,
1989), individuals presenting with this disease are now less likely to
be colonized with H. pylori; more often, their condition is linked to
non-steroidal anti-inflammatory drug use or to low-dose aspirin
(Musumba et al., 2012; Sung et al., 2009).
H. pylori-induced peptic ulceration occurs in the context of pre-
existing chronic superficial gastritis, but is associated with increased
gastric acid secretion and a T helper 1 (Th1) polarized immune
response, compared with individuals with isolated superficial
gastritis (D’Elios et al., 1997; Shimada et al., 2002).
Frequently, individuals with PUD exhibit antral predominant
gastritis, which leads to enhanced gastrin secretion (see Glossary,
Box 1). In turn, this stimulates the parietal cells of the gastric corpus
(Fig. 2) to secrete more acid (McColl et al., 1997), leading
to mucosal ulceration. Eradication of H. pylori is reportedly
sufficient to suppress excess gastrin secretion (McColl et al., 1991),
which is an important component of the healing process of
H. pylori-associated peptic ulcers.
Gastric adenocarcinoma and its precursor lesions
In 2012, gastric cancer was the fifth commonest malignancy
worldwide, and the third commonest cause of cancer-related death,
with over 720,000 deaths worldwide caused by the disease (Ferlay
et al., 2013). H. pylori colonization is the single biggest risk factor
for gastric carcinogenesis and is a risk factor in at least 80% of cases
of gastric cancer (Graham, 2015). However, as only a very small
percentage of people infected with H. pylori go on to develop gastric
cancer, understanding why those individuals do so is a key aim of
future studies in this field.
Other risk factors linked to gastric cancer (Fig. 3) fall into two main
groups. The first consists of potentially modifiable exogenous risk
factors, such as dietary salt and nitrosamine intake (Jakszyn et al., 2006;
Mendez et al., 2007; Wang et al., 2009), H. pylori virulence factors
(Yamaoka, 2010), non-Helicobacter gastric microbiota (Dicksved
et al., 2009; Lofgren et al., 2011) and smoking status (La et al., 2009).
The second groupconsists of unalterable host genetic, or intrinsic, risk
factors. Amongst these genetic factors are polymorphisms at loci
encoding cytokines and their receptors (Persson et al., 2011), stromal
remodelling proteins, such as matrix metalloproteinases (Tang et al.,
2008), and prostate stem cell antigen (PSCA), which in the context of
gastric pathology, acts as a tumour suppressor gene (Garcia-Gonzalez
et al., 2015; Ichikawa et al., 2015; Mou et al., 2015).
The development of gastric cancer occurs through a stereotypical
pathological pathway (Fig. 3, and Glossary, Box 1), which was first
proposed well before the identification of H. pylori (Correa et al.,
1975). Over the course of several decades, some individuals with
chronic superficial gastritis develop gastric atrophy, characterized by
the patchy loss of parietal cells in the gastric corpus mucosa. This
decreases gastric acid secretion, leading to higher intraluminal pH,
decreased somatostatin secretion and consequent gastrin secretion. In
addition to stimulating acid secretion from parietal cells, gastrin also
enhances proliferation in the gastric epithelial stem cell zone (Burkitt
et al., 2009), leading to an increase in epithelial cell turnover.
A proportion of people with established gastric atrophy develop
intestinal-type metaplasia (see Glossary, Box 1) of the gastric
mucosa over time, where oxyntic glands (see Glossary, Box 1) are
Pit
Isthmus
Neck
Base
Pit
Gland
Antral gland
Human
Mouse
Antrum
Antrum
Duodenum
Duodenum
Oesophagus
Oesophagus
Corpus
Fundus
Cardia
Corpus
Fore-stomach
Corpus gland
Surface mucous cell
Stem cell
Mucous neck cell
Parietal cell
Endocrine cell
Chief cell
Key
Fig. 2. The anatomy of the human
and mouse stomach. A schematic of
the anatomy of the human and mouse
stomach and the structure of gastric
glands. Two types of columnar mucosa
line the human stomach: the antrum is
lined with antral glands, whilst the
corpus and fundus arelined with deeper
oxyntic, or corpus glands (see
Glossary, Box 1). The murine stomach
has areas that are analogous to the
human stomach, including antral and
corpus glands, and it also has a
forestomach lined with squamous
epithelium. Stem cells that reside at the
base of the gland generate the antral
gland. Following asymmetric cell
division in the stem cell zone, daughter
cells migrate upwards towards the
gastric lumen and differentiate into
mucous neck, surface mucous and
endocrine cells. In corpus glands, the
stem cell niche is located at the isthmus
of the gland. Cells migrate upwards
from the stem cellzone and differentiate
into surface mucous cells. Other cells
migratedown the gland and differentiate
into acid-secreting parietal cells,
endocrine cells, or zymogen-secreting
(see Glossary, Box 1) chief cells.
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replaced by CDX2 (caudal-type homeobox 2)-expressing glandular
units, which are morphologically similar to the intestinal crypt.
Intestinal metaplasia in the stomach is linked to gastric dysplasia
(see Glossary, Box 1); up to 20% of affected individuals with
intestinal metaplasia have concurrent dysplasia (den Hoed et al.,
2011). Gastric epithelial dysplasia is associated with an at least
10-fold increased risk of developing gastric cancer (You et al.,
1999), but it has been difficult to represent this risk accurately from
population-based studies.
Several studies have assessed the strategy of testing for, and
eradicating, H. pylori in populations at a high risk of developing
gastric cancer. Unfortunately, a recent well-designed meta-analysis
confirmed a relatively poor outcome for this strategy. The
eradication of H. pylori in this study reduced the risk of
developing gastric cancer by about one-third [odds ratio (OR),
0.66; 95% CI, 0.46-0.95] (Ford et al., 2014). However, when
individuals with pre-existing pre-neoplastic gastric pathology
(defined as the presence of gastric atrophy, intestinal metaplasia
or dysplasia) were considered, there was no evidence that
eradication of H. pylori decreased the risk of gastric cancer (OR,
0.86; 95% CI, 0.47-1.59). For this highest risk group, therefore,
there are currently no effective therapeutic strategies.
MALT lymphoma
Gastric extranodal marginal zone lymphomas of mucosa-associated
lymphoid tissue (MALT lymphomas) are B-cell lymphomas that
develop within the mucosa-associated lymphoid tissue of the
stomach. The incidence rate of gastric MALT lymphoma in the USA
was estimated to be 3.8 in 1,000,000 individuals between 2001 and
2009, making it a rare outcome of H. pylori infection (Khalil et al.,
2014). In the only published systematic review of this condition,
79% of 1844 reported cases of MALT lymphoma were associated
with H. pylori infection (Asenjo and Gisbert, 2007; Gisbert and
Calvet, 2011).
As with other haematological malignancies, characteristic
cytogenetic profiles have been described for MALT lymphoma.
Amongst the most well characterized is the formation of the MALT1-
API2 fusion oncogene by the t (11:18) translocation. This results in
the expression of API2 (encoding the cellular inhibitor of apoptosis
2) under the control of the MALT1 promoter (Rosebeck et al., 2011).
MALT1 encodes mucosa-associated lymphoid tissue lymphoma
translocation protein 1, which is essential for the activation and
proliferation of T- and B-lymphocytes, and also plays a fundamental
role in NF-κB activation. One of the downstream effects of this
fusion protein is enhanced cleavage of NIK (NF-κB-inducing
kinase), which is a critical regulator of alternative pathway NF-κB
signalling (Merga et al., 2016).
Human infections with other Helicobacter species
Whilst infection of the gastric mucosa with H. pylori is by far the
most frequently observed gastric infection in humans, non-H. pylori
Helicobacter (NHPH) species infections of human hosts have been
identified since at least the mid-1990s. The identification of
these organisms remains a challenge, and relies on molecular
microbiological techniques that are not routinely available.
Although NHPH infections are frequently reported to occur in
association with gastritis, different studies have yielded conflicting
results as to their significance (Flahou et al., 2013; Liu et al., 2015).
Understanding the contribution of NHPH species to gastritis is
further complicated by the occurrence of mixed NHPH infections,
by the heterogeneity of NHPH strains, by the nomenclature of
these species, and the inability to cultivate many of them. For
example, long spiral-shaped bacteria that were first recognized as
microscopically different from H. pylori were isolated from human
gastric biopsies and named Gastrospirillum hominis (McNulty
et al., 1989). These organisms were subsequently reclassified as
Helicobacter heilmannii based on 16S RNA analysis, of which
there are at least two strains (Heilmann and Borchard, 1991). Many
species of spiral-shaped NHPHs have since been found in the
stomachs of animals (discussed in more detail below).
The most robust data for the pathogenicity of NHPH involve
MALT lymphoma formation. Helicobacter heilmannii-associated
MALT lymphoma was first described in 2000. Following this, a
large study examining the prevalence of MALT lymphoma in
263,680 H. pylori-infected and 543 NHPH-infected people,
demonstrated an odds ratio of 2.2 (95% CI, 1.1-4.5) for
developing gastric MALT lymphoma in individuals infected with
Box 1. Glossary
Anthroponosis: Infectious disease transmitted to a non-human species
from humans.
CagA: Helicobacter pylori virulence factor. The secreted component of a
type IV secretion system (the cag pathogenicity island) that is associated
with more severe gastric pathology.
Correa model: A model of gastric carcinogenesis first proposed by
Pelayo Correa et al. in 1975, describing the development of gastritis,
gastric atrophy, gastric intestinal-type metaplasia, dysplasia and gastric
intestinal-type adenocarcinoma.
Dysplasia: Replacement of normal gastric mucosa with structurally
abnormal tissue with potentially abnormal proliferation, disordered
arrangement of cells within the tissue, and structurally abnormal cells.
Gastric organoid/gastroid: An organoid generated from gastric
epithelial stem cells.
Gastrin: A hormone produced in the stomach that stimulates the
production of acid by parietal cells, mainly through its interaction with
histamine-secreting ECL cells. In addition to its role in stimulating gastric
acid secretion, gastrin is also a growth factor and stimulates gastric
epithelial cell proliferation.
Gastritis: Inflammation of the epithelial lining of the stomach.
Hypergastrinemia: An elevated level of the hormone gastrin in the
bloodstream. This is observed particularly in the context of gastric pre-
neoplasia, where the growth factor function of gastrin is thought to
promote increased epithelial cell proliferation.
Metaplasia: Replacement of one differentiated epithelial tissue by
another, which is abnormal for that anatomical site.
Microaerophilic: A term that describes a bacterium adapted to survive
in an environment with reduced partial oxygen pressure.
Organoid: A primary epithelial cell culture (usually 3D) that contains
healthy, proliferating epithelial stem cells, which can be expanded and
passaged multiple times, and generates daughter cells representative of
all lineages generated by the stem cell in vivo.
Oxyntic gland: An acid-secreting gland that consists of an epithelial
monolayer with a proliferative stem cell zone towards the upper third of
the gland. Asymmetric proliferation in this region generates daughter
cells that migrate both up and down the gland and differentiate into
mucous neck cells, parietal cells, chief cells and enteroendocrine cells.
Pars oesophagea: A small area of non-glandular squamous mucosa
near the oesophageal opening present in the stomachs of some animals
that is analogous to the oesophageal mucosa in humans.
Polymorphonuclear leukocyte infiltration: The recruitment of multi-
lobulated white blood cells (including neutrophils, basophils and
eosinophils) into an epithelial tissue. Indicative of active inflammation
in the tissue.
Zoonosis: Infectious disease in a human transmitted from an animal
host.
Zymogen: The inactive form of a digestive enzyme, for example
pepsinogen, which is an inactive form of the proteolytic enzyme, pepsin.
Pepsinogen is activated by gastric acid secreted by parietal cells.
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NHPH rather than with H. pylori (Stolte et al., 2002). This organism
has also been described in association with individuals with chronic
gastritis (Heilmann and Borchard, 1991). These observations
suggest that NHPHs play a role in the development of human
disease, and might in some cases be as pathogenic as H. pylori. The
additional challenge of identifying these organisms suggests that
there could be a group of individuals with gastric pathology due to
unidentified NHPHs, representing an unmet clinical need.
Naturally occurring gastric Helicobacter infections in non-
human mammals
Whilst the association of H. pylori with humans has been extensively
studied over the past 40 years, significantly less has been published
on the association of H. pylori, or other Helicobacter species with
different mammalian hosts. The data that are available suggest a
spectrum of pathogenicity for different Helicobacter species, as well
as a spectrum of susceptibility for gastric pathology in different host
organisms. A better understanding of these comparative biological
responses may, in the future, offer insights into the mechanisms that
underlie human disease.
Forty-five species of Helicobacter have been detected by PCR in
faecal samples from 150 vertebrate species, demonstrating their
colonization of the digestive system of a wide range of domesticated
and wild vertebrate species (Schrenzel et al., 2010). Spontaneous
gastric colonization by NHPH has also been demonstrated in several
mammalian species (Table 1), leading to speculation that all
mammals harbour one or more Helicobacter species as part of their
natural gastric flora (Brown et al., 2007).
When examining the evidence for NHPH-induced gastric lesions
in animal species, various factors need to be considered, such as
differences in gastric morphology, and in the distribution and site of
gastric Helicobacter strains among some animal species and
humans. Furthermore, the sequential pathological lesions that lead
to adenocarcinoma formation in humans, as described by Correa’s
model (see Glossary, Box 1), have rarely been established in clinical
veterinary species (Amorim et al., 2016), in which gastric cancer is
rare. Exceptions to this include: ferrets colonized by Helicobacter
mustelae, which undergo a similar sequence of pathology that leads
to gastric adenocarcinoma (Fox et al., 1990, 1997); Mongolian
gerbils infected with H. pylori, which develop some specific pre-
neoplastic lesions, including intestinal metaplasia (Honda et al.,
1998b); and Syrian hamsters infected with Helicobacter aurati
(Patterson et al., 2000).
In veterinary medicine, dogs and cats frequently undergo gastric
biopsy to investigate unresolved gastrointestinal disease. In both
species, NHPHs are frequently observed, and genetic studies show
that these organisms are often present as a mixed infection of
different NHPHs (Priestnall et al., 2004) with H. felis,H.
heilmannii,Helicobacter bizzozeronii and Helicobacter salomonis
being the most commonly identified species (Canejo-Teixeira et al.,
2014; Priestnall et al., 2004; Van den Bulck et al., 2005).
Although NHPHs, including H. felis, are commonly identified in
dogs and cats, and are associated with gastritis (Shiratori et al., 2016)
and gastric MALT lymphoma in humans (Stolte et al., 2002), genetic
studies have shown limited evidence for zoonosis (see Glossary,
Box 1). The most frequently isolated H. heilmannii from dogs and cats
are distinct from the type 1 H. heilmannii identified in human MALT
lymphoma (Priestnall et al., 2004). However, there is evidence of
anthroponosis (see Glossary, Box 1) in the H. pylori infection of cats;
infection has only been reported in cat colonies that live in proximity
to humans (Canejo-Teixeira et al., 2014; Handt et al., 1994).
In dogs, lymphoplasmacytic gastritis is commonly observed in
association with NHPHs (Neiger and Simpson, 2000). However,
there is no evidence to link conclusively NHPH infection with this
gastric pathology. Indeed, NHPH infection is present in 67-86% of
clinically healthy dogs, and in 61-100% of animals presenting with
chronic vomiting (Amorim et al., 2016). A small-scale study
reported that NHPHs are found in association with all cases of
canine gastric polyps (Taulescu et al., 2014). Similarly, NHPHs
have been found in the stomach of 42-100% of healthy cats, and in
53-76% of those presenting with clinical signs of gastrointestinal
disease (Norris et al., 1999). It is hypothesized that these infections
play a role in the development of feline gastric lymphoma
(Bridgeford et al., 2008).
Helicobacter suis is detected in the stomach of up to 80% of pigs
at the time of slaughter, with ulceration and hyperkeratosis of the
Helicobacter
pylori
colonisation
Normal
gastric
epithelium
Superficial
gastritis
Gastric
adenocarcinoma
Dysplasia
Intestinal
metaplasia
SPEM
Atrophic
gastritis
Diet Smoking
Bacterial
virulence
Hypergastrinaemia
Stromal
remodelling ProliferationInflammation Apoptosis
Host genotype
Endogenous risk factors
Endogenous risk factors
Fig. 3. H. pylori infection and
progression to gastric cancer.
A schematic demonstrating the
pathological progression of H. pylori-
induced gastric pre-neoplasia, and
highlighting endogenous risk factors for
progression towards gastric cancer.
SPEM, spasmolytic polypeptide-
expressing metaplasia.
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pars oesophagea (see Glossary, Box 1) present in 20-90% of
slaughtered pigs (De Bruyne et al., 2012). Although some studies
have associated H. suis with increased severity of gastritis and with
reduced weight gain in pigs (De Bruyne et al., 2012), gastritis is
likely to be multifactorial and also involves feed particle size, highly
fermentable carbohydrates and stress factors. Interestingly, H. suis
is the most commonly isolated NHPH found in human stomachs,
suggesting the potential for zoonotic transmission.
Helicobacter-like DNA has also been isolated from the stomach
of thoroughbred horses (Contreras et al., 2007), although the role of
Helicobacter in gastritis and gastric ulceration in horses is unclear.
Horses possess a much larger proportion of non-glandular
squamous mucosa than do pigs, which constitutes the proximal
half of the stomach mucosa, and gastric ulceration is present in up to
86% of training racehorses (Begg and O’Sullivan, 2003). The high
proportion of horses suffering from ulceration that undergo
strenuous exercise suggests that stress, management and training
practices are likely risk factors (Murray et al., 1996). Ulceration
occurs most commonly in the non-glandular portion of the stomach,
close to the transition of the non-glandular and glandular stomach,
although pyloric ulceration is also observed in 47% of horses (Begg
and O’Sullivan, 2003).
Up to 100% of adult ferrets harbour gastric Helicobacter
mustelae, (Fox et al., 1990); however, this organism is rarely
found in ferrets of less than 6 weeks of age (Fox et al., 1988). The
incidence of gastric ulceration in ferrets varies between 1.4 and 35%
(Andrews et al., 1979), and H. mustelae has also been associated
with adenocarcinoma (Fox et al., 1997) and gastric lymphoma
(Erdman et al., 1997) in this species.
Captive rhesus macaques are commonly infected with H. pylori,
(Drazek et al., 1994), and non-human primates have been used as
models of H. pylori infection. Indeed, rhesus macaques in social
housing rapidly acquire H. pylori from other infected individuals
(Solnick et al., 2003). Neonatal rhesus macaques are more
commonly infected with H. pylori when born to infected mothers,
suggesting that close contact in the peripartum period is important
for bacterial transmission (Solnick et al., 2003), potentially via an
oral-oral route. (Solnick et al., 2006). The induced pathology in
rhesus macaques is also very similar to that observed in humans
with H. pylori infection (Haesebrouck et al., 2009). However, no
NHPH species have been uniquely associated with non-human
primate gastric colonization, although H. suis has been
demonstrated in captive mandrills (Papio sphinx), cynomolgus
monkeys (Macaca fascicularis), and in a rhesus macaque (Macaca
mulatta) from a zoo (Haesebrouck et al., 2009). The question of
whether these NHPHs are implicated in the development of gastritis
in non-human primates remains unknown. More recently, a study
identified a high incidence of gastric adenocarcinoma in a captive
colony of sooty mangabeys (Cercebus atys) (Sharma et al., 2011).
This colony has subsequently been shown to be heavily colonized
with H. suis by both fluorescence in situ hybridization and 16S
ribosomal RNA sequencing (Esmail et al., 2016). This is the first
evidence of naturally occurring Helicobacter associated with gastric
carcinogenesis in a non-human primate.
In vivo models of Helicobacter-induced gastric pathology
Because of the breadth of potential pathological outcomes that can
follow an H. pylori infection, no single animal model can replicate
all of the pathological outcomes of this condition. However, as we
discuss in more detail below, models do exist that can replicate
each of the potential outcomes of H. pylori infection in humans
(Fig. 4).
Models of superficial gastritis
The acute phase of Helicobacter gastritis has rarely been the focus
of research, partly because few cases are reported in the human
literature. Where data have been published, they have focused on
defining the bacterial and host factors that influence Helicobacter
colonization and the acute cytokine milieu induced by these
bacterial infections.
Historic studies in gnotobiotic pigs demonstrated that the urease
enzyme produced by Helicobacter bacteria (Eaton and Krakowka,
Table 1. Naturally occurring gastric Helicobacter infections and the associated host and human pathology
Helicobacter
species Natural host Natural host lesions
Implicated in human
gastric pathology?
H. suis Pig, macaque, mandrill (Haesebrouck et al.,
2009)
In pigs, associated with gastric ulceration of the pars
oesophagea (De Bruyne et al., 2012)
Yes
H. felis Dog, cat (Priestnall et al., 2004) rabbit,
(Haesebrouck et al., 2009), cheetah
(Terio et al., 2005), mouse
Associated with gastritis in dogs and cats Yes
H. bizzozeronii Dog, cat (Priestnall et al., 2004) Associated with gastritis in dogs and cats Yes
H. salomonis Dog, cat, rabbit (Haesebrouck et al., 2009) Associated with gastritis in dogs and cats Yes
H. heilmannii Dog, cat, wild felidae, non-human primates
(Haesebrouck et al., 2009)
In cats, hypothesized to be associated with gastric
lymphoma (Bridgeford et al., 2008)
Yes
H. baculiformis Cat (Baele et al., 2008) Associated with gastritis No
H. cynogastricus Dog (Van den Bulck et al., 2006) Associated with gastritis No
H. bovis Cattle (De Groote et al., 1999) No known pathology No
H. mustelae Ferret Gastritis, gastric pre-malignant lesions and gastric
adenocarcinoma (Fox et al., 1997) and gastric MALT
lymphoma (Erdman et al., 1997)
No
H. aurati Syrian hamster (Patterson et al., 2000) Gastric adenocarcinoma with gastritis and intestinal
metaplasia
No
H. acinonychis Cheetah (Eaton et al., 1993), tiger Associated with gastric ulcers and erosions in tigers
(Cattoli et al., 2000)
No
H. cetorum Whale, dolphin (Harper et al., 2002) Associated with gastric and oesophageal ulceration in
whales and dolphins
No
H. pylori Human, Rhesus macaque (Dubois et al., 1994),
cat (Canejo-Teixeira et al., 2014)
Peptic ulcer disease, gastric adenocarcinoma and
gastric MALT lymphoma in humans; gastritis in dogs
and cats
Yes
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1994) and their functioning flagellae are essential for effective
colonization of the gastric mucosa (Eaton et al., 1992).
Experimental infections of cats with H. pylori have shown
that active colonization of the gastric mucosa occurs readily, and
that a chronic gastritis can follow (Fox et al., 1995). Independently,
researchers characterized the acute cytokine response of cats born
into a colony of H. pylori-infected animals. In this study
(Straubinger et al., 2003), the researchers compared animals born
into their H. pylori-infected colony with specific-pathogen-free
(SPF) animals from elsewhere. They demonstrated that animals in
the infected colony became colonized with H. pylori passively by
14 weeks of age, and that this was associated with an immune
response dominated by the expression of cytokines IFN-γ, IL-1α,
IL-1βand IL-8. As with the other large animal models of H. pylori
infection, these experiments benefit from the availability of serial
endoscopic evaluation, but are challenging to perform because of
the ethical and cost limitations associated with using large animals
in experimental procedures.
More recently, Mongolian gerbils have been used to investigate
host factors that allow optimal gastric colonization (Bücker et al.,
2012). Gastrostomies were performed under terminal anaesthesia to
place an intraluminal pH probe, and an auto-titrator into the
stomach. The authors used this system to recapitulate the effect on
gastric pH of a meal whilst simultaneously inoculating H. pylori.
Three physiological conditions were replicated using this apparatus.
First, it replicated the gastric pH profile observed in newborn human
infants, who have a persistently elevated gastric pH due to the large
buffering capacity of a milk-based diet and relatively low gastric
acid secretion. Second, it replicated the pH profile of young
children, in whom gastric pH is transiently elevated as a result of the
buffering capacity of a high-milk diet, but then lowered to pH 1-2 by
gastric acid secretion. And finally, it replicated the profile of adults,
in whom luminal pH remains low throughout the post-prandial
period because of high acid secretion and the relatively low
buffering capacity of food. The pH profile similar to that of young
children enhanced the ability of H. pylori to colonize the stomach up
to 15-fold, supporting other evidence that the commonest mode of
H. pylori colonization is through vertical transmission from parent
to child (Suerbaum and Josenhans, 2007). This model provides
the potential to look at other elements of gastric microbiotal
colonization. For instance, does an achlorhydric stomach offer a
niche for colonization with other non-Helicobacter organisms, and
if so, does co-infection with H. pylori help or hinder this process?
The immediate host response to colonization with Helicobacter
has also been modelled by the introduction of H. pylori-derived
lipopolysaccharide into the stomachs of Sprague-Dawley rats. This
induced an inflammatory cell infiltrate typified by lymphocyte
infiltration, and increased gastric epithelial cell apoptosis over the
course of 4 days. Rather than recapitulating the pathology observed
in acute human H. pylori infection, the pathological description of
this model was more in keeping with the pathology observed in
people with chronic superficial gastritis (Slomiany et al., 1998).
These studies have remained relatively niche areas of
investigation, and to date have not been replicated by other groups.
Significant research questions remain, particularly regarding the
initial host responses to H. pylori exposure.
Models of gastric ulceration
In vivo models of gastric ulceration induced by Helicobacter
infection alone are limited to gnotobiotic pigs, Mongolian gerbils
and isolated reports of murine gastric ulceration. Several groups
have independently shown that gnotobiotic pigs develop ulcers at
the junction between the squamous epithelium of the pars
oesophagea and columnar mucosa of the true stomach, following
infection with Helicobacter species harvested from commercially
reared pigs (Krakowka et al., 1995, 2005; Kronsteiner et al., 2013).
These models have helped to confirm the association between
Helicobacter colonization and peptic ulceration, but have not been
adopted more widely for mechanistic studies.
Mongolian gerbils reportedly develop a wide spectrum of
Helicobacter-induced pathologies, including gastric ulceration.
For example, Honda et al. (1998a) reported that 4 out of 5 gerbils
developed gastric ulceration 6 months after colonization with the
CagA
+
(see Glossary, Box 1) H. pylori strain, ATCC-43504.
Independently, Ogura and colleagues (2000) identified gastric
ulceration in 22 out of 23 gerbils infected with the TN2 strain of H.
pylori for 62 weeks. When the H. pylori virulence factor CagE was
deleted, none of the 22 infected animals developed gastric
ulceration in the same timescale, demonstrating the utility of
Mongolian gerbils for modelling gastric ulceration, and the value of
this model for characterizing the virulence factors of H. pylori.
Most of the literature reporting H. pylori colonization of mice
suggests that colonization is usually transient, and if persistent, that
it is often associated with only mild gastritis (Lachman et al., 1997).
In contrast, Kaur et al. (2014) reported a model of gastric ulceration
following colonization of female C57BL/6 mice with H. pylori
DSMZ 10242 for 8 weeks. This study reported multifocal gastric
antral ulceration with relatively deep ulcers at 8 weeks, which, if left
untreated did not heal. Whilst this study contrasts with much of the
literature, it might reflect how factors in an individual laboratory, in
particular the baseline microbiota, can influence the outcome of
infection in different institutions.
A more established method for investigating the effect of
H. pylori on gastric ulceration has been to investigate the impact
of H. pylori infection on chemically induced gastric ulcers. For
instance, rats administered acetic acid to the serosal surface of the
stomach develop ulcers that heal over several weeks. Gastric
colonization with both CagA
+
(ATCC 43504) (Bui et al., 1991) and
CagA
−
(AH69) (Li et al., 1997) strains of H. pylori, impairs the
healing of acetic acid-induced ulcers in Sprague-Dawley rats. In this
H. pylori
colonization
Superficial gastritis
Mongolian gerbils
colonized with H. pylori
Gnotobiotic piglets
infected with H. suis
Peptic ulcer
disease
Gnotobiotic piglets
infected with H. suis
MALT lymphoma
H.felis or H.heilmannii
infection of Balb/c mice
Gastric
adenocarcinoma
H. pylori infection of
Rhesus macaques
H.heilmannii infection
of C57BL/6 mice
H. pylori or H. felis
infection of INS-Gas
mice
H. felis infection of
C57BL/6 mice
Mongolian gerbils
infected with H. pylori
or H. felis
Fig. 4. Modelling the pathological outcomes of Helicobacter infection.
A schematic of the principal pathological outcomes of Helicobacter infection in
humans, annotated with details of the best-characterized in vivo models for
these conditions.
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model, extracted H. pylori surface proteins (Brzozowski et al.,
1999) also impaired the healing of gastric ulcers, suggesting that a
response to bacterial components, and not necessarily to the
presence of live Helicobacter, can impair mucosal healing
following gastric injury (Brzozowski et al., 1999).
Models of gastric adenocarcinoma and its precursor lesions: large
and small mammals
Gastric carcinogenesis has been the most extensively studied
outcome of H. pylori infection, and it has the most diverse array
of in vivo models, several of which are used in laboratories
across the world. Much of the original work investigating the
pathogenesis of Helicobacter-induced gastric cancer was performed
in large animals.
Large animal models of gastric pathology offer the opportunity to
perform serial endoscopic biopsies during an experiment. This
allows investigators to document the temporal development of
gastric pre-neoplasia in individual animals, and has been adopted in
beagle dogs (Rossi et al., 1999) and Rhesus monkeys (Liu et al.,
2009).
In one such study (Rossi et al., 1999), conventionally housed
dogs were infected with a CagA
+
strain of H. pylori (SPM326s) and
underwent endoscopic evaluation 1, 2, 4, 8, 12, 18 and 24 weeks
after infection. From 8 weeks, chronic superficial gastritis
developed, with progressive changes observed in the gastric
mucin composition, in keeping with functional gastric atrophy.
The authors interpreted this as the development of early pre-
neoplastic pathology. The study also demonstrated progression
towards atrophic gastritis; however, it is not possible to predict
whether more-advanced pre-neoplastic lesions would subsequently
develop in this model. This study was also compromised by a lack of
detail about the pre-infection gastric microbiota of the animals used
in this study; they were demonstrated to be H. pylori seronegative,
but pre-infection gastric colonization was not assessed for H. pylori
or for other NHPHs.
H. pylori infection of rhesus macaques has been studied in
combination with the administration of the ethylating agent,
N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). ENNG is similar
to N-nitroso compounds present in traditional Far Eastern diets, and
is proposed to be a potential dietary risk factor for developing
gastric cancer (Seel et al., 1994). Rhesus macaques were observed
over 5 years following H. pylori colonization, and gastroscopy
was performed quarterly. Neither the continuous administration of
ENNG in food ad libitum, nor H. pylori infection alone induced
gastric pre-neoplasia. However, the administration of both
agents together induced intestinal-type metaplasia after 2 years of
treatment, and more advanced neoplasia, including high-grade
dysplasia, in one animal after 5 years. This study demonstrates
synergy between ENNG and H. pylori, but the study design was
unable to determine whether both agents are essential for gastric
carcinogenesis, or whether one carcinogen accelerated the effect of
the other (Liu et al., 2009).
Although studies using large animal models provided insights
into the development of Helicobacter-induced gastric cancer, they
have not been widely adopted because of the need for specialized
animal facilities and the associated high costs. As a result, rodent
models are most commonly used. In particular, Mongolian gerbil
and murine models have been used frequently in different
laboratories to investigate diverse aspects of gastric carcinogenesis.
Both mice and gerbils develop stereotypical, pre-neoplastic
pathology in response to Helicobacter infection. Whilst the
pathways observed in humans and gerbils are similar, there is a
difference during the metaplastic phase of gastric pre-neoplasia in
mice. In humans and gerbils, the commonest metaplasia is
intestinal-type metaplasia. This is characterized by the presence of
goblet cells and by the expression of appropriate intestinal markers,
such as trefoil factor 3 (TFF3) and mucin 2 (MUC2) (Ectors and
Dixon, 1986), as well as by the intestinal differentiation regulating
transcription factor CDX2 (Barros et al., 2011; Silberg et al., 2002).
Another metaplastic lesion, defined as spasmolytic polypeptide-
expressing metaplasia (SPEM), is less frequently identified in
people with gastric pre-neoplasia. It is characterized by a phenotype
similar to the secreting Brunner’s glands of the intestine, or to deep
antral gland cells that express MUC6 and trefoil factor 2 (TFF2, or
spasmolytic polypeptide) (Weis and Goldenring, 2009). In gerbils,
the distribution of these lesions is similar to that observed in humans
(Yoshizawa et al., 2007), whereas C57BL/6 mice infected with
H. felis develop a predominantly SPEM response, with little or no
evidence of intestinal-type metaplasia (Shimizu et al., 2016; Weis
and Goldenring, 2009).
When colonized by H. pylori and by several other NHPH strains,
including H. felis,H. bizzozeroni and H. suis (De Bock et al.,
2006a,b; Liang et al., 2015; Nakamura et al., 2007), Mongolian
gerbils reportedly develop advanced pre-malignant lesions.
However, the use of this organism is complicated by problems
of experimental reproducibility. For example, Watanabe and
colleagues (1998) reported that 5 out of 5 gerbils infected with
the CagA
+
H. pylori strain TN2GF4 in a standard animal house
environment for 52 weeks developed intestinal metaplasia, and
10 of 27 gerbils infected for 62 weeks developed invasive
adenocarcinomas. In contrast, Elfvin et al. (2005) studied gerbils
that were colonized with either H. pylori SS1 or TN2GF4 and
maintained in a SPF facility for up to 18 months. None of
these animals developed invasive adenocarcinoma, and only 2 of
5 infected animals at 12 months, and 3 of 10 infected animals at
18 months, developed intestinal metaplasia.
Because the Mongolian gerbil is outbred, the genetic backgrounds
of animals supplied to different laboratories up to a decade apart, will
have been divergent. The conditions within different animal units
might also have contributed to differences in these studies,
particularly differences in diet and resident microbiota, which
could influence host pathology. These observations demonstrate
some of the challenges of comparing in vivo studies performed in
different environments, and challenge the received wisdom that
experimental results should always be reproducible in different
settings (discussed further in Justice and Dhillon, 2016; Schofield
et al., 2016). Fully understanding why these apparently similar
experiments resulted in divergent outcomes could offer insights into
the mechanisms that drive the development of gastric pre-neoplasia
in the gerbil.
The Mongolian gerbil has been particularly useful for
identifying Helicobacter-specific and environmental factors that
influence the development of gastric cancer. It has been used to
demonstrate the carcinogenic potential of both H. pylori
(Watanabe et al., 1998) and H. felis (Court et al., 2002; De Bock
et al., 2006a,b), and that H. bizzozeronii and H. salomonis
(De Bock et al., 2006b) are less likely to induce gastric pre-
neoplasia in this species. In addition, several studies have
identified that high-salt diets promote the development of H.
pylori-induced gastric pre-neoplastic pathology in gerbils (Bergin
et al., 2003; Kato et al., 2006; Nozaki et al., 2002), consistent with
human epidemiological studies (Wang et al., 2009).
The Mongolian gerbil has also been used to adapt H. pylori
strains to a rodent environment. This has been demonstrated best by
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the passage of H. pylori B128, derived from a patient with gastric
ulceration, through a male gerbil for 3 weeks. H. pylori was
subsequently re-cultured from this animal’s stomach and described
as H. pylori strain 7.13, which is more pathogenic than strain B128.
Six of eight gerbils infected with H. pylori 7.13 developed gastric
adenocarcinoma 8 weeks after inoculation, in comparison to none in
the B128 group (Franco et al., 2005). The same researchers
demonstrated that this phenotype was essentially reproducible, and
preventable by eradication of H. pylori (Romero-Gallo et al., 2008).
The 7.13 strain has been further characterized by genome
sequencing (Asim et al., 2015), providing data that will help to
advance our future understanding of H. pylori pathogenicity.
The laboratory mouse is the other rodent used extensively to
model gastric pre-neoplasia. H. pylori colonization of C57BL/6
inbred mice leads to gastritis with epithelial cell hyperplasia, but this
does not progress to dysplasia or to cancer (Lee et al., 1997).
However, the colonization of C57BL/6 mice with H. felis is
consistently shown to lead to gastric pre-neoplasia, and when
infections have persisted for 13-15 months, adenocarcinomas have
been reported (Fox et al., 2002). This outcome is specific to the
C57BL/6 genetic background.
Other strains of inbred mouse, including Balb/c, respond
differently to H. felis infection.This strain can be colonized by
H. felis, but does not develop gastric pre-neoplastic pathology
(Wang et al., 1998). The mechanisms underlying these differences
between mouse strains are attributable to differences in immune
response. C57BL/6 mice demonstrate a Th1-polarized immune
response, whilst Balb/c mice have a more Th2-polarized response to
H. felis colonization, which allows infection to persist but does not
promote chronic epithelial disruption (Wang et al., 1998). The
B6129 mouse, generated by crossing C57BL/6 with 129S6/SvEv, is
particularly sensitive to Helicobacter-induced pathology triggered
by H. felis. In these mice, gastric intraepithelial neoplasia developed
8 months after H. pylori infection whereas malignant tumours
developed after ∼15 months (Rogers et al., 2005).
In addition to the models of gastric cancer induced solely
by Helicobacter infection, several chemically induced gastric
cancer models are accelerated by co-infection with H. pylori.
These include mice and Mongolian gerbils infected with H. pylori
and treated with N-methyl-N-nitrosourea, (MNU), and Mongolian
gerbils infected with H. pylori and treated with the carcinogen
methylnitronitrosoguanidine (MNNG). In Mongolian gerbils, the
co-administration of H. pylori with MNNG accelerated the
carcinogenic process, leading to invasive cancer in 60-80% of
animals treated for 1 year (Maruta et al., 2001, 2000; Tatematsu
et al., 1998). In mice, the effect of co-administering MNU and
H. pylori is less clear, with contrasting reports of either synergy
between the two stimuli (Han et al., 2002) or no additional effect
above chemical carcinogen alone (Nakamura et al., 2002).
Transgenic mouse models of gastric carcinogenesis
Several transgenesis strategies have been used to study gastric
carcinogenesis. These include the induction of spontaneous
gastric atrophy, the expression of H. pylori pathogenicity factors,
and the overexpression of known oncogenes in the gastric
mucosa. Transgenic animals have also been used to explore the
role of specific molecular pathways that potentially modulate
gastric carcinogenesis. A complete description of the genetically
engineered mouse models (GEMMs) used in gastric carcinogenesis
research is beyond the scope of this article, and so we refer readers to
another recent review for more information (Jiang and Yu, 2017).
Here, we focus on the best characterized of these models.
Amongst the most established examples of transgenically
induced gastric atrophy is the INS-Gas mouse model [also
known as FVB-tg(rl1-hinsgas) (Wang and Brand, 1992)]. This
mouse expresses a human gastrin mini-gene under the control of
the rat insulin promoter. It constitutively expresses gastrin in
β-cells of the pancreatic islets, resulting in the constitutive over-
production of amidated gastrin. These animals are born with
increased numbers of parietal cells and at birth hypersecrete acid,
but over the first 5 months of life, parietal cell numbers fall to that
of wild-type animals. Over longer periods, profound gastric
atrophy occurs, and a proportion of these animals develop
spontaneous gastric cancers (Wang et al., 2000). Helicobacter
infection accelerates the gastric pathology observed in this mouse;
85% of INS-Gas mice infected with H. felis developed gastric cancers
7 months after infection (Wang et al., 2000). Similarly, when INS-Gas
mice are infected with H. pylori,they exhibit significantly more severe
gastric inflammation and dysplasia relative to uninfected controls.
Interestingly, INS-Gas males are more severely affected by H. pylori
infection than are female mice, for reasons that are unknown, and in
the same study, mice exposed to high dietary salt levels had more-
severe gastric pathology relative to untreated controls (Fox et al.,
2003).
More recently, the INS-Gas mouse has been used to characterize
the role that the non-Helicobacter microbiome has during gastric
carcinogenesis. INS-Gas mice bred and maintained in a germ-free
environment did not develop spontaneous gastric pre-neoplasia,
while otherwise germ-free mice infected with H. pylori developed
fewer tumours than did H. pylori-infected SPF mice (Lofgren et al.,
2011). Subsequently, the same group demonstrated that a limited
group of three bacterial species is sufficient to restore the phenotype
of SPF mice in otherwise germ-free mice (Lertpiriyapong et al.,
2014), and that co-infection with the intestinal roundworm
Heligmosomoides polygyrus can protect INS-Gas mice against
H. pylori-induced gastric pre-neoplasia (Whary et al., 2014). This
protection was associated with an increase in the number of cells
positive for forkhead box P3 (FOXP3) –a master regulator of
regulatory T-cell (T
reg
) development –in the gastric corpus of mice,
suggesting a possible shift in the T
reg
immune response to H. pylori
infection.
The CEA/SV40T L5496 mouse expresses the SV40T proto-
oncogene under the control of a truncated carcinoembryonic antigen
(CEA) promoter. Gastric tumours formed in all mice, with dysplasia
evident as early as 37 days postnatally and with animals becoming
moribund due to gastric tumour burden by 100-130 days postnatally
(Thompson et al., 2000).
Mice transgenically deficient for mutT homolog-1 (Mth1)are
unable to process oxygen free radicals, which cause DNA damage,
and are susceptible to several spontaneous tumours, including
gastric tumours, which develop over ∼18 months; 14% (13 of 93) of
Mth1-null mice developed gastric tumours, compared with 4% (4 of
90) of littermate controls (Tsuzuki et al., 2001).
In Trefoil factor 1 (Tff1)-deficient mice, the structure of both the
antral and corpus mucosa is markedly altered from birth. By 5
months of age, all Tff1-null mice exhibit adenomatous changes in
the gastric mucosa, and 30% have established adenocarcinomas
(Lefebvre et al., 1996).
The C57BL/6J-tg(H/K_ATPase/hIL-1β) mouse overexpresses
human interleukin 1B (IL1B) under the control of the H
+
/K
+
ATPase β-subunit promoter, leading to expression of human IL1-β
exclusively in parietal cells. In this model, 15% of male mice develop
spontaneous tumours at 14 months of age, and disease severity is
markedly increased by infection with H. felis (Tu et al., 2008).
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Transgenic mutation of the IL6 co-receptor, Gp130, leads to a
severe gastric phenotype in which gastric antral adenomas
spontaneously develop over the first 6-8 weeks of life, and
subsequently grow and spread to include the gastric fundus by
48 weeks of age (Judd et al., 2004; Tebbutt et al., 2002). Whilst this
animal does not exhibit the classical pre-neoplastic pathology
induced by Helicobacter infection, it may have relevance to
H. pylori-mediated disease because CagA status has been shown to
influence gp130-mediated switching between the MAPK/ERK
(stimulated by the tyrosine phosphatase SHP2) and JAK-STAT
signalling cascades in vitro (Lee et al., 2010). Signalling through
these mechanisms influences a number of cellular processes that are
altered in cancer, including regulation of cell proliferation and
apoptosis, invasion and angiogenesis. Disruption of these pathways,
either by direct mutagenesis or through other mechanisms, has been
identified in many different tumour types (Zhang et al., 2015).
Transgenic expression of the H. pylori virulence factor CagA is
oncogenic to the gastric mucosa, both when expressed
constitutively throughout the animal under the control of the
β-actin promoter [C57BL/6-tg(CAG-CagA
Hs
)], and when limited to
the gastric mucosa under the control of the H
+
/K
+
ATPase β-subunit
promoter [C57BL/6J-tg(HK-CagA
Hs
)]. In both cases, animals
developed gastric hypertrophy by 12 weeks of age, and over
3 years, developed gastric dysplasia or occasionally gastric
adenocarcinomas (Ohnishi et al., 2008).
Models of MALT lymphoma
Several factors make Helicobacter-induced MALT lymphoma
challenging to model. It is a rare outcome of H. pylori infection;
it develops after a prolonged, complex interaction among the
bacteria, host epithelium and host immune system; and the
commonest, relatively indolent form of MALT lymphoma is
challenging to diagnose pathologically. These issues are
compounded by the fact that natural Helicobacter-induced MALT
lymphomas have not been reported in commonly used laboratory
species. Consequently, in some cases, only one research group has
assessed the models described below, and substantial gaps remain in
our knowledge of the mechanisms involved in H. pylori-induced
gastric MALT lymphoma formation.
Within these studies, there is also heterogeneity in the criteria
used to report MALT lymphoma formation. Most studies describe
lymphoepithelial lesions as a pathognomic event, signifying the
initiation of lymphomagenesis. Other studies used evidence of
monoclonal lymphoid aggregate formation as a surrogate for the
development of MALT lymphoma.
Experimental induction of MALT lymphoma by H. pylori was
reported in a single study of conventionally housed beagle dogs.
This study described the formation of monoclonal lymphoid
aggregates in the gastric mucosa of dogs infected with H. pylori
strain SPM326s (CagA
+
) for 6 months. This study was not extended
to later time points, and in the absence of epithelial destruction or
evidence of genetic instability, the association with MALT
lymphoma remains somewhat tenuous (Rossi et al., 1999). In
addition, the pre-existing gastric Helicobacter status of the dogs at
the study onset was not evaluated, although seropositivity for
H. pylori was excluded. It is therefore not possible to conclude
whether the observed phenotype was due to H. pylori infection in
isolation or to a synergy between H. pylori and other gastric
Helicobacter species.
Several NHPHs have been reported to induce either MALT
lymphoma or precursor lesions in laboratory conditions. Two of six
Mongolian gerbils infected with the H. suis strain, HS5, for 9 months
developed lymphoepithelial lesions (Flahou et al., 2010), whilst
outbred Swiss, and inbred Balb/c and C57BL/6, mice infected with a
variety of NHPH strains developed lymphoepithelial lesions.
In one of these studies, Enno et al. (1995) reported that 25% of
Balb/c mice colonized with H. felis for 22-26 months developed
advanced lymphoepithelial lesions, and a further 5% had early
lymphoepithelial lesions. These findings have since been
independently replicated, and in the same study, a variety of
H. heilmannii strains isolated from various sources, including
mandrill monkeys and bobcats, also induced gastric MALT
lymphomas over a similar timescale (O’Rourke et al., 2004).
A commonly used model of gastric carcinogenesis is the long-term
infection of C57BL/6 micewith H. felis; however, these mice have not
been reported to develop gastric MALT lymphoma. They do,
nevertheless, develop gastric lymphoepithelial lesions and low-
grade MALT lymphomas when colonized for 1 year with a candidatus
H. heilmannii isolated from the stomach of a cynomolgus monkey
(Nakamura et al., 2007). More recently, we have demonstrated that
50% (3/6) of C57BL/6 mice lacking the c-Rel NF-κBsubunit
developed early lymphoepithelial lesions when colonized with
H. felis for 12 months (Burkitt et al., 2013). This pathology has not
previously been reported in the C57BL/6/H. felis model, suggesting
that signalling through the c-Rel NF-κB subunit could influence the
regulation of gastric MALT lymphoma formation.
Overall, these data support the hypothesis that NHPHs play a
specific role in the development of MALT lymphoma, and since
several of the typical H. pylori virulence factors are not expressed in
these Helicobacter species, this suggests that novel bacterial factors
might be important in the development of gastric MALT lymphoma.
Future work in this field needs to incorporate models of genetic
instability, in addition to morphological criteria, to strengthen the
quality of data generated from these models.
Ex vivo models of Helicobacter infection of the stomach
To date, most studies investigating the mechanisms that underlie
H. pylori-induced gastric pathology have relied on in vivo models.
Many of these models require prolonged exposure to Helicobacter
and use relatively large numbers of animals, raising questions of
animal welfare and cost. In addition, studying the interaction of
two whole organisms (and increasingly the rest of the microbiome
to which H. pylori contributes) generates hugely complex systems.
Some studies have tried to address this complexity by using
elegant transgenic mouse systems, for example, by using tissue-
specific transgenesis. However, these systems remain highly
complex, and genetic manipulation can introduce further
complexity, either through gene expression in a suboptimal
location, or through off-target effects of the drugs used to induce
genetic recombination.
There is, therefore, a need for better in vitro or ex vivo models
of H. pylori-associated pathology. Over the past 6 years, the
development of first murine, and subsequently human, three-
dimensional, primary gastric gland cultures called gastric organoids
or ‘gastroids’(see Glossary, Box 1) has opened up the prospect of
using untransformed, gastric tissue in culture to model the
development of gastric pathology.
Ex vivo gastric mucosal culture models
In order to generate gastric glandular units in culture, it must first be
possible to isolate primary material from an organism, to passage
this material in vitro, and then demonstrate its ability to differentiate
into the different cell types of the gastric epithelium. Ideally, it
should also be possible to store the cultures in the laboratory and
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Disease Models & Mechanisms

reconstitute them, deriving reproducible results from cultures that
have been stored or not stored.
Over the past decade, culture systems have been developed that
fulfil these criteria. The key discovery came with the identification
of LGR5 as a marker of gastrointestinal stem cells (Barker et al.,
2010, 2007). The identification of this WNT-signalling family
member as a key marker of gastrointestinal stem cells led rapidly to
the development of primary culture systems that support these cells
primarily through the optimization of WNT signalling. Since these
discoveries, several groups have established gastric organoid
systems using slightly different approaches (McCracken et al.,
2014; VanDussen et al., 2015), as described below.
Long-lived gastric epithelial cultures derived from primary gastric
tissue
The generation of self-renewing gastric gland cultures was first
described by Barker, using methodology developed from earlier
intestinal organoid models (see Glossary, Box 1) established by the
same group. This method uses LGR5
+
stem cells extracted from the
gastric antrum of mice expressing green fluorescent protein (GFP)
under the control of the Lgr5 promoter as starting material. These
cells are supported in vitro in a 3D matrix together with recombinant
growth factors that together recapitulate the stem cell niche. In
addition to activating the WNT pathway, TGFβpathway signalling
is suppressed and gastrin 17 (GAST) and fibroblast growth factor 10
(FGF10) are added as gastroid-specific growth factors. During the
initial phase of culture, the ROCK inhibitor Y-27632 is added to the
growth medium to prevent anoikis. This methodology established
LGR5
+
cells as gastric antral stem cells; intriguingly, an effective
marker for the gastric corpus stem cell remains elusive.
Subsequent protocols have established similar methods for the
establishment of murine gastric organoid cultures from both antrum
and corpus using non-enzymatically dissociated gastric glands as
starting material (Mahe et al., 2013), and for organoids based on
gastric tissue samples taken at the time of gastric resection (Bartfeld
and Clevers, 2015; Schlaermann et al., 2016). These cultures
generate spherical cultures that maintain a 3D structure in culture
(Fig. 5).
A modification of this protocol uses conditioned medium from
the L-WRN cell line, which secretes human WNT3A, noggin
(NOG) and r-spondin 3 (RSPO1) (VanDussen et al., 2015). This
system has been shown to allow cultures to be established from
small samples taken during an endoscopic examination of the
stomach, rather than requiring samples to be excised during surgery,
and could be more cost effective because of the high cost of
recombinant growth factors. However, the use of this cell line limits
the control that an experimentalist can have on the culture system; in
particular, gastric organoids grown using this method are exposed to
particularly high levels of WNT3A, making the cultures more
proliferative and less likely to differentiate than gastric organoids
established using recombinant growth factors.
Most reports of gastrointestinal organoid culture systems to date
have retained the 3D structure. However, an increasing number of
studies use 3D organoids as the source material to generate
epithelial monolayers on collagen-coated glass or plastics. This
technique offers different opportunities for quantification and
observation of morphology, which in some cases might be easier
to relate back to more-established 2D cancer cell cultures
(Schlaermann et al., 2016).
Long-lived gastric epithelial cultures derived from induced
pluripotent stem cells
Human gastric organoids have recently been generated from both
human embryonic stem cell lines and induced pluripotent stem cell
(iPSC) lines (McCracken et al., 2014). These stem cells were first
differentiated into definitive endoderm before the induction of the
foregut marker SOX2, by exposure to WNT3A, FGF4 and noggin.
They were further differentiated into ‘antral’- and ‘corpus’-type
cultures by exposure to retinoic acid and subsequently differentiated
into mature organoids using epidermal growth factor (EGF).
Both antral- and corpus-type gastric organoids generated from
such cultures are similar to their originating tissues, as shown by
microarray and by gene set enrichment analyses (McCracken et al.,
2014). Morphologically, antral gland type organoids also contain
identifiable epithelial and endocrine cell types. Corpus-type
organoids do not possess parietal cells; however, other markers of
Surface mucous cell
Stem cell
Mucous neck cell
Parietal cell
Endocrine cell
Chief cell
Gastric glands
Gastroid
A
BCD
Key
Fig. 5. Gastric organoid culture and differentiation. Diagrams and images showing the maturation of gastric organoids. (A) Freshly digested gastric corpus
glands from a C57BL/6 mouse. (B) Glands 24 h after harvesting that have formed immature organoids and have a small spherical appearance. (C) On day 3 of
culture, the immature organoids have expanded and can be passaged. (D) Following passage, the organoids retain their spherical appearance and continue to
grow. Images from authors’own laboratories. Scale bars: 250 µm.
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gastric corpus tissue, including expression of pepsinogens and
ghrelin, are detectable.
The effect of Helicobacter infection on cultured gastric organoids
Several groups have investigated the impact of H. pylori infection
on gastric organoid cultures. The most reproduced finding is that
epithelial cell proliferation is enhanced by direct mucosal
interaction with H. pylori. This has been demonstrated by the
microinjection of H. pylori into 3D fundic gland organoids derived
from both mice and humans (Bartfeld et al., 2015; Bertaux-Skeirik
et al., 2015), and into human pluripotent stem cell-derived gastric
organoids (McCracken et al., 2014).
H. pylori infection also induces morphological changes in 2D
gastric-organoid-derived monolayers grown on collagen-coated
glass or plastics, with epithelial cells taking on a hummingbird
morphology (Schlaermann et al., 2016). This response was CagA
dependent and appears to be analogous to the SHP2-mediated
hummingbird morphology previously described in gastric cancer
cell lines (Higashi et al., 2002). This change in morphology was
associated with the activation of the classical NF-κB signalling
pathway; this pathway is also implicated in the response of 3D
gastric organoids to H. pylori microinjection (Schlaermann et al.,
2016; Schumacher et al., 2015). In gastric cancer cell lines, this
morphology is associated with a more aggressive, invasive
phenotype (Chang et al., 2016) and epithelial-mesenchymal
transition (Snider et al., 2008).
Sigal et al. (2015) have established an organoid-formation assay
in which they quantified the percentage of viable organoids formed
from a preparation. Using this assay, they verified their own
observation that the antral LGR5
+
stem cell zone expands in
response to H. pylori infection. This provides a novel method for
quantifying the abundance of antral stem cells.
A further study investigating the interactions between H. pylori
and human gastric cells in the context of organoids has
demonstrated that H. pylori can sense nanomolar concentrations
of urea, and use this as a chemoattractant. This study made use of the
observation that whilst most organoids form with the luminal
surface of the epithelium facing inwards, a small proportion form
with an ‘inside-out’structure. This allowed the authors to observe
epithelial and bacterial interactions, and in particular the adherence
of H. pylori to cell-cell junctions (Huang et al., 2015).
Conclusions
An ever more diverse array of laboratory models exists to explore
H. pylori-induced pathology. The course of infection and plethora
of outcomes in current in vivo models are probably too complex to
understand fully using current technology. The new generation of
ex vivo models offer opportunities for researchers to be more
systematic in their approach; however, at present the models also
risk being reductionist. Over time we need to develop ex vivo
systems that can be interrogated systematically, but which
incorporate key elements of in vivo models, including host
epithelial, mesenchymal and immune compartments, and both
Helicobacter and non-Helicobacter microbiota.
Currently available gastric organoid models have focused largely
on the development of organoids from healthy animals and humans,
which have then been infected with H. pylori. However, future
studies will need to develop models that mimic the development of
other gastric epithelial pathologies in culture. In particular, the
development of 3D models of gastric atrophy and metaplasia will
allow researchers to perform experiments to compare the effect of
developing gastric epithelial pathology in vivo and ex vivo. Being
able to make these comparisons will allow better mechanistic
studies to be performed in the relatively simple organoid systems
and verified in whole animals, avoiding the pitfalls of a reductionist
scientific approach.
The results of these studies will begin to provide better data that
segregate epithelial events from immune and mesenchymal driven
changes in the stomach. Developing these models presents a major
challenge for the future but, if successful, they are likely to permit
the design and evaluation of new therapeutic strategies for patients
who currently have no meaningful treatment options.
Competing interests
The authors declare no competing or financial interests.
Funding
This research received no specific grant from any funding agency in the public,
commercial or not-for-profit sectors.
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