1-1 Helicobacter pylori
Helicobacter pylori is a well-known gastric-parasitical pathogen. Since 1982, Dr.
Marshell swallowed ten hundred million of H. pylori personally to prove that the persistence of H. pylori in stomach can result in some kind of gastric diseases, and lead a new epoch in human gastroenterology. H. pylori has been found in human in all parts of the world, with over half of the world’s population infected with H. pylori. In developing countries, 70-90 % of the population caries H. pylori. In 20-30 % of cases, the end result of the infection can be life-threatening (1). Many publishes revealed that H. pylori infection was associated with acute or chronic gastritis, peptic ulcer, gatroduodenal ulcer, and gastric cancer development, which led to H. pylori becoming classified as a classⅠcarcinogen by the World Health Organization. The complete process of H. pylori invasion, infection, and proliferation is really complex. Host immune surveillance system plays an essential role in pathogen elimination at early or late stage. However, H. pylori seems not be effectively cleared by host immune system and persist in host stomach for a half life time. Recently, scientists work hard to investigate many subversives of H. pylori and found that H. pylori can utilize multiple factors to protect them living in host stomach where a horrible environment for most pathogens is.
1-1.1 The morphology of Helicobacter pylori
H. pylori organisms are spiral, microaerophilic, gram-negative bacteria that colonizes the
gastric mucosa of humans. In gastric biopsy specimens, H. pylori organisms are 2.5 to 5.0 μm long and 0.5 to 1.0 μm wide; there are four to six unipolar sheathed flagella, which are essential for bacterial motility. Moreover, the surface of individual bacteria may be linked to gastric epithelial microvilli by thread-like extensions of the glycocalyx (2, 3). Interestingly, H.
pylori is classified as a noninvasive bacterial organism because it typically does not traverse
the epithelial barrier (4). Nevertheless, the bacterium is able to induce strong immune responses in such environment and results in some kind of gastric disease.
1-1.2 H. pylori-associated diseases
Colonization with H. pylori is not a disease in itself but a condition that affects the relative risk of developing various clinical disorders of the gastrointestinal tracts.
1-1.2.1 Acute gastritis:
Several reports showed that the acute phase of colonization with H. pylori may be associated with transient nonspecific dyspeptic symptoms, such as fullness, nausea, and vomiting, and with great inflammation of stomach mucosa. This phase is often associated with hypochlorhydria and it is unclear whether this initial colonization can be cleared spontaneously and prevents gastritis occurrence (5, 6).
1-1.2.2 Chronic gastritis:
Colonization with H. pylori always results in infiltration of the gastric mucosa with neutrophilic and mononuclear cells. H. pylori colonization relates to the chronic active gastritis, and other H. pylori-associated disorders result from this chronic inflammatory process. When colonization becomes persistent, a close correlation exists between the level of acid secretion and the distribution of gastritis. In subjects with intact acid secretion, H. pylori in particular colonizes the gastric antrum, where few acid-secretory-parietal cells are present.
The pattern is associated with an antrum-predominant gastritis. Subjects with impaired acid secretion have a distribution of bacteria in antrum and corpus. The corpus bacteria in corpus are in closer contact with the mucosa, leading to a corpus-predominant pangastritis (7).
1-1.2.3 Peptic ulcer disease:
Duodenal ulcers (peptic ulcer) are defined as mucosal defects with a diameter of at least 0.5 cm penetrating through the muscularis mucosa. Duodenal ulcers usually occur in the duodenal bulb, which is the area most exposed to gastric acid. Both gastric and duodenal ulcer diseases are strongly related to H. pylori. It was showed that approximately 95 % of duodenal ulcers and 85 % of gastric ulcers occurred in the presence of H. pylori infection (8).
1-1.2.4 Atrophic gastritis, intestinal metaplasia, and gastric cancer:
Chronic H. pylori-induced inflammation can eventually lead to loss of normal mucosal architecture, with destruction of gastric glands and replacement by fibrosis and intestinal-type
epithelium. The risk atrophic gastritis depends on the distribution and pattern of chronic active inflammation (9). Patients with decreased acid output show a more rapid progression towards atrophy (10). It was reported that the risk of gastric cancer development via the sequence of atrophy and metaplasia, and the development of atrophy and cancer in the presence of H. pylori is related to host and bacterial factors, which influence the severity of the chronic inflammatory responses (6).
1-1.2.5 Gastric MALT lymphoma:
The gastric mucosa does not normally contain lymphoid tissue, after H. pylori infection, a lymphoid infiltrate appears, which constitute a chronic gastritis. In certain cases the lympoid tissue can be organized as lymphoid follicles. MALT lymphoma emerges from these lymphoid structures (11). The in vitro experiment showed that T lymphocytes sensitized for H.
pylori produce cytokines which stimulate B lymphoid proliferation. It is a B cell lymphoma
with a very unusual pathogenesis and evolution which slowly progresses and stays localized in the stomach for a long time (12).
1-1.3 The virulence factors resulting in H. pylori colonization and pathogenicity 1-1.3.1 The cag PAI and Cag A protein:
An intact cag PAI, which is associated with severe disease (13), encodes 31 proteins, which form a type IV secretion system capable of directly transferring bacterial proteins to the
cytoplasm of target cells, and it can stimulate human gastric epithelium cells to secrete IL-8, a mediator of serious gastric inflammation. (reviewed in Ref.(14)). Recntly, Nalini Ramarao et al. further investigated that type IV transporter of H. pylori is essential in preventing phagocytosis. The H. pylori cag PAI mediates the translocation of an effector protein, CagA, into gastric epithelial cells, and might also be directly involved in loosening of tight junctions (15). Furthermore, infection with H. pylori strains possessing CagA is associated with an increased risk of developing adenocarcinoma of the stomach (16).
1-1.3.2 VacA:
VacA is a secreted protein toxin, which causes vacuolar degeneration of epithelial cells in vitro and gastric epithelial erosion in vivo. In addition, VacA can loosen tight junctions in monolayers of polarized epithelial cells (17). A recent study showed that VacA alters the intracellular trafficking of proteins, increases the permeability of polarized epithelial cells, inhibits the process of antigen presentation, forms anion-selective channels in lipid bilayers, and interferes with cytoskeleton-dependent cell functions (18).
1-1.3.3 Urease:
The H. pylori urease can break down urea (CN2H4O) to form NH3 and CO2, which buffer the microenvironment and the cytosol of the bacteria (19). Furthermore, H. pylori urease is a potent stimulus of mononuclear phagocyte activation and inflammatory cytokines production from immune cells and gastric epithelium cells (20) and the urease activity is also toxic to
human gastric epithelium cells (21).
1-1.3.4 NAP:
Helicobacter pylori neutrophil activating protein is also called HP-NAP. The main
evidence supporting a role for HP-NAP in virulence is the ability to activate neutrophils to produce oxygen free radicals and adhere to cultured endothelial cells (22), however, oxygen free radicals production will result in gastric tissue damage in the future. NAP released by bacterial lysis directly interacts with neutrophils, monocytes, and mast cells, resulting in the activation of their inflammatory functions (23).
1-1.3.5 Arginase:
H. pylori produces an arginase that uses arginine to produce urea and L-ornithine.
Furthermore, bacterial arginase allows H. pylori to evade the immune response by down-regulate eukaryotic NO production (24).
1-1.3.6 Cell wall and lipopolysaccharide:
Urease and HspB, a homolog of the GroEL protein of Escherichia coli, are abundant in outer membrane proteins (OMP) preparations. Urease and HspB are located strictly within the cytoplasm in early log phase cultures of H. pylori (25). However, in late-log-phase cultures, urease and HspB become associated with the bacterial surface in a novel manner. These cytoplasmic proteins are released by bacterial autolysis and become adsorbed to the surface of intact bacteria due to the unique characteristics of the outer membrane. The
lipopolysaccharide (LPS) of H. pylori has low biological activity, a property which may aid in the persistence of infection. H. pylori LPS disrupts the gastric mucus coat by interfering with the interaction between mucin and its mucosal receptor (26). However, the outstanding feature of the H. pylori LPS is its low proinflammatory activity.
1-1.3.7 Heat shock protein 60:
H. pylori HSP60 has been shown to play a role in the adherence and attachment of H.
pylori to gastric epithelium and induce IL-8 secretion from human gastric epithelial cells (27,
28). In the immune cells, H. pylori HSP60 can induce IL-6 and IL-8 from macrophages and monocytes, respectively (29, 30). Chronic gastritis is initiated and maintained by cytokines that are secreted by gastric epithelial cells and macrophages. Interleukin 8 (IL-8) is one of the principal mediators of the inflammatory response. Moreover, Kobayashi et al. showed that development of lymphoid tissue in patients with MALT lymphoma was associated with HSP60 (31).
1-1.4 Interactions between H. pylori and immune cells
Neutrophils: Neutrophils are recruited when H. pylori initially colonizes the human stomach, and the gastric mucosal inflammatory response that occurs in the persistent H. pylori infection is characterized by infiltration of neutrophils. Several specific H. pylori factors are known to interact with neutrophils and modulate their function (32).
Mast cells: In vitro experiments indicate that whole H. pylori bacteria and various H.
pylori components can activate mast cells. One H. pylori factor that can activate mast cells is
VacA (33). VacA can induce mast cell chemotaxis and can stimulate mast cell expression of multiple proinflammatory cytokines, including IL-1, TNF-α, IL-6, IL-13, and IL-10 (34).
VacA induces degranulation of the mast cell line but does not induce degranulation of murine bone marrow-derived mast cells. HP-NAP also can activate mast cells, resulting in IL-6 production. Activation of mast cells by H. pylori may contribute to the inflammatory response associated with H. pylori infection (35).
Macrophages: Contact between macrophages and intact H. pylori bacteria or H. pylori components results in macrophage activation and secretion of numerous cytokines and chemokines. Macrophage recognizes the intact H. pylori by mediating TLR2 or TLR4.
Ingested H. pylori cells have at least some ability to resist intracellular killing. Another mechanism of H. pylori escaping from macrophage killing is by blocking the production of nitric oxide. This effect is mediated by H. pylori arginase, which competes with nitric oxide synthase for arginine. In addition to resisting killing by macrophages, in vitro experiments indicated that H. pylori can induce macrophage apoptosis (36). H. pylori-induced apoptosis of macrophages may result in impaired innate and adaptive immune responses.
Dendritic cells: In response to H. pylori, monocyte-derived human DCs express costimulatory molecules and major histocompatibility complex class II proteins (37), which
results in increased efficiency of antigen presentation. Similar to several other bacterial pathogens, H. pylori can bind to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC-specific lectin (38). Interactions between H. pylori antigens and DC-SIGN may contribute to suppress the inflammation.
B lymphocytes: H. pylori is reported to have several inhibitory effects on B lymphocytes.
In one study, H. pylori VacA interfered with the prelysosomal processing of tetanus toxin in Epstein-Barr virus-transformed B cells, and the ability of these cells to stimulate human CD4+ T cells were impaired in the presence of VacA. VacA inhibited the Ii-dependent pathway of antigen presentation mediated by newly synthesized MHC class II molecules but did not affect the pathway dependent on recycling MHC class II (39). Expression of CagA in B cells is reported to inhibit interleukin-3-dependent B-cell proliferation by inhibiting JAK-STAT signaling, which may result in inefficient antibody production and reduced cytokine expression (40).
T lymphocytes: One report indicated that H. pylori can have proapoptotic effects on T cells (41), but most of the observed effects occur in the absence of cell death. Coincubation of
H. pylori with T cells results in diminished expression of IL-2 and IL-2 receptor (CD25),
inhibition of activation-induced proliferation, and cell cycle arrest (42). The effects of H.
pylori on T cells are mediated by several different bacterial factors, one of which is VacA.
VacA interferes with the activity of nuclear factor of activated T cells (NFAT), a transcription
factor that regulates immune response genes, in Jurkat T cells, resulting in inhibition of IL-2 expression and G1/S cell cycle arrest (42). In addition to VacA and arginase, an uncharacterized low-molecular-weight protein of H. pylori has been reported to inhibit proliferation of T lymphocytes. This low-molecular-weight H. pylori factor is reported to block cell cycle progression at the G1 phase (43).
1-1.5 Immune activation and cell damage by H. pylori infection
The human gastric luminal pH is < 2, which prevents the proliferation of bacteria within the gastric lumen. H. pylori penetrates the gastric mucus layer after entering host stomach and thereby encounters a less acidic environment. H. pylori typically does not traverse the epithelial barrier, and it is classified as a noninvasive bacterial organism (4). Nevertheless, the bacterium is able to induce strong pro-inflammatory responses in these cells. Since H. pylori adherence, the production of a vacuolating cytotoxin and bacterial enzymes all contribute to epithelial damage. H. pylori infection, irrespective of their cag PAI phenotype leads to chronic gastric inflammation in the host. Recruitment and activation of immune cells in the underlying mucosa involves H. pylori chemotaxins, epithelial-derived chemokines such as IL-8 and pro-inflammatory cytokines liberated by mononuclear phagocytes (TNF-α, IL-1 and IL-6) as part of non-specific immunity. Moreover, gastric epithelial cells up-regulate expression of major histocompatibility complex (MHC) class II and costimulatioy molecules
on mococytes, macrophages, and dendritic cells in the gastric mucosa also play an important role in antigen presentation to activate adaptive immune cells activation (44). However, the infiltrated immune cells-induced inflammation response appears to be a primary cause of the damage to gastric surface epithelial layers and finally resulted in gastritis, peptic ulcer disease, and gastric cancer (40).
1-1.6 Immune subversion by H. pylori
Once arriving at the gastric epithelium, H. pylori must face the rapid onslaught of effector cells of the strong immune response. To overcome continually intense attack, H.
pylori utilize some virulence factors to break host immune defense and successfully escape
from killing by effector cells. In the innate immune stage, H. pylori first attack by nitrogen oxide (NO), which is an important component of innate immunity and an effective antimicrobial agent. To avoid killing by NO, H. pylori produces an arginase to regulate NO synthesis. Arginase can convert L-arginine to urea and L-ornithine, because L-ornithine is also used by iNOS to produce NO so that arginase can compete with iNOS for their substrate to decrease NO production. Even though H. pylori was unfortunately ingested by professional phagocytes, it is capable to resist phagocytic killing. Phagocytosis of H. pylori by macrophages becomes a large megasomes, which result from homotypic phagosome fusion and subsequent macrophage apoptosis might enable the escape of the bacteria. Interestingly,
the LPS of H. pylori is at least 1000-fold less active than E. coli LPS. The VacA protein of H.
pylori is contributed to disrupt host adaptive immune response. Clear evidence has recently
been obtained for VacA in suppression of T-cell response. Sundrud et al. showed that VacA inhibits human peripheral blood lymphocytes proliferation by TCR-CD28 co-stimulation by interfering with IL-2-dependent cell cycle progression. Gastric MALT lymphoma results from the uncontrolled polyclonal expansion of IgM memory B cells, T-cells inactivation might also contribute to the abnormal B-cell growth. Moreover, Cag A is also capable of preventing B-cell apoptosis by inhibiting p53 accumulation, which might involved in development of MALT lymphoma.
1-1.7 Chronic infection
Levels of numerous cytokines, including gamma interferon (IFN-γ), tumor necrosis factor (TNF), IL-1β, IL-6, IL-7, IL-8, IL-10, and IL-18, are increased in the stomachs of H.
pylori-infected humans compared to uninfected humans (45). These cytokines have great
effect on immune cells activity and attract these effector cells to the inflammation site. The concentration of various types of leucocytes was detected in gastric mucosal biopsies from human infected with H. pylori. Lymphocytes (both T cells and B cells), macrophages, neutrophils, mast cells, and dendritic cells (DCs) are usually present at the inflammation area and play an important role in antigen presentation (46). The relative abundance of
IFN-γ-producing T cells and the relative scarcity of IL-4-producing gastric T cells in the setting of H. pylori infection, it has been concluded that H. pylori infection leads to a Th1-polarized response (47). The chronic gastric mucosal inflammatory response to H. pylori probably reflects the combined effects of a cellular immune response and an ongoing stimulation of an innate immune response.
1-2 Human innate immunity
The human immune system defends against a spectrum of microbial pathogens, in terms of environmental prevalence, rang from common to rare. Invasion by common environmental microbes is prevented by constitutive innate immune defense in mucosal and epithelial tissues.
Upon infection with highly virulent pathogens, auxiliary innate defenses are induced to combat the pathogens. Neutrophils, monocytes, macrophages, and dendritic cells are important cellular mediators of innate immune defense.
1-2.1 The characterizations of innate immune cells
Most cellular components of immune system derive from bone marrow. The typical developmental pathway begins with pluripotent bone marrow stem cells that give rise to progenitors that follow a variety of differentiation pathways to become mature cells with defined effector functions.
1-2.1.1 Monocytic cells:
Newly produced monocyte are released into blood where they circulate for 1-3 days before entering tissues to differentiate into mature resident macrophage (48).
1-2.1.1.1 Human monocyte subsets:
In humans, circulating monocytes are divided into two subsets on the basis of the expression of CD14, a component of the lipopolysaccharide (LPS) receptor complex, and CD16, the FcγRIII immunoglobulin receptor (49). These monocyte subsets express distinct chemokine, immunoglobulin, adhesion, and scavenger receptors (50). CD14+CD16− (CD14+) monocytes are large, ~ 18 μm in diameter, and represent ~ 80%–90% of circulating monocytes. In contrast, CD14−CD16+ (CD16+) monocytes are smaller, ~14 μm in diameter, and constitute ~ 10% of circulating monocytes.
1-2.1.1.2 Monocytes differentiation:
Under inflammatory conditions, monocyte production in the bone marrow is increased and after released into the circulation monocyte are rapidly recruited to sites of injury and infection where they differentiate into inflammatory macrophage (51). Furthermore, monocytes can also give rise to dendritic cells (DCs) in vitro and in vivo, and microbial infection triggers in vivo monocyte differentiation into specialized DC populations.
1-2.1.1.3 Bacterial infection:
Circulating monocytes are increasingly implicated as essential players in defense against
a range of microbial pathogens. Monocytes kill bacteria by producing reactive nitrogen intermediates (RNIs) and reactive oxygen intermediates (ROIs) and through the action of phagolysosomal enzymes (52, 53).
1-2.1.2 Macrophage:
Macrophage is a part of mononuclear phagocyte system and is professional antigen presenting cells for adaptive immunity. Mononuclear phagocytes migrate out from bone marrow, circulate briefly in the blood as monocytes, and then enter into the tissues and inflammatory foci where they differentiate into macrophages. Macrophages, which is a heterogeneous population of phagocytic cells found throughout the body that originate from the mononuclear phagocytic system (54).
1-2.1.3 Neutrophils granulocytes:
Neutrophils are abundant in blood, where they have a short half-life if they are not recruited to a site of inflammation by specific chemokines and cytokines. Once recruited to an inflammatory site, neutrophils migrate rapidly from blood to tissue, which is otherwise devoid of neutrophils (55). In response to inflammatory stimuli, neutrophils migrate from the circulating blood to infected tissues, where they efficiently bind, engulf, and inactivate bacteria. Phagocytosed bacteria are killed rapidly by proteolytic enzymes, antimicrobial proteins, and reactive oxygen species (56).
1-2.1.4 Dendritic cells:
Dendritic cells (DC) are one of the most potent antigen presenting cells (APCs) of the immune system and are thought to be crucial for the initiation of primary T cell-mediated immune responses (57). Resting DCs capture and process soluble or particulate antigens in late endosomal and lysosomal compartments that are rich in major histocompatibility
Dendritic cells (DC) are one of the most potent antigen presenting cells (APCs) of the immune system and are thought to be crucial for the initiation of primary T cell-mediated immune responses (57). Resting DCs capture and process soluble or particulate antigens in late endosomal and lysosomal compartments that are rich in major histocompatibility