Emerging role of IL-23/IL-17 axis in H pylori-associated pathology

Abstract

Colonization of stomach by H pylori is followed by a marked infiltration of the mucosa with polymorphonuclear leukocytes, macrophages, and lymphocytes that very often remains asymptomatic, but in some circumstances can lead to the development of gastroduodenal ulceration, gastric carcinoma, and mucosa-associated lymphoid tissue lymphoma. The molecular mechanisms by which H pylori triggers and maintains the local immune response are complex, but there is evidence that cytokines produced by both immune and non-immune cells contribute to amplify the ongoing inflammation. H pylori infection is associated with a marked mucosal induction of T helper (Th) type 1 and Th17-type cytokines that is governed by specific antigen-presenting cell-derived molecules, such as interleukin (IL)-12 and IL-23. In this paper, we will review the available data on the expression and role of IL-23 and IL-17 in H pylori-related gastritis.

Key Words: IL-23, IL-17, H pylori

INTRODUCTION

H pylori is a spiral-shaped Gram-negative flagellate bacterium that colonizes the human gastric mucosa and chronically infects more than half of the human population. Infection is inversely correlated with socioeconomic conditions. Most new H pylori infections occur in children, but the lack of specific H pylori-related clinical signs makes difficult to define the mode of transmission[1]. H pylori survives within the gastric mucus layer despite the acidic microenvironment, that limits the growth of most bacteria. This primarily relies upon the ability of H pylori to secrete a large amount of urease that breaks down urea into carbon dioxide and ammonia, the latter buffering its environment. Most H pylori organisms remain in the mucus layer, even though a small proportion adheres to the mucosal epithelial cells and rarely invades the mucosa[2]. Moreover, H pylori can inject into the epithelial cells bacterial products that modify epithelial cell functions[3].

IL-17 IS OVER-PRODUCED IN H pylori-COLONIZED GASTRIC MUCOSA

H pylori infection causes a marked infiltration of the gastric mucosa with neutrophils, macrophages, and lymphocytes. Most H pylori-infected patients are asymptomatic, but H pylori-driven gastritis can lead to the development of gastroduodenal ulcers, gastric carcinoma, and mucosa-associated lymphoid tissue lymphoma[4]. The level of inflammation increases the risk of disease, but it does not seem to influence which disease develops. In contrast, this is thought to be largely influenced by the pattern of gastric inflammation. In particular, antral gastritis is associated with increased stimulated acid production and predisposes to duodenal ulceration, while corpus-predominant or pan-gastritis is associated with reduced acid production and predisposes to gastric ulcer and gastric adenocarcinoma[5]. There is also evidence that the degree of gastric infiltration by neutrophils correlates with the development of gastro-duodenal ulcerations, and this is in part dependent on the release of damaging inflammatory mediators such as reactive oxygen species[6,7]. Because neutrophils are short-lived, they must be constantly recruited into the infected mucosa from circulation. Antigens released by H pylori can stimulate endothelial cells, macrophages and epithelial cells to make huge amounts of chemokines, such as interleukin (IL)-8 and growth-regulated oncogene-alpha, that produce a chemotactic gradient for the migration of neutrophils into the gastric mucosa[8-11]. It is also known that infections with specific H pylori strains that possess the cag pathogenicity island (cag+) induce significantly higher levels of chemokines than do cag-strains[12]. Both macrophages and epithelial cells also synthesize neutrophil-recruiting chemokines in response to lamina propria mononuclear cell (LPMC)-derived molecules. In this context, we and others have recently shown that IL-17, a key regulator of neutrophil chemotaxis, is produced in excess in H pylori-infected stomach[13-15]. By real-time PCR and Western blotting it was shown that IL-17 up-regulation occurs at both RNA and protein levels in H pylori-infected biopsies in comparison to uninfected biopsies either with or without gastritis[13,14]. Notably, among H pylori-positive patients, the gastric mucosa at the site of ulcers contains more IL-17 than the non-ulcerated mucosa of the antrum[15]. Several observations suggest that IL-17 plays a decisive role in the neutrophil recruitment to the H pylori-infected gastric mucosa. First, IL-17 levels correlate with the number of neutrophils infiltrating the Hp-infected mucosa[15]. Second, both gastric LPMC and epithelial cells express IL-17 receptors and are functionally capable of responding to IL-17 by secreting IL-8[14-16]. Consistently, conditioned media of gastric epithelial cells stimulated with IL-17 enhance the migration of peripheral blood neutrophils, and this effect is inhibitable by a blocking anti-IL-8, but not anti-IL-17 antibody (Figure 1)[14]. Functional analysis of intracellular pathways involved in the induction of IL-8 synthesis by IL-17 revealed that IL-17 activates ERK1/2 MAP kinases in gastric epithelial cells, and that pharmacologic blockade of this pathway significantly inhibits IL-8 secretion[16]. These findings are in line with the demonstration that activated ERK1/2 and IL-8 are more pronounced in gastric epithelial cells isolated from H pylori-infected biopsies in comparison to uninfected controls, and that neutralization of endogenous IL-17 in ex vivo cultures of H pylori-infected gastric biopsies down-regulates the expression of activated ERK1/2 and IL-8[16]. Finally, IL-17 expression positively correlates with IL-8 content in H pylori-colonized biopsies[15].

Figure 1 The figure illustrates some of the putative functions of IL-17 in the human gastric mucosa. During H pylori infection, IL-17 is produced by both lamina propria (LP) T (CD4+ and CD8+) and non- T cells, through a process that could be positively regulated by IL-1, IL-21, and IL-23. IL-17 stimulates both epithelial cells (EC) and LP antigen presenting cells (APC) to make IL-8, thereby enhancing the recruitment of blood neutrophils (N) into the mucosa. Additionally, IL-17 increases the production of inflammatory cytokines, such as IL-1, IL-6, and TNF by LP APC, as well as it stimulates fibroblasts to secrete matrix metalloproteinases (MMPs), a family of proteases that can cause mucosal degradation.

Besides its effects on IL-8 synthesis, IL-17 exerts additional immune-regulatory functions which could influence the magnitude and/or severity of H pylori-related gastritis. For example, IL-17 stimulates the production of IL-1, IL-6, and TNF-α by both immune and non-immune cells[17], and induces fibroblasts to make matrix metalloproteinases (MMPs)[18]. MMPs are a family of proteases that can cleave multiple components of the extracellular matrix, thereby contributing to the mucosal damage[19] (Figure 1).

IL-23 CONTROLS IL-17 PRODUCTION IN THE HUMAN GASTRIC MUCOSA

IL-17 was originally named cytotoxic T lymphocyte-associated-8 (CTLA-8), subsequently IL-17, and more recently IL-17A, since it is one of six related members belonging to the IL-17 family (IL-17A-F)[20]. IL-17 was initially described at the message level as a product of human blood activated CD4+ memory T cells. Subsequent studies have shown that IL-17 can be also made by activated CD8+ T cells, TCRγδ+ T cells, and neutrophils[20]. More recently, it was shown that IL-17 is produced by a specific subset of CD4+ T cells, termed T helper (Th) 17-cells, that are distinct from, and antagonized by the classical Th1 or Th2 cells[21]. Th17-cells produce also, but to a lesser extent, TNF-α, IL-6, IL-17F, IL-22, and granulocyte macrophage-colony stimulating factor[22,23]. Flow-cytometry analysis of IL-17 production in gastric LPMC isolated from biopsies of H pylori-infected patients showed that CD4+ T cells are a major source of IL-17, even though CD8+ T cells and CD3-negative cells were also positive for IL-17 (Figure 1)[13].The molecular pathways governing the development of Th17-cells in humans have not been yet elucidated, but studies in murine systems indicate that Th-17 cell differentiation is driven by IL-6 and TGF-β1[24,25]. There is also evidence that expansion and survival of Th17-cells require additional factors, such as IL-23[25]. IL-23 is a heterodimeric protein that is composed by the p40 subunit of IL-12 and a specific subunit, termed IL-23/p19. The functional IL-23 heterodimer is produced by activated dendritic cells (DC), monocytes and macrophages[26]. We have recently shown that IL-23 protein is produced in excess in H pylori-colonized mucosa. RNA transcripts for both p40 and p19 subunits were also up-regulated in biopsies from H pylori-infected patients, indicating that IL-23 is regulated at the transcriptional level in this condition[13]. These results confirm and expand on data of previous studies showing that H pylori enhances IL-23 secretion by monocyte-derived DC[27], and that H pylori neutrophil-activating protein

(H pylori-NAP), a member of a broad super-family of ferritin-like proteins, induces IL-23 production by neutrophils and monocytes[28,29]. Functional studies also revealed that IL-23 enhances IL-17 synthesis by normal gastric LPMC, and that blockade of endogenous IL-23 activity in cultures of LPMC isolated from H pylori-infected biopsies down-regulates IL-17 production[13]. The exact molecular mechanism by which IL-23 regulates IL-17 in H pylori-infected mucosa remains to be ascertained. Notably, neutralization of endogenous IL-23 by a blocking anti-IL-23/p19 antibody in cultures of LPMC isolated from H pylori-infected biopsies attenuates the expression of active Stat3. Moreover, in normal gastric LPMC, exogenous IL-23 enhances the activation of Stat3, and pharmacologic inhibition of Stat3 suppresses IL-17 production induced by IL-23[13]. Taken together, these results suggest that Stat3 plays a key role in the IL-23-driven IL-17 production during H pylori infection. This well fits with the demonstration that Stat3 is essential for the induction and expansion of IL-17-producing cells in response to cytokine stimulation both in vitro and in vivo[30]. Such an effect could rely on the ability of Stat3 to bind the promoter of IL-17 gene and enhance its transcriptional activity[31], and/or favor the induction of RORγt, a master regulator of Th17-cell differentiation[32], and the expression of IL-23R.

IL-17 synthesis may be regulated by additional cytokines other than IL-23. IL-1R1-deficient mice fail to mount a robust Th17 response, and IL-1R1-deficient cells do not produce IL-17 in response to IL-23[33]. Since H pylori infection enhances the production of IL-1 at the gastric level[34], it is tempting to speculate that this cytokine may act in concert with IL-23 in enhancing IL-17. IL-17 synthesis is also increased by IL-15 in cultures of human and murine CD4+ T cells[35]. However, the fact that IL-15 expression is down-regulated in H pylori-infected biopsies argues against a role for IL-15 in the control of IL-17 production during H pylori-related gastritis[36]. Th17 cell differentiation is also enhanced by IL-21[37,38], a T-cell derived cytokine that is produced in excess in H pylori-colonized stomach[39].

THE ROLE OF IL-23 IN AMPLIFYING H pylori-DRIVEN TH1-IMMUNE RESPONSE

During H pylori infection, there is a pronounced specific acquired immune response, characterized by generation of antibodies, and differentiation and activation of effector T cells. Although this later includes both a Th1 and a Th2 component, mucosal cytokine profiles imply Th1 predominance[40], and the number of cells producing interferon (IFN)-γ, the key Th1 cytokine, in the H pylori-infected human gastric mucosa correlates with the severity of gastritis[41]. Animal models also suggest that the extent of Th1 differentiation is important in pathogenesis. Mice with a predominant Th1 response develop more gastric inflammation during H pylori colonization than those with a Th2 response[42-43]. Gastric inflammation and atrophic changes are abrogated in the absence of IFN-γ[44], while IFN-γ infusion into mice, even in the absence of H pylori infection, induces pre-cancerous gastric atrophy, metaplasia and dysplasia[45]. IL-12-deficient mice have also reduced gastric inflammatory infiltration and are unable to clear H pylori infection[46].

Several virulence factors are reported to promote Th1 responses, including the plasticity region locus jhp0947-jhp0949 which is associated with duodenal ulcer disease[47] and the H pylori-NAP[29]. The Th1/Th2 balance is also influenced by phase-variable expression of Lewis blood-group antigens and genomic DNA recombination[48,49].

IL-12 is supposed to be one of the major Th1-inducing factors in H pylori-colonized gastric mucosa[50], even though IL-23 may contribute to expand the ongoing Th1 cell response[26]. Indeed, blockade of endogenous IL-23 by anti-IL23/p19 in cultures of LPMC isolated from H pylori-colonized biopsies reduces IFN-γ secretion, and stimulation of normal gastric LPMC with IL-23 enhances IFN-γ production. These data are in accordance with the demonstration that IL-23 activates Stat4 and enhances IFN-γ production in cultures of human and murine memory T cells[26], and that in two models of H. hepaticus-triggered T cell-dependent colitis, IL-23 enhances both IFN-γ and IL-17 responses that together synergize to trigger severe intestinal inflammation[51,52].

IL-23 and gastric cancer

As pointed out above, H pylori is a major factor in the induction of gastritis and its progression to pre-neoplastic lesions and non-cardia gastric cancer. Despite the high prevalence of H pylori infection, the risk of gastric cancer in H pylori-infected patients is, however, estimated to be approximately 1%-3%. This indicates that infection per se is not sufficient to induce the progression to gastric neoplasia and that additional bacterial and host factors are required[2]. A detailed description of such factors is beyond the scope of this review. In this context it is, however, noteworthy that accumulating evidence would seem to suggest that IL-12 and IL-23 are important mediators in the process that links H pylori infection to gastric cancer. Indeed, polymorphisms of IL-12p40 and p35 genes enhance the risk of non-cardia gastric cancers in H pylori-infected patients[53]. Moreover, high levels of IL-23 have been documented in human gastric cancers[53]. Nonetheless, no functional study has so far mechanistically linked the activity of IL-12 and IL-23 to gastric cancer. Additionally, studies in murine models of epithelial cancers have shown that IL-23, but not IL-12, is essential for sustaining the tumor-promoting inflammatory process and counteracting the ability of cytotoxic CD8+ T cells to infiltrate tumors[54].

CONCLUSIONS

Although bacterial virulence factors are important in conditioning the outcome of the H pylori-driven infection, it is the host attempt to clear the bacterium that causes an exaggerated and inappropriately counter-regulated immune response that may eventually cause tissue damage. Emerging experimental evidence suggests that IL-23/IL-17 pathway is an important driving force the ongoing gastric inflammation in H pylori-infected patients. However, further studies will be required to establish the exact contribution of each of these cytokines in the H pylori-associated gastric pathology. The availability of strains of mice deficient either for IL-23 subunits or IL-17 should provide valuable models to specifically address these issues.