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Helicobacter pylori, asthma and allergy

Mario Milco D'Elios, Gaia Codolo, Amedeo Amedei, Paola Mazzi, Giorgio Berton, Giuseppe Zanotti, Gianfranco Del Prete, Marina De Bernard
DOI: http://dx.doi.org/10.1111/j.1574-695X.2009.00537.x 1-8 First published online: 1 June 2009


Bronchial asthma and allergic diseases are orchestrated by T-cells producing T-helper type 2 (Th2) cytokines, such as interleukin-4 (IL-4) and IL-5, and are inhibited by Th1 responses. Helicobacter pylori has chronically infected the human population for c. 100 000 years and preferentially elicits a Th1 mucosal immune response with the production of interferon-γ and IL-12. Among several bacterial factors, the neutrophil-activating protein of H. pylori (HP-NAP) not only plays a key role in driving Th1 inflammation but it is also able to inhibit Th2 responses in vitro and in vivo in allergic bronchial asthma, in humans and mice. Both systemic and mucosal administrations of HP-NAP are successful in reducing eosinophilia, immunoglobulin E and systemic Th2 cytokines at the bronchial level. Thus, these results identify HP-NAP as a candidate for novel strategies for the prevention and treatment of allergic diseases.

  • Helicobacter pylori
  • asthma
  • allergy
  • cytokines
  • chemokines
  • immune modulation


Helicobacter pylori is a Gram-negative bacterium that chronically infects the stomach of >50% of the human population and represents the major cause of gastroduodenal pathologies (Warren & Marshall, 1983; Parsonnet et al., 1991; Wotherspoon et al., 1991; Blaser, 1993). Helicobacter pylori gastric colonization is typically followed by mucosa infiltration of polymorphonuclear leukocytes, macrophages and T-helper type 1 (Th1) lymphocytes, with active production of interleukin-12 (IL-12) and interferon-γ (IFN-γ) (D'Elios et al., 1997).

An inverse association between the H. pylori infection and the frequency of allergic asthma has been reported recently (Blaser et al., 2008); however, the absence of a convincing molecular mechanism, besides the epidemiological data, in support of such an observation, represents a limitation of this study and has raised several criticisms (Wjst, 2008).

Bronchial asthma and allergic diseases are characterized by Th2 inflammation, which is strongly inhibited by IL-12 and IFN-γ. Recently we demonstrated that in allergic asthmatic patients, the typical Th2 responses can be redirected toward Th1 by the neutrophil-activating protein of H. pylori (HP-NAP) (Amedei et al., 2006). Furthermore, the in vivo administration of HP-NAP prevents the typical eosinophil accumulation in the lung and the increase of serum immunoglobulin E (IgE) in a mouse model of allergic asthma (Codolo et al., 2008b). These results provide the possibility that HP-NAP might be a part of the molecular mechanism underlying the negative association between H. pylori infection and allergy, corroborating the epidemiological observations with a plausible scientific explanation. This intriguing issue will be discussed in the present review, which focuses on the immunopathological basis of bronchial asthma, allergy and H. pylori infection. Finally, the potential use of HP-NAP as a new tool for the prevention and treatment of asthma and allergy will also be considered.

Th1/Th2 responses in health and diseases

During the course of evolution, the immune system had to continuously shape and refine its mechanisms of defense against pathogens. In response to different microorganisms, specialized types of specific responses allow the recognition and elimination of infectious agents. Viruses, which grow within the infected cell, can be successfully eliminated only by killing their host cells by CD8+ class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes, which recognize viral antigens synthesized within infected cells and present on their surface in the context of class I MHC molecules. In contrast, most microbial antigens are endocytosed by antigen-presenting cells (APC), processed and presented preferentially in association with MHC class II molecules to CD4+ class II MHC-restricted Th cells. CD4+ T cells aid B cells in the production of antibodies, which challenge extracellular microorganisms or neutralize their exotoxins (humoral immunity). Some microorganisms such as mycobacteria, however, can survive within macrophages in spite of the microbicidal activity of these cells unless CD4+ Th cells reactive to mycobacterial antigens activate macrophage production of reactive oxygen intermediates, nitric oxide and tumor necrosis factor (TNF)-α, leading to the microorganism's destruction (cell-mediated immunity). Most immune responses against pathogens involve both arms of the immune system (humoral and cell-mediated immunity) acting in concert.

During the effector-specific immune response, different patterns of cytokine profiles are characteristic of certain Th-cell subsets, whose polarized forms are Th1 and Th2 cells (Mosmann et al., 1986; Del Prete et al., 1991). Th1 cells producing IFN-γ and TNF-β elicit macrophage activation and B-cell production of opsonizing and complement-fixing antibodies, whereas Th2 cells producing IL-4, IL-5, IL-10 and IL-13 induce the production of high levels of antibodies, including IgE, and eosinophilia. The two types of effector responses are usually distinct and mutually exclusive. Between polarized Th1 and Th2 cells, a heterogenous population of T-cell subsets simultaneously producing a peculiar combination of Th1/Th2 cytokines (collectively defined as Th0 cells) participate in the immune response (D'Elios & Del Prete, 1998). A new subset of Th cells, named Th17 cells, producing IL-17 alone or in combination with IFN-γ, has been identified recently (Weaver et al., 2006). Th17 cells play a critical role in protection against microbial challenges, particularly extracellular bacteria and fungi (Bettelli et al., 2007). Further, some of the autoimmune responses formerly attributed to Th1 cells, such as experimental autoimmune encephalomyelitis, collagen-induced arthritis, Lyme arthritis and some forms of inflammatory bowel disease, have now been shown to be mediated, at least in part, by Th17 cells (Codolo et al., 2008a; Romagnani, 2008).

The factors responsible for the Th-cell polarization into a predominant Th1 or Th2 profile have been extensively investigated. Considerable evidence suggests that Th1 and Th2 cells develop from the same Th cell precursor under the influence of mechanisms associated with antigen presentation (Kamogawa et al., 1993). Both environmental and genetic factors influence the Th1 or Th2 differentiation by determining the ‘leader cytokine’ in the microenvironment of the responding Th-cell. IL-4 is the most powerful stimulus for Th2 differentiation, whereas IL-12 and IFNs favor Th1 development (Swain et al., 1990; Trinchieri, 2003). A role has been shown for the site of antigen presentation, the physical form of the immunogen, the type of adjuvant and the dose of antigen (Constant & Bottomly, 1997). Potent adjuvants and microbial products induce Th1-dominated responses because they stimulate IL-12 production. IFN-γ and IFN-α favor Th1 development by enhancing IL-12 secretion by macrophages and by maintaining the expression of functional IL-12 receptors on Th cells (Szabo et al., 1995).

Th1- and Th2-dominated responses not only provide different strategies of protection against pathogens but also play a role under some pathological conditions (Table 1). Studies in humans and gene-targeted mice have clearly shown that Th1-dominated responses are effective in protection against several microorganisms and usually drive their clearance. However, if the pathogen persists, a chronically ongoing Th1 response may lead to inflammatory tissue damage. Extracellular infectious agents would be more efficiently counteracted by a combination of Th2 and Th1 cytokines, as in Th0 cell activation. Th2-dominated responses are usually observed during infections by intestinal nematodes. Because IL-4, IL-10 and IL-13 inhibit the development of Th1 cells and macrophage activation, Th2 responses prevent the extensive inflammatory tissue injury that would eventually result from Th1- and macrophage-mediated responses to such complex parasites. A shift toward a Th2-dominant response may also occur in some immune responses against microorganisms, when the Th1 response fails to clear the infection rapidly (D'Elios & Del Prete, 1999).

View this table:
Table 1

Human pathological conditions associated to Th1 and Th2 predominant responses

H. pylori infectionAllergic diseases
AtherosclerosisVernal conjunctivitis
Organ-specific autoimmunityParasitic infections
Acute allograft rejectionSystemic sclerosis
Crohn's diseaseSome hypereosinophilic syndromes
Some recurrent abortionsChronic graft vs. host disease
SarcoidosisSome patients with AIDS

Bronchial asthma and Th2-driven inflammation

Asthma, defined by the World Health Organization as a ‘chronic inflammatory disease of the airways,’ is a complex disorder characterized by airway hyper-responsiveness to a variety of specific and nonspecific stimuli, and mucus hypersecretion by goblet cells. The increased use of bronchoscopy, along with bronchoalveolar lavage (BAL) and bronchial biopsies, provided important tools for research on asthma. The histopathological characteristics of bronchial asthma, even a mild one, is represented by epithelial shedding, basal membrane thickening, inflammatory infiltrates consisting of T lymphocytes and accumulation of activated eosinophils.

Immunological and molecular studies of bronchial biopsies and BAL samples obtained in baseline disease or taken after natural or ‘experimentally’ induced asthma exacerbations have shown the in vivo relevance of T cells, inflammatory cells and the related cytokine network in the pathogenesis of different variants of bronchial asthma: allergic, occupational or nonallergic. Asthma is driven and maintained by bronchial persistence of a subset of chronically activated memory T cells, previously sensitized against allergenic, occupational, or viral antigens. Limiting dilution T-cell cloning techniques and in situ hybridization studies showed that activated T cells and related cytokines could be identified in biopsies derived from all major variants of asthma.

In allergic asthmatic patients, allergen exposure induces a predominant activation of CD4+ Th2 lymphocytes in the airways, able to overexpress several Th2 cytokines, including IL-4 and IL-5 (Robinson et al., 1992; Del Prete et al., 1993). Moreover, the degree of IL-5 expression at the bronchial level is associated with disease severity both in atopic and in nonatopic asthma (Kon & Kay, 1999). IL-5 and granulocyte macrophage colony-stimulating factor (GM-CSF) can be considered the most important cytokines for eosinophil accumulation in asthmatic inflammation. Th2 cytokines in bronchial asthma are produced not only by CD4+ but also by CD8+ T cells, which contribute to the genesis of asthma and to the clinical expression of the disease (Betts & Kemeny, 2009).

Helicobacter pylori-driven Th1 response and HP-NAP

In H. pylori infection, a predominant activation of Th1 cells, with the production of IFN-γ, IL-12, IL-18, IL-23 and TNF-α, occurs in vivo in the stomach in human and animal models (Mohammadi et al., 1996; D'Elios et al., 1997; Luzza et al., 2000; Mattapallil et al., 2000; Rossi et al., 2000; Tomita et al., 2001; Vivas et al., 2008). Complex and fascinating mechanisms are responsible for the mucosal Th1 polarization (D'Elios et al., 1997, 2005; Del Giudice et al., 2001). Stimulation of neutrophils, monocytes and dendritic cells with HP-NAP resulted in prompt and remarkable upregulation of IL-12 and IL-23 mRNA expression and protein secretion, via Toll-like receptor 2 (TLR2) activation. In the gastric mucosa of H. pylori-infected patients, a considerable proportion of Th cells show significant proliferation to different H. pylori antigens, including HP-NAP, CagA, urease, VacA and heat shock proteins. HP-NAP drives the production of high levels of IFN-γ and TNF-α by antigen-specific gastric Th cells and induces a powerful cytotoxic activity, thus promoting a polarized Th1 response (Amedei et al., 2006).

We recently investigated the immune-modulating activities of HP-NAP in relation to the structure of the protein. HP-NAP structurally belongs to the Dps (DNA protecting protein under starved conditions) family (Grant et al., 1998). It consists of 12 identical subunits arranged in a dodecameric shell with 32 symmetry (Fig. 1a) (Zanotti et al., 2002). The resulting complex, which is about 90 Å in diameter, surrounds a large central cavity, whose function is to host Fe atoms in the form of mixed iron oxides (Tonello et al., 1999). In addition, 12 Fe ions are structurally bound in the inner part of the shell, contributing to its structural stability and possibly representing the sites for iron oxidation. Because each subunit in the dodecamer interacts with the neighboring subunit considerably, this quaternary arrangement is very stable and resistant to denaturation. For this reason, it can be assumed that the immunogenic properties of this protein, which are in general not shared by other members of the Dps family, depend on the characteristics of the protein surface. Because the surface of the dodecamer is characterized by the presence of a large number of positively charged residues (Fig. 1b), a property that is peculiar to HP-NAP within the Dps family, it has been hypothesized (Zanotti et al., 2002) that this basic trait could be responsible for neutrophil activation, in a manner similar to other chemokines (Laurence et al., 2001). Moreover, it has been demonstrated recently that neutrophil activation is stimulated by structural elements that are localized within the C-terminal region of the HP-NAP subunit (Kottakis et al., 2007).

Figure 1

HP-NAP. (a) Ribbon representation of the HP-NAP dodecamer. Each subunit is represented with a different color. Red spheres show the positions of the 12 structural Fe ions. (b) Surface of the dodecamer of HP-NAP. Positively and negatively charged residues on the surface are colored: Arg and Lys, blue; His, cyan; Glu and Asp, red. Overall, the surface presents a prevalence of positively charged residues; the pore in the center of the picture, which is the postulated entrance for the Fe ions, is characterized by negative charges (Glu1145, Asp126 and 127).

The crystal structure of the extracellular domain of TLR2 (the receptor engaged by HP-NAP on the surface of cells with innate immunity) in association with TLR1 and a lipopeptide, and also that of TLR4 in complex with MD-2 (Kim et al., 2007), has been resolved recently (Jin et al., 2007). The extracellular domain of TLRs shares a common structural feature, represented by a repetition of 16–28 leucine-rich repeat modules (Matsushima et al., 2007). The binding of ligands to these extracellular domains triggers a rearrangement of the receptor, which in turn induces the recruitment of specific adaptor proteins to the intracellular domains (O'Neill & Bowie, 2007). The surface of both TLR2 and TLR4 is heavily charged and the complex of TRL4 with MD-2 is stabilized by electrostatic and hydrogen bond interactions (Kim et al., 2007). Because the surface of HP-NAP is also heavily charged, the TLR4-MD2 complex may possibly represent a prototype of the complex of TLR2 with HP-NAP.

Helicobacter pylori and bronchial asthma: is HP-NAP the molecular explanation for the inverse association?

The severity and incidence of asthma have increased drastically in the developed nations over the last decades. Although the underlying reason is still unknown, epidemiological studies and experimental data provided evidence suggesting that infectious diseases can influence the development of allergic disorders (Strachan, 1989). An inverse correlation has been demonstrated between the onset of allergic disorders and the incidence of infections (Herz et al., 2000). This phenomenon can be explained by the inhibition of allergic Th2 inflammation by Th1 responses elicited by infectious agents, able to induce the production of IFN-γ, IL-12, IL-18 and IL-23 (Herz et al., 2000; Wohlleben & Erb, 2001). This view is supported by studies showing that development of asthma can be prevented in animals by administration of alive or killed bacteria or their components, which induce Th1 responses (Wohlleben & Erb, 2006).

Interestingly, on the basis of large epidemiological studies, recently, a consistent negative association between H. pylori infection and the presence of allergic disorders, such as asthma and rhinitis, has been proposed (Chen & Blaser, 2007, 2008; Blaser et al., 2008). Although it is undoubtedly an interesting theory, no convincing molecular mechanism has been proposed to support it, and this was the principal reason for the criticism raised.

Our recent studies, carried out with the HP-NAP, may help understand this complex issue. We have shown that addition of a culture of HP-NAP to allergen-induced T-cell lines derived from allergic asthmatic patients led to a drastic increase in IFN-γ-producing T cells and a decrease in IL-4-secreting cells, thus resulting in a redirection of the immune response from a Th2 to a Th1 phenotype (Amedei et al., 2006). These results suggest that HP-NAP might be the key element responsible for the decrement of allergy frequency in H. pylori-infected patients.

HP-NAP as a candidate for the prevention and treatment of bronchial asthma

To address whether HP-NAP, on the basis of its immune-modulating activity, could be beneficial for the prevention and treatment of bronchial asthma, it was administered via the intraperitoneal or the intranasal route using a mouse model of allergic asthma induced by inhaled ovalbumin (OVA). For this, groups of nine C57BL/6j, wild type or tlr2−/−, mice were treated with saline, or with OVA alone, or with OVA plus intraperitoneal HP-NAP or with OVA plus mucosal HP-NAP. In both systemic and mucosal protocols, mice were treated with OVA according to a standardized procedure consisting of a first phase of sensitization with OVA intraperitoneally and a second phase of induction of the allergic response with aerosolized OVA on day 8 and finally exposed to aerosolized antigen on days 15–18 (Gonzalo et al., 1996). Control animals were injected with phosphate-buffered saline (PBS) alone and then exposed to aerosolized PBS. In the systemic protocol, mice were treated with intraperitoneal HP-NAP on day 1, whereas in the mucosal protocol mice received intranasal HP-NAP on days 7 and 8 (Codolo et al., 2008a, b).

After priming and a repeated aerosol challenge with OVA, Th2 responses were induced in the mouse lung. Moreover, following OVA treatment, eosinophils were recruited and activated in bronchial airways, and serum IgE levels increased, and the elicited Th2 response correlated with the appearance of airway hyper-responsiveness. Both systemic and mucosal administration of HP-NAP strongly inhibit the development of airway eosinophilia and bronchial inflammation (Fig. 2). Likewise, HP-NAP treatment strongly affected the lung cytokine release, reducing the production of IL-4, IL-5 and GM-CSF (Fig. 3). Systemic HP-NAP also significantly resulted in both the reduction of total serum IgE and an increase of IL-12 plasma levels. However, no suppression of lung eosinophilia and bronchial Th2 cytokines was observed in TLR-2 knock-out mice following HP-NAP treatment (Codolo et al., 2008a, b) (Fig. 4).

Figure 2

Intraperitoneal and intranasal administration of HP-NAP inhibited the development of airway eosinophilia in OVA-sensitized animals. On day 1, mice were sensitized with OVA alone, or with OVA plus systemic HP-NAP (Sy HP-NAP) or with intranasal aerosolized HP-NAP (Mu HP-NAP) on days 7 and 8, and then exposed to aerosolized OVA, followed by a repeated challenge from days 15 to 18. Control animals were injected with saline alone and then exposed to aerosolized PBS. On day 18, cytocentrifuge preparations from BAL of the different groups of animals were stained to calculate the proportions of eosinophils. Absolute counts of eosinophils (±SD) were calculated from the number of total cells in the BAL. ***P<0.01 vs. mice treated with OVA alone. OVA, ovalbumin.

Figure 3

Intraperitoneal and intranasal administration of HP-NAP resulted in reduced Th2 accumulation in the airway lumen. BAL samples were collected from control, OVA, OVA plus intraperitoneal HP-NAP (OVA+Sy HP-NAP) and OVA plus intranasal. HP-NAP (OVA+Mu HP-NAP)-treated animals on day 18, and cytokines were assayed in the cell-free supernatants with a Bio-Plex cytokine assay. Mean values (±SD) are reported. Cytokines were undetectable (<1 pg mL−1) in samples from control mice. *P<0.05 vs. OVA alone treated mice. OVA, ovalbumin.

Figure 4

Schematic representation of HP-NAP activities in asthma and allergic diseases. Following mucosal or systemic administration, HP-NAP, via production of IL-12 and IL-23, inhibits allergic inflammation and redirects Th2 toward Th1 responses.

It is worth noting that in another mouse model of Th2-mediated disease, such as Trichinella spiralis infection, HP-NAP was also able to enhance endogenous IL-12 and IFN-γ response in vivo and to exert a powerful anti-Th2 activity, targeting both the IL-5-induced eosinophilia and the IL-4-mediated hyper-IgE responses induced by parasitic infection (Del Prete et al., 2008).

Concluding remarks

Asthma is one of the most common chronic diseases in industrialized countries and consists of airway inflammation, bronchial hyper-responsiveness and airway obstruction. Typical pathological features include infiltration of the airways by activated lymphocytes, particularly Th2 cells and eosinophils. The reason why the severity and incidence of asthma has dramatically increased in the developed nations over the last decades is unknown; however, epidemiological studies and experimental data provided evidence suggesting that infectious diseases, such as H. pylori infection, can influence the development of allergic disorders. This phenomenon can be explained by the inhibition of the allergic Th2 inflammation by Th1 responses elicited by infectious agents, able to induce the production of IFN-γ, IL-12, IL-18 and IL-23. HP-NAP, by acting on both neutrophils and monocytes via TLR2 agonistic interaction, significantly contributes toward inducing an IL-12- and IL-23-enriched milieu, and represents a key bacterial factor able to drive the differentiation of antigen-stimulated T cells toward a polarized Th1 phenotype. HP-NAP has the potential to redirect the in vitro allergen-specific T-cell response from a predominant Th2 to a Th1 response. Furthermore, HP-NAP administration in vivo resulted in inhibition of the typical Th2-mediated bronchial inflammation of allergic bronchial asthma. Thus, altogether, these results support the view that the increased prevalence and severity of asthma and allergy in western countries may be related, at least in part, to the decline of H. pylori infection, which is able to induce a long-lasting Th1 background, and suggest HP-NAP as an important candidate for novel strategies of the prevention and treatment of asthma and allergic diseases.


M.M.D'E., M.de B., A.A., and G.D.P. are among the applicants of EU Patent 05425666.4 for HP-NAP as a potential therapeutic agent in cancer, allergic and infectious diseases. The remaining authors have no conflict of interest.


We thank Ente Cassa di Risparmio di Firenze, Istituto Superiore di Sanità, the Ministry of Health, the Ministry of University and Scientific Research, the University of Florence, the University of Padua (Progetto di Ateneo) and the Associazione Italiana per la Ricerca sul Cancro (AIRC) for providing financial support for our studies.


  • Editor: Leif Andersen


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