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Evaluation of the Efficacy of the <em>H. pylori</em> Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

University of Padua

The Helicobacter pylori neutrophil-activating protein (HP-NAP) is a virulence factor of H. pylori that stimulates in neutrophils high production of oxygen radicals and adhesion to endothelial cells. We report here that HP-NAP is a TLR2 agonist able to induce the expression of IL-12 and IL-23 by neutrophils and monocytes. Addition in culture of HP-NAP, as an immune modulator, to antigen-induced T cell lines resulted in a remarkable increase in the number of IFN-gamma-producing T cells and decrease of IL-4-secreting cells, thus shifting the cytokine profile of antigen-activated human T cells from Th2 to a Th1 cytotoxic phenotype. We also found that in vivo HP-NAP elicited an antigen-specific Th1-polarized T cell response in the gastric mucosa of H. pylori-infected patients. These data indicate HP-NAP as an important factor of H. pylori able to elicit cells of the innate immune system to produce IL-12 and IL-23, and they suggest it as a new tool for promoting Th1 immune responses.

The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses.

The Helicobacter pylori neutrophil-activating protein (HP-NAP) is able in vitro to elicit IL-12 and IL-23 production via agonistic interaction with toll-like receptor 2, and to promote Th1 polarization of allergen-specific T-cell responses. This study was aimed to assess whether systemic/intraperitoneal and/or mucosal HP-NAP administration inhibited the Th2-mediated bronchial inflammation using a mouse model of allergic asthma induced by inhaled ovalbumin (OVA). Systemic HP-NAP delivery markedly reduced the lung eosinophilia in response to repeated challenge with aerosolized OVA. Likewise, the production of IL-4, IL-5 and GM-CSF was significantly lower in the bronchoalveolar lavage of animals treated with systemic HP-NAP plus OVA than that of animals treated with OVA alone. Systemic HP-NAP also significantly resulted in both reduction of total serum IgE and increase of IL-12 plasma levels. Mucosal administration of HP-NAP was equally successful as the systemic delivery in reducing eosinophilia, IgE and Th2 cytokine levels in bronchoalveolar lavage. However, no suppression of lung eosinophilia and bronchial Th2 cytokines was observed in toll-like receptor 2-knock-out mice following HP-NAP treatment. These results identify HP-NAP as a candidate for novel strategies of prevention and treatment of allergic diseases.

The neutrophil-activating protein of Helicobacter pylori down-modulates Th2 inflammation in ovalbumin-induced allergic asthma.

The Helicobacter pylori neutrophil-activating protein (HP-NAP) is able to induce IL-12 expression by cells of innate immunity and to shift to T(H)1 human allergen-specific T(H)2 cells in vitro.

Immunosuppression of TH2 responses in Trichinella spiralis infection by Helicobacter pylori neutrophil-activating protein.

Human Lyme arthritis caused by Borrelia burgdorferi is characterized by an inflammatory infiltrate that consists mainly of neutrophils and T cells. This study was undertaken to evaluate the role of the innate and acquired immune responses elicited by the neutrophil-activating protein A (NapA) of B burgdorferi in patients with Lyme arthritis.

Borrelia burgdorferi NapA-driven Th17 cell inflammation in lyme arthritis.

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-gamma 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 and allergy.

Reply to letter by Nardelli and Schell commenting on the pathogenesis of Lyme arthritis.

NapA from Borrelia burgdorferi is a member of the Dps-like protein family with specific immunomodulatory properties; in particular, NapA is able to induce the expression of IL-23 in neutrophils and monocytes, as well as the expression of IL-6, IL-1β, and transforming growth factor beta (TGF-β) in monocytes, via Toll-like receptor (TLR) 2. Such an activity on innate immune cells triggers a synovial fluid Th17 response. Here we report the crystal structure of NapA, determined at 2.6Å resolution, which shows that the quaternary structure of the protein is that of a dodecamer with 23 symmetry, typical of the proteins of the family. We also demonstrate that the N- and C-terminal tails, which are flexible and not visible in the crystal, are not relevant for its pro-Th17 activity. Based on the crystal structure and on the comparison with the structure of the orthologous protein from Helicobacter pylori, HP-NAP, we hypothesize that the charge distributions on the two proteins' surfaces are responsible for the interaction with TLR2 and for the different behaviors in modulating the immune response.

Structure and immunomodulatory property relationship in NapA of Borrelia burgdorferi.

Current evidence indicates an inverse association between Helicobacter pylori and asthma and allergy. H. pylori is a Gram-negative bacterium which represents the major cause of peptic ulcer and gastric cancer, and preferentially elicits a T helper (Th)-1 response. Many H. pylori factors, such as the neutrophil-activating factor of H. pylori (HP-NAP), are able to drive Th-1 polarization and to display a powerful inhibition of allergic Th-2 response. This article proposes an overview of the actual knowledge about the effects of H. pylori on asthma and allergy. Special attention has been drawn to HP-NAP as a potential novel strategy for the prevention and treatment of asthma and atopy.

The effect of Helicobacter pylori on asthma and allergy.

Intravesical Bacillus Calmette-Guérin (BCG) is the gold standard treatment for intermediate and high-risk non-muscle-invasive bladder cancer. BCG therapy is the most successful example of immunotherapy in cancer. Unfortunately, the treatment-related side effects are still relevant. Furthermore, non-responder patients are candidate to radical cystectomy in the absence of valuable alternative options. These aspects have prompted the search for newer biological response modifiers (BRM) with a better benefit/side effects ratio. The toll-like receptor (TLR) 2 ligand, Helicobacter pylori protein HP-NAP, has been shown to deserve a potential role as BRM. HP-NAP is capable of driving the differentiation of T helper (Th) 1 cells, both in vitro and in vivo, because of its ability to create an IL-12-enriched milieu. Herein, we report that local administration of HP-NAP decreases tumour growth by triggering tumour necrosis in a mouse model of bladder cancer implant. The effect is accompanied by a significant accumulation of both CD4+ and CD8+ IFN-γ-secreting cells, within tumour and regional lymph nodes. Noteworthy, HP-NAP-treated tumours show also a reduced vascularization due to the anti-angiogenic activity of IFN-γ induced by HP-NAP. Our findings strongly indicate that HP-NAP might become a novel therapeutic "bullet" for the cure of bladder tumours.

HP-NAP inhibits the growth of bladder cancer in mice by activating a cytotoxic Th1 response.

Patients with non-muscle invasive bladder cancer recurrence after 2 induction courses of BCG are eligible for radical cystectomy. So, in the last years research to discover new drugs for the management of non-muscle invasive bladder cancer recurrence after failure of first and second line therapy is ongoing. In accordance to the results obtained with BCG, whose mechanism depends on the induction of the T helper 1 (TH1) immune response, we investigated the activity of a Toll-like receptor (TLR) 2 ligand, named Helicobacter Pylori Neutrophil Activating Protein (HP-NAP), that we recently demonstrated being able of enhancing the differentiation of Th1 cells, both in vitro and in vivo, because of its ability to create an IL-12 enriched milieu.

[Therapy for non-muscle invasive bladder cancer: HP-NAP].

Lyme arthritis (LA) is characterized by infiltration of inflammatory cells, mainly neutrophils (polymorphonuclear cells [PMNs]) and T cells, into the joints. This study was undertaken to evaluate the role of the neutrophil-activating protein A (NapA) of Borrelia burgdorferi in eliciting inflammation and in driving the adaptive immune response.

Orchestration of inflammation and adaptive immunity in Borrelia burgdorferi-induced arthritis by neutrophil-activating protein A.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. It is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. Another feature is represented by the formation in these cells of inclusions called Lewy bodies (LB), principally constituted by fibrillar α-synuclein (αSyn). This protein is considered a key element in the aetiology of a group of neurodegenerative disorders termed synucleinopathies, which include PD, but the cellular and molecular mechanisms involved are not completely clear. It is established that the inflammatory process plays a crucial role in the pathogenesis and/or progression of PD; moreover, it is known that aggregated αSyn, released by neurons, activates microglia cells to produce pro-inflammatory mediators, such as IL-1β. IL-1β is one of the strongest pro-inflammatory cytokines; it is produced as an inactive mediator, and its maturation and activation requires inflammasome activation. In particular, the NLRP3 inflammasome is activated by a wide variety of stimuli, among which are crystallized and particulate material. In this work, we investigated the possibility that IL-1β production, induced by fibrillar αSyn, is involved the inflammasome activation. We demonstrated the competence of monomeric and fibrillar αSyn to induce synthesis of IL-1β, through TLR2 interaction; we found that the secretion of the mature cytokine was a peculiarity of the fibrillated protein. Moreover, we observed that the secretion of IL-1β involves NLRP3 inflammasome activation. The latter relies on the phagocytosis of fibrillar αSyn, followed by increased ROS production and cathepsin B release into the cytosol. Taken together, our data support the notion that fibrillar αSyn, likely released by neuronal degeneration, acts as an endogenous trigger inducing a strong inflammatory response in PD.

Triggering of inflammasome by aggregated α-synuclein, an inflammatory response in synucleinopathies.

Neisseria meningitidis is a human pathogen that can cause fatal sepsis and meningitis once it reaches the blood stream and the nervous system. Here we demonstrate that a fragment, released upon proteolysis of the surface-exposed protein Neisserial Heparin Binding Antigen (NHBA), by the bacterial protease NalP, alters the endothelial permeability by inducing the internalization of the adherens junction protein VE-cadherin. We found that C2 rapidly accumulates in mitochondria where it induces the production of reactive oxygen species: the latter are required for the phosphorylation of the junctional protein and for its internalization that, in turn, is responsible for the endothelial leakage. Our data support the notion that the NHBA-derived fragment C2 might contribute to the extensive vascular leakage typically associated with meningococcal sepsis.

The C2 fragment from Neisseria meningitidis antigen NHBA increases endothelial permeability by destabilizing adherens junctions.

BAFF is a crucial cytokine that affects the activity of both innate and adaptive immune cells. It promotes the expansion of Th17 cells in autoimmune disorders. With this study, we investigated the BAFF/Th17 responses in Helicobacter pylori-induced gastritis in humans. Our results show that the mucosa from Helicobacter(+) patients with chronic gastritis is enriched in IL-17 and BAFF, whereas the two cytokines are weakly expressed in Helicobacter(-) patients with chronic gastritis; moreover, the expression of both BAFF and IL-17 decreases after bacteria eradication. We demonstrate that BAFF accumulates in macrophages in vivo and that it is produced by monocyte-derived macrophages in vitro, after Helicobacter stimulation. Application of BAFF on monocytes triggers the accumulation of reactive oxygen species that are crucial for the release of pro-Th17 cytokines, such as IL-23, IL-1β, and TGF-β. Moreover, BAFF directly promotes the differentiation of Th17 cells. In conclusion, our results support the notion that an axis BAFF/Th17 exists in chronic gastritis of Helicobacter(+) patients and that its presence strictly depends on the bacterium. Moreover, we demonstrated that BAFF is able to drive Th17 responses both indirectly, by creating a pro-Th17 cytokine milieu through the involvement of innate immune cells, and directly, via the differentiation of T cells toward the specific profile. The results obtained in this study are of great interest for Helicobacter-related diseases and the development of novel therapeutic strategies based on the inhibition of the BAFF/IL-17 response.

Gaia Codolo

Department of Biology

University of Padua

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model