Helicobacter pylori Neutrophil Activating Protein (HP-NAP) Enhances the Anti-Leishmanial Activity of Canine Macrophages Against Leishmania infantum

Abstract

Leishmania infantum is the etiological agent of visceral leishmaniasis (VL) and is linked to cases of cutaneous leishmaniasis in dogs. Dogs often develop severe systemic disease and serve as the primary reservoir of L. infantum. Although several vaccine candidates are under development, no vaccine for visceral leishmaniasis has been approved for human use to date. Chemotherapeutic treatment is hampered by toxicity, cost, and the emergence of parasite-resistant strains. Immunotherapy, combining chemotherapy with modulation of Th1 responses, is a promising therapeutic approach. Helicobacter pylori neutrophil-activating protein (HP-NAP), an immunomodulatory protein from Helicobacter pylori, is known to promote Th1 immune responses. A Th1 response activates macrophage promoting parasite killing, while a Th2 response favors disease progression. Macrophages are central for infection, either eliminating parasites (Th1 response) or supporting their persistence (Th2 response). IL-12 is a crucial cytokine in driving Th1 immunity and counteracting Th2 responses. We therefore investigated the role of HP-NAP in an in vitro model of L. infantum macrophage infection. Canine monocyte-derived macrophages from seven dogs were incubated with L. infantum promastigotes. More than 85% of macrophages from all donors were infected, with approximately seven amastigotes per cell. HP-NAP treatment significantly reduced all infection parameters and induced IL-12 production. Collectively, these findings suggest that HP-NAP may represent a promising candidate for adjuvant immunotherapies and vaccine development against L. infantum.

1. Introduction

Leishmaniasis is a vector-borne, zoonotic parasitic disease of major veterinary and public health importance [1]. It is caused by protozoa of the genus Leishmania, transmitted to vertebrate hosts by phlebotomine sandflies. Human leishmaniasis is classified as a Neglected Tropical Disease (NTD), primarily present in tropical and subtropical regions, but also endemic in some parts of Europe, particularly within the Mediterranean basin. Leishmania infantum is the most prevalent species in this area, responsible for both canine and human diseases. Human leishmaniasis manifests in three distinct clinical forms: cutaneous, mucocutaneous and visceral leishmaniasis (VL). VL, caused by L. infantum and L. donovani, is the most severe form with high mortality in symptomatic cases if left untreated [2].

Dogs infected with L. infantum exhibit a wide spectrum of clinical manifestations, ranging from asymptomatic carriers, still capable of transmitting the parasites to the insect vector [3], to severe visceral disease, characterized by a high parasite burden in multiple organs. They represent the primary peridomestic reservoir of L. infantum and play a central role in the zoonotic transmission cycle of the parasite to humans [4].

Leishmania spp. exists as the extracellular promastigote stage in the insect vector, whereas in the vertebrate host, it transforms into an intracellular amastigote form, primarily within macrophages, which represent both the main host cells where the parasite survives and proliferates and the effector cells capable of mediating parasite killing [5]. Resistance to infection is typically associated with a strong Th1-type cell-mediated immune response, characterized by activation of M1 macrophages and production of inflammatory cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and Interleukin 12 (IL-12). The pro-inflammatory response of M1 macrophages is characterized by the production of reactive oxygen species (ROS) and nitric oxide (NO), which are toxic to Leishmania parasites. In contrast, disease progression is associated with a predominant Th2-type exaggerated response and activation of M2 macrophages, marked by elevated levels of antibodies and cytokines such as TGF-β, but limited parasite control [6]. The balance between protective Th1-driven and non-protective Th2-oriented immune responses determines disease outcome, influencing whether infected dogs remain subclinically infected or develop clinical signs of canine leishmaniasis (CanL) [7].

Despite extensive research and the demonstration of effective cellular immunity, no effective vaccine currently exists for either human or canine leishmaniasis. Multiple vaccine candidates and adjuvants have been tested for CanL, but to date, only two vaccines have been registered and are commercially available. However, current evidence indicates that their protective efficacy remains weak [8,9]. Also, the range of adjuvants currently available remains limited.

Treatment of both human and CanL remains challenging due to the limited number of available drugs, many of which are toxic, costly and increasingly compromised by parasite resistance. None of the available drugs alone are fully effective; therefore, the aim of the treatment is both to reduce the parasite load and concomitantly to enhance Leishmania-specific cell-mediated immunity [10]. Recent guidelines recommend that all dogs diagnosed with CanL should be treated with anti-leishmanial drugs and with immunostimulants or with a combination of both [11,12].

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a virulence factor of H. pylori initially identified for its ability to enhance neutrophil adhesion to the endothelium and to induce the release of reactive oxygen species (ROS) by neutrophils, hence its designation as a neutrophil activating protein [13]. Subsequent work revealed that HP-NAP acts as a TLR2 agonist on neutrophils and monocytes, inducing the production of IL-12 and IL-23, thereby creating a cytokine milieu that supports Th1 polarization of antigen-specific T cells [14]. In in vivo models of allergy, cancer, and helminth infections (such as Trichinella spiralis), HP-NAP has been shown to redirect immune responses from a Th2-biased profile toward a Th1 phenotype, a shift that is critical for controlling disease progression and shaping protective immunity [15,16,17,18,19]. Despite this clear immunomodulatory potential, its application in other Th1-dependent contexts, such as leishmaniasis, remains largely unexplored. In our current work, we demonstrate that HP-NAP reduces the infection index of L. infantum-infected canine macrophages and stimulates the production of IL-12, supporting its application as an adjuvant immunotherapy agent in canine leishmaniasis.

2. Materials and Methods

2.1. Reagents

Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), HEPES and L-glutamine were obtained from EuroClone (Pero, Italy). Schneider Drosophila Medium, Giemsa stain, Griess reagents (sulphanilamide, napthtylenthylenediamine dihydrochloride, phosphoric acid [H₃PO₄]), SDS and N,N-dimethylformamide were purchased from Sigma-Aldrich (Milan, Italy). Canine IL-12 and IL-10 ELISA kits were obtained from R&D Systems (Minneapolis, MN, USA).

HP-NAP was cloned, expressed, and purified from Bacillus subtilis to avoid lipopolysaccharide (LPS) contamination, as described previously [20].

2.2. Cultivation of L. infantum

The promastigote stage of L. infantum (MHOM/TN/80/IPT1) (WHO international reference strain, kindly provided by Dr. M. Gramiccia and Dr. T. Di Muccio, ISS, Roma, Italy) was cultured at 24 °C in Schneider’s Drosophila Medium supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine and 20 mM HEPES (pH 7.4). Stationary-phase parasite cultures were obtained after incubation at 24 °C for 5 days. Cultures were routinely screened for Mycoplasma spp. contamination by PCR.

2.3. HP-NAP Toxicity on L. infantum Promastigotes

The complete medium used for the anti-promastigotes activity assay was RPMI-1640 (EuroClone) supplemented with 10% heat-inactivated fetal bovine serum (EuroClone), 20 mM HEPES pH 7.4, and 2 mM L-glutamine. The parasite suspension (5 × 10⁶ parasites/mL) was dispensed into 96-well round-bottom microplates, and HP-NAP (10–20 µg/mL), diluted in complete medium, or complete medium alone, were added to the wells in triplicate. After 72 h of incubation at 23 °C, 20 μL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution (5 mg/mL in PBS) was added to each well. After 3 h in the dark, the supernatants were discarded and the formazan crystals dissolved in 100 μL of lysing buffer (20% w/v of a solution of SDS 40% of N,N-dimethylformamide in H₂O). The plates were then read on a Synergy 4 (Biotek^®, Agilent Technologies Italia, S.p.a, Cernusco sul Naviglio, Milan, Italy) microplate reader at a test wavelength of 550 nm and at a reference wavelength of 650 nm. Promastigotes were also counted by light microscopy. See Supplementary Figure S1 for the effect of HP-NAP on promastigote viability.

2.4. Isolation of Monocyte-Derived Canine Macrophages

Blood collected from healthy blood donor dogs (N = 7) of different breeds, middle age, and both sexes, was used (Table S1). The blood was collected during routine blood sampling for annual control. According to the University of Milan animal use regulations, formal ethical approval was not needed as dogs were sampled with the informed consent of the owners during routine visits for prophylactic reasons, and the owners gave their consent for the use of excess blood after routine testing in further studies (EC decision 29 October 2012, renewed with the protocol n 02-2016). Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood samples through density gradient centrifugation. Briefly, the whole blood was diluted in RPMI-1640 at a 1:1 ratio, overlaid on Ficoll-Hypaque (Sigma-Aldrich, Italy) at a 2:1 ratio of Ficoll:blood and centrifuged at 543× g for 30 min at RT. The PBMC ring was collected and washed with RPMI-1640. Isolated PBMC were plated at a density of 2 × 10⁵ cells in Nunc- Lab-Tek chamber slides (Sigma-Aldrich, Milan, Italy) and allowed to adhere for 2 h at 37 °C in an atmosphere of 5% CO₂. Cells were then washed twice with phosphate-buffered saline (PBS) to discard non-adherent cells and then treated with 0.1 µM of PMA (Sigma-Aldrich, Milan, Italy) for 72 h to achieve differentiation into macrophages, as described in the literature for dog monocytes [21].

2.5. Infection of Canine Macrophages

After differentiation, cells were washed with PBS and infected with stationary-phase L. infantum promastigotes at a macrophage: promastigote ratio of 1:10. After 3 h, cell monolayers were washed with PBS to remove non-internalized promastigotes and incubated in the presence of HP-NAP (10 or 20 µg/mL) for 72 h. “Uninfected” refers to macrophages cultured in medium alone, without infection. “Control” refers to infected macrophages cultured without HP-NAP treatment. All HP-NAP-treated macrophages were first infected with L. infantum before incubation with the protein. At the end of the incubation, the supernatants were collected and stored at −20 °C. Slides were fixed with 100% methanol and stained with Giemsa. Uninfected and infected cells were counted in random fields using an inverted light microscope (Nikon Europe B.V.1181 VX Amstelveen, The Netherlands). The number of parasites/cell was determined by examining three different wells for each treatment group. The results are expressed as percent of infected macrophages and mean number of amastigotes/cell, and the infectivity index was calculated according to the following Equation [22,23]:

Infectivity Index = (% Infected macrophages) × (mean number of amastigotes/cell)

2.6. Determination of Inflammatory Mediators

The levels of IL-12 or IL-10 in the supernatants of stimulated canine macrophages were determined using commercial ELISA kits following the manufacturing instructions.

2.7. Griess Assay for Nitric Oxide Determination

The presence of nitrites in the supernatants was evaluated using the Griess assay, as previously described [24]. The Griess reagents consisted of a mixture of equal parts of Reagent A (1% [w/v] sulphanilamide) and Reagent B (0.1% [w/v] naphthylethylenediamine dihydrochloride, and 2.5% [w/v] phosphoric acid). Briefly, 100 μL of each supernatant was mixed with an equal volume of Griess reagent mixture and, after 10 min of incubation at room temperature, nitrite levels were measured spectrophotometrically (Synergy 4 microplate reader, Biotek, GE) at 540 nm. A standard curve of NaNO₂ was prepared.

2.8. Statistical Analysis

All data were obtained from at least three independent experiments, and the results are shown as mean ± standard deviations. Differences among three or more groups were analyzed for statistical significance by using ordinary one-way ANOVA test followed by post hoc Dunnett’s multiple comparison test. Differences between two groups were analyzed by unpaired t-test. Statistical significance was set at p < 0.05. Statistical analysis was performed using GraphPad Prism 8.0.2 Software (San Diego, CA, USA).

3. Results

3.1. In Vitro Activity of HP-NAP Against Intracellular Amastigotes in Canine Macrophages

Canine-monocyte derived macrophages, isolated from seven different dog donors, were used for this study. Macrophages were infected with L. infantum promastigotes for 3 h and subsequently treated with HP-NAP for 72 h. At the end of the treatment the percentage of infected macrophages, the mean number of amastigotes per infected macrophage and the infectivity index of each sample were calculated.

Canine macrophages exhibited strong phagocytic activity, with a mean of 85 ± 11% (range: 66–98%) of infected macrophages. The infection burden was also high, with more than 80% of macrophages containing more than five intracellular parasites per cell. A reduction in the number of infected macrophages was observed after treatment with 10 or 20 µg/mL of HP-NAP. Representative images of Giemsa-stained infected macrophages treated or not with HP-NAP (20 µg/mL) are shown in Figure 1, panels A and B. Figure 1C shows the % of infected macrophages in control and in HP-NAP-treated cells. Treatment with HP-NAP resulted in a reduction in infection, with inhibition effects of 9% (range 15.1–4.6%) and 21% (range: 19.7–6.4%) at 10 and 20 µg/mL of HP-NAP, respectively. A drop in the number of intracellular amastigotes per macrophage was also observed after treatment with HP-NAP (Figure 1C). The mean number of amastigotes per cell in the controls was 7.2 (range 9.7–5.9), whereas it decreased to 5.45 (range 5.3–5.6) and 4.7 (range 3.8–5.6) following treatment with HP-NAP at 10 and 20 µg/mL, respectively, with a significant reduction observed at the 20 µg/mL concentration (p = 0.011) (Figure 1D). Consequently, the infection index (see Section 2.5 for details) also decreased following treatment with HP-NAP, with a significant reduction at 20 µg/mL concentration (p = 0.005) (Figure 1E). The percentage of inhibition was 42.6 (range 33.2–50.7%). The direct effect of HP-NAP was also evaluated against the promastigote stage. A slight and not significant decrease in promastigote viability was observed after treatment with HP-NAP at 20 µg/mL (Figure S1, Supplementary Materials).

3.2. In Vitro Production of IL-12 by L. infantum-Infected Canine Macrophages

IL-12 was measured in the cell supernatants of control and HP-NAP- treated cells. Macrophages from two out of seven dogs did not produce IL-12 in any of the tested conditions. Constitutive production of IL-12 varied considerably among different donors, with levels ranging from 0.1 to 14.02 pg/mL. Following L. infantum infection, mean IL-12 levels approximately doubled compared to uninfected cells, although they remained low and the difference was not statistically significant. However, the production of IL-12 was significantly enhanced in the presence of 20 μg/mL HP-NAP (p = 0.013) (Figure 2A). The mean fold increase was 8, ranging between 2.5 and 30 among the five different donors.

Macrophages from three dog donors were also stimulated with two different doses of HP-NAP, and a dose-dependent induction of IL-12 was observed, although the differences did not reach statistical significance (Figure 2B)

Nitric oxide production was also evaluated. Only macrophages from two out of seven donors stimulated with L. infantum produced detectable amounts of nitric oxide (4.67 and 4.92 µM), but only a negligible increase in NO was measured after treatment with HP-NAP 20 μg/mL (5.26 and 5.85 µM) (Supplementary Figure S2). IL-10 levels in all the experiments and samples were below the detection limits of the assay.

4. Discussion

Macrophages are key cells in Leishmania infection, as they are capable of either eliminating the parasite or supporting its survival. They also produce cytokines that shape the adaptive immune response: a Th1-polarized profile, characterized by high levels of IL-12, IFN-γ, and TNF-α, promotes parasite clearance, whereas a Th2-skewed response, with elevated IL-10 and IL-4, is detrimental [25]. Therefore, immunomodulators that drive Th1 polarization have the potential to enhance and accelerate parasite elimination and are actively sought. In this preliminary study, we used canine monocyte-derived macrophages infected with L. infantum to investigate the effects of HP-NAP, a potent immune modulator, on parasite survival and cytokine production.

The data show that canine macrophages phagocytose Leishmania promastigotes with very high efficiency. The percentage of infected macrophages was 85%, with an average of 7.2 parasites per macrophage. This high infection rate has already been reported in previous studies [26]. Macrophages of human, murine, and canine origin indeed show distinct susceptibility to L. infantum infection [26], likely due to species-specific receptor repertoires and cell polarization-mediated expression patterns [27]. Furthermore, since infection was assessed 72 h post-infection, the high proportion of infected macrophages may reflect the inability of canine macrophages to control parasite replication over time, thereby allowing substantial parasite proliferation. Treatment with HP-NAP at a concentration of 20 µg/mL, but not at 10 µg/mL, significantly inhibited parasite replication, resulting in a decreased infectivity index.

The immunomodulatory activity of HP-NAP in the same range of doses has already been reported in previous studies [17]. However, to our knowledge, this is the first study evaluating the activity of HP-NAP against L. infantum amastigotes. Two key mediators for the control of Leishmania infection are represented by reactive oxygen species (ROS) and nitric oxide (NO), leading to the elimination of intracellular parasites. ROS are released during the macrophage oxidative burst, while NO is produced through the induction of nitric oxide synthase (iNOS). It has previously been demonstrated that HP-NAP can induce ROS production by neutrophils and monocytes [13,28]. Since our experiments showed no difference in the induction of NO by HP-NAP, it is plausible that the reduced macrophage infection values observed after HP-NAP treatment were due to ROS induction by macrophages. ROS were not directly measured in this work, but further studies are planned to investigate the mechanisms of intracellular parasite reduction. We tend to exclude the direct effects of HP-NAP on Leishmania parasites, since the results shown in Supplementary Figure S1 indicate that HP-NAP does not significantly affect promastigote viability under cell-free conditions. This further supports the idea that the antileishmanial activity observed is mediated by macrophage activation by HP-NAP.

HP-NAP is a TLR2 ligand [29], and a protective role for TLR2 agonists in L. infantum infections has been reported [30]. Indeed, TLR2 signaling in macrophages leads to the production of several pro-inflammatory cytokines and other molecules, including, for example, IL-12, TNF-α, and iNOS. Increased expression of TLR2 has been observed on canine monocytes and macrophages from dogs naturally infected with Leishmania, suggesting its involvement in the immune response to the parasites [31]. Our data are consistent with the above observations, as treatment of L. infantum-infected macrophages with HP-NAP induced a significant, dose-dependent increase in IL-12 production. Induction of IL-12 plays an important role in promoting protective Th1 immune response; however, Leishmania is known to inhibit macrophage IL-12 production as a survival strategy [32,33]. Therefore, modulation of IL-12 production by HP-NAP may represent a potential approach to enhance the host immune responses and to counterbalance the immunosuppression induced by the parasite.

It must be acknowledged that a limitation of this study is represented by the small number of dog donors that have been investigated. This may be responsible for the high dog-to-dog variation seen for cytokine production. Some biological variability was expected since the dogs were not matched for age, sex or breeding; however, the fact that the parasite infectivity and the effect of HP-NAP were similar in all the dogs examined, points toward details in the assay conditions (doses and kinetic of the stimulant, for example) that need to be investigated further and will be addressed in the near future with a larger set of animals.

These results envisage a possible use of HP-NAP as an immunostimulatory tool to be included in candidate vaccine preparation or combined with anti-parasitic drugs.