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.

Keywords: Helicobacter pylori-neutrophil activating protein (HP-NAP); Leishmania infantum; canine macrophages; cytokines; leishmaniasis.

Figure 1

L. infantum infection of primary canine macrophages. Upon isolation from peripheral blood and differentiation, canine monocyte-derived macrophages were infected with L. infantum promastigotes. “Control” refers to infected macrophages cultured without HP-NAP treatment. After 3 h, cell monolayers were washed with PBS and incubated in complete medium alone or in the presence of HP-NAP (10 µg/mL in N = 4 donors or 20 µg/mL in N = 5 donors) for 72 h. Representative optical images of Giemsa-stained canine macrophages of Control (A) and HP-NAP 20 µg/mL treated macrophages (B) using a 100× immersion oil objective. Scale bar: 25 µm. The percent of infected macrophages and the number of amastigotes/cell were quantified through Giemsa staining. (C) Percent of infected macrophages. (D) Mean number of intracellular amastigotes within infected macrophages. (E) Infectivity index calculated as % Infected macrophages × number of amastigotes/cell. Data are presented as mean ± SD. Statistical analysis: Ordinary one-way ANOVA followed by Dunnett’s post hoc test comparing each condition to Control; *, p < 0.05; **, p < 0.01.

Figure 2

IL-12 production in culture supernatants of primary canine macrophages infected with L. infantum and treated with HP-NAP. (A) IL-12 levels measured in supernatants of macrophages from N = 5 dogs under the following conditions: uninfected macrophages, infected untreated macrophages (control), and infected macrophages treated with HP-NAP 20 µg/mL. (B) IL-12 levels measured in supernatants of macrophages from N = 3 dogs under the following conditions: uninfected macrophages, infected untreated macrophages (control), and infected macrophages treated with HP-NAP 10 or 20 µg/mL. Data are presented as mean ± SD. Statistical analysis: Ordinary one-way ANOVA followed by Dunnett’s post hoc test comparing each condition to uninfected macrophages; *, p < 0.05.