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This study aimed to investigate the role of neutrophils in canine leishmaniosis by assessing neutrophil activation and its relationship with different states of L. infantum infection and antibody and IFN-γ production. Dogs were categorized into five groups: healthy-seronegative (n = 25), healthy-seropositive (n = 21), LeishVet-stage I (n = 25), Leishvet-stage II (n = 41), and LeishVet-stage III–IV (n = 16). Results of the nitroblue tetrazolium reduction test (NBT) showed significantly higher neutrophil activation in stage I (median:17.17, range: [7.33–31.50]%) compared to in healthy-seronegative (4.10 [1.20–18.00]%), healthy-seropositive (7.65 [3.98–21.74]%), stage II (6.50 [1.50–28.70]%), and stage III–IV (7.50 [3.00–16.75]%) groups (p < 0.0001). Healthy-seropositive dogs also displayed higher values than all groups except stage I. Stages II and III–IV did not show significant differences compared to healthy-seronegative. Regarding IFN-γ, stage I dogs had higher concentrations (median:127.90, range: [0–3998.00] pg/mL) than healthy-seronegative (0 [0–109.50] pg/mL) (p = 0.0002), stage II (9.00 [0–5086.00] pg/mL) (p = 0.045), and stage III–IV (3.50 [80.00–548.80] pg/mL) (p = 0.02) dogs. Stage II dogs showed increased IFN-γ compared to healthy-seronegative dogs (p = 0.015), while stage III–IV dogs had no significant differences compared to healthy-seronegative dogs (p = 0.12). Healthy-seropositive dogs had elevated IFN-γ concentrations compared to healthy-seronegative dogs (p = 0.001) and dogs in stage III–IV (p = 0.03). In conclusion, neutrophil activation was higher in dogs with mild disease and healthy-seropositive dogs, and a relationship between neutrophil activation and the production of IFN-γ was found.

Dogs are the main reservoir for Leishmania infantum, manifesting from a subclinical to a fatal disease. Limited treatments are available, although new antiparasitics and immunomodulators are pursued. Polyhexamethylene biguanide (PHMB) has a broad antimicrobial spectrum, including antiparasitic activity. Here, we evaluated the potential for Toll-like receptor agonists (TLRa) and PHMB alone, and as polyplex nanoparticles containing PHMB and TLR4 or TLR9 agonists, to selectively kill L. infantum. Susceptibility of L. infantum promastigotes to PHMB, miltefosine, and allopurinol was performed, and the half-maximum inhibitory concentrations (IC50) were determined. Then, DH-82 cells were infected and treated with PHMB alone or combined with TLR4a (MPLA-SM) or TLR9a (CpG ODNs) and allopurinol alone. The IC50 values of L. infantum promastigotes were PHMB (1.495 µM), miltefosine (9.455 µM), and allopurinol (0.124 µM). After infection, treated DH-82 cells displayed a lower percentage (p = 0.0316), intensity (p = 0.0002), and index of infection (p = 0.0022) when compared to non-treated cells. PHMB induced lower percentage of infection alone (p = 0.043), in combination with TLR9a (p = 0.043), and with TLR4a (p = 0.0213). Supernatants were collected and used to measure TNF-α and IL-6 levels. Increased TNF-α was observed after PHMB plus TLR4a, relative to uninfected and infected untreated macrophages (p = 0.043). PHMB combined with TLR4a shows promise as a potential anti-L. infantum drug combination, as well as inducer of proinflammatory response, as demonstrated by decreased infection and increased TNF-α production.