Resistance of Leishmania (Viannia) Panamensis to Meglumine Antimoniate or Miltefosine Modulates Neutrophil Effector Functions

Abstract

Leishmania (Viannia) panamensis (L. (V.) p.) is the main causative agent of cutaneous leishmaniasis in Colombia and is usually treated with either meglumine antimoniate (MA) or miltefosine (MIL). In recent years, there has been increasing evidence of the emergence of drug-resistance against these compounds. Neutrophils are known to play an important role in immunity against Leishmania. These cells are rapidly recruited upon infection and are also present in chronic lesions. However, their involvement in the outcome of infection with drug-resistant Leishmania has not been examined. In this study, human and murine neutrophils were infected in vitro with MA or MIL drug-resistant L. (V.) p. lines derived from a parental L. (V.) p. drug-susceptible strain. Neutrophil effector functions were assessed analyzing the production of reactive oxygen species (ROS), the formation of neutrophil extracellular trap (NET) and the expression of cell surface activation markers. Parasite killing by neutrophils was assessed using L. (V.) p. transfected with a luciferase reporter. We show here that MA and MIL-resistant L. (V.) p. lines elicited significantly increased NET formation and MA-resistant L. (V.) p. induced significantly increased ROS production in both murine and human neutrophils, compared to infections with the parental MIL and MA susceptible strain. Furthermore, neutrophils exposed to drug-resistant lines showed increased activation, as revealed by decreased expression of CD62L and increased expression of CD66b in human neutrophils yet presented higher survival within neutrophils than the drug-susceptible strain. These results provide evidence that parasite drug-susceptibility may influences neutrophil activation and function as well as parasite survival within neutrophils. Further investigaton of the inter-relationship of drug susceptibility and neutrophil effector function should contribute to better understanding of the factors involved in susceptibility to anti-Leishmania drugs.

TLR7 Sensing by Neutrophils Is Critical for the Control of Cutaneous Leishmaniasis

“…Neutrophil purity was assessed using CytoSpin and Quick-Fix staining and was established to be > 95%. Human neutrophils were isolated as previously described (Regli et al, 2018), from venous blood of healthy volunteers by Polymorphoprep gradient. The purity was > than 95%.…”

“…Neutrophils were seeded on Poly-L-Lysine coated coverslips and exposed for 4 hours with either L. major PMA (Phorbol-12-myristate-13-acetate, Sigma), PMA + DNase (Sigma) or without stimulus. Subsequently, cells were fixed with 4% PFA (Paraformaldehyde, Sigma) and stained with rabbit anti-human MPO (Dako) primary antibody and Alexa Fluor-488 goat anti-rabbit secondary antibody (Life Technologies) as previously described (Regli et al, 2018). Briefly, coverslips were mounted on glass slides using a DAPI containing mounting medium (Molecular Probes) and analyzed by confocal microscopy (ZEISS LSM 510).…”

Profiles of Local and Systemic Inflammation in the Outcome of Treatment of Human Cutaneous Leishmaniasis Caused byLeishmania(Viannia)

“…The involvement of neutrophils in the therapeutic outcome of CL remains unknown. However, recent findings suggest that antimony-as well as miltefosine-resistant L. (V.) panamensis elicits significantly higher neutrophil activation, reflected by increased neutrophil extracellular trap (NET) formation, CD66b expression, and reactive oxygen species (ROS) production, yet promotes enhanced parasite survival within neutrophils compared to infections with the drug-susceptible counterpart (44). These results suggest that neutrophils could contribute to sustained inflammation during drug-resistant Leishmania infections, potentially favoring treatment failure.…”

Leishmania Spp-Host Interaction: There Is Always an Onset, but Is There an End?

“…The interaction between NETs and macrophages favor the survival and persistence of parasites since it stimulates the M2 profile of macrophages, which are susceptible to L. amazonensis infection but unable to kill the parasite (Guimarães-Costa et al, 2017). Recently, L. panamensis resistant strains to miltefosine and meglumine antimoniate were described (Regli et al, 2018). These strains were able to modulate neutrophils stimulating the release of NETs and ROS production and survived better within neutrophils than the drug-susceptible strains (Regli et al, 2018).…”

“…Recently, L. panamensis resistant strains to miltefosine and meglumine antimoniate were described (Regli et al, 2018). These strains were able to modulate neutrophils stimulating the release of NETs and ROS production and survived better within neutrophils than the drug-susceptible strains (Regli et al, 2018). Altogether, these published data suggest that the induction of NET formation may lead to escape from extracellular effector immune mechanisms, facilitating the access of a permissive cell to parasite infection and persistence.…”