Ex vivo host and parasite response to antileishmanial drugs and immunomodulators

Abstract

Background: Therapeutic response in infectious disease involves host as well as microbial determinants. Because the immune and inflammatory response to Leishmania (Viannia) species defines the outcome of infection and efficacy of treatment, immunomodulation is considered a promising therapeutic strategy. However, since Leishmania infection and antileishmanial drugs can themselves modulate drug transport, metabolism and/or immune responses, immunotherapeutic approaches require integrated assessment of host and parasite responses.

Methodology: To achieve an integrated assessment of current and innovative therapeutic strategies, we determined host and parasite responses to miltefosine and meglumine antimoniate alone and in combination with pentoxifylline or CpG 2006 in peripheral blood mononuclear cells (PBMCs) of cutaneous leishmaniasis patients. Parasite survival and secretion of TNF-α, IFN-γ, IL-10 and IL-13 were evaluated concomitantly in PBMCs infected with Luc-L. (V.) panamensis exposed to meglumine antimoniate (4, 8, 16, 32 and 64 μg SbV/mL) or miltefosine (2, 4, 8, 16 and 32 μM HePC). Concentrations of 4 μM of miltefosine and 8 μg SbV/mL were selected for evaluation in combination with immunomodulators based on the high but partial reduction of parasite burden by these antileishmanial concentrations without affecting cytokine secretion of infected PBMCs. Intracellular parasite survival was determined by luminometry and cytokine secretion measured by ELISA and multiplex assays.

Principal findings: Anti- and pro-inflammatory cytokines characteristic of L. (V.) panamensis infection were evaluable concomitantly with viability of Leishmania within monocyte-derived macrophages present in PBMC cultures. Both antileishmanial drugs reduced the parasite load of macrophages; miltefosine also suppressed IL-10 and IL-13 secretion in a dose dependent manner. Pentoxifylline did not affect parasite survival or alter antileishmanial effects of miltefosine or meglumine antimoniate. However, pentoxifylline diminished secretion of TNF-α, IFN-γ and IL-13, cytokines associated with the outcome of infection by species of the Viannia subgenus. Exposure to CpG diminished the leishmanicidal effect of meglumine antimoniate, but not miltefosine, and significantly reduced secretion of IL-10, alone and in combination with either antileishmanial drug. IL-13 increased in response to CpG plus miltefosine.

Conclusions and significance: Human PBMCs allow integrated ex vivo assessment of antileishmanial treatments, providing information on host and parasite determinants of therapeutic response that may be used to tailor therapeutic strategies to optimize clinical resolution.

Fig 1. Kinetics of parasite and immunologic responses in the absence of drugs.

(A) Kinetics of infection evaluating the parasites: monocyte ratios of 10:1 and 20:1 (left panel), and the parasite burden (right panel) in PBMCs vs macrophages alone (infection ratio 10:1) are shown. Parasite survival is expressed as bioluminescence produced by luciferase activity in relative light units (RLU). Mean signal of uninfected cells: 135.3 ± 23.7 RLU. (B) Kinetics of cytokine secretion (TNF-α, IL-10, IL-13, IFN-γ) over the 72 hours of treatment. Data are based on at least 4 patients and expressed as means ± SEM.

Fig 2. Concentration-dependent effect of miltefosine and meglumine antimoniate on parasite survival and cytokine secretion.

(A) Parasite survival and (B) Cytokine secretion after 96 hours of exposure to increasing concentrations of miltefosine (HePC) and meglumine antimoniate (Sb^V). TNFα, IL-10, IFNγ and IL-13 were evaluated in supernatants of PBMCs infected with L. (V) panamensis. Data are based on at least 6 patients and presented as mean ± SEM of the parasite burden or cytokine secretion compared to infected control without drug. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Fig 3. Kinetics of parasite survival and cytokine secretion in response to 4 μM miltefosine and 8 μg Sb^V/mL as meglumine antimoniate in the ex vivo PBMC model.

(A) Parasite survival and (B to E) cytokine secretion (IL-13, TNF-α, IL-10, IFN-γ) over 96 h. Mean values ± SEM for PBMCs from 5 patients. ** p ≤ 0.01, control vs both drugs.

Fig 4. Effect of anti-leishmanial drugs, immunomodulators and their combinations on parasite burden.

(A, C and E) Dose response for pentoxifylline alone, combined with 8 μg Sb^V/ml meglumine antimoniate or 4 μM miltefosine. (B, D and F) Dose response for CpG alone, combined with 8 μg Sb^V/ml meglumine antimoniate or 4 μM miltefosine. Data are presented as means ± SEM of the parasite burden compared to infected control cultures without drugs for PBMCs from 10 patients. * p< 0.05.

Fig 5. Dose-response of cytokine secretion by PBMCs to pentoxifylline and anti-leishmanial-pentoxifylline combinations.

Cells from 10 patients were infected at a parasite: monocyte ratio of 10:1. Cytokine levels (IL-10, IL-13, TNF-α, IFN-γ) were determined after exposure for 96 h to different concentrations of pentoxifylline (PTX), (A) alone, combined with (B) 8 μg Sb^V/ml meglumine antimoniate or (C) 4 μM miltefosine. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.0001, **** p ≤ 0.0001.

Fig 6. Dose-response of cytokine secretion by PBMCs to CpG, and anti-leishmanial- CpG combinations.

Cells from 10 patients were infected at a parasite: monocyte ratio of 10:1. Cytokine levels (IL-10, IL-13, TNF-α, IFN-γ) were determined after exposure for 96 h to different concentrations of CpG (A) alone, combined with (B) 8 μg Sb^V/ml meglumine antimoniate or (C) 4 μM miltefosine. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01.