# The activity of miltefosine combined with oral probiotics in a mouse model of cutaneous leishmaniasis

## TL;DR

This study shows that combining a probiotic blend with a lower dose of miltefosine improves treatment outcomes in a mouse model of cutaneous leishmaniasis.

## Contribution

The novel finding is that a multistrain probiotic enhances the efficacy of miltefosine in treating leishmaniasis.

## Key findings

- PB8 probiotic combined with miltefosine reduced lesion size by 74% compared to vehicle-treated mice.
- The combination reduced parasite load by 76% and 87% via imprinting and qPCR, respectively.
- PB8 cotreatment lowered serum CCL2 levels, suggesting reduced inflammation and disease exacerbation.

## Abstract

Leishmaniasis, caused by the protozoan Leishmania, is a neglected tropical disease (NTD) with diverse clinical forms, the most common being cutaneous leishmaniasis (CL). Antileishmanial treatments rely on a small arsenal of chemoherapeutic agents, which are outdated, toxic, and increasingly ineffective due to drug resistance. New antileishmanial treatments and/or adjunctive therapies are warranted.

Given the role of microbiota in modulating host immunity, we explored whether probiotics (PB8-multistrain probiotic blend, or Lactobacillus rhamnosus GG, LGG-single strain) alone or in combination with the reference drug miltefosine (ML) could improve clinical outcomes against Leishmania amazonensis infection in a BALB/c mouse model for CL.

Mice were administered probiotics [gavage, 109 colony-forming units (CFU)] for a 7-day pre-treatment before infection, followed by another 14-day probiotic treatment with ML co-administration. Paw lesions were measured using a digital calliper, and parasite loads were determined through lesion imprinting and quantitative polymerase chain reaction (qPCR). The potential immunoregulatory effects of probiotic administration on the mouse serum cytokine profiles were investigated via flow cytometry.

Probiotics alone reduced lesion size slightly, with PB8 achieving a 32% and LGG a 10% reduction at the endpoint (47-50 days post-infection, dpi). The combination of PB8 with a suboptimal ML dose (4 mg/kg/day) reduced the lesion size by 74% compared to the vehicle-treated mice, while ML alone achieved 53%. These findings were corroborated by amastigote quantification via imprinting (light microscopy) and qPCR: PB8 plus ML reduced parasite load by 76% and 87%, respectively. Multiplex cytokine analysis [interferon (IFN)-γ, interleukin (IL)-6, IL-10, IL-12p70, tumour necrosis factor (TNF) and chemokine CCL2] showed reduced serum CCL2 in PB8-cotreated groups. This suggests that PB8 could modulate serum cytokine levels to mitigate the risk of excessive inflammation, as elevated CCL2 is linked to disease exacerbation through monocyte recruitment. Our findings demonstrate the potential effect of probiotic administration to enhance antileishmanial efficacy of antiparasitic drugs.