Department of Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon.
Centre Hospitalo-Universitaire, Université de Montpellier, Montpellier, France.
PLoS Negl Trop Dis. 2018 Nov 21;12(11):e0006854. doi: 10.1371/journal.pntd.0006854. eCollection 2018 Nov.
Cutaneous Leishmaniasis (CL) is a parasitic infection classified by the WHO as one of the most uncontrolled spreading neglected diseases. Syria is endemic for Leishmania tropica and Leishmania major, causing CL in the Eastern Mediterranean. The large-scale displacement of Syrian refugees exacerbated the spread of CL into neighboring countries. Therapeutic interventions against CL include local, systemic and physical treatments. The high risk for drug-resistance to current treatments stresses the need for new therapies. Imiquimod is an immunomodulatory drug with a tested efficacy against L. major species. Yet, Imiquimod efficacy against L. tropica and the molecular mechanisms dictating its potency are still underexplored. In this study, we characterized the effect of Imiquimod against L. tropica and L. major, and characterized the molecular mechanisms dictating its anti-leishmanial efficacy against both strains. We also investigated the potency and molecular mechanisms of an Imiquimod analog, EAPB0503, against these two strains. We have tested the effect of Imiquimod and EAPB0503 on macrophages infected with either L. major, L. tropica strains, or patient-derived freshly isolated L. tropica parasites. The anti-amastigote activity of either drugs was assessed by quantitative real time PCR (RT-PCR) using kinetoplast specific primers, confocal microscopy using the Glycoprotein 63 (Gp63) Leishmania amastigote antibody or by histology staining. The mechanism of action of either drugs on the canonical nuclear factor kappa- B (NF-κB) pathway was determined by western blot, and confocal microscopy. The immune production of cytokines upon treatment of infected macrophages with either drugs was assessed by ELISA. Both drugs reduced amastigote replication. EAPB0503 proved more potent, particularly on the wild type L. tropica amastigotes. Toll-Like Receptor-7 was upregulated, mainly by Imiquimod, and to a lesser extent by EAPB0503. Both drugs activated the NF-κB canonical pathway triggering an immune response and i-NOS upregulation in infected macrophages. Our findings establish Imiquimod as a strong candidate for treating L. tropica and show the higher potency of its analog EAPB0503 against CL.
皮肤利什曼病（CL）是一种寄生虫感染，世界卫生组织将其列为最不受控制的传播性被忽视疾病之一。叙利亚是利什曼原虫热带株和利什曼原虫主要种的流行地区，导致东地中海地区出现 CL。叙利亚难民的大规模流离失所加剧了 CL 在邻国的传播。针对 CL 的治疗干预包括局部、全身和物理治疗。目前治疗方法存在耐药性的高风险，这凸显了对新疗法的需求。咪喹莫特是一种免疫调节剂药物，已被证明对利什曼原虫主要种具有疗效。然而，咪喹莫特对利什曼原虫热带株的疗效及其决定其效力的分子机制仍未得到充分探索。在这项研究中，我们描述了咪喹莫特对利什曼原虫热带株和利什曼原虫主要种的作用，并描述了决定其对这两种菌株抗利什曼病疗效的分子机制。我们还研究了咪喹莫特类似物 EAPB0503 对这两种菌株的效力和分子机制。我们已经测试了咪喹莫特和 EAPB0503 对感染利什曼原虫主要种、利什曼原虫热带株或患者来源的新鲜分离的利什曼原虫热带株的巨噬细胞的影响。通过使用动基体特异性引物的实时定量 RT-PCR（RT-PCR）、使用 Glycoprotein 63 (Gp63) Leishmania 无鞭毛体抗体的共聚焦显微镜或组织学染色，评估了两种药物的抗无鞭毛体活性。通过 Western blot 和共聚焦显微镜确定了两种药物对经典核因子 kappa-B (NF-κB) 途径的作用机制。通过 ELISA 评估了感染巨噬细胞用两种药物治疗后的细胞因子免疫产生情况。两种药物均减少了无鞭毛体的复制。EAPB0503 更为有效，特别是对野生型利什曼原虫热带株。Toll-Like Receptor-7 上调，主要由咪喹莫特引起，其次是 EAPB0503。两种药物均可激活 NF-κB 经典途径，在感染的巨噬细胞中引发免疫反应和 i-NOS 上调。我们的研究结果确立了咪喹莫特作为治疗利什曼原虫热带株的有力候选药物，并表明其类似物 EAPB0503 对抗 CL 的效力更高。
