Antileishmanial Effects of Synthetic Eh PIb Analogs Derived from the Entamoeba histolytica Lipopeptidephosphoglycan

Abstract

With an estimated number of new cases annually of approximately 1.4 million, leishmaniasis belongs to the most important parasitic diseases in the world. Nevertheless, existing drugs against leishmaniasis in general have several drawbacks that urgently necessitate new drug development. A glycolipid molecule of the intestinal protozoan parasite Entamoeba histolytica and its synthetic analogs previously showed considerable immunotherapeutic effects against Leishmania major infection. Here, we designed and synthesized a series of new immunostimulatory compounds derived from the phosphatidylinositol b anchor of Entamoeba histolytica (EhPIb) subunit of the native compound and investigated their antileishmanial activity in vitro and in vivo in a murine model of cutaneous leishmaniasis. The new synthetic EhPIb analogs showed almost no toxicity in vitro. Treatment with the analogs significantly decreased the parasite load in murine and human macrophages in vitro. In addition, topical application of the EhPIb analog Eh-1 significantly reduced cutaneous lesions in the murine model, correlating with an increase in the production of selected Th1 cytokines. In addition, we could show in in vitro experiments that treatment with Eh-1 led to a decrease in mRNA expression of arginase-1 (Arg1) and interleukin 4 (IL-4), which are required by the parasites to circumvent their elimination by the immune response. The use of the host-targeting synthetic EhPIb compounds, either alone or in combination therapy with antiparasitic drugs, shows promise for treating cutaneous leishmaniasis and therefore might improve the current unsatisfactory status of chemotherapy against this infectious disease.

High Content Analysis of Macrophage-Targeting EhPIb-Compounds against Cutaneous and Visceral Leishmania Species

“…The infection rates and the number of parasites per infected cell obtained by the image analysis sequence also increases proportionally with the MOI ( Figure 2 C). Therefore, we conclude that our HCS assay is adequate to perform a phenotypic, multispecies screening for the analysis of anti-leishmanial efficacy of the lately described immunostimulatory Eh PIB-compounds [ 24 ].…”

“…Mouse primary macrophages were prepared as described previously [ 23 , 24 , 30 , 31 ]. Briefly bone marrow-derived macrophages (BMDMs) were isolated from the tibiae and femurs of 8–12 week old male C57BL/6J mice and cultured for 10 days in Iscove’s Modified Dulbecco’s Medium (IMDM, Sigma-Aldrich, Hamburg, Germany) supplemented with 10% heat inactivated FCS (Sigma-Aldrich, Hamburg, Germany), 5% horse serum (Sigma-Aldrich, Hamburg, Germany), 1% L-glutamine (Biochrom, Berlin, Germany), 50 µg/mL Gentamicin (Sigma-Aldrich, Hamburg, Germany), and 10% LADMAC supernatant as a source of macrophage colony-stimulating factor [ 31 , 32 ].…”

“…In this study, the Eh PIb-compounds Eh-1, Eh-2, Eh-3, Eh-4, Eh-5, and Eh-6 used for the treatment were synthesized in a convergent synthetic route and have been shown to present anti-leishmanial activity against L. major [ 24 ]. Compound stocks (1 mg/mL) in DMSO were stored at −20 °C.…”

“…Treatment with Eh PIb-compounds of murine and human macrophages infected with L. major in vitro resulted in a decreased parasite load. In addition, a topical application of one selected compound (Eh-1) reduced cutaneous lesions in a murine model of experimental cutaneous leishmaniasis [ 24 ].…”

Programmed Cell Death Ligand (PD-L)-1 Contributes to the Regulation of CD4+ T Effector and Regulatory T Cells in Cutaneous Leishmaniasis

“…Infectivity of parasites was maintained through animal passage and parasites were used for infection after maximum five passages in culture. Viable parasites were counted, washed with PBS (pH 7.0), and the concentration was adjusted to 3 × 10 6 parasites in 10 µL volume, as described previously in other publications (37)(38)(39). Mice were kept under isoflurane/oxygen, the left ear was exposed, and 10 µL parasitedose were injected.…”