THP-1 cell line derived macrophage, THP-1 macrophage infected with K133 wild type Leishmania donovani and THP-1 macrophage infected with laboratory generated miltefosine resistant K133 Leishmania donovani parasite.
Leishmania donovani, a causative organism for visceral leishmaniasis is considered as master manipulator of host macrophages. Leishmania adopts several strategies to assure their survival and proliferation within host cell. We investigated differential gene expression in THP-1 derived macrophage after infecting with L. donovani parasite. A field isolate K133 of L. donovani (MHOM/IN/2000/K133) was derived from bone marrow aspirates of VL patient and propagated in medium M199. Isolate K133 was made resistant to miltefosine in vitro by stepwise exposure to increasing drug (miltefosine; MIL) concentration and designated here as LdMIL-R. K133WT parasite is designated here as LdMIL-S. Human monocytic cell line, THP-1, was maintained in RPMI-1640 supplemented with 10 percent FCS, penicillin (100 U/ml) and streptomycin (100 mg/ml). In order to induce differentiation into macrophages, THP-1 cells were seeded (30000 cells/well) into 96 well tissue culture plate and treated with 50 ng/ml 4alpha-Phorbol 12-myristate 13-acetate (PMA) for 18 h. After stimulation with PMA, the cells displayed macrophage characteristics such as morphological alterations and adherence to culture plates. THP-1 macrophage was infected with either LdMIL-S (T1) or LdMIL-R (T2) parasite. Total RNA was isolated from uninfected macrophages (C), LdMIL-S infected macrophage (T1) and LdMIL-R infected macrophage (T2). Total RNA was reverse transcribed to cDNA, library was prepared and RNA sequencing was done using Illumina HiSeq 2500 for differential gene expression study. This study will help in understand of host factors responsible for Leishmania pathogenesis. Less...