Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B

The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2 − (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further,study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophag

lnflammation amplified by a viral endosymbiont revealed extensive lymphatic connections facilitating metastatic dissemination of intracellular and free Leishmania parasites

Leishmaniasis is a neglected tropical disease, caused by the human protozoan parasite Leishmania Spp. Given its prevalence in East Africa, South America, Southeast Asia, and the Mediterranean countries, it is a major diffusing global health problem currently. With an estimated 700000 to 1 million new cases annually, it affects more than 12 million people worldwide right now and causing over 20000 deaths per year. Leishmaniasis is spread to humans by the bite of a female phlebotomine sandfly vector and can have various outcomes, which itself depends on the species of the infecting parasite, immune-competence of the host, and various environmental factors. Cutaneous leishmaniasis (CL) is the most common form that can have further have various outcomes, and sometimes progress to a more severe form where parasite metastasize to form secondary lesions, leading to disseminated or mucocutaneous leishmaniasis (DCL or MCL). L guyanensis (Lgy) causes CL but 5-10% of all infections result in metastatic complications due to the presence of the cytoplasmic Leishmania RNA Virus (LRV1). The double-stranded RNA (dsRNA) within LRV is recognized by host Toll-like receptor 3 (TLR3) and has been linked with disease exacerbation, severe disease chronicity, treatment failure, clinical relapse along with mounting a potent tissue-damaging proinflammatory response. Our lab established the LRV1-exacerbated metastatic model of lfng-1- mice with LgyLRV1+ infections (as compared to LgyLRV1-), which correlated with extremely reduced IFN-y levels observed in human patients. We, therefore, aimed to unravel the dissemination process and define the major cell type(s) which transport these infective parasites to distant secondary lesion sites, thereby exhibiting severe metastasis. Our results corroborated that Lgy infection leads to severe inflammation of the primary inoculation site and is further exacerbated by the presence of LRV1. Moreover, Lgy parasites can hijack lymphatic vessels as efficient routes for the egress of infected cells to escape the local infection site for colonizing draining and non-draining lymph nodes. These infective parasites are disseminated initially through the lymph and efferent lymphatics, causing severe inflammation and infection of the draining LNs sequentially, to ultimately enter the circulatory system for systemic spread. Moreover, contrary to reported studies that Leishmania transport can occur only intracellularly by various phagocytes, we observed that Lgy parasites disseminated not only intracellularly but mostly as a free parasite; or by getting stuck to the surface of various migrating myeloid cells and even as amastigote clumps through lymph and blood. lnterestingly, they were also able to subvert even the phagocytic clearance by different resident and recruited immune cells and rather colonized the LNs for further growth and multiplication. This led to the intense infection of the blood which further disseminated these infective Lgy parasites to distant organs, favorable low-temperature niches, and skin extensively, causing the formation of newer lesions and permitting further transmission. We thus concluded that Lgy parasites could hijack the lymphatic network to reach the blood for a successful systemic spread and can disseminate in association with cells or even extracellularly on their own as free parasites

Role of ROS and nitric oxide generation on in-vitro leishmanicidal effect of KalsomeTM10 on intracelluar amsastigotes.

<p>Macrophages, infected with promastigotes, for 24 h were pretreated with ROS scavangers like NAC (10 mM), PEG-Cat (500 units) and PEG-SOD (500 units) and nitric oxide inhibitor L-NMMA (2 mM) for 30 mins and then treated with KalsomeTM10 (0.5 μg/ml) for 1h. After washing, incubation for 48 h post drug treatment agan in the prescence of these scavangers and inhibitor was carried out and then the infected macrophages were geimsa stained and counted on a light microscope to determine infectivity index (Number of amastigotes per 100 macrophages) that was represented as bar graph. Results are presented as means + SD; n = 2.<i>***P</i><0.0001.</p

In-vitro leishmanicidal effect of KalsomeTM10 on peritoneal macrophages infected with promastigotes of <i>L</i>. <i>donovani</i>.

<p>Macrophages, infected with promastigotes, were treated with different concentrations of KalsomeTM10 for 72 h post infection. The infected macrophages on coverslips were geimsa stained and counted on a light microscope (a). Number of amastigotes per 100 macrophages was represented as bar graph. Results are presented as means + SD; n = 3. Infected macrophages untreated (b i) and treated with 10 ng/ml (b ii), 50 ng/ml (b iii), 100 ng/ml (b iv), 500 ng/ml (b v) and 1000 ng/ml (b vi) of KalsomeTM10, were giemsa stained and imaged on a light microscope. Pictorial representation of three independent experiments is depicted.</p

Scanning electron microscopy of promastigotes treated with KalsomeTM10.

<p>Promastigotes were either left untreated (a) or treated with 5.0 μg/ml of Kalsome TM10 (b) for 2 h and then analysed for surface topology. SEM micrographs show rounded and distorted shape in KalsomeTM10 treated promastigotes compared to controls. Scale bars: 5 μM. Images are representative of three independent experiments.</p

Analysis of cell cycle arrest in <i>L</i>. <i>donovani</i> promastigotes treated with KalsomeTM10.

<p>Promastigotes, either left untreated (a i) or treated with 2.5 μg/ml (a ii) and 5.0 μg/ml (a iii) of kalsomeTM10 for 2 h and untreated (b i) or treated with 2.5 μg/ml (b ii) and 5.0 μg/ml (b iii) of KalsomeTM10 for 4 h, were analysed for cell cycle through flow cytometry and plotted as histograms which are representative of three independent experiments. (c) Mean % Sub G0 cells represented as bar graphs.<i>**P</i><0.001, <i>***P</i><0.0001.</p

Analysis of genomic DNA fragmentation in KalsomeTM10, Ambisome and amphotericin B treated intracellular amastigotes analysed through TUNEL assay.

<p><i>L</i>. <i>donovani</i> infected murine peritoneal macrophages were either left untreated (a) or treated with KalsomeTM10 (b) at 0.5 μg/ml, Ambisome (c) at 1.0 μg/ml and amphotericin B (d) at 2.0 μg/ml. After 48 h, infected macrophages were stained using TUNEL and analysed by fluorescence microscopy. Total nuclei are visualized in red (PI) and TUNEL-positive nuclei are stained in green (FITC). The images are representative of three independent experiments.</p

Generation of lipid peroxidation products on treatment with KalsomeTM10.

<p>Promastigotes were treated with different concentrations of kalsomeTM10 for 2 h in the absence (a) and presence of NAC (b), and lipid peroxidation products were determined flourometrically. Results are presented as means + SD; n = 3. <i>P</i><0.05.</p

ROS and mitochondrial super-oxide production, and depletion of GSH pool in KalsomeTM10 treated promastigotes.

<p>Promastigotes incubated at different time points with KalsomeTM10 at 2.5 μg/ml (a i) and 5.0 μg/ml (a ii), with or without NAC (1 mM and 10 mM). Cellular ROS was measured by bioluminescence assay. Results are presented as means + SD; n = 3. Promastigotes, untreated (b i and ii) and treated with 2.5 μg/ml of KalsomeTM10 for 30 min (b iii) and 1 h (b iv), and treated with 5.0 μg/ml of KalsomeTM10 for 30 min (b v) and 1 h (b vi), were stained with mitosox, and analysed by flow cytometry. The histograms are representative of three independent experiments. (c) Bar graphs representing mean % superoxide⁺ cells. (d) Promastigotes treated with 2.5 μg/ml of KalsomeTM10 at different time points and GSH concentration determined flourometrically. Results are presented as means + SD; n = 3. <i>*P</i><0.01, <i>**P</i><0.001.</p

PS externalization in KalsomeTM10 treated promastigotes.

<p>Promastigotes, untreated (a and b) and treated with 2.5 μg/ml (c) and 5.0 μg/ml (d) of KalsomeTM10 for 1 h, and treated with 2.5 μg/ml (e) and 5.0 μg/ml (f) of KalsomeTM10 for 2 h, were co stained with annexin V-FITC and PI, and analysed by flow cytometry. The dot plots are representative of one of the three independent experiments. (g) Bar graphs representing mean % PS⁺/PI⁻ cells.<i>**P</i><0.001.</p