Host cholesterol influences the activity of sterol biosynthesis inhibitors in Leishmania amazonensis

A significant percentage of exogenous cholesterol was found in promastigotes and amastigotes of all studied species of Leishmania, suggesting a biological role for this molecule. Previous studies have shown that promastigotes of Leishmania uptake more low-density lipoprotein (LDL) particles under pharmacological pressure and are more susceptible to ergosterol inhibition in the absence of exogenous sources of cholesterol. This work shows that the host’s LDL is available to intracellular amastigotes and that the absence of exogenous cholesterol enhances the potency of sterol biosynthesis inhibitors in infected macrophages. A complete understanding of cholesterol transport to the parasitophorous vacuole can guide the development of a new drug class to be used in combination with sterol biosynthesis inhibitors for the treatment of leishmaniases.


Leishmaniasis
Leishmaniases are caused by parasites of the genus Leishmania; they are unicellular eukaryotes that belong to the family Trypanosomatidae. The disease presents with several clinical manifestations, such as cutaneous, mucocutaneous, and visceral manifestations, and it occurs in the old and new worlds. More than 90% of visceral leishmaniasis cases worldwide have occurred in seven countries (Brazil, Ethiopia, India, Kenya, Somalia, South Sudan, and Sudan). In addition, Brazil is among the countries where more than 90% of cases of cutaneous leishmaniasis have been reported worldwide. (1,2) Since the 1940s, two commercially available formulations of pentavalent antimonials have been available for the treatment of leishmaniasis: N-methylglucamine antimoniate (meglumine antimoniate) (Glucantime ® ) and sodium stibogluconate (Pentostam ® ). Miltefosine, the only licensed oral drug for the treatment of leishmaniasis, has been employed for cutaneous and visceral leishmaniasis in some countries, such as India, Germany, and Colombia; (1,2,3) however, resistant cases have been reported, with increased failure in the treatment of visceral leishmaniasis. (3) Other drugs, such as pentamidine, amphotericin B, and paromomycin, are used in some circumstances, such as in cases of resistance to first-line treatment, even though they have significant toxic effects. (1,2) Sterol biosynthesis in Leishmania spp.
Sterols are structural lipids and are present in the membranes of most eukaryotic cells. Cholesterol is the essential mammalian cell sterol, but plants, fungi, and some protozoa synthesise other closely related sterols instead of cholesterol using the same synthetic pathway up to the 2,3-squalene epoxide step. (4) Leishmania spp. produce ergosterol and other 24-alkylated sterols (ergostane-based sterols) that are considered essential for both promastigote and amastigote forms. (5,6,7) The sterol biosynthetic pathway of Leishmania spp. and Trypanosoma cruzi has been deduced from studies with [ 14 C]-acetate and [ 14 C]-mevalonate and from the identification of intermediate sterols that accumulate after treatment with various sterol biosynthesis inhibitors. (5,8) The discovery of these intermediates suggested that sterol biosynthesis in Leishmania spp. occurs similarly to that in fungi, offering an opportunity for the development of targeted chemotherapy in the sterol biosynthetic pathway. (4,9) In fact, the inhibition of several enzymes of this pathway, such as HMG-CoA reductase, squalene epoxidase, lanosterol C-14 demethylase, and C-24 methyltransferase, by statins (simvastatin, lovastatin, and atorvastatin), allylamines (terbinafine), azoles (ketoconazole, miconazole, posaconazole, and itraconazole) and azasterols causes the death of Leishmania spp. (5,10,11,12,13,14) Therefore, the obvious conclusion is that some of these drugs, which have already been successfully used in clinics against fungal infections, could be alternatives for the treatment of leishmaniases. (5,9)

Uptake of exogenous cholesterol
Although Leishmania spp. synthesise ergostane-derived sterols endogenously, they also obtain exogenous cholesterol, mainly through a low-density lipoprotein (LDL) receptor that is conserved in several trypanosomatids. This receptor is located in the flagellar pocket, and studies have shown that the cholesterol obtained by this parasite comes from the uptake and metabolism of LDL, which mainly contains cholesterol and cholesterol esters. (15) Leishmania amazonensis promastigotes, under pharmacological pressure from sterol biosynthesis inhibitors (ketoconazole, miconazole, and simvastatin), endocytose more LDL from the culture medium. They are also more sensitive to these inhibitors when they are deprived of exogenous cholesterol sources, suggesting that cholesterol uptake may play a compensatory role when the ergosterol biosynthetic pathway is inhibited. (16,17) Disturbance of sterol biosynthesis by itraconazole and ketoconazole was also analysed in L. mexicana amastigotes, and this life cycle stage was shown to have greater sensitivity to these molecules. Treatment of amastigotes with itraconazole and ketoconazole was also shown to cause increased amounts of exogenous cholesterol, as seen in the untreated promastigote forms. (18)

Leishmaniasis and cholesterol metabolism
Lipid alterations in plasma have been demonstrated in some infectious diseases, and cholesterol has been studied in many parasitic infections. (19) Specifically, in leishmaniasis, two study approaches are used globally: the correlation of systemic cholesterol homeostasis with the disease and the relationship of host cell cholesterol with the infection. The first approach is derived from observations that patients with visceral leishmaniasis have hypocholesterolemia. The second approach is derived from the importance of lipid rafts for the entry of the parasite into the macrophage and the presentation of antigens. Patients with visceral leishmaniasis have a decrease in serum LDL-cholesterol, leading to hypocholesterolemia, which is mainly evident in this form of the disease. (20,21) The two study approaches overlap in some respects but differ essentially in that macrophages per se are not responsible for cholesterol homeostasis, a role played mainly by hepatocytes.
Ghosh and colleagues (22) showed that hypercholesterolemia offers protection against infection caused by L. donovani, whereas hypocholesterolemia makes mice more susceptible to infection with the parasite. (22) Recent works have shown that the hypocholesterolemia observed in L. donovani infection is due to the release of metalloprotease GP63 by the parasites infecting Kupffer cells in the liver. GP63 cleaves dicer (endonuclease RNAse III) in hepatocytes, reducing the expression of miR122 and leading to reduced production of cholesterol. miR122 is a posttranscriptional regulator (miRNA) that is abundantly expressed in the liver and modulates various functions. It comprises more than 70% of the miRNA in the liver and is mainly responsible for homeostasis and lipid metabolism. The downstream events of GP63 release affect cholesterol homeostasis, leading to progression of the infection due to failure in antigen presentation. (23,24) However, the mechanisms of transport of GP63 from the parasitophorous vacuole of Kupffer cells to hepatocytes and the expression of GP63 by amastigotes remain to be elucidated. Corroborating these data, parallel studies have shown that lipoproteins can modulate the cellular immune response in leishmaniasis. (25) Other studies have shown that macrophages infected with L. donovani have significantly decreased membrane cholesterol. The administration of cholesterol in infected hamsters offers substantial protection, showing the importance of cholesterol in the macrophage membrane for correct antigen presentation and immune system activation. (26,27) On the other hand, the results published by Fernandes and colleagues (28) revealed that infection by L. major significantly increases the migration of inflammatory cells to atherosclerotic lesions and promotes atherogenesis. The process of cholesterolemia accompanies this increase. These effects of L. major primary infection are a consequence of the stimulation of the immune system, specifically stimulation of the inflammatory components of atherosclerosis, which are activated by parasites in macrophages. (28) There are some controversial results regarding the modulation of host cholesterol homeostasis, but there seems to be no doubt that Leishmania infection alters host cell cholesterol metabolism.

Availability of LDL particles for intracellular amastigotes
LDL is the primary extracellular carrier of cholesterol, playing an essential physiological role in cell function and the regulation of metabolic pathways. Macrophages have LDL receptors that endocytose these particles under normal conditions. Under pathological conditions, such as hyperlipidemia, genetic disorders, and oxidative stress, specific components of LDL are oxidised, increasing its uptake. (29) Semine and colleagues (30) showed cholesterol trafficking in macrophages infected by L. mexicana. Infected macrophages increase LDL-cholesterol uptake and simultaneously decrease the level of NPC intracellular cholesterol transporter 1 (NPC1), which is responsible for late lysosomal cholesterol efflux, leading to retention of exogenous cholesterol in the parasitophorous vacuole (PV). Cholesterol concentrates around the parasites, forming a coating directly proportional to the number of parasites. (30) Furthermore, the parasites take up cholesterol from the host cell membrane. Cholesterol sequestration occurs soon after the onset of infection; this helps explain the cholesterol-dependent process in macrophages that influences the function of the immune response, such as antigen presentation. (30) This increase in cholesterol retention in the PV may be related to the decrease in cholesterol in the macrophage membrane, decreasing antigen presentation and contributing to disease progression.
Other works have demonstrated that infected macrophages alter their cholesterol metabolism by increasing expression of pathway enzymes, increasing LDL receptors, and decreasing ABC transporters that efflux cholesterol, thus leading to an accumulation of cholesterol within these cells. (31,32) The accumulated cholesterol may be used in some way by the parasite, since the presence of cholesterol in amastigotes has already been demonstrated. (18,33) Since the central cell affected in L. amazonensis infection is the macrophage, and it has LDL receptors, we studied whether amastigotes use LDL particles. To assess the availability of LDL to intracellular amastigotes, peritoneal macrophages were infected with L. amazonensis-GFP and incubated with LDL-Alexa 594 or infected with L. amazonensis and incubated with LDL-gold.
First, for the confocal microscopy experiments, peritoneal macrophages from Swiss mice (1.0 x 10 6 /mL) were plated and adhered to Lab-teks (Thermo-Fisher) for 1 hour and infected with 3.0 x 10 6 /mL metacyclic promas-tigotes of L. amazonensis-GFP (MHOM/BR/77/Josefa strain) for 72 h at 37ºC. Macrophages were washed and incubated with serum-free medium 24 h before incubation with LDL-labeled Alexa Fluor ® 594 AcLDL (Invitrogen, Life Technologies Corporation). LDL particles (red) can be visualised very close to the intracellular amastigotes, probably inside the PV (Fig. 1). The PV has proteins that can transport molecules from or to the cytoplasm. In addition to this active transport, intracellular vesicles, such as those formed by LDL endocytosis, can fuse with PV, discharging its intravesicular content.
For the transmission electron microscopy experiments, peritoneal macrophages from Swiss mice (1.0 x 10 6 /mL) were plated, adhered to small Petri dishes for 1 hour, and infected with 3.0 x 10 6 /mL metacyclic promastigotes of L. amazonensis (strain MHOM/BR/77/ LTB0016) for 3 h. After this period, the plates were washed with serum-free medium and incubated for 72 h at 37ºC. Four hours before the end of the final incubation period, the plates were incubated with colloidal gold-labeled LDL (34) at a 1:10 dilution. After this period, infected and uninfected macrophages were washed with phosphate buffered saline (PBS) and fixed for 40 min in a 2.5% glutaraldehyde solution diluted in 0.1 M sodium cacodylate buffer, pH 7.4. (35) Ultrathin sections were collected, stained with uranyl acetate and lead citrate, and examined in a Jeol 1011 transmission electron microscope (Tokyo, Japan) at Plataforma de Microscopia Eletrônica at Fiocruz. LDL purification was performed as described by Poumay and Ronveaux, (36) with some modifications.
The confocal microscopy results were corroborated by transmission electron microscopy. Fig. 2A-B shows the distribution of LDL particles in uninfected macrophages. LDL-gold particles (dark spots) were endocytosed and stored in endocytic vacuoles to be processed and used by the host cell. In Fig. 2C-D, macrophages infected with L. amazonensis were incubated with LDLgold particles. The endocytic vacuoles containing the LDL particles fused with the PV. We also observed the presence of free LDL particles inside the PV, even at very close distances to the amastigotes, suggesting adhesion to the parasite membrane. These images demonstrate that intracellular amastigotes have access to LDL and, therefore, may be obtaining their content, mainly cholesterol. The parasite may use the cholesterol of the LDL particles inside the PV, since the presence of cholesterol in amastigotes has already been demonstrated. (18,33)

Influence of exogenous cholesterol on the leishmanicidal activity on intracellular amastigotes
After observing that intracellular amastigotes access LDL endocytosed by macrophages, we evaluated the effect of the absence of exogenous cholesterol sources on the leishmanicidal activity of ketoconazole and miconazole. Murine peritoneal macrophages (1.0 x 10 6 cells/ mL) were infected with 3 x 10 6 promastigotes/mL of L. amazonensis for 3 hours and incubated in RPMI medium with cholesterol-free Nutridoma ® (Roche) or foetal bovine serum (FBS) in the absence or presence of miconazole or ketoconazole (0-16 µM) for 72 h. After incubation, the slides were stained with fast panoptic, and the infection index was determined by counting under an optical microscope. The infection index was calculated using the formula: % infected macrophages x nº of amastigotes/nº total of macrophages.
The parasites became more sensitive to the leishmanicidal activity of ketoconazole (Fig. 3A) and miconazole (Fig. 3B)   The experiments were carried out in triplicate, n = 3. The graphs are representative of an experiment with the standard deviation value. The graphs and IC 50 values were obtained using the GraphPad Prism 7 program. Student's t test, *p < 0.5, **p > 0.001. sence of exogenous cholesterol can influence the activity of ergosterol biosynthesis inhibitors. Intracellular amastigotes were more sensitive to ketoconazole and miconazole when incubated in cholesterol-free medium, suggesting that even in the intracellular environment, L. amazonensis uses cholesterol to replace ergosterol in an attempt to maintain its membrane properties.

Concluding remarks
In conclusion, the data presented here suggest that intracellular amastigotes access the host's LDL and that the availability of this pool of cholesterol can be used by the parasite to overcome the effect of sterol biosynthesis inhibitors. A complete understanding of this phenomenon can guide the development of a new drug class to be used in combination with sterol biosynthesis inhibitors for the treatment of leishmaniases.