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Retinoic Acid Increases Cellular Cholesterol in Leishmania donovani-Infected Macrophages in an mTOR-Independent Manner

ABSTRACT Infection with Leishmania donovani reduces cellular cholesterol and thus deprives the host cells by inhibiting its synthesis and uptake. Changes in cholesterol levels increase the chance of attachment and internalization of L. donovani in macrophages (Mϕ). Retinoic acid (RA), an important micronutrient, restores the lysosomal uptake of cholesterol in L. donovani-infected Mϕ. Importantly, mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) increases the cellular cholesterol level by increasing expression of sterol regulatory element-binding protein 2 (SREBP2). Whether the efficacy of RA in L. donovani-infected Mϕ is mediated by mTOR is not yet established. Moreover, there are contradicting reports suggesting potential activation and inhibition of mTOR in L. donovani-infected Mϕ. Intrigued by this, we attempted to understand the RA-mediated restoration of cholesterol as well as the possible roles of mTORC1, if any. Our findings suggest that L. donovani infection impairs the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), uptake of low-density lipoprotein receptor (LDLR), and secretion of ATP-binding cassette transporter (ABCA1) in Mϕ. L. donovani infection possibly impairs mTORC1 formation, as it inhibits the expression of regulatory-associated protein of mammalian target of rapamycin (RAPTOR). Importantly, all these are restored upon RA supplementation. RA also restores the levels of SREBP2 in L. donovani-infected Mϕ, resulting in increased cellular cholesterol and thus reducing the parasite burden. When mTORC1 was inhibited, RA exerted a similar response in L. donovani-infected Mϕ; i.e., it restored cholesterol levels and reduced the parasite burden. In summary, RA restores cholesterol levels in L. donovani-infected Mϕ and reduces the parasite burden in an mTOR-independent manner. IMPORTANCE People who reside in regions where leishmaniasis is endemic and who lack proteins, iron, zinc, and vitamin A in their diet are more prone to develop visceral leishmaniasis (VL) as a full-blown disease. Vitamin A deficiency favors the development of a parasitic infection in the human host, and the WHO recommends administering 200,000-IU doses to VL patients on admission. Additionally, Leishmania entry and its survival inside the host are achieved by utilizing host cholesterol, as all trypanosomatids lack de novo synthesis of sterol. We have already shown that RA regulates cellular cholesterol levels associated with an efficient immune response. A deficiency of retinoic acid (RA) favors the parasite in Leishmania donovani-infected macrophages by downregulating the immune response. In the present work, we observed that RA restores cellular cholesterol levels in Leishmania donovani-infected macrophages. This study proposes using RA as an immune potentiator along with standard therapy.

leishmaniasis (MCL) (1). VL is prevalent in Brazil, China, Ethiopia, India, Iraq, Kenya, Nepal, Somalia, and Sudan and is caused by Leishmania donovani in the Indian subcontinent. If not treated promptly, it can be fatal. Drug resistance and lack of new effective drugs are challenges which need global attention. Though the number of VL cases has dropped during the last few years globally, however, 50,000 to 90,000 new cases still occur every year, and over 600 million people are at risk (2)(3)(4).
The nutritional status of people at risk plays an important role in overall immune status and in the initial response to encountering the parasite. Once the infection is established in host cells and chemotherapy begins, the immune response, which is affected by nutritional status, restricts the parasite through drug-immune interphase. Since the Leishmania parasite targets the malnourished population, which has overall low immunity, therapeutic advancement may not be possible unless we target malnutrition in a general or specific manner. This is possibly the reason that primary infection often progresses to full-blown disease (3), and treatment may also fail due to poor immune responses in malnourished VL patients. Our earlier findings show low levels of retinoic acid (RA) in VL patients, and an in vitro infection with L. donovani shows decreased endogenous conversion of RA from retinol, a dietary form of vitamin A (5). This results in poor immune responses in infected macrophages (Mf ) showing increased expression of arginase-1 and interleukin 10 (IL-10). Our recent findings also show that RA restores the cellular cholesterol by increasing its lysosomal uptake (via NPC-1 and NPC-2 proteins) in L. donovani-infected Mf (6). In extrahepatic cells such as Mf , cellular cholesterol is maintained in three possible ways; synthesis, uptake, and efflux. Briefly, cellular synthesis from acetyl coenzyme A (acetyl-CoA) is regulated by the enzyme 3-hydroxy-3methylglutaryl-CoA (HMG-CoA) reductase (HMGCR), and cellular uptake is mediated by lowdensity lipoprotein receptor (LDLR) and regulated by PCSK9. Cellular efflux of cholesterol in the form of high-density lipoprotein (HDL) is routed through ABCA1 present on the surface of Mf . Their status is not yet completely understood in L. donovani-infected Mf .
In general, it is believed that RA, an active metabolite of vitamin A, performs various biological functions, including immune response regulation through interaction with retinoic acid response element (RARE)-binding receptors. Our findings showed the presence of RARE sites upstream of npc-1 and npc-2 genes, which are involved in lysosomal uptake of cholesterol (6). Interestingly, we have also shown that RA exerts its effect on cellular cholesterol level in an mTORC1 (mammalian [or mechanistic] target of rapamycin complex 1)-dependent manner in Mf . However, not much is known about its role in L. donovani-infected Mf . Moreover, there are contradictory reports on the status of the mTORC1 pathway in L. donovani infection. One report suggests increased phosphorylation at Ser2148 and Ser2481 in the mTOR kinase domain in L. donovani-infected Mf (7). In contrast, another report suggests inhibition of mTOR activity in L. donovani-infected Mf (8). Thus, the status of mTOR pathway in L. donovani-infected Mf and its potential involvement in RA-mediated restoration of cellular cholesterol need to be revisited. In the present work, we attempted to clarify this in an L. donovani-infected mouse macrophage line.

RESULTS
RA consumption increased in L. donovani-infected macrophages. At first, we attempted to check whether RA supplementation is used by the L. donovani-infected Mf or whether cellular entry and uptake of RA in Mf are unaffected by L. donovani infection. Thus, we measured the levels of residual RA in the culture supernatants of uninfected and L. donovani-infected Mf (Fig. 1a). The cultures were supplemented with 10 mg/mL of RA. Findings showed more consumption of RA in L. donovani-infected Mf , suggesting intactness of cellular uptake/entry of RA (Fig. 1b).
L. donovani infection impairs the synthesis, uptake, and efflux of cholesterol in M/. We measured the expression of HMGCR, ABCA1, and LDLR in L. donovani-infected Mf in order to measure synthesis, efflux, and uptake of cholesterol, respectively. Our findings showed a significant decrease in the mRNA expression of HMGCR (Fig. 2a) as well as ABCA1 (Fig. 2b) in L. donovani-infected Mf , suggesting impaired synthesis and efflux. The mRNA expression of LDLR gene was also decreased in L. donovani-infected Mf (Fig. 2c). Furthermore, PCSK9, a protein involved in the delivery of LDL complex to the lysosomal compartment for degradation, was also measured under similar conditions. Our findings showed decreased mRNA expression of PCSK9 in L. donovani-infected Mf (Fig. 2d).
L. donovani infection inhibits the expressions RAPTOR and SREBP2 in M/. It is established that the mTORC1 pathway regulates the expressions of SREBP2. Moreover, there are contradictory reports on the status of mTOR pathways in L. donovani-infected Mf . The experiments were performed in three independent sets, and each set was performed in duplicate. The difference between two conditions is considered significant if the P value is less than 0.05. Thus, we were interested to measure the mRNA expression of RAPTOR, which encodes a regulatory protein of mTORC1, and SREBP2, which encodes a cholesterol-sensing transcription factor initially sequestered in the endoplasmic reticulum and involved in expression of regulatory genes in cholesterol biosynthesis and uptake. Our findings showed a significant decrease in the mRNA expression of RAPTOR (Fig. 3a) and SREBP2 (Fig. 3b) in L. donovani-infected Mf . Furthermore, significant depletion of cellular cholesterol was also observed in infected Mf through biochemical estimation (Fig. 3c). To further validate this, we showed loss of cholesterol in L. donovani-infected Mf through a fluorescence-based study using the dye filipin III (Fig. 3d to f). To detect the parasite burden under similar conditions, we measured kinetoplast DNA (kDNA) expression, which is used to indicate parasite load. Our finding showed significant increases in parasite loads in infected Mf (Fig. 3g).
RA restores the cellular cholesterol levels in infected M/ and reduces the parasite load. We have already demonstrated that RA increases the mRNA expression of npc-1 and npc-2 genes, which are involved in lysosomal transfer of cholesterol, and restores expression in L. donovani-infected Mf (6). Since we observed decreases in the mRNA expression of RAPTOR and SREBP2 genes in L. donovani-infected Mf , we measured the levels of these genes after treating the infected cells with RA. Our findings showed restoration of the expression of RAPTOR (Fig. 4a) and SREBP2 (Fig. 4b) upon RA supplementation postinfection. When we supplemented infected cells with RA, we found increases in the levels of cellular cholesterol over those of untreated infected cells (Fig. 4c). Importantly, parasitic load was significantly reduced upon RA treatment of L. donovani-infected Mf (Fig. 4d). RA-driven restoration of cholesterol in L. donovani-infected M/ is not mediated through the mTORC1 pathway. Our earlier findings confirmed that RA increased the cellular cholesterol and is not mediated through mTOR pathway (9). Moreover, we had observed increased mRNA expression of RAPTOR, possibly indicating the effective assembly of mTORC1, upon RA supplementation of L. donovani-infected Mf . Intrigued by this, we measured the expression of genes involved in the pathway under similar conditions along with an mTORC1 pathway inhibitor, i.e., rapamycin. First, we detected the mRNA expression of genes involved in cholesterol biosynthesis and uptake, and we found unaltered or marginally increased mRNA expression of SREBP2 and LDLR in RA-supplemented L. donovani-infected cells upon treatment with rapamycin. Rapamycin treatment did not cause any substantial change in the expression of PCSK9 mRNA in RA-supplemented L. donovani-infected Mf (Fig. 5a). This suggests that mTORC1 may not be involved in the RA-mediated increase of cellular cholesterol levels in L. donovani-infected Mf . To confirm this, we measured cellular cholesterol levels and parasite loads upon rapamycin treatment of RA-supplemented and L. donovani-infected Mf . Findings showed increased cholesterol levels (Fig. 5b) and decreased parasite loads under this condition (Fig. 5c). The difference between two conditions is considered significant if the P value is #0.05.

DISCUSSION
L. donovani, as it cannot synthesize cholesterol de novo, utilizes host cholesterol for early establishment of infection and impairing the immune response (10). Cholesterol is found at low levels in VL patients (11) and is a key component of the eukaryotic plasma membrane that regulates structural stability, fluidity, and maintenance (12). Its concentration in extrahepatic cells (i.e., Mf ) is tightly regulated in the cell as well as in subcellular organelles. It is either endogenously synthesized inside the cells from acetyl-CoA using a complex pathway regulated by HMG-CoA reductase or acquired from endocytosing LDL through LDLR expressed on the surfaces of cells. Few proteins, such as NPC-1 and NPC-2, are required to capture cholesterol in the lysosome and subsequently transfer it to the other side of membrane (13). It can also be secreted in the form of HDL through the ABCA1 surface transporter (14). In our findings, it is evident that L. donovani infection impairs the synthesis (HMGCR), uptake (LDLR), and efflux (ABCA1) of cellular cholesterol in infected Mf . Many of the genes encoding these regulatory proteins/receptors/transporters are transcriptionally governed by SREBP2, which is sequestered in the endoplasmic reticulum (ER). This protein senses the levels of cholesterol, and its expression increases in a feedback manner. Thereafter, it is released from the ER and transported to the nucleus for expression of various genes, including LDLR, etc. (15). We have shown here that L. donovani infection reduces this significant transcription factor in Mf , which possibly impairs the expression of HMGCR, LDLR, and ABCA1. We have already shown that the L. donovani infection decreases the levels of cellular cholesterol in Mf by impairing the mRNA expression of npc-1 and npc-2 (6). In the same study, we showed improvement in the levels of cellular cholesterol in L. donovani-infected Mf upon RA treatment, which resulted in reduced parasite burden (6). In another part of our study in healthy uninfected Mf , RA was shown to restore the levels of cellular cholesterol in an mTOR-independent manner (9).
In general, under cholesterol-depleted conditions, mTOR, which is a key regulator of cell growth, proliferation, and immune response, is activated. mTOR complex 1 (mTORC1), one of two forms of the complex in mammalian cells, assists in regulating cholesterol synthesis along with several other cellular processes, such as autophagy, protein synthesis through eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), p70S6 kinase (p70S6K) activation, etc. (16). In the presence of nutrients and growth factors, Akt/PKB, PIP3 kinase signaling pathways, etc., activate mTORC1. Expression of RAPTOR, encoding an important regulatory protein of mTORC1, is also controlled and poses an additional regulatory step in addition to phosphorylation in the functioning of mTORC1 proteins. In Leishmania infection, the status of the mTOR pathway is not clear. One report suggests increased phosphorylation of serine 2148 The experiments were performed in three independent sets, and each set was performed in triplicate. The difference between two conditions is considered significant if the P value is #0.05. and 2481 residues in the mTOR kinase domain (7); however, another report suggests that inactivation of the mTOR pathway due to cleavage of the mTOR complex and activation of 4E-BP1 supports proliferation of Leishmania (17). We believe that assembly of various proteins in the complex is a first as well as a prerequisite step for the functioning of mTORC1 pathway prior to phosphorylation of serine residues in the kinase domain. Hence, we measured the expression of RAPTOR in L. donovani-infected cells and found a significant decrease in its mRNA expression. An intact as well as activated mTORC1 induces the release and expression of SREBP2 protein, which in turn activates the expression of various genes involved in cholesterol biosynthesis and uptake. This explains the decrease in the mRNA expression of LDLR in L. donovani-infected Mf , which is in accordance with the findings of low cholesterol in L. donovani-infected Mf .
It has always been of utmost importance to look for a substance, preferably a micronutrient, which potentiates the immune function of infected Mf . RA, in our earlier findings, showed an increase in the levels of cellular cholesterol. More importantly, RA overcomes the L. donovani-driven loss of cellular cholesterol and reverts it to optimum levels. Additionally, in the present study, we found that L. donovani infection compromises the expression of SREBP2, possibly resulting in reduced expression of HMGCR, ABCA1, and LDLR. This suggests impaired cholesterol biosynthesis and uptake.
In summary, RA entry into the infected cell is not affected, and it restores the immune response. RA increases the levels of cellular cholesterol in Mf , which is mTOR independent, as rapamycin abrogates the RA-driven increase in cellular cholesterol (9). However, in L. donovani-infected cells, inhibition of mTORC1 by rapamycin did not reverse the RA-mediated increase in expression of SREBP2 and LDLR mRNA and thus the cellular cholesterol. This suggests that RA, in L. donovani-infected Mf , stimulates the mRNA expression of these genes involved in cholesterol synthesis and uptake in an mTOR-independent manner. One possibility which cannot be excluded is that RA binds to RAR-RXR and directly activates the expression of genes in an mTOR-independent manner. To corroborate this possibility, we explored the presence of RARE sites and/or binding of RAR-RXR complex upstream of genes such as SREBP2 and LDLR. It has been reported that retinoic acid receptor binds to the rs57217136 domain of LDLR (18). Furthermore, mRNA expression of SREBP2 and LDLR increased upon all-trans-retinoic acid (ATRA) treatment in MCF-7 cells (19). Thus, direct binding of RA through the RAR-RXR complex cannot be ruled out.
After 1 Â 10 6 Mf were allowed to adhere in each well of the culture plate for 6 h in a 5% CO 2 incubator at 37°C, nonadherent cells were removed and L. donovani promastigotes were added to adherent Mf for 12 h at a 10:1 (L. donovani to Mf ) ratio. Once infection was established, unbound parasites were washed off with incomplete medium. Upon completion of the experiment, the parasite load was measured quantitatively using kDNA gene expression (6).
Treatment with RA and rapamycin. Before RA supplementation, infected cells were treated with 50 nM rapamycin (catalog no. R0097; TCI, Japan) for 2 h (20). Subsequently, 30 mg/mL of RA (equivalent to 100 mM; ATRA) (catalog no. R2625; Sigma-Aldrich, USA) was given for 12 h as per the experimental layout (21). This nonphysiological dose of RA was purposely chosen to observe its therapeutic effect on Mf . Following the completion of experiments, cells were harvested for RNA and genomic DNA (gDNA) isolation.
RNA and cDNA preparation. Total RNA was isolated using TRIzol (catalog no. 9108; TaKaRa Bio, USA) by adding 1 mL to harvested cells for a minimum period of 1 to 2 h. Subsequently, chloroform was added in a 10:2 (TRIzol to chloroform) ratio, mixed properly, and incubated for 10 min at room temperature. The suspension was centrifuged at 21,952 Â g for 20 min at 4°C. After centrifugation, the uppermost layer containing RNA was transferred to a fresh microcentrifuge tube, and 500 mL of isopropanol was added. The tube was kept at room temperature for 10 min and then centrifuged at 21,952 Â g for 20 min at 4°C. The precipitated RNA pellet was washed with 75% ethanol at least twice at 6,300 Â g for 5 min at 4°C. After that, the RNA pellet was resuspended in 20 mL of nuclease-free water (catalog no. ML064; Himedia, India) (22).
RNA was quantified using a Picogene Nanodrop instrument, and 1 mg RNA was used to prepare cDNA. For cDNA preparation, 1 mL oligo(dT) (catalog no. S0131; Thermo Fisher Scientific, USA) primers, 1 U/mL murine