C57BL/6 and DBA/2 DLs hosting live L. am amastigotes show only minor transcriptional modifications compared to control DLs
DLs were derived from GM-CSF-responsive progenitors (otherwise known to be present in the bone marrow cell suspensions prepared from the femurs of C57BL/6 and DBA/2 mouse inbred strains) as previously described for DLs derived from BALB/c mouse bone marrow 
. More than 97% of the cells in these cultures expressed CD11c in parallel to CD11a and CD11b (data not shown). Two different cell subsets were defined by flow cytometry (FCM) analysis. The first did not express surface MHC II molecules, and consequently was not considered as bona fide
DLs (subset 1, figure S1A
). The second did express surface MHC II molecules, albeit at various intensities. These are DL phenotypic signatures per se (subset 2, figure S1A
). The latter features were thus used for all subsequent DL FCM analyses, as well as for DL sorting.
The cultures were either left in medium alone (ctrl condition) or places in contact with live L. am
amastigotes at a ratio of 5 amastigotes per cell (L. am
condition) and either processed or analysed 24 hours later. Combined with the DL features mentioned above, transgenic L. am
amastigotes expressing fluorescent Ds
Red2 protein were used for the specific detection and sorting of all DLs harbouring live L. am
amastigotes, without further fixation and permeabilization. Thus, ctrl DLs and DLs hosting live Ds
Red2 L. am
amastigotes were sorted based on their expression of MHC II molecules and MHC II molecules plus Ds
Red2 fluorescence, respectively (Figure S1B
). This sorting strategy constitutes a major improvement over all previously published methods for an in-depth comparative characterization of ctrl DLs versus DLs harbouring live Ds
amastigotes (Figure S1B
A genome-wide transcriptional analysis based on Affymetrix technology was performed on sorted DLs from both mouse strains. It highlighted that DLs hosting live L. am amastigotes showed only minor transcriptional modifications compared to ctrl DLs, and this both in term of frequency and magnitude. In fact, out of 28, 853 mouse genes, only 858 and 932 were captured with differential expression at the 5% significance level in C57BL/6 and DBA/2 DLs, respectively. These numbers correspond to only 1.6% and 1.8% of genes modulated in C57BL/6 and DBA/2 DLs harbouring live L. am amastigotes.
Cholesterol uptake and generation of cholesteryl ester
perpetuation is known to be strictly reliant upon cholesterol provided by the host organism, we set about investigating whether cellular cholesterol uptake, trafficking and the complex systems regulating cholesterol delivery may be modified in DLs hosting live L. am
amastigotes. Mammalian cells acquire cholesterol from three sources: i) from de novo
synthesis in the ER, iii) from low-density lipoprotein (LDL)-derived cholesterol and iii) from the cholesterol/cholesteryl ester (CE) cycle. Our data showed that, in contrast to BALB/c mouse bone marrow-derived macrophages hosting proliferating L. am
, DLs harbouring live L. am
amastigotes did not show any fluctuations in the many enzyme-coding transcripts involved in the de novo
biosynthesis of cholesterol. The only exception to this was mvd
in DLs of C57BL/6 origin (). We noted that cholesterol hydroxylase, which is known to convert cholesterol to 25-hydroxycholesterol/25-HC, was up-regulated. It should here be noted that 25-HC can act on the SCAP/SREBP complex - involved in cholesterol synthesis - by suppressing the SREBP maturation process 
, and that any increase in 25-HC might prevent cholesterol synthesis. By contrast, we also noted that transcripts encoding for molecules involved in the uptake and transport of exogenous cholesterol and cholesterol storage were up-modulated (). For instance, L. am+
DLs showed concerted up-modulation of multiple surface receptors () that may promote the uptake of cholesterol, either specifically or after LDL endocytosis. Cholesterol esterification in L. am+
C57BL/6 DLs appeared to be promoted through increased transcription of cytosolic thiolase acat2
(, ). This increased CE generation may also be promoted by the higher FA content resulting from increased FA uptake and transport, as described previously. The absence of any modulation of aadacl
transcript encoding for cholesteryl ester hydrolase (CEH), and the down-modulation of lipa
in L. am+
C57BL/6 and DBA/2 DLs, respectively, () suggested that CE hydrolysis is prevented. No transcriptional signatures were detected except the increased transcription of cav-1
that could account for cholesterol delivery to cell surface caveolae and promote its efflux out of the L. am+
Transcriptional signatures accounting for cholesterol metabolism in dendritic leukocytes (DLs) hosting live L. amazonensis amastigotes.
C57BL/6 DLs hosting live DsRed2 L. am amastigotes showing cholesterol storage transcriptional signatures.
DLs hosting live L. am amastigotes contain more cytosolic LBs than control DLs
We noted increased transcriptional expression of genes coding for LB surface proteins, in particular cav-1
. Since no evidence was found of any promotion of de novo
synthesis of cholesterol and TAG, we cultured DLs hosting or not hosting live L. am
amastigotes in the presence of oleic acid – a mono-unsaturated LCFA - bound to BSA and compared LB number and size in L. am+
versus L. am−
DLs. Briefly, DLs were places in contact with Ds
Red2-LV79 amastigotes for 3 hours and cultured in the presence of 200 µm oleate/BSA for the next 21 hours. Thirty minutes before FCM analyses of unfixed control (Ctrl) and L. am+
DLs, a fluorescent lipophilic probe - BODIPY 493/503 dye - was added to the cultures: its incorporation into the highly hydrophobic ester core of LBs allowed the latter to be evidenced by FCM or EFM as green organelles ( and ). Parasitized DLs were detected by the orange fluorescence of intracellular Ds
amastigotes. shows the outcome of a representative FCM experiment performed on C57BL/6 mice, and the collective results of 7 independent experiments. Similar results were obtained for DBA/2 DLs (data not shown). White histogram () represents background mean fluorescence emitted by ctrl DLs (here mean fluorescence intensity (mfi) was 214). This value was increased (, mfi
716, black histogram) in the oleate-free DLs containing Ds
amastigotes fraction. A statistical analysis showed that the quantity of neutral lipids was significantly higher in L. am+
than in both L. am−
DLs from the same culture (, grey histogram), and ctrl DLs (, white bars) (p<0.01 and p<0.01, ). In the oleate only-treated sample (, white histogram), mfi reached 454, proof that the neutral lipids content was higher than in ctrl DLs ()). This increase was significant (white bars, p<0.04, ). When exposed to live amastigotes plus oleate (), DLs contained an even higher neutral lipids content: L. am+
DLs gave a higher mfi (, black histogram: 1014) than both L. am−
DLs from the same culture (, grey histogram: 443) and DLs treated by oleate alone (, white histogram: 454). This increase was significant (p<0.005) in both cases, and also significant when compared to L. am+
DLs from the oleate-free sample (black bars, p<0.04, ). Altogether, these findings indicate that the presence of intracellular amastigotes was associated with increased neutral lipids contents in oleate-treated and untreated DLs. However, since amastigotes have their own LBs, this increased fluorescence could be due to LBs from the host, from the parasite, or from both, and FCM analyses cannot differentiate between them. We therefore conducted apotome analyses of our DL samples by EFM (). No LBs were seen in control cultures (image 1), but in all types of DL cultures, i.e. in the presence of L. am
(image 2), oleate only (image 3) or L. am
plus oleate (image 4). LBs from oleate-free L. am+
DLs were located in the periphery relative to the amastigote nucleus. LBs from oleate-treated L. am+
DLs were located similarly or could also be found far from the parasites. LBs differed in size and number depending on culture conditions: they were most numerous in the oleate-treated L. am+
DLs, followed by oleate-treated DLs, and oleate-free L. am+
DLs. These EFM analyses were unable to determine precisely whether the LBs were of host or parasite origin. To overcome these FCM and EFM limitations, cell cultures were analysed by TEM imaging.
FCM detection of lipid bodies (LBs) in control C57BL/6 DLs and C57BL/6 DLs hosting DsRed2 L. am amastigotes.
EFM detection of lipid bodies (LBs) in C57BL/6 DLs.
Osmium tetroxide is highly reactive with unsaturated FA and can therefore be used to reveal the presence of lipid esters derived from the LB core 
. Since our transcriptomic analysis focused specifically on host cell response, we analysed the number, size and precise location of LBs in the cytoplasm of DLs. These LBs were of low electron density i.e. light grey and uniform in appearance. LBs were observed in only 9.7% of ctrl DLs () and in roughly the same proportion of DLs cultured with L. am
(6.8%) (), which was consistent with the lack of any promotion of de novo
TAG and cholesterol synthesis suggested by the transcriptomic analysis. Altogether, these findings indicate that the increased BODIPY staining obtained in FCM and EFM analyses of ctrl and L. am+
DLs was due to LBs from the parasites, not the host. By contrast, significant numbers of LBs were detected in the cytoplasm of DLs when oleate was added to both L. am−
and L. am+
samples (). LBs were detected at a higher frequency in oleate-treated L. am+
DLs (). The total number () and area () of LBs in host cell cytoplasm were significantly higher under this condition. These observations indicate that the reprogramming of lipid metabolism in DLs harbouring L. am
amastigotes may be phenotypically reflected 24 hours post-infection in the presence of oleic acid. Similar results were obtained for DBA/2 DLs (Figure S4
). Interestingly, a high number of the resulting LBs were seen to be in intimate contact with the membrane of PVs () and parasites ().
TEM detection and analysis of lipid bodies (LBs) in live L. am amastigote-hosting C57BL/6 mouse DLs.
Visualization of contact site with lipid bodies.
In conclusion, our study shows that neutral lipid metabolism was rapidly reprogrammed in GM-CSF responsive mouse DLs hosting live L. am
amastigotes. Whatever the origin - C57BL/6 or DBA/2 mouse - of the bone marrow from which the DLs were generated, this DL reprogramming showed similar features at both the transcriptional and morphological levels, with many LBs being detected in DL cultures to which oleic acid had been added. Parasitized DLs showed coordinated transcriptional modulations that correlated in part to pparγ
up-regulation and promoted the generation and storage of neutral lipids: TAG and cholesteryl esters. The generation of these lipids was singular since not derived from de novo
synthesis but from increased import of key constituents, i.e. FAs and cholesterol, from the extracellular milieu, and up-modulation of transcripts involved in their (re-) esterification, such as TAG and CE (). When live L. am
amastigote-hosting DLs were exposed to oleate
, LBs were located in close proximity to PV, and some established close contacts with the PV membrane. No direct fusion of LB phospholipid monolayer with the PV bilayer membrane was evidenced by the methods used in our study. But in a Leishmania
we can speculate that LBs store neutral lipids that could be further scavenged by L. am
amastigotes otherwise shown to be bound to the PV membrane. These LBs could constitute an essential source of both triacylglycerol and cholesterol. As a precursor of major phospholipids such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, TAG from host DL may not only be hydrolysed to provide the live amastigotes with DAG, but could also be important for the synthesis of their key membrane components. CE from host DL may be used to provide the live amastigotes with cholesterol and CE since it is known that the cholesterol present in Leishmania
parasites is not a product of de novo
sterol biosynthesis, but is derived from the host (see for review: 
). Moreover, FAs could be used by amastigotes to produce energy via FA β-oxidation, a process that is known to occur in Leishmania
and involves several putative enzymes that have been detected by sequencing of the L. major
. β-oxidation is particularly pronounced in amastigotes versus promastigotes, the former relying on FA and amino acids as their main sources of energy 
. FA like oleate can be involved in the synthesis of polyunsaturated FA (PUFA) through elongases and desaturases, as evidenced in L. major
(for review: 
). FA and cholesterol can be used in L. am
amastigotes for energy and lipid storage, through TAG and CE synthesis, and to generate cytosolic LBs. This de novo
glycerolipid synthesis involves the activity of several enzymes such as G-3-P acyltransferase (Lm
GAT) described in L. major
, and GPAT activity expressed in different Leishmania
Summary of the subversion of neutral lipid metabolism in C57BL/6 DLs hosting live L. am amastigotes.
A recent report has suggested a novel mechanism by which exogenous lipids can affect DC function. Indeed, the increased uptake of FAs- that leads to TAG accumulation in LBs - has been shown to reduce antigen processing and presentation to effector T cells 
. Interestingly, in vitro
DLs hosting L. am
show altered responsiveness to exogenous stimuli, impaired differenciation and migration, and a low capacity to prime naive CD4+T cells 
. It is not clear how the accumulated lipid could interfere with antigen handling in DCs (for review, 
). Further investigations should be conducted to determine whether LB-loaded DLs hosting live L. am
amastigotes can prime and re-activate regulatory T lymphocytes that are reactive to unique peptides delivered from persistent amastigotes. It will be also important to identify the mechanism by which FAs and other lipids might affect DL function and to determine how lipid accumulation relates to and links with membrane, cytosolic and nuclear sites of action of FAs and other lipids on DLs.
Altogether, once loaded with LBs, the DLs hosting live L. am amastigotes, may indirectly promote the amastigote-driven remodeling of rodent skin as a dynamic niche where two L. am developmental stages durably co- persist: i) those undergoing “controlled” proliferation and those pre-adapted to blood-feeding female sand flies, namely, the next host population upon which L. am perpetuation is dependent. Pharmacological agents and transgenic mice are now available to clarify the direct role played by these metabolic active DL organelles in L. am replication and/or in the persistence of non-cell-cycling amastigotes.