L. amazonensis amastigotes subvert MΦ as host cells where they enter a cell-cycling phase lasting several days (Fig. ). We compared the transcriptomes of amastigote-free MΦ and amastigote-harbouring MΦ 24 h after the uptake of amastigotes carefully purified from nude mouse lesions. At this time-point amastigotes were multiplying within a huge PV (Fig. ) and their population size had almost doubled (Fig. ). Among the 45,101 probe-sets of the Mouse430_2 GeneChip, 1,248 (2.77%) were displaying features of differential expression at the 5% significance level (Fig. , see Additional file
1). Of these, 1,206 matched Ingenuity Pathway Analysis database version 5.5.1 which represented 898 genes with a known function. About 80% of these genes were incorporated into either Ingenuity's canonical pathway or biological network (
i.e. their products interact with other molecules in Ingenuity's knowledge base). The symbols of the modulated genes are specified in the text (fold change [FC] values between brackets), while their full names are given in Additional file
1. Furthermore, comparable FC values were obtained between the Affymetrix technology and the Real Time quantitative Polymerase Chain Reaction (RTQPCR) method (Table ) [
3].
| Table 1List of differentially expressed genes between L. amazonensis-harbouring MΦ and parasite-free MΦ. |
Though transcriptional changes due to the phagocytic uptake process
per se – known to occur mostly within the first 2 hours post particle addition – cannot be completely excluded, the MΦ transcript modulation – detected at 24 h post the amastigote addition – very likely reflects MΦ reprogramming due to the presence of cell cycling amastigotes within giant PV. Indeed, in our experimental conditions, no extracellular amastigotes could be evidenced in the MΦ culture (a) after a brief centrifugation step and (b) one hour contact with adherent MΦ indicating that the phagocytic uptake of
L. amazonensis amastigotes is a rapid and efficient process. Furthermore, it is worth mentioning that the size of the amastigote population hosted within the MΦ PV rapidly expands within the first 24 h (Fig. ) [
4]. Using also mouse bone marrow-derived MΦ as host cells for
Leishmania, Gregory and coworkers demonstrated that the gene expression profiles of control MΦ and MΦ that have phagocytosed latex beads 24 h before were very similar. They evidenced a statistically significant difference for only 15 probe sets. None of the 29 corresponding probe sets in the mouse 430 DNA Affymetrix gene chip was present in the list of 1248 modulated probe sets observed in presence of
L. amazonensis amastigotes. Thus, these data strongly support our conclusion that the gene expression profile observed 24 h after the phagocytosis of
L. amazonensis amastigotes was due to the presence of intracellular cell-cycling parasites.
L. amazonensis amastigotes set up an optimal sub cellular niche
Modulation of MΦ genes encoding vacuolar proton ATPase sub-units
Within their host cells,
L. amazonensis amastigotes are known to multiply efficiently in the acidic environment of the MΦ PV [
1]. In presence of amastigotes, we observed an up-regulation of the gene expression of eight isoforms of the V0 and V1 sub-units of the MΦ vacuolar proton ATPase (
atp6V0a1,
atp6V0c,
atp6V0d2,
atp6V1a,
atp6V1c1,
atp6V1d,
atp6V1g1 and
atp6V1h: +1.27 < FC < +2.32) [
5]. This could contribute to the sustained acidification of the PV lumen which has been shown to be important at least for the optimal amastigote nutrient acquisition [
6,
7].
Coordinated modulation of MΦ lipid metabolism
The most relevant biological networks fitting our dataset were strongly associated to the function "lipid metabolism", the most significant canonical metabolic pathway being "biosynthesis of steroids" (
p-value = 1.31e-02). Indeed, several up-regulated genes (Fig. , Table ) were involved i) in cholesterol uptake (
ldlr: + 4.68), ii) in cholesterol transport (
fabp4: + 6.42 and
stard4: + 2.31) and iii) in sterol biosynthesis (
hmgcs1,
hmgcr,
mvd,
idi1,
fdps,
fdft1,
sqle,
lss,
cyp51,
sc4mol,
hsd17b7,
sc5d and
dhcr24: +1.95 < FC < +4.30). Worth is mentioning the most up-regulated gene encoding type II deiodinase (
dio2, + 25.92), an enzyme converting intracellular thyroxin (T4) to tri-iodothyronine (T3), the more active form of thyroid hormone. It has previously been demonstrated in mouse hepatocytes that the molecular basis for the connection of T3 and cholesterol metabolism involves the master transcriptional activator of the aforementioned genes, namely
srebf2 (+ 1.84) the promoter of which contains a thyroid hormone response element [
8]. Furthermore, thyroid hormone receptors can activate transcription of target genes upon T3 binding and this could be facilitated by co-activators
ncoa4 (+ 1.65) and
brd8 (+ 1.08). Interestingly, opposite to
dio2, the most down-regulated gene was cholesterol-25-hydrolase (
ch25h: -6.57), an enzyme acting downstream this pathway by breaking down cholesterol and by synthesizing a co-repressor of
srebf2 transcriptional activation [
9]. Upstream this pathway, several up-regulated genes involved in glycolysis could also contribute to increase the supply of acetate (
acsl3, adhfe1, akr1a1, aldoa, aldoc, eno2, hk2, hk3, ldha, pfkl, pkg1 and
pkm2: +1.13 < FC < +2.61). Of note was the down-regulation of genes encoding enzymes competing i) with
hmgcs1 for acetate (
acaca: -1.32) and ii) with
aldoa and
aldoc for fructose, 1-6, biphosphate, which is needed to produce glyceraldehyde-3-phosphate upstream the sterol biosynthesis pathway (
fbp1: -2.16). In addition, the up-regulation of the transcription factor encoded by
atf3 (+ 1.77) was consistent with the down-modulation of
fbp1. These data suggest that the available intracellular pool of sterol-synthesis molecular intermediates was maintained by a gene expression program relying on a coordinated regulation at both the transcriptional level by
srebf2,
atf1 (+ 1.84) and
atf3, and also most likely at the post-transcriptional level by
insig1 (+ 2.62) encoding a sterol-sensing protein that regulates the intracellular cholesterol level [
10].
The expression of several genes involved in the fatty acid biosynthesis pathway was also up-regulated with the modulation of
ppap2b (+ 8.53),
scd1 (+ 2.68),
scd2 (+ 2.45) and
acsl3 (+ 2.09). Moreover, genes encoding fatty acid binding proteins that play a role in fatty acid uptake and transport were up-regulated (
fabp3: + 2.29,
fabp4: + 6.42 and
fabp5: + 1.57). Extracellular lipolysis was down-modulated (
lipe: -2.20,
lpl: -1.44 and
apoc2: -1.63), while intracellular catabolism of triglycerides mediated via
mgll was up-regulated (+ 3.40). Fatty acid transport to peroxisome was diminished with
abcd2 down-modulation (-2.11). Since this was not described neither for
L. major nor
L. donovani [
11], this could be unique for the
L. mexicana complex, all sub-species of which multiply within giant communal PV [
1]. Indeed, previous experimental work performed with
L. mexicana [
12,
13], which is very close to
L. amazonensis (both share the same distinctive feature to multiply within a communal PV), has shown that amastigotes could take advantage of the MΦ sterol biosynthesis pathway to produce ergosterol.
These data were in agreement with the sterol biosynthesis machinery of the MΦ host cell being exploited by the cell-cycling amastigotes for both their own cell membrane sterols, in particular ergosterol and the PV membrane sterol-dependent remodelling. Indeed, cholesterol availability might play a role in the formation of the PV lipid rafts [
14] that could be involved in the control of fusion events leading to the sustained remodelling of the huge communal PV membrane where the aforementioned proton pump components are regularly delivered.
Modulation of MΦ polyamine metabolism
Polyamines (
e.g. putrescine) derived from arginine catabolism are essential compounds for amastigote growth [
15]. Using the Affymetrix technology we failed to detect, at the 5% significance threshold, arginase-2 (
arg2) and ornithine decarboxylase-1 (
odc1), two enzymes leading to the formation of polyamines through arginine catabolism. Indeed, while for
arg2 the raw fluorescence intensity values were below or close to the background level, for
odc1 the raw fluorescence intensities before data processing displayed only a slight increase (+ 1.21) in presence of amastigotes (see Additional file
1). However, the up-regulation of SLC7A2 (+ 4.14) in MΦ hosting amastigotes was a strong incentive for monitoring the abundance of
arg2 and
odc1 transcripts with a validated RTQPCR method. Using this method we did detect a slight variation of the expression of
arg2 (+ 1.91) and
odc1 (+ 1.18) (Table ). Therefore, in presence of amastigotes,
arg2 could favour arginine transformation into ornithine, the latter being catalyzed in turn by
odc1 to generate putrescine (Fig. ).
ODC1-antizyme plays a role in the regulation of polyamine synthesis by binding to and inhibiting ODC1. The transcript abundance of
azin1 encoding ODC1-antizyme inhibitor-1 was higher (+ 1.96) when amastigotes were present, so that this inhibitor might prevent antizyme-mediated ODC1 degradation. Of note, ornithine could also be generated from proline by
p4ha2 (+ 2.27), and putrescine from spermine and spermidine by the successive action of
sat1 (+ 1.47) and
maoa (+ 2.56). Spermidine synthase (
srm) and spermine synthase (
sms), two enzymes catalyzing the reverse reactions leading to the formation of spermine from putrescine, were not detected with Affymetrix (5% threshold), although their transcript abundance decreased in presence of amastigotes (-1.22 and -1.38, respectively; see Additional file
1). No gene expression modulation was detected with Affymetrix for
nos2 (5% threshold) that encodes a competing enzyme for arginine substrate leading to the production of microbe-targeting nitric oxide derivatives (fluorescence intensity was below the background level, see Additional file
1), and only a slight up-regulation was detected with RTQPCR (+ 1.28) (Table ). The present data further extend former observations [
15,
16], and highlight a coordinated gene expression modulation that sustains a metabolic flux leading to the biosynthesis of putrescine from arginine and proline
via ornithine, and from spermine and spermidine.
L. amazonensis amastigotes set up an optimal dermis niche
Decreased expression of genes involved in the entry of non leishmanial micro-organisms as well as in the sensing and processing of microbial molecules
Several genes involved in classical and alternate complement component pathways were down-regulated (
c1qa,
c1qb,
serping1,
c3,
c4b,
cfh,
c5ar1 and
pros1: -2.80 < FC < -1.35) as well as some genes of the Toll-like receptor signalling pathway (
tlr2,
tlr7,
tlr8,
cd14,
mapk14,
c-fos and
nfkbia: -3.11 < FC < -1.61. Furthermore, the negative regulator
tollip also was up-regulated (+ 1.69). These pathways are known to contribute to the entry of micro-organisms and the sensing/processing of microbial molecules. In presence of the intracellular cell-cycling amastigotes these biological processes would be restricted, if not prevented. Indeed, it is conceivable that non-
Leishmania micro-organisms or microbial molecules might trigger a different MΦ transcriptional program that could interfere with the one already set up by
L. amazonensis amastigotes for their multiplication. Nevertheless, it has recently been demonstrated that the other
L. amazonensis developmental stage, the promastigote, was still able to enter MΦ already hosting amastigotes, to transform into amastigote and to multiply efficiently within the PV [
17].
The above data suggested that
L. amazonensis amastigotes were able to control MΦ expression of the early complement components, the proteolytic products of which are known to be pro-inflammatory. This complement component pathway down-modulation was also recently described for human MΦ housing
L. major parasites [
18]. The down-modulation of the Toll-like receptor pathway also suggested prevention of the inflammatory process signalling. At this stage, although some anti-inflammatory genes were not up-modulated (
il10: -2.97 and
il10ra: -2.16) the gene expression modulation for the majority of the listed genes involved in inflammatory processes showed that the presence of cell-cycling amastigotes imposed an immune unbalance favouring the shaping of a counter-inflammatory and safe dermis niche for these parasites (
il1rn,
il1b,
il11ra1,
il17rb, il18, socs6, cd200, nfkbia, relB, c-fos and
anxA1, an inhibitor of phospholipase A2 mediated-inflammation: 1.41 < | FC | < 4.19).
Decreased expression of genes involved in the chemokine-dependent MΦ traffic
The down-modulation of the expression of genes encoding chemokine receptors (
ccr2,
ccr3,
cx3cr1 and
cmklr1: -2.65 < FC < -1.83) suggested that amastigote-harbouring MΦ were less responsive to chemo-attractant gradients and thus less amenable to enter into the afferent lymphatics. This is consistent with the dominant residence of
L. amazonensis-hosting MΦ in the skin. In favour of this possible reduced emigration of MΦ from the dermis niche was the down-regulation of
itga4 (-2.06) encoding an integrin shown to contribute to the lymphatic adhesion/transmigration [
19]. It is beyond the scope of this article to discuss about more than a dozen of chemokine receptor ligands the gene expression of which was modulated (see Additional file
1). Indeed, the interpretation is not that straightforward because of the complexity of their partial overlapping functions and/or common receptors.
Decreased expression of genes involved in the cellular communication with leukocytes prone to display parasite-damaging functions
The modulation of several transcripts indicated a prevention of MΦ communication with leukocytes that could be rapidly recruited such as NK lymphocytes, and T-lymphocytes. For instance, H60 is one of the ligand able to efficiently activate NK-lymphocytes by binding to the NKG2D receptor (encoded by
klrk1). In presence of amastigotes, the
h60 MΦ expression was down-modulated (-2.07), suggesting the prevention of this "immune synapse" by which parasitized MΦ and NK lymphocytes can communicate. Interestingly, NKG2D receptor engagement by H60 ligand in MΦ, that normally leads to the production of MΦ leishmanicidal molecules such as NO and TNF-α [
20], could be impaired in MΦ hosting amastigotes since the expression of
klrk1 gene was also down-modulated (-1.72). Besides, the gene expression of the co-stimulatory molecule CD86 was reduced (-1.83), while that of the inhibitory receptor CD274 (also referred to as B7-H1) was increased (+ 1.93). In addition, the transcript abundance of the co-stimulatory molecules ICAM1 (-1.75), ICAM2 (-1.85) and LFA-1 (or integrin-alpha L, – 2.0) was also reduced. The down-modulation of several genes involved in antigen presentation by MHC class II molecules was recently discussed for human MΦ housing
L. major parasites [
18]. This data suggested plausible reduced effectiveness of this other "immune synapse" involving TCR-dependent signalling by which MΦ and T-lymphocytes can communicate. Consistent with this was the reduced transcription level in MΦ hosting
L. amazonensis amastigotes of
h-2ma (-1.88) and of
ifngr1 (-1.83 FC) that encodes the receptor for IFNγ, a cytokine secreted by both activated NK- and T-lymphocytes and involved upstream the MHC class II gene up-regulation.
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