Leishmania infection activates PPAR gene expression. In (a) and (b), BALB/c mice were infected i.v. with 10⁷ stationary phase promastigotes of L. donovani for 4 weeks, and then liver and spleen, respectively, were harvested. In (c), peritoneal exudate cells (PECs) were obtained from the peritoneum of normal BALB/c mice, infected with L. donovani at 1 : 10 ratio, and then harvested for RNA isolation after 2 days. PPAR activation was measured by real-time RT-PCR with normalization to 18S or actin RNA, and modulation was compared and expressed relative to the uninfected control using the delta-delta Ct method. The infected groups (black bars) showed higher levels of gene expression in liver (a), spleen (b), and PECs (c) in comparison to uninfected controls (open bars). Statistical analysis was performed by Mann-Whitney U test, and a P  value of less than 0.05 was considered as significant.

Blocking PPARγ activation with an antagonist reduces L. donovani infectivity. In (a), peritoneal exudate cells were infected by L. donovani using a 1 to 10 : PEC to promastigote ratio. The cultures were incubated with 4 ng/mL of IL-4 for 24 hours, then total RNA was harvested, and RT-PCRs were performed to quantify the copies of PPARγ and β-actin mRNA as described in Adapala and Chan [16]. In (b), PECs from C57/BL6 mice were attached to cover slips and infected with L. donovani promastigotes at 1 : 5 ratio. After 20 to 24 hours, 5 ng/mL of IL-4 and various concentration of SR202 were added. The infection was allowed to develop at 37°C in a 5% CO₂ incubator for another 3 days. Then, the cover slips were fixed in methanol and stained with Diff-Quik. The degree of parasite burden was determined by enumeration under a microscope in a double-blind manner by at least two individuals. Uninfected macrophages, infected macrophages, and the number of amastigotes in these macrophages were counted. The result is reported as amastigotes/macrophage, and each data point was derived from counting at least about one hundred macrophages or one hundred infected macrophages, as appropriate. (c) shows the levels of nitric oxide in PECs that were similarly infected with L. donovani promastigotes, except that IFNγ was added instead of IL-4 to stimulated inducible nitric oxide synthase expression. On day 5, the amount of nitric oxide was determined with Griess reagent. Shown are the relative levels of nitrite, oxidized form of nitric oxide, in the culture supernatants. In (d), SR202 was added to freshly harvested, uninfected PECs and bone marrow cells at various concentrations. After 3 days, the number of live cells was determined by counting with trypan blue. In (e), SR202 was added to promastigotes, and after 5 days of proliferation, the number of parasites was determined by counting under a microscope. The results shown are representative of three independent experiments.

Chemical structure of curcumin.

Curcumin induces PPARγ mRNA expression and reduces iNOS mRNA expression in infected macrophages. In (a), PECs of BALB/c mice were infected with L. donovani promastigotes, and then 10 μM of curcumin (brown bar) or vehicle control (0.1% acetone) was added. After 2 days, the cells were harvested for RNA isolation to determine the level of PPARγ and β-actin expression by real-time RT-PCR. In (b), murine RAW264.7 cells were infected with L. donovani for 16–20 hours. Then, different concentrations of curcumin were added. Thirty minutes after curcumin treatment, IFNγ was added to activate the macrophages. At 5 hours after the addition of IFNγ, the cells were harvested, mRNA was extracted, and conventional RT-PCR was performed. The gel shows the end point PCR-amplified iNOS (496 bp) and β-actin cDNAs products. In (c), nonelicited PECs from resistant C3H and susceptible BALB/c strains were cultured with L. donovani promastigotes in wells that contained cover slips for a period of 16–20 hours; then curcumin and IFNγ and TNFα were added. On day 4-5, the coverslips were stained to enumerate the percent of infected macrophages and number of amastigotes per macrophage under a microscope, similar to steps described in Figure 3.

Kinetics of PPAR expression and parasitic infection. BALB/c mice were intravenously injected with 10⁷ stationary phase promastigotes of L. donovani 1S. At two-week intervals, groups (n = 5) were sacrificed, and their liver and spleen were harvested. PPAR expression was determined by real-time RT-PCR as described in legend of Figure 1. The amount of parasites in the organs was determined by limiting dilution analysis according to the procedure of Titus and colleagues [17]. Cells were plated into wells of 96-well plates at a range of concentrations, according to the number of red blood cells in the liver and splenocytes in the spleen suspensions and then incubated at 27°C to allow the parasites to transform from intracellular amastigotes into promastigotes. After 2-3 weeks of proliferation, the number of wells that shows parasite growth was scored under a microscope, and the L-Calc software for limiting dilution analysis (provided by Stem Cell Technology, Vancouver, Canada) was used to determine the frequency of parasite. Normalization was based on the number of red blood cells in the liver cultures (a) and the number of splenocytes in the spleen cultures (b).

Morphology of the peritoneal macrophages after SR202 treatment. Micrograph of cover slips from the cultures described in Figure 3(b). The black arrows point to macrophages with phagolysosomes filled with amastigotes. The black arrows point to infected macrophages and the red arrow points to infected macrophages with phagolysosomes cleared of amastigotes.

Activation of PPAR enhances parasitic infection. Mice were infected with L. donovani. One group was fed with 0.2 mL freshly prepared curcumin solution, given by oral gavage every other day throughout the course of the study. Curcumin was dissolved to a solution of 7.52 mg/mL in 0.1 N NaOH and immediately brought to pH 7.2 by diluting to a concentration of 11.1 μg/mL in PBS. The second group received phosphate buffered saline (PBS) in the same manner. At 4 weeks, the peak of hepatic infection (as shown in Figure 2), livers, and spleens were harvested for DNA and RNA extractions. PPAR and iNOS expression were determined by real-time RT-PCR and normalized to 18S RNA. Parasite load in the liver and spleen was determined by the real-time PCR procedure which was that of Nicolas and colleagues [45], except the reaction occurred in SYBR Green I PCR master mix (from Superarray). DNA, at 40 ng per reaction, was denatured at 95°C for 10 min, and then Leishmania kinetoplast DNA (kDNA) was amplified in a thermal cycler (Rotor-gene 6.0, from Corbett). The number of copies of kDNA per μg of DNA was determined using a standard curve that was established with the cloned PCR product. Filled diamonds were curcumin-treated, and clear circles were saline controls. N indicates the number of mice in each group. Statistic analysis was performed by ANOVA after the data underwent natural log transformation as described in Adapala and Chan [16]. A P value of <0.05 was considered as significant.

Scheme of Leishmania interaction with mediators of the resolution process during inflammation. When infected sandflies bite, promastigotes enter the neutrophils and macrophages that are recruited to the inflamed site of injection. The parasite can, by itself, activate the infected host cells to produce PPAR activators. This includes PPARγ agonists such as the bioactive lipids 15d-PGJ₂ and LXA₄ from the arachidonic acid pathways. Engulfment of apoptotic neutrophils and IL-4 from T helper 2 cells can activate PPARγ as well. Activation of PPARγ polarizes the host macrophage towards the alternatively activated macrophage (M2) phenotype, which would produce arginase to divert substrate from iNOS and thus reduce the production of nitric oxide. As such, the parasite can survive and multiply within the host's macrophages, and the infection becomes chronic.