PPAR Research / 2012 / Article / Fig 3

Review Article

Peroxisome Proliferator-Activated Receptor-γ-Mediated Polarization of Macrophages in Leishmania Infection

Figure 3

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.
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