NO production by macrophages stimulated with IFN-γ + Leishmania is inhibited by LXR ligands.

<p>BMDM were preincubated overnight with LXR (GW  =  GW3965) and RXR (LG  =  LG268) ligands, with or without IFN-γ, prior to a 1 hour <i>in vitro</i> infection with stationary-phase <i>L. chagasi/infantum</i>. Amount of NO secreted in 24 hours by wild-type BMDM stimulated with <i>IFN-γ + Leishmania</i> in the absence of ligand was normalized to 100%. L-NNMA is a NOS inhibitor. Error bars represent standard deviations of triplicate NO measurements. Results are representative of at least 3 independent experiments; ** denotes p<0.005.</p

Gene expression in wild-type and LXR-deficient tissues and macrophages following Leishmania infection.

<p>A. Wild-type and LXRα-deficient mice were intravenously challenged with 1×10⁷ live, stationary-phase <i>L. chagasi/infantum</i> parasites, and RNA was prepared from livers and spleens 24 and 72 hours following infection. Real-time RT-PCR was performed with SYBR primers specific for LXRα and LXRβ. B. BMDM were preincubated overnight with IFN-γ, infected with stationary-phase <i>Leishmania</i> for 1 hour, washed to remove extracellular parasites, and incubated for an additional 2 hours. RNA was harvested, and levels of various genes were determined by real-time RT-PCR. Error bars represent standard deviations of replicate infections; ** denotes p<0.005. Results are representative of at least 3 independent experiments.</p

LXR-deficient macrophages produce increased levels of NO following exposure to Leishmania and IFN-γ in vitro.

<p>A. BMDM from individual wild-type and DKO (Sv129xC57Bl/6) mice were stimulated <i>in vitro</i> with stationary-phase <i>L. chagasi/infantum</i> promastigotes for the times indicated, in the absence (left) or presence (right) of IFN-γ pre-treament. B. BMDM (left) or peritoneal macrophages (right) from wild-type (solid bars) or LXR-DKO (empty bars) mice (C57Bl/6 background) were infected with stationary-phase <i>L. chagasi/infantum</i> (with or without IFN-γ pre-treatment) for 24 hours <i>in vitro</i>. Supernatants were removed and levels of NO were quantitated by the Griess method. Error bars represent standard deviations of triplicate NO measurements. Results are representative of at least 3 independent experiments; * denotes p<0.05, ** p<0.005.</p

LXR-deficient mice display enhanced resistance to Leishmania infection of the liver and spleen.

<p>Wild-type or LXR-deficient C57Bl/6 mice (except for (B), which were the mixed Sv129xC57Bl/6 background) were challenged with 1×10⁷ live, stationary-phase <i>L. chagasi/infantum</i> parasites, and livers and spleens harvested at various time points. A–B. Parasite loads in the livers at d28 were quantitated by microscopy. Leishman-Donovan Units (LDU)  =  [(# amastigotes/# mononuclear cells) multiplied by liver weight in mg]. C–F. Parasite loads in liver (C, E) and spleen (D, F) were quantitated by qPCR of genomic DNA from wild-type (black bars) and LXRα^−/− (white bars) tissue. Levels are expressed as # parasites/µg tissue. Groups consisted of 7–9 mice/group for A–D, 5 mice/timepoint for E–F. Results are representative of at least 3 independent experiments. Error bars represent standard error; * denotes p<0.05, ** p<0.005, p-value for <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000886#pntd-0000886-g001" target="_blank">Fig. 1D</a>  = 0.06, comparisons to wild-type.</p

LXR-deficient macrophages are infected similarly, but kill Leishmania more efficiently than wild-type macrophages treated with IFN-γ.

<p>Bone marrow-derived macrophages (BMDM) were infected with stationary-phase, (A) CFSE-labeled or (B) unlabelled <i>L. chagasi/infantum</i> at an MOI of 10∶1 for 1 hour, and 2 hours later percentages of infected cells (CFSE-positive events) were determined by (A) flow cytometry (WT uninfected  =  gray dotted line; WT infected  =  black solid line; LXRα^−/− infected  =  gray solid line; LXR-DKO infected  =  black dotted line) or (B) microscopy (the number of parasites per cell was also assessed by microscopy). C. BMDM were infected with stationary-phase, unlabelled <i>L. chagasi/infantum</i> at an MOI of 20∶1 for 2 hours in the presence or absence of IFN-γ (overnight pretreatment), and immediately, 24, or 48 hours later were Giemsa-stained for microscopic quantitation of numbers of amastigotes per cell. Similar results were found in least 3 independent experiments; error bars represent standard error; * denotes p<0.01.</p