Palmitoylethanolamide induces microglia changes associated with increased migration and phagocytic activity: involvement of the CB2 receptor

Abstract

The endogenous fatty acid amide palmitoylethanolamide (PEA) has been shown to exert anti-inflammatory actions mainly through inhibition of the release of pro-inflammatory molecules from mast cells, monocytes and macrophages. Indirect activation of the endocannabinoid (eCB) system is among the several mechanisms of action that have been proposed to underlie the different effects of PEA in vivo. In this study, we used cultured rat microglia and human macrophages to evaluate whether PEA affects eCB signaling. PEA was found to increase CB2 mRNA and protein expression through peroxisome proliferator-activated receptor-α (PPAR-α) activation. This novel gene regulation mechanism was demonstrated through: (i) pharmacological PPAR-α manipulation, (ii) PPAR-α mRNA silencing, (iii) chromatin immunoprecipitation. Moreover, exposure to PEA induced morphological changes associated with a reactive microglial phenotype, including increased phagocytosis and migratory activity. Our findings suggest indirect regulation of microglial CB2R expression as a new possible mechanism underlying the effects of PEA. PEA can be explored as a useful tool for preventing/treating the symptoms associated with neuroinflammation in CNS disorders.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

PEA induces CB2R expression via PPAR-α activation. (A) Real-time PCR analyses of CB2m-RNA in cultured microglia following incubation with PEA (1, 10 and 100 nM for 24 hours), using 18S as a loading control. Data are shown as mean ± SD (n = 6–8). *P < 0.05 compared to vehicle-treated cells. One way ANOVA, post-hoc Tukey’s. (B) Representative western blot image and related quantification showing the expression of CB2R in microglial cell lysates following incubation with vehicle, PEA (100 nM), GW6471 (10 µM) + PEA, or GW6471 alone, using β-actin as a loading control. Data are shown as mean ± SD (n = 6–8). *P < 0.05 compared to vehicle-treated cells, °P < 0.05 compared to PEA-treated cells. One way ANOVA, post-hoc Tukey’s. (C) CB2R expression (red) in Iba-1 labeled microglia (green) following incubation with vehicle, PEA (100 nM), GW7647 (1 µM), IFNγ (300 U/ml). Scale bar = 25 μm. (D) PPAR-α expression (green) in microglia cells (red) following incubation with vehicle, PEA (100 nM), GW7647 (1 µM), IFNγ (300 U/ml). Scale bar = 25 μm. (E) Representative western blot image and related quantification showing the expression of CB2R in microglial cell lysates incubated for 24 hours with vehicle, PEA (100 nM), GW7647 (1 µM), IFNγ (300 U/ml), in presence of siRNA or non-targeting siRNA. Data are shown as mean ± SD (n = 6). *P < 0.05 compared to vehicle, °P < 0.05 compared to relative control within non-targeting siRNA group. One way ANOVA, post-hoc Tukey’s. (E’) Real-time PCR analyses of PPAR-α/HPRT ratio in PPAR-α silencing (siRNA) or non-targeting siRNA conditions, compared with vehicle. Data are shown as means ± SD (n = 6). *P < 0.05, One way ANOVA, post-hoc Tukey’s. (F) Representative staining and related quantification of microglia cells in Iba-1 labeled microglia incubated for 24 hours with PEA (100 nM), GW7647 (1 µM) or IFNγ (300 U/ml), compared with vehicle-treated cells. Morphological evaluations measured as a percentage of activated cells over the total cell number. Data are shown as mean ± SEM (n = 6). ***Indicates statistically significant values (P < 0.0001) vs. vehicle-treated cells. One way ANOVA, post-hoc Tukey’s. Full-length gels are showed in Supplementary Figs S6 and S7.

Figure 2

Cnr2 is directly regulated by PEA-mediated PPAR-α activation: bioinformatic analysis and chromatin immunoprecipitation assay. (A) A schematic alignment of the human and rat Cnr2. The black boxes correspond to regions of high sequence homology between the two species contaning the putative PPARα/RXR sites indicated as small black rectangles in the lower part below the schematic. The PPARα/RXR matrix used for the analysis is also shown in the upper part. (B) Average data of the relative amount of the PPARα -immunoprecipitated DNA in HEK293 cells transfected with either control (pCDNA3) or murine PPARα and RXR encoding plasmids. Data are from three to six separate experiments and normalized relative to the input DNA, using the 2−ΔΔCt formula. The inset shows a representative agarose gel electrophoresis of the qPCR products obtained from PAX7-immunoprecipitated DNA for each experimental condition. Data are shown as mean ± SEM (n = 3) of three independent experiments conducted in triplicate. *p value ≤ 0.05 vscontrol condition (pcDNA-transfected cells), obtained using the unpaired T-TEST for statistical analyses.

Figure 3

PEA enhances phagocytosis and intracellular killing of P. gingivalis by microglial cells. Phagocytosis assay (A) and intracellular survival evaluation (B) were performed in the presence of PEA (100 nM), or GW6471 (10 µM) + PEA, or AM630 (100 nM) + PEA, or GW6471 alone. The number of bacteria ingested (at 90 min) or number of bacteria recovered (at 270 min) by the control group (vehicle) were considered to be 100%. Data are shown as means ± SEM (n = 6). *P < 0.05 compared to vehicle, °P < 0.05 compared to PEA. One way ANOVA, post-hoc Tukey’s.

Figure 4

PEA induces microglia migration towards a 2-AG source. (A) Image-based detection of 2-AG (100 µM) chemoattractive effect on cultured microglial cells after different treatments. Panel shows the migratory effect induced by PEA (100 nM) or GW7647 (1 µM), alone or in the presence of GW6471 (10 µM) or AM630 (100 nM), as compared to the control group (vehicle). Representative data of microglial cell time-lapse migration recorded at starting point (0 min), 30 and 90 min from the 2-AG exposure. (B) The quantification indicates the percentage of microglia cells affected by 2-AG chemoattractive movements vs total cell number at 90 min. Data are shown as means ± SEM (n = 6–8). *P < 0.05 compared to vehicle and °P < 0.05 compared to PEA or GW7647. One way ANOVA, post-hoc Tukey’s. Scale bar = 50 µm.