CCL2 Inhibition of Pro-Resolving Mediators Potentiates Neuroinflammation in Astrocytes

Abstract

The chemokine CCL2 participates in multiple neuroinflammatory processes, mainly through the recruitment of glial cells. However, CCL2 has also been proven to exert different types of actions on these cells, including the modification of their response to inflammatory stimuli. In the present study we analyzed the effect of CCL2 on the resolution of inflammation in astrocytes. We observed that genetic removal of CCL2 increases the expression of the enzymes responsible for the synthesis of specialized pro-resolving mediators arachidonate 15-lipoxygenase and arachidonate 5-lipoxygenase in the brain cortex of 5xFAD mice. The expression of FPR2 receptor, known to mediate the activity of pro-resolving mediators was also increased in mice lacking CCL2.The downregulation of these proteins by CCL2 was also observed in cultured astrocytes. This suggests that CCL2 inhibition of the resolution of inflammation could facilitate the progression of neuroinflammatory processes. The production of the pro-inflammatory cytokine IL-1beta by astrocytes was analyzed, and allowed us to confirm that CCL2 potentiates the activation of astrocytes trough the inhibition of pro-resolving pathways mediated by Resolvin D1. In addition, the analysis of the expression of TNFalpha, MIP1alpha and NOS2 further confirmed CCL2 inhibition of inflammation resolution in astrocytes.

Keywords: CCL2; LPS; MCP-1; astrocytes; inflammation; resolvin.

Figure 1

CCL2 deficiency increases the expression of pro-resolution mediators. FPR2, 15-LOX and 5-LOX mRNA concentrations were analyzed in brain cortex samples from WT, 5xFAD, CCL2-KO and 5xFAD/CCL2-KO mice. Data are means ± SE of n = 6 replicates per group. *** p < 0.001, ** p < 0.01 vs. WT.

Figure 2

CCL2 prevents the expression of pro-resolution markers. Astrocyte cultures were incubated with or without CCL2 (100 ng/mL). Twenty-four hours later, the whole medium was replaced in all wells, maintaining CCL2 where it was used for pre-treatment and adding LPS (0.1 g/mL) to indicated groups. Six hours later, RNA was isolated and 15-LOX, 5-LOX, FPR2 and GPR32 mRNA levels determined by RT-PCR. Data are means ± SEM of n = 6 replicates per group. *** p < 0.001, ** p < 0.01 vs. C. ^ϕϕϕ p < 0.001, ^ϕϕ p < 0.01 vs. LPS. ϕ.

Figure 3

CCL2 induces production of astrocytic inflammatory markers. (A) Astrocyte cultures were incubated with or without CCL2 (100 ng/mL). Twenty-four hours later, the whole medium was replaced in all wells, maintaining CCL2 where it was used for pre-treatment and adding LPS (0.1 g/mL) to indicated groups. Forty-eight hours later, RNA was isolated and IL-1β mRNA levels determined by RT-PCR. Data are means ± SEM of n = 6 replicates per group. *** p < 0.001 vs. C-C. ^ϕ p < 0.05 vs. LPS. (B) IL-1β concentration in the media was measured by ELISA. Data are means ± SEM of n = 6 replicates per group. *** p < 0.001 vs. C. ^ϕ p < 0.05 vs. LPS. (C) Cytosolic lysates were examined for the presence of IL-1β and β-actin protein by Western blot analysis. The gels shown are representative of experiments done on three separate astrocyte preparations. (D) Densitometric analysis of the bands. AU: arbitrary units relative to C. *** p < 0.001 vs. C. ^ϕ p < 0.05 vs. LPS. (E) Astrocyte cultures were incubated with vehicle (EtOH 0.037%), RvD1 100 nM (dissolved in EtOH 0.037%) or RvD1+CCL2 (100 ng/mL). Twenty-four hours later, the whole medium was replaced in all wells, maintaining RvD1 and CCL2 where it was used for pre-treatment and adding LPS (0.1 g/mL) to all groups except the control one. Forty-eight hours later, RNA was isolated and IL-1β mRNA levels determined by RT-PCR. Data are means ± SEM of n = 6 replicates per group. *** p < 0.001 vs. C. ^ϕϕ p < 0.01 vs. LPS. ^δδδ p < 0.001 vs. LPS+RvD1. (F) Astrocyte cultures were incubated with or without CCL2 (100 ng/mL). Twenty-four hours later, the whole medium was replaced in all wells, maintaining CCL2 where it was used for pre-treatment and adding LPS (0.1 g/mL) to indicated groups. Forty-eight hours later, RNA was isolated and TNFα, MIP1α and NOS2 mRNA levels determined by RT-PCR. Data are means ± SEM of n = 6 replicates per group. *** p < 0.001 vs. C-C. ^ϕϕϕ p < 0.001, ^ϕ p < 0.05 vs. LPS.