Central activation of the fatty acid sensor GPR120 suppresses microglia reactivity and alleviates sickness- and anxiety-like behaviors

Abstract

G protein-coupled receptor 120 (GPR120, Ffar4) is a sensor for long-chain fatty acids including omega-3 polyunsaturated fatty acids (n-3 PUFAs) known for beneficial effects on inflammation, metabolism, and mood. GPR120 mediates the anti-inflammatory and insulin-sensitizing effects of n-3 PUFAs in peripheral tissues. The aim of this study was to determine the impact of GPR120 stimulation on microglial reactivity, neuroinflammation and sickness- and anxiety-like behaviors by acute proinflammatory insults. We found GPR120 mRNA to be enriched in both murine and human microglia, and in situ hybridization revealed GPR120 expression in microglia of the nucleus accumbens (NAc) in mice. In a manner similar to or exceeding n-3 PUFAs, GPR120 agonism (Compound A, CpdA) strongly attenuated lipopolysaccharide (LPS)-induced proinflammatory marker expression in primary mouse microglia, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and inhibited nuclear factor-ĸB translocation to the nucleus. Central administration of CpdA to adult mice blunted LPS-induced hypolocomotion and anxiety-like behavior and reduced TNF-α, IL-1β and IBA-1 (microglia marker) mRNA in the NAc, a brain region modulating anxiety and motivation and implicated in neuroinflammation-induced mood deficits. GPR120 agonist pre-treatment attenuated NAc microglia reactivity and alleviated sickness-like behaviors elicited by central injection TNF-α and IL-1β. These findings suggest that microglial GPR120 contributes to neuroimmune regulation and behavioral changes in response to acute infection and elevated brain cytokines. GPR120 may participate in the protective action of n-3 PUFAs at the neural and behavioral level and offers potential as treatment target for neuroinflammatory conditions.

Keywords: Anxiety; Behavioral neuroimmunology; Endotoxemia; Ffar4; Free fatty acid receptor; Locomotion; Neuropharmacology; Nucleus accumbens; Sickness behaviors.

Fig. 1

Central GPR120 expression and microglial anti-inflammatory function. A Distribution of GPR120 (free fatty acid receptor 4, Ffar4) mRNA in mouse brain (whole: whole brain, NAc: nucleus accumbens, DS: dorsal striatum, Amy: amygdala, Hippo: hippocampus, PFC: prefrontal cortex, Hypo; hypothalamus) (n = 3–5/group). B GPR120 mRNA expression in primary murine microglia (MG), neurons (Neu), and astrocytes (Ast) (n = 5). C GPR120 mRNA expression in human fetal microglia (MG), neurons (Neu), and astrocytes (Ast) (n = 3–4). Effect of GPR120 agonism on D Ffar4 and E Iba-1 mRNA expression on LPS-stimulated primary microglia (n = 6–11/group). Proinflammatory cytokine mRNA expression on LPS-stimulated primary cultured microglia pre-treated with F CpdA or G unsaturated FAs (OA; oleic acid, ALA; α-linolenic acid, EPA; eicosapentaenoic acid, DHA; docosahexaenoic acid). H Cytokine protein levels in culture medium (n = 3–5/group). I Representative image of NFκB translocation after LPS-treatment with or without CpdA pre-treatment. Scale bar, 20 µm. NFκB intensity in J nuclear and K cytoplasmic compartments. L Ratio of nuclear/cytoplasmic NFκB (n = 63, from 3 cover slips). Data are expressed as the mean ± SEM. One-way ANOVA with post hoc Sidak multiple comparison test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 vs Veh LPS

Fig. 2

Central GPR120 agonism reduces LPS-induced hypolocomotion and anxiety-like behavior. A Experimental design of LPS-CpdA in vivo study 1. B Total distance travelled, C time spent, and D number of entries made into open arms of the elevated-plus maze (EPM). E Total distance travelled, F time spent, and G number of entries in the light compartment of the light–dark box. Effect of GPR120 agonism on LPS-induced H Iba-1, I TNF-α, J IL-1β, and K IL-6 mRNA expression in the nucleus accumbens (NAc). n = 7–8/group. Mean ± SEM; *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001

Fig. 3

GPR120 agonism attenuates nucleus accumbens microglial reactivity. A GPR120 (red) expression in NAc microglia (Tmem119, green) detected by RNAscope in situ hybridization in adult mice. Scale bar, 20 µm. B The effect of IL-1β and/or TNF-α application on Iba-1, TNF-α, and IL-1β mRNA expression in primary microglia derived from NAc. C Time course of cytokine -induced proinflammatory and microglial marker mRNA expression. Effect of GPR120 agonist pre-treatment on cytokine-induced D Iba-1, E TNF-α, F IL-1β, G IL-6, and H Ccl2 mRNA expression (n = 4). I Representative images of NAc microglia in slice preparations derived from mice ICV-injected with the TNF-α and IL-1β mixture with/without ICV CpdA pre-treatment. Scale bar, 20 µm. J Number of Iba-1 positive cells (n = 3). K Iba-1 intensity, L cell area, M cell perimeter, N circularity, O roundness, and P branches (n = 41–47 microglia/n = 3 brain). Mean ± SEM; one-way ANOVA with post hoc Sidak multiple comparison test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 vs Veh

Fig. 4

Central GPR120 agonism protects against central cytokine-induced behavioral responses. A Experimental design of central cytokine in vivo study 2. B Total distance travelled, C time spent, and D number of entries made in open arms of the elevated-plus maze (EPM) test. E Total distance travelled, F time spent, and G number of entries made in the light compartment of the light–dark box (LDB) test. H Total distance travelled, I number of entries in mouse zone, J duration of time spent in mouse zone in the 3-chamber (3-CT) social interaction test. n = 11–14/group. Mean ± SEM; one-way ANOVA with post hoc Sidak multiple comparison test;*p ≤ 0.05, **p ≤ 0.01

Fig. 5

Working model of GPR120 stimulation modulation of acute neuroinflammation and associated behavioral changes. GPR120 agonism attenuates LPS or cytokines-induced microglial activation and cytokine production. Sickness- or anxiety-like behaviors caused by systemic LPS injection and central cytokine infusion are rescued by ICV GPR120 agonist treatment