Activating cannabinoid receptor 2 alleviates pathogenesis of experimental autoimmune encephalomyelitis via activation of autophagy and inhibiting NLRP3 inflammasome

Abstract

Aims: Activation of cannabinoid receptor 2 (CB2R) has been reported to ameliorate the pathogenesis of experimental autoimmune encephalomyelitis (EAE). In this study, we examined whether autophagy is involved in the beneficial effect of CB2R on EAE and explored the mechanism with a focus on inflammasome activation.

Methods: EAE severity was analyzed with clinical score and histological score stained by hematoxylin and eosin or luxol fast blue in spinal cord. Immunoblot analysis was conducted to detect proteins of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-related caspase-1 (Casp-1) and the maturation of interleukin (IL)-1β as well as autophagy-related light chain 3 (LC3), and Beciln 1 both in vivo and in vitro. Reverse transcription and real-time PCR were used to detect mRNA of NLRP3, IL-1β and Casp-1. Autophagy-related gene 5 (ATG5)-specific siRNA was transiently transfected in BV2 microglia, and immunofluorescence staining was carried out to detect the expression of NLRP3, caspase recruitment domain (ASC), and pro-caspase-1.

Results: The current data indicated that deleting CB2R decreased the expression of LC3-II/LC3-I ratio, Beclin 1 and increased caspase-1 activation and IL-1β production in the spinal cord of EAE mice, whereas activation of CB2R with a specific agonist HU-308 induced inverse effects. Further study indicated that HU-308 could promote autophagy and inhibit expression and activation of NLRP3 inflammasome in BV2 microglia. Blocking autophagy by ATG5-specific siRNA dismissed the effort of CB2R in mediating NLRP3 inflammasome in vitro.

Conclusion: Collectively, our results demonstrated for the first time that CB2R plays a protective role in EAE through promoting autophagy and inhibiting NLRP3 inflammasome activation.

Keywords: Autophagy; Cannabinoid receptor 2; EAE; NLRP3 inflammasome.

Conflict of interest statement

The authors declare no competing financial interests.

© 2014 John Wiley & Sons Ltd.

Figures

Figure 1

CB2R activation alleviates clinical symptoms and CNS infiltration in EAE mice. EAE mice were treated with HU‐308 (0.3, 1 and 3 mg/kg/day, i.p.) or vehicle (Veh) from days 3 postimmunization (PI) and were maintained on drug for the duration of the study. (A) Clinical signs were assessed daily by researchers as described in Methods. 1 and 3 mg/kg HU‐308 significantly reduced the peak severity and cumulative clinical score of EAE (n = 16 per group). ###P < 0.001 vs Veh. (B) H&E staining and (C) Luxol fast blue staining of paraffin sections of spinal cords isolated from normal, vehicle, or HU‐308 (1 mg/kg)‐treated EAE mice on day 17. (D–E) Quantification of CNS infiltrates and the amount of demyelination presented in B and C. (n = 6 per group). ***P < 0.001 versus normal, ###P < 0.001 versus Veh.

Figure 2

CB2R mediates inflammasome activation in EAE mice. (A–B) Spinal cords were isolated from EAE mice on day 10 PI and then lysed with buffer. Caspase 1 (Casp‐1) activation and IL‐1β production were analyzed using Western blotting. CB2R knock‐out (KO) increased Casp‐1 activation and IL‐1β production, whereas activation of CB2R with HU‐308 induced inverse effect (n = 6 per group). **P < 0.01 versus wild‐type (WT); ##P < 0.01 versus Veh. (C–D) Spinal cords and brains were isolated from EAE mice, and levels of NLRP3 mRNA were analyzed with real‐time PCR. CB2R‐KO increased levels of NLRP3 mRNA in both spinal cord and brain, whereas activation of CB2R with HU‐308 induced inverse effect (n = 6 per group). **P < 0.01 versus WT, ##P < 0.01 versus Veh.

Figure 3

CB2R increases autophagy in CNS. Spinal cords were isolated from EAE mice on day 10 PI, and then lysed with buffer. (A) CB2R‐KO decreased the expression of LC3‐II/LC3‐I ratio and Beclin 1 (n = 6 per group). **P < 0.01 versus WT. (B) Activation of CB2R with HU‐308 significantly increase the expression of LC3‐II/LC3‐I ratio and Beclin 1 (n = 6 per group). ##P < 0.01 versus Veh.

Figure 4

CB2R agonist HU‐308 induces autophagy and inhibits NLRP3 inflammasome activity in BV2 microglia. BV2 microglia were pretreated with vehicle or HU‐308 (10 μM) for 10 min and then stimulated with LPS (100 ng/mL) and ATP (1 mM). (A–C) BV2 microglia were lyzed and expression of LC3‐II/LC3‐I ratio and Beclin 1 were analyzed using Western blotting. HU‐308 significantly increase the expression of LC3‐II/LC3‐I ratio and Beclin 1 (n = 6 per group). **P < 0.01 versus Normal; ##P < 0.01 versus LPS/ATP. (D–F) Cells were housed and mRNA levels of NLRP3, Casp‐1 and IL‐1β were analyzed with real‐time PCR. HU‐308 significantly decrease the mRNA levels of NLRP3, Casp‐1, and IL‐1β (n = 6 per group). **P < 0.01 versus Normal; #P < 0.05 versus LPS/ATP; ##P < 0.01 versus LPS/ATP.

Figure 5

Blocking autophagy by ATG5 siRNA inhibits the effect of CB2R on NLRP3 inflammasome formation in vitro. (A) Confocal images representing the colocalization of NLRP3 (green) with ASC or caspase‐1 (red) in cultured BV2 microglia. (B) Quantitative analysis showing the fold change in Pearson coefficient correlation (PCC) for the colocalization of NLRP3 with ASC and NLRP3 with caspase‐1 (n = 6 per group). **P < 0.01 versus control, ##P < 0.01 versus veh, &&P < 0.01 versus HU‐308.