Activating α7 nicotinic acetylcholine receptor inhibits NLRP3 inflammasome through regulation of ß-arrestin-1.

AIMS: To evaluate whether activating α7 nicotinic acetylcholine receptor (α7nAChR) could inhibit the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome through regulation of ß-arrestin-1 in monocyte/macrophage system, thus contributing to the control of neuroinflammation. METHODS: The protein levels of NLRP3, caspase-1 (Casp-1) p20 and proCasp-1, interleukin-1ß (IL-1ß) p17 and proIL-1ß, IL-18 and proIL-18 were measured using Western blotting. The mRNA levels of Casp-1 and IL-1ß were detected by real-time PCR (RT-PCR). The colocalization and interaction of NLRP3 protein and ß-arrestin-1 were measured by immunofluorescence staining and immunoprecipitation. RESULTS: The expression of ß-arrestin-1 was significantly increased and colocalized with CD45-positive cells in spinal cord of experimental auto-immune encephalomyelitis (EAE) mice when compared with the sham mice, which was attenuated by pretreatment with PNU282987, a specific α7nAChR agonist. PNU282987 also significantly inhibited the activation of NLRP3 inflammasome and thus decreased the production of IL-1ß and IL-18 both in lipopolysaccharide (LPS)/ATP-stimulated BV2 microglia in vitro and spinal cord from EAE mice in vivo, while inverse effects were observed in α7nAChR knockout mice. Furthermore, overexpression of ß-arrestin-1 attenuated the inhibitory effect of PNU282987 on NLRP3 inflammasome activation in LPS/ATP-stimulated BV2 microglia. PNU282987 inhibited the interaction between ß-arrestin-1 and NLRP3 protein in vitro. CONCLUSIONS: The present study demonstrates that activating α7nAChR can lead to NLRP3 inflammasome inhibition via regulation of ß-arrestin-1 in monocyte/microglia system.

Autophagy Plays an Important Role in Anti-inflammatory Mechanisms Stimulated by Alpha7 Nicotinic Acetylcholine Receptor.

Alpha7 nicotinic acetylcholine receptor (α7nAChR) has been reported to alleviate neuroinflammation. Here, we aimed to determine the role of autophagy in α7nAChR-mediated inhibition of neuroinflammation and its underlying mechanism. Experimental autoimmune encephalomyelitis (EAE) mice and lipopolysaccharide-stimulated BV2 microglia were used as in vivo and in vitro models of neuroinflammation, respectively. The severity of EAE was evaluated with neurological scoring. Autophagy-related proteins (Beclin 1, LC3-II/I, p62/SQSTM1) were detected by immunoblot. Autophagosomes were observed using transmission electron microscopy and tandem fluorescent mRFP-GFP-LC3 plasmid was applied to test autophagy flux. The mRNA levels of interleukin-6 (IL-6), IL-1ß, IL-18, and tumor necrosis factor-α (TNF-α) were detected by real-time PCR. We used 3-methyladenine (3-MA) and autophagy-related gene 5 small interfering RNA (Atg5 siRNA) to block autophagy in vivo and in vitro, respectively. Activating α7nAChR with PNU282987 ameliorates EAE severity and spinal inflammatory infiltration in EAE mice. PNU282987 treatment also enhanced monocyte/microglia autophagy (Beclin 1, LC3-II/I ratio, p62/SQSTM1, colocalization of CD45- or CD68-positive cells with LC3) both in spinal cord and spleen from EAE mice. The beneficial effects of PNU282987 on EAE mice were partly abolished by 3-MA, an autophagy inhibitor. In vitro, PNU282987 treatment increased autophagy and promoted autophagy flux. Blockade of autophagy by Atg5 siRNA or bafilomycin A1 attenuated the inhibitory effect of PNU282987 on IL-6, IL-1ß, IL-18, and TNF-α mRNA. Our results demonstrate for the first time that activating α7nAChR enhances monocyte/microglia autophagy, which suppresses neuroinflammation and thus plays an alleviative role in EAE.

Activation of Cannabinoid Receptor 2 Ameliorates DSS-Induced Colitis through Inhibiting NLRP3 Inflammasome in Macrophages.

Activation of cannabinoid receptor 2 (CB2R) ameliorates inflammation, but the underlying mechanism remains unclear. In the present study, we examined whether activation of CB2R could suppress the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome. In peritoneal macrophages isolated from C57BL/6 mice, LPS/DSS challenge for 24 h increased the expression of the components of NLRP3 inflammasome NLRP3, Casp-1 p20/Casp-1 p45 ratio, proIL-1ß and IL-1ß and also enhanced autophagy (LC3-II/LC3-I ratio, Beclin-1 and SQSTM1). Pretreatment of peritoneal macrophages with HU 308, a selective CB2R agonist, attenuated LPS/DSS-induced NLRP3 inflammasome activation, but further enhanced autophagy. In comparison with wild-type (WT) control, peritoneal macrophages from CB2R knockout (KO) mice had more robust NLRP3 inflammasome activation and attenuated autophagy upon LPS/DSS challenge. Knockdown autophagy-related gene 5 (Atg5) with a siRNA in peritoneal macrophages attenuated the inhibitory effects of HU 308 on LPS/DSS-induced NLRP3 inflammasome activation in vitro. In vivo, HU308 treatment attenuated DSS-induced colitis mice associated with reduced colon inflammation and inhibited NLRP3 inflammasome activation in wild-type mice. In CB2R KO mice, DSS-induced inflammation and NLRP3 inflammasome activation were more pronounced than those in WT control. Finally, we demonstrated that AMPK-mTOR-P70S6K signaling pathway was involved in this CB2R-mediated process. We conclude that activation of CB2R ameliorates DSS-induced colitis through enhancing autophagy that may inhibit NLRP3 inflammasome activation in macrophages.