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

Abstract

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 β-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-1β (IL-1β) p17 and proIL-1β, IL-18 and proIL-18 were measured using Western blotting. The mRNA levels of Casp-1 and IL-1β were detected by real-time PCR (RT-PCR). The colocalization and interaction of NLRP3 protein and β-arrestin-1 were measured by immunofluorescence staining and immunoprecipitation.

Results: The expression of β-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-1β 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 β-arrestin-1 attenuated the inhibitory effect of PNU282987 on NLRP3 inflammasome activation in LPS/ATP-stimulated BV2 microglia. PNU282987 inhibited the interaction between β-arrestin-1 and NLRP3 protein in vitro.

Conclusions: The present study demonstrates that activating α7nAChR can lead to NLRP3 inflammasome inhibition via regulation of β-arrestin-1 in monocyte/microglia system.

Keywords: NLRP3 inflammasome; neuroinflammation; α7nAChR; β-arrestin-1.

Figure 1

β‐arrestin‐1 expression in spinal cord of the sham and experimental auto‐immune encephalomyelitis (EAE) mice. (A,B) Total RNA and protein in spinal cord of the sham and EAE mice were collected, and the mRNA and protein levels of β‐arrestin‐1 were analyzed using real‐time PCR (RT‐PCR) and Western blot. Compared with the sham group, both mRNA and protein levels of β‐arrestin‐1 were elevated in EAE mice (n = 6 per group). **P < 0.01 vs sham. (C) Spinal cords of the sham and EAE mice were fixed in 4% (w/v) paraformaldehyde overnight and then paraffin‐embedded and sectioned. The colocalization of β‐arrestin‐1 and CD45‐positive cells was stained by immunofluorescence assay. The colocalization of β‐arrestin‐1 (red) and CD45‐positive cells (green) was significantly increased in spinal cord of EAE mice compared with the sham mice (n = 6 per group). **P < 0.01 vs sham

Figure 2

α7 nicotinic acetylcholine receptor (α7nAChR) regulated the expression of β‐arrestin‐1 in experimental auto‐immune encephalomyelitis (EAE) mice and LPS/ATP‐stimulated BV2 microglia. Spinal cords of the sham and EAE mice as well as BV2 microglia were collected and lysated, and the expression of β‐arrestin‐1 was measured using Western blot. (A) Compared with the vehicle group, treatment with PNU282987 significantly decreased the expression of β‐arrestin‐1 in spinal cords of EAE mice (n = 6 per group). PNU, PNU282987. **P < 0.01 vs sham; ^## P < 0.01 vs vehicle. (B) The expression of β‐arrestin‐1 in spinal cords of α7nAChR KO mice was increased compared with those of WT mice (n = 6 per group). **P < 0.01 vs sham; ^## P < 0.01 vs WT. (C) PNU282987 dose‐dependently inhibited the expression of β‐arrestin‐1 in BV2 microglia challenged by LPS/ATP (n = 6 per group). PNU, PNU282987. **P < 0.01 vs control; ^## P < 0.01 vs vehicle

Figure 3

α7 nicotinic acetylcholine receptor (α7nAChR) regulates NLRP3 inflammasome activation in experimental auto‐immune encephalomyelitis (EAE) mice. Spinal cords were isolated from the sham and EAE mice on day 12 PI and then lysated with buffer. The levels of NLRP3 protein, Casp‐1 p20/proCasp‐1, IL‐1β p17/proIL‐1β, and IL‐18/proIL‐18 were analyzed using Western blot. (A‐B) Treatment with PNU282987 (0.1 mg/kg) significantly decreased the levels of NLRP3 protein, Casp‐1 p20/proCasp‐1, IL‐1β p17/proIL‐1β, and IL‐18/proIL‐18 compared with the vehicle group (n = 6 per group). PNU, PNU282987. **P < 0.01 vs sham; ^## P < 0.01 vs vehicle. (C‐D) Compared with the WT mice, the levels of NLRP3 protein, Casp‐1 p20/proCasp‐1, IL‐1β p17/proIL‐1β, and IL‐18/proIL‐18 were significantly increased in α7nAChR KO mice (n = 6 per group). **P < 0.01 vs sham; ^# P < 0.05 vs WT; ^## P < 0.01 vs WT

Figure 4

α7 nicotinic acetylcholine receptor (α7nAChR) regulates NLRP3 inflammasome activation in LPS/ATP‐stimulated BV2 microglia. BV2 microglia were pretreated with vehicle or PNU282987 (0.1, 1 or 10 μmol/L) for 10 minutes before stimulated with LPS (100 ng/mL) and ATP (1 mmol/L) for 12 hours. (A‐E) Cells were lysed, and the levels of NLRP3 protein, Casp‐1 p20/proCasp‐1, IL‐1β p17/proIL‐1β, and IL‐18/proIL‐18 were analyzed using Western blot. Compared with the vehicle group, treatment with PNU282987 significantly decreased the levels of NLRP3 protein, Casp‐1 p20/proCasp‐1, IL‐1β p17/proIL‐1β, and IL‐18/proIL‐18 (n = 6 per group). PNU, PNU282987. **P < 0.01 vs control; ^# P < 0.05 vs vehicle; ^## P < 0.01 vs vehicle. (F‐G) Cells were collected, and mRNA levels of Casp‐1 and IL‐1β were analyzed by RT‐PCR. Compared with the vehicle group, PNU282987 significantly decreased the mRNA levels of Casp‐1 and IL‐1β (n = 6 per group). PNU, PNU282987. **P < 0.01 vs control; ^# P < 0.05 vs vehicle; ^## P < 0.01 vs vehicle

Figure 5

β‐arrestin‐1 mediates the inhibitory effects of α7 nicotinic acetylcholine receptor (α7nAChR) on the NLRP3 inflammasome activation. BV2 microglia were transfected with the control lentivirus (LV‐GFP) or β‐arrestin‐1 overexpression lentivirus (LV‐β‐arr‐1) for 72 hours and then treated with vehicle or PNU282987 (10 μmol/L) for 10 minutes followed by the stimulation with LPS (100 ng/mL) and ATP (1 mmol/L) for 12 hours. BV2 microglia were lysated, and expression of NLRP3 protein, Casp‐1 p20/proCasp‐1, IL‐1β p17/proIL‐1β and IL‐18/proIL‐18 was analyzed using Western blot. (A‐E) Compared with LV‐GFP group, overexpression of β‐arrestin‐1 significantly attenuated the inhibitory effects of PNU282987 on the levels of NLRP3 protein, Casp‐1 p20/proCasp‐1, IL‐1β p17/proIL‐1β, and IL‐18/proIL‐18 (n = 6 per group). PNU, PNU282987. **P < 0.01 vs control; ^## P < 0.01 vs LPS/ATP

Figure 6

PNU282987 suppresses the interaction of β‐arrestin‐1 and NLRP3 protein in LPS/ATP‐stimulated BV2 microglia. BV2 microglia were treated with vehicle or PNU282987 (10 μmol/L) for 10 minutes and then were left without stimulation or stimulated with LPS (100 ng/mL) and ATP (1 mmol/L) for 12 hours. (A‐B) β‐arrestin‐1, NLRP3 protein, and nuclei (with DAPI) were stained and then observed with confocal laser scanning microscope. LPS/ATP stimulation increased the colocalization of NLRP3 protein (red) and β‐arrestin‐1 (green), which was significantly inhibited by treatment with PNU282987 (n = 6 per group). PNU, PNU282987. *P < 0.05 vs control; ^## P < 0.01 vs LPS/ATP. (C) BV2 microglia were lysated and then the interaction of β‐arrestin‐1 and NLRP3 protein was detected by immunoprecipitation. LPS/ATP stimulation increased the bind of NLRP3 protein and β‐arrestin‐1, which was inhibited by treatment with PNU282987. Three independent experiments were repeated. PNU, PNU282987

Figure 7

Schematic illustration of α7 nicotinic acetylcholine receptor (α7nAChR) inhibits NLRP3 inflammasome activation. Upon LPS/ATP stimulation, β‐arrestin‐1 binds with NLRP3 protein and facilitates the formation and activation of NLRP3 inflammasome complex. Activation of α7nAChR with PNU282987 not only could decrease the expression of β‐arrestin‐1, but also inhibit the bind of β‐arrestin‐1 with NLRP3 protein, which subsequently suppresses the NLRP3 inflammasome activation