Anti-inflammatory effects of astroglial α7 nicotinic acetylcholine receptors are mediated by inhibition of the NF-κB pathway and activation of the Nrf2 pathway

Abstract

Background: α7 nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous system and are reported to have neuroprotective properties. α7 nAChRs are expressed on astrocytes, which are key regulators of neuroinflammation and oxidative stress in several neurodegenerative diseases. However, the anti-inflammatory and antioxidant properties of astroglial α7 nAChRs are not well studied. Therefore, we evaluated the role of astroglial α7 nAChR activation in neuroinflammation.

Methods: Anti-inflammatory and antioxidant effects of α7 nAChR activation were evaluated in an in vitro mouse model of neuroinflammation using lipopolysaccharide (LPS) in primary astrocyte cultures. α7 nAChR anti-inflammatory effects on the NF-κB pathway were evaluated using ELISA, gene expression analysis, immunofluorescence, and western blotting. Antioxidant effect of α7 nAChR activation on expression profiles of canonical Nrf2 target genes was examined by quantitative PCR and western blotting. The role of the Nrf2 pathway in α7 nAChR-mediated anti-inflammatory response was evaluated using Nrf2 knockout astrocytes. Brain ex vivo NF-κB luciferase signals were evaluated after treatment with an α7 nAChR agonist in lipopolysaccharide (LPS)-injected NF-κB luciferase reporter mouse model.

Results: Astrocytes treated with the α7 nAChR partial agonist (GTS21) showed significantly reduced LPS-mediated secretion of inflammatory cytokines and this effect was reversed by the α7 nAChR antagonist methyllycaconitine (MLA) and by knockdown of α7 nAChR expression with a short hairpin RNA. Further, α7 nAChR activation blocked LPS-mediated NF-κB nuclear translocation indicating that the observed anti-inflammatory effect may be mediated through inhibition of the NF-κB pathway. Treatment with GTS21 also upregulated canonical Nrf2 antioxidant genes and proteins suggesting antioxidant properties of α7 nAChR in astrocytes. Using an astrocyte conditioned media approach, we demonstrated reduction in neuronal apoptosis when astrocytes were pretreated with GTS21. Finally, in an in vivo neuroinflammation model using LPS in NF-κB luciferase reporter mice, we demonstrated reduction in LPS-induced NF-κB activity and pro-inflammatory cytokines with GTS21 treatment in brain tissue.

Conclusion: Our results suggest that activating astroglial α7 nAChRs may have a role in neuroprotection by decreasing inflammation and oxidative stress, and therefore could have therapeutic implication for disease modifying treatments of neurodegenerative diseases.

Keywords: Astrocytes; NF-κB pathway; Nrf2 pathway; α7 nicotinic acetylcholine receptors.

Fig. 1

Characterization of an in vitro model of neuroinflammation and expression of α7 nAchRs in mouse astrocytes. a These are representative images of mouse astrocytes using the Cellomics ArrayScan XTI high-content analysis system for automated image acquisition of 20 fields for each well at × 20 magnification. In each field, two channels were captured to image nuclei (DAPI) and astrocyte marker (GFAP). The ArrayScan Target Activation bioapplication was used for processing and analyzing the images to quantify GFAP positive cells as percentage of total cell counts using DAPI within a well. This was found to be more than 90%. b LPS (60 ng/ml) treatment for 3 h increased nuclear translocation of p65 subunit of NF-κB in astrocytes as a marker of acute inflammation phase. c LPS (60 ng/ml) treatment for 24 h resulted in significant increase in pro-inflammatory cytokines, TNF and IL-6, downstream of NF-κB activation in astrocytes, *p < 0.05 as compared with untreated (t test). Error bars represent SD (n = 4). d Stimulation of astrocytes with LPS (60 ng/ml) for 24 h increased cell processes substantially as compared to the control cells. e α7 nAChR subunit mRNA expression observed in cultured astrocytes (three replicates). f Binding of fluorescently labeled α-bungarotoxin was observed in astrocytes. Pretreatment with 200 μm nicotine (α7 nAchR agonist), significantly inhibited binding of fluorescently labeled α-bungarotoxin shown by decrease in fluorescence confirming expression of α7 nAchR in these cells

Fig. 2

α7 nAchR activation reduced LPS-mediated pro-inflammatory cytokine secretion in astrocytes. a Pretreatment with GTS21 (α7 nAchR agonist) for 1 h resulted in a dose-dependent reduction in both IL-6 and TNF-α secretion as compared to LPS treated alone for 24 h, *p < 0.05 compared with LPS (60 ng/ml) treatment (one-way ANOVA with adjustment for multiple comparisons using Dunnett’s test). Error bars represent SD (n = 4). b Pretreating the cultured astrocytes for 1 h with 1 μm α7 nAchR antagonist (MLA) resulted in blockage of anti-inflammatory response of 20 μm GTS21, as measured by secretion of pro-inflammatory cytokines TNF-α and IL-6 in astrocytes treated with LPS for 24 h. *p < 0.05 compared with LPS 60 ng/ml treatment, ^# p < 0.05 compared to respective agonist treatment alone (both using one-way ANOVA with adjustment for multiple comparisons using Tukey’s test). Error bars represent SD (n = 4). c Knocking down α7 nAchR expression with lenti short hairpin RNA in astrocytes resulted ~ 75% gene knockdown, which significantly blocked the anti-inflammatory response of GTS21 (30 μm) in astrocytes treated with LPS for 24 h. Error bars represent SD (n = 2)

Fig. 3

α7 nAchR activation resulted in inhibition of the NF-κB signaling pathway and astrogliosis. a Pretreatment with GTS21 (15 and 30 μm) resulted in reduction of phosphorylated form of IκBα in LPS (60 ng/ml)-treated astrocytes for 30 min. b Treatment of astrocytes with LPS (60 ng/ml) for 3 h caused robust nuclear translocation of NF-κB and pretreatment with α7 nAchR agonist GTS21 (30 μm) for 1 h caused significant reduction in NF-κB nuclear translocation, *p < 0.05 (one-way ANOVA with adjustment for multiple comparisons using Dunnett’s test for the comparison of LPS alone group and GTS21+LPS group as percentage of LPS alone group). Error bars represent SD (n = 6 wells, 50 fields/well). c Treatment with GTS21 (30 μm) reduced the number of processes in LPS (60 ng/ml for 24 h)-activated astrocytes suggesting reduction in reactive astrogliosis. d Pretreatment of GTS21 with 7.5, 15, and 30 μm for 1 h resulted in a dose-dependent decrease in LPS (60 ng/ml for 72 h)-induced levels of nitrites further confirming anti-inflammatory properties of α7 nAchR activation. *p < 0.05 compared with control, ^# p < 0.05 compared with LPS, 60 ng/ml treatment (both using one-way ANOVA with adjustment for multiple comparisons using Tukey’s test). Error bars represent SD (n = 6)

Fig. 4

α7 nAchR activation reduced LPS-induced inflammatory genes in astrocytes. a LPS-mediated inflammatory genes were measured using TaqMan® OpenArray® Mouse Inflammation Panel covering 632 inflammatory genes, and changes were plotted on a volcano plot with log fold changes on x-axis and log of corrected p values on y-axis. Red vertical lines indicate boundaries for fold changes. Green dots indicate at least 1.5-fold lower expression and red dots indicate at least 1.5-fold higher expression (both statistically significant at p < 0.05). Gray dots, plotted below the horizontal line, indicate statistically non-significant changes. Pretreatment of astrocytes with GTS21 (30 μm) for 1 h followed by LPS (60 ng/ml) treatment for 24 h resulted in a substantially lower number of inflammatory genes upregulation (fewer red dots in the lower panel) indicating a robust anti-inflammatory effect of α7 nAchR activation. b A gene set enrichment analysis was performed using the software package Generally Applicable Gene-set Enrichment (GAGE) in R Bioconductor based on the 214 differentially expressed inflammatory genes with GTS21 treatment to identify KEGG pathways enriched by the downregulation of inflammatory genes in the GTS21 plus LPS-treated samples compared to LPS alone treated samples. This plot shows the NF-κB signaling pathway, which was statistically significantly downregulated with GTS21 treatment (p value 0.04), along with specific genes that were downregulated in green shaded boxes

Fig. 5

α7 nAchR activation induced upregulation of canonical Nrf2 antioxidant genes. a Pretreatment with GTS21 (15 and 30 μm) for 1 h resulted in significant upregulation of canonical Nrf2 antioxidant genes HO1, TXNRD1, and GCLC, in astrocytes treated with LPS for 24 h. α7 nAchR antagonist MLA (1 μm) significantly reduced this effect indicating the specificity of this response. *p < 0.05 compared with LPS, 60 ng/ml treatment, ^# p < 0.05 compared with GTS21 30 μm + LPS 60 ng/ml (both using one-way ANOVA with adjustment for multiple comparisons using Tukey’s test). Error bars represent SD (n = 4). b A significant increase in protein levels of HO1, NQO1, and TXNRD1 upon GTS21 (30 μm) treatment was noted using western blot in astrocytes treated with LPS for 24 h, and this effect was blocked by the 1 μm α7 nAchR antagonist MLA. *p < 0.05 compared with LPS 60 ng/ml treatment, ^# p < 0.05 compared with GTS21 30 μm + LPS 60 ng/ml treatment (both using one-way ANOVA with adjustment for multiple comparisons using Tukey’s test). Error bars represent SD (n = 3)

Fig. 6

Antioxidant and anti-inflammatory responses of α7 nAchR activation are Nrf2 dependent. a Pretreatment with GTS21 (15 and 30 μm) resulted in significant upregulation of canonical Nrf2 antioxidant genes HO1, TXNRD1, GCLC, and OSGIN1 in wild-type astrocyte cultures treated with LPS (60 ng/ml) for 24 h, but not in astrocytes from Nrf2 knockout mice. *p < 0.05 compared with LPS (60 ng/ml) treatment (one-way ANOVA with adjustment for multiple comparisons using Dunnett’s test). Error bars represent SD (n = 4). b One hour pretreatment with GTS21 (15 and 30 μm) in astrocytes isolated from Nrf2 knockout mice treated with LPS (60 ng/ml) for 24 h resulted in substantially reduced blockage of pro-inflammatory cytokine, TNF-α, secretion (~ 22%) as compared with astrocytes cultured from wild-type mice (~ 63%). *p < 0.05 compared with LPS (60 ng/ml) treatment (one-way ANOVA with adjustment for multiple comparisons using Dunnett’s test). Error bars represent SD (n = 4)

Fig. 7

α7 nAchR activation reduced inflammatory astrocyte-mediated neuronal apoptosis. a Representative images of caspase activation in neurons treated with astrocyte conditioned media in the presence of LPS (60 ng/ml) for 24 h with or without 1 h GTS21 pretreatment (30 μm). b Quantification of caspase activation upon GTS21 (30 μm) pretreatment for 1 h in LPS (60 ng/ml)-activated astrocyte conditioned media showed significantly lowered levels of caspase 3/7 in neuronal cultures, and this effect was significantly blocked upon addition of 1 μm MLA, *p < 0.05 compared with control astrocyte conditioned media. *p < 0.05 compared with control (one-way ANOVA with adjustment for multiple comparisons using Dunnett’s test). Error bars represent SD (n = 5 wells, 15 fields per well)

Fig. 8

α7 nAchR activation reduced NF-κB luciferase signal and downregulated pro-inflammatory genes downstream of NF-κB signaling pathway in brain. a Representative images of NF-κB-dependent luminescence in one hemisphere of the brains (n = 5 mice per group) acquired on the IVIS Spectrum instrument (Perkin Elmer, Hopkinton, MA), using the Perkin Elmer proprietary software, Living Image (v4.3.1). Luciferin was injected intraperitoneally at 150 mg/kg for ex vivo brain imaging, which was conducted after euthanizing the animal and harvesting the brain. b NF-κB-dependent luminescence in the brains of NF-κB luciferase reporter mice was measured by imaging. A significant increase in NF-κB luciferase signal was observed with LPS (1.7 mg/kg) treatment, which was reduced in the presence of GTS21 (25 mg/kg) treatment. *p < 0.05 compared with LPS treated (one-way ANOVA with adjustment for multiple comparisons using Dunnett’s test). Error bars represent SD for n = 10 mice/group from two independent experiments. c RNA analysis of the other brain hemisphere from NF-κB luciferase reporter mice treated with LPS demonstrated statistically significant reductions in gene expression of inflammatory cytokines, TNF-α and IL-6 with 5 mg/kg and 25 mg/kg GTS21 treatment and IL-1β with 25 mg/kg GTS21 treatment, *p < 0.05 compared with LPS treatment (one-way ANOVA with adjustment for multiple comparisons using Dunnett’s test). Error bars represent SD for n = 5 mice/group. d Expression of OSGIN1 and HO1, which are downstream antioxidant genes of Nrf2 pathway, was significantly increased with GTS21 treatment, *p < 0.05 compared with PBS treatment (one-way ANOVA with adjustment for multiple comparisons using Dunnett’s test). Error bars represent SD for n = 5 mice/group