Varenicline Prevents LPS-Induced Inflammatory Response via Nicotinic Acetylcholine Receptors in RAW 264.7 Macrophages

Abstract

The cholinergic anti-inflammatory pathway plays an important role in controlling inflammation. This study investigated the effects of varenicline, an α7 nicotinic acetylcholine receptor (α7nAChR) agonist, on inflammatory cytokine levels, cell proliferation, and migration rates in a lipopolysaccharide (LPS)-induced inflammation model in RAW 264.7 murine macrophage cell lines. The cells were treated with increasing concentrations of varenicline, followed by LPS incubation for 24 h. Prior to receptor-mediated events, anti-inflammatory effects of varenicline on different cytokines and chemokines were investigated using a cytokine array. Nicotinic AChR-mediated effects of varenicline were investigated by using a non-selective nAChR antagonist mecamylamine hydrochloride and a selective α7nAChR antagonist methyllycaconitine citrate. TNFα, IL-1β, and IL-6 levels were determined by the ELISA test in cell media 24 h after LPS administration and compared with those of dexamethasone. The rates of cellular proliferation and migration were monitored for 24 h after drug treatment using a real-time cell analysis system. Varenicline decreased LPS-induced cytokines and chemokines including TNFα, IL-6, and IL-1β via α7nAChRs to a similar level that observed with dexamethasone. Varenicline treatment decreased LPS-induced cell proliferation, without any nAChR involvement. On the other hand, the LPS-induced cell migration rate decreased with varenicline via α7nAChR. Our data suggest that varenicline inhibits LPS-induced inflammatory response by activating α7nAChRs within the cholinergic anti-inflammatory pathway, reducing the cytokine levels and cell migration.

Keywords: cytokine; inflammation; migration; proliferation; varenicline; α7nAChR.

FIGURE 1

LPS-induced increase in inflammation markers in RAW264.7 cells. Shown are IL-1β (A); IL-6 (B), TNFα (C) levels in response to increasing LPS concentrations. Data are expressed as mean ± S.E.M. (**p < 0.01; ***p < 0.001 vs. the control group, n = 5–7, One-way ANOVA with post hoc Tukey–Kramer multiple comparisons test or Student’s t-test).

FIGURE 2

Effects of varenicline on LPS-induced cytokine levels in RAW 264.7 macrophages. Shown are membrane-based antibody arrays of 14 mouse cytokines found in supernatants of the control group (A), 4 μg/ml LPS-induced (B) and LPS-induced macrophages in the presence of 1 µM varenicline (VAR) (C). Each sample dot corresponds to a specific cytokine released from the same cell population used in other experiments (pooled data, n = 5–7). Bar graphics show averaged pixel intensities of each dot in duplicate. The table below the right panel shows the numerical annotations of the relevant cytokines detected in the left membranes (1: reference spot).

FIGURE 3

Effects of varenicline on LPS-induced IL-1β, IL-6, and TNFα elevations via nAChR. Shown are the effects of varenicline on 4 μg/ml LPS-induced IL-1β (A), IL-6 (B), and TNFα (C) levels and the comparison with dexamethasone. Data are expressed as mean ± S.E.M. (**, p < 0.01; ***, p < 0.001 vs. the control group; ^† p < 0.05; ^†† p < 0.01, ^††† p < 0.001 vs. the LPS group, n = 5–7, One-way ANOVA with post hoc Tukey–Kramer multiple comparisons test or Student’s t-test). VAR: varenicline, DEX: dexamethasone.

FIGURE 4

Effects of varenicline on LPS-induced IL-1β, IL-6, and TNFα elevations in the presence or absence of nAChR antagonists. Shown are 4 μg/ml LPS-elevated IL-1β (A); IL-6 (B), and TNFα (C) levels in the absence or presence of varenicline (VAR, 1 μM), mecamylamine (MEC, 50 μM) and methyllycaconitine (MLA, 1 μM). Data are shown as mean ± S.E.M. (**p < 0.01; ***p < 0.001 vs. the control group, ^† p < 0.05; ^†† p < 0.01, ^††† p < 0.001 vs. LPS group; ^‡ p < 0.05, ^‡‡‡ p < 0.001 vs. LPS + VAR, n = 5–7, One-way ANOVA with post hoc Tukey–Kramer multiple comparisons test or Student’s t-test).

FIGURE 5

Effects of varenicline on LPS-induced cell proliferation in the presence or absence of nAChR antagonists. Shown are line graphs drawn from the averaged data points of the real-time proliferation assay tracings (A) and the cumulative data (B). RAW 264.7 cells were treated with varenicline (VAR, 1 μM) in the presence or absence of mecamylamine (MEC, 50 μM) and methyllycaconitine (MLA, 1 μM) prior to lipopolysaccharide (LPS, 4 μg/ml) administration at 24th hour. Then, cell proliferation rates were monitored for 24 h after the treatments. Data are expressed as mean ± S.E.M. (***p < 0.01 vs. the control group ^† p < 0.05; ^†† p < 0.01, ^††† p < 0.001 vs. the LPS group, n = 5–7, One-way ANOVA with post hoc Tukey–Kramer multiple comparisons test or Student’s t-test). CI: cell index.

FIGURE 6

Effects of varenicline on LPS-induced cell migration in the presence or absence of nAChR antagonists. Shown are line graphs drawn by the averaged data points of real-time migration assay tracings (A) and the cumulative data (B). RAW 264.7 cells were treated with varenicline (VAR, 1 μM) in the presence or absence of mecamylamine (MEC, 50 μM) and methyllycaconitine (MLA, 1 μM) prior to lipopolysaccharide (LPS, 4 μg/ml) administration at the 24th hour. Then, cell migration rates were monitored for 24 h after the treatments. Data are expressed as mean ± S.E.M. (*p < 0.05; **p < 0.01 vs. the control group; ^† p < 0.05 vs. the LPS group; ^‡ p < 0.05, ^‡‡ p < 0.01 vs. LPS + VAR group, n = 5–7, One-way ANOVA with post hoc Tukey–Kramer multiple comparisons test or Student’s t-test).