Anti-inflammatory effects of the neurotransmitter agonist Honokiol in a mouse model of allergic asthma

Abstract

Chronic airway inflammation is a hallmark of asthma, an immune-based disease with great societal impact. Honokiol (HNK), a phenolic neurotransmitter receptor (γ-aminobutyric acid type A) agonist purified from magnolia, has anti-inflammatory properties, including stabilization of inflammation in experimentally induced arthritis. The present study tested the prediction that HNK could inhibit the chronic inflammatory component of allergic asthma. C57BL/6 mice sensitized to and challenged with OVA had increased airway hyperresponsiveness to methacholine challenge and eosinophilia compared with naive controls. HNK-treated mice showed a reduction in airway hyperresponsiveness as well as a significant decrease in lung eosinophilia. Histopathology studies revealed a marked drop in lung inflammation, goblet cell hyperplasia, and collagen deposition with HNK treatment. Ag recall responses from HNK-treated mice showed decreased proinflammatory cytokines in response to OVA, including TNF-α-, IL-6-, Th1-, and Th17-type cytokines, despite an increase in Th2-type cytokines. Regulatory cytokines IL-10 and TGF-β were also increased. Assessment of lung homogenates revealed a similar pattern of cytokines, with a noted increase in the number of FoxP3(+) cells in the lung. HNK was able to alter B and T lymphocyte cytokine secretion in a γ-aminobutyric acid type A-dependent manner. These results indicate that symptoms and pathology of asthma can be alleviated even in the presence of increased Th2 cytokines and that neurotransmitter agonists such as HNK have promise as a novel class of anti-inflammatory agents in the treatment of chronic asthma.

Figure 1

Protocols used for mouse models of allergic asthma and treatment with HNK.

Figure 2

Effect of HNK on acute (A) and chronic (B) mouse models of allergic asthma. Female C57Bl/6 mice remained naïve (Control) or were sensitized/challenged to OVA ± treatment with HNK during the challenge phase. Control and OVA-sensitized mice not receiving HNK were treated with vehicle control, as described in Materials and Methods. Airway hyperactivity to methacholine, as measured by an index of resistance in the major conducting airways (R_N, Newtonian resistance), was assessed 48 h after the final allergen challenge. Each bar represents the mean ± SEM of central airway resistance readings (10 per methacholine dose) from individual mice. A. n = 4 for Control; n = 4 for OVA; n = 4 for HNK (challenge). B. n = 4 for Control, OVA, and HNK. Statistical analysis: p ≤ 0.05 (*); p ≤ 0.001 (**) for OVA vs. Control or HNK at indicated concentrations of methacholine.

Figure 3

Effect of HNK on OVA-mediated lung/airway inflammation. A–B. Representative hematoxylin and eosin (H&E) stained tissue sections of lungs from mice in (A) acute or (B) chronic models of allergic asthma. C–D. Representative ABPAS stained tissue sections of lungs from mice in (C) acute or (D) chronic models of allergic asthma. E. Representative Masson Trichrome stained tissue sections of lungs from mice in the chronic model of allergic asthma.

Figure 4

Effect of HNK on OVA-mediated cell recruitment to the lung. Post-mortem, lungs from mice in the (A) acute or (B) chronic models of allergic asthma were lavaged as described in Materials and Methods. Total and differential cell counts were obtained. Data are presented as total number or % of cells in the lavage fluid. Each bar represents the mean ± SEM. A–B. n = 4 for Control, OVA, and HNK (challenge). Statistical analysis (total # of cells): p ≤ 0.001 (**); p ≤ 0.0001 (***) for OVA vs. Control or HNK

Figure 5

Effect of HNK treatment on lung homogenate cytokines. Lung samples from mice in (A) acute or (B) chronic asthma models were homogenized and cytokines were assessed by multiplex ELISA as described in Materials and Methods. Each bar represents the mean ± SEM. IFN-γ was below the level of detection in lung homogenates from mice in the acute asthma model. A–B. n = 4 for Control, OVA, and HNK (challenge). Statistical analysis: p ≤ 0.05 (*) for OVA vs. Control or HNK

Figure 6

Effect of HNK treatment on OVA-specific responses in ex vivo splenocyte cultures. Single cell suspensions of spleens from mice in (A) acute or (B) chronic asthma models were cultured with OVA and assessed for proliferation (3H incorporation, CPM) and cytokine production (by ELISA), as described in Materials and Methods. Each bar represents the mean ± SEM (cytokines) or the mean ± SEM of triplicate wells from individual mice (proliferation). Splenocytes cultured in medium (Med) alone induced minimal proliferative and cytokine (data not shown) responses. A–B. n = 4 for Control, OVA, and HNK (challenge). Statistical analysis: : p < 0.05 (*); p < 0.01 (**) for OVA vs. Control or HNK

Figure 7

Effect of HNK treatment on total and OVA-specific serum Ig in the acute model of allergic asthma. Sera were obtained from female C57BL/6 mice on the penultimate day of the protocol, as outlined in Fig. 1. Levels of Ig isotypes were determined by ELISA, as described in Materials and Methods. Each bar represents the mean + SEM of triplicate wells of sera from individual mice that were serially diluted from 1:16,000 to 1:256,000 (total) or 1:4,000 to 1:15,000 (OVA). n = 4 for Control, OVA, and HNK (challenge). Statistical analysis: p ≤ 0.05 (*); p ≤ 0.01 (**); p ≤ 0.001 (***) for OVA vs. Control or HNK

Figure 8

Effect of HNK treatment on total and OVA-specific serum Ig in the chronic model of allergic asthma. Sera were obtained from female C57BL/6 mice on the penultimate day of the protocol, as outlined in Fig. 1. Levels of Ig isotypes were determined by ELISA, as described in Materials and Methods. Each bar represents the mean + SEM of triplicate wells of sera from individual mice that were serially diluted from 1:16,000 to 1:256,000 (total) or 1:4,000 to 1:15,000 (OVA). n = 4 for Control, OVA, and HNK (challenge). Statistical analysis: p ≤ 0.05 (*); p ≤ 0.01 (**); p ≤ 0.001 (***) for OVA vs. Control or HNK

Figure 9

GABA_A-dependent effect of in vitro HNK treatment on CD40-mediated cytokine response and transcription factor activation in mouse B cell lines. CH12.LX (AB) cells were cocultured with Hi-5 mCD154 (mCD40) vs Hi5-WT (control), in the absence or presence of HNK ± bicuculline, as described in Materials and Methods. Cytokines in culture supernatants were assessed by ELISA at 4 h (TNF-α, A) or 48 h (IL-6, B). Data points represent mean ± SEM of triplicate wells from 2 separate experiments. M12.4.1 cells (1.5 e7/ml) were transiently transfected with 10 μg 4X NF-κ B (C), 40 μg 7X AP-1 (D), or 40 μg 4X C/EBPβ (E) and 1 μg Renilla luciferase reporter plasmids by electroporation. Cells were rested on ice, then incubated an additional 6 (NF-κ B) or 24 (AP-1, C/EBPβ ) hours with insect cell stimuli specific for mCD40, in the absence or presence of HNK ± bicuculline, as described in Materials and Methods. Relative luciferase activity of Hi-5 mCD154 (mCD40) vs Hi5-WT (control) was calculated as the mean ± SEM of duplicate samples from two independent experiments. No cell death was seen in the cultures. Statistical analysis: p ≤ 0.05 (*); p ≤ 0.01 (**); p ≤ 0.001 (***) for HNK vs. vehicle or bicuculline.

Figure 10

GABA_A-dependent effect of in vitro HNK treatment on CD3 or CD3/CD28- mediated cytokine response and transcription factor activation in 2B4.11 cells. 2B4.11 cells (1e6/ml) were cultured in the presence of plate bound anti-CD3ε(CD3) ± soluble anti-CD28 (CD3/CD28) vs. Medium/Isotype Control (Med/IC), in the absence or presence of HNK ± bicuculline, as described in Materials and Methods. Cytokines in culture supernatants were assessed by ELISA at 48 h (IL-2, A) or 72 h (IFN-γ , B; IL-17, C; IL-13, D). Data points represent mean ± SEM of triplicate wells from 2 separate experiments. Alternatively, 2B4.11 cells (1.5 e7/ml) were transiently transfected with 40 μg 4XNFAT (E), pSTAT4 (F), pSTAT1 (G), or pGATA-3 (H), and 1 μg Renilla luciferase reporter plasmids by electroporation. Cells were rested on ice, then incubated an additional 24 hours with insect cell stimuli specific for CD3 ± CD28, in the absence or presence of HNK ± bicuculline, as described in Materials and Methods. Relative luciferase activity of CD3 or CD3/CD28 vs. Medium/IC (control) was calculated as the mean ± SEM of duplicate samples from two independent experiments. No cell death was seen in the cultures. Statistical analysis: p ≤ 0.01 (**); p ≤ 0.001 (***) for HNK vs. vehicle or bicuculline.