GABAA receptor α4-subunit knockout enhances lung inflammation and airway reactivity in a murine asthma model

Abstract

Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABA_AR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABA_ARs, particularly isoforms containing α₄-subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABA_AR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABA_AR α₄-subunit global knockout (KO; Gabra4^0/0 ) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation (P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice (P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration (P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4⁺ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4⁺ cells. These data suggest that the GABA_AR α₄-subunit plays a role in immune cell function during allergic lung sensitization. Thus GABA_AR α₄-subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.

Keywords: eosinophil; flexiVent; house dust mite antigen; lymphocyte; organ bath.

Fig. 1.

Gel electrophoresis images from RT-PCR survey of γ-aminobutyric acid A receptor (GABA_AR) subunit expression in mouse primary CD4⁺ lymphocytes. mRNA was detected for multiple GABA_AR subunits, including α₄ and all other subunits needed to form a functional channel. The full results are summarized in Table 2. Marker: basepair length marker; Blank: PCR reaction devoid of cDNA (negative control); CD4: murine CD4⁺ lymphocyte; Brain: murine brain (positive control).

Fig. 2.

In vivo mouse airway resistance and lung compliance during a graded inhaled methacholine challenge. A: house dust mite (HDM) sensitization led to a significantly increased central airway resistance (Rn) in both wild-type (WT) and Gabra4 knockout (KO) mice compared with their corresponding nonsensitized controls (*P < 0.01). Notably, HDM-sensitized Gabra4 KO mice were significantly more reactive than HDM-sensitized WT mice (#P < 0.05). B: HDM-sensitized Gabra4 KO mice also demonstrated increased lung compliance (Crs) compared with both HDM-sensitized WT mice (#P < 0.05) and nonsensitized Gabra4 KO mice (*P < 0.01). Areas under the curves were compared by ANOVA with Bonferroni post hoc comparisons. Data are means ± SE; n = 8 mice.

Fig. 3.

Ex vivo tracheal ring organ bath experiments with HDM-sensitized mice. A: acetylcholine (ACh) dose-response curve for tracheal rings from WT and Gabra4 KO mice. Contraction forces as a percentage of maximal acetylcholine-induced contraction are not significantly different. B: absolute contraction force generate by 1 mM ACh is also not significantly different between groups. C: relaxation of an acetylcholine EC₅₀ contraction by the β-agonist isoproterenol was not significantly different between tracheal rings from WT and Gabra4 KO mice. Data are means ± SE; ns, not significant by Student’s t-test; n = 4 mice for all.

Fig. 4.

Hematoxylin and eosin-stained lung sections from HDM-sensitized (A) WT and (B) Gabra4 KO mice. KO mice exhibit enhanced perivascular and peribronchial inflammatory infiltration after 3 wk of intranasal HDM sensitization. C: lung inflammation was quantified using the lung composite lung inflammation score (16), which demonstrated Gabra4 KO lungs were significantly more inflamed than WT lungs. D: HDM-sensitized Gabra4 KO mice have a significantly larger proportion of invading eosinophils than HDM-sensitized WT mice. Data are means ± SE; n = 4 mice. *P < 0.05 by ANOVA with Bonferroni post hoc comparisons.

Fig. 5.

A: representative figures from flow cytometry analyses of mouse lung immune cells. Forward scatter/side scatter (FSC/SSC) plots of lung digests from nonsensitized (PBS) and HDM-sensitized WT and Gabra4 KO and mice are presented at top. The black circles contain cells with FSC (size) and SSC (granularity) values characteristic of lymphocytes. HDM-sensitized Gabra4 KO mice have a decrease in lymphocytes and an increase in granulocytes (area outline in red at top right) based on FSC/SSC analysis. Despite a decreased percentage of lymphocytes in the total lung digest, those CD4⁺ cells present in HDM-sensitized Gabra4 KO mice express increased markers of T-cell activation/memory (CD62L^LOW/CD44^HIGH). This is represented in the bottom right quadrants in A and is quantified in B. Data are means ± SE; n = 4 mice. **P < 0.01 by Student’s t-test.

Fig. 6.

In vitro CD4⁺ cell cytokine production. Follow stimulation with anti-CD3/28 coated beads, Gabra4 KO and WT CD4⁺ lymphocyte cell culture supernatant concentrations of several cytokines were measure at multiple time points over 96 h. Gabra4 KO CD4⁺ cells produced significantly higher concentrations of multiple cytokines (pro- and anti-inflammatory) at multiple time points. ANOVA with Bonferroni post hoc comparisons. Data are means ± SE; n = 3 mice. *P < 0.05. ***P < 0.001.

Fig. 7.

In vitro CD4⁺ cell proliferation. Following stimulation with anti-CD8/28 beads for 72 h, Gabra4 KO and WT CD4⁺ cell proliferation was assayed by fluorescein diacetate dilution. As demonstrated in the representative tracings (A; black tracing is WT, and gray tracing is Gabra4 KO) and as quantified by proliferation index (B), Gabra4 KO CD4⁺ cells proliferated significantly more than WT cells. Data are means ± SE; n = 3 mice. **P > 0.01 by Student’s t-test.