Elsevier

Cellular Immunology

Volume 332, October 2018, Pages 7-23
Cellular Immunology

Research paper
Autocrine GABA signaling distinctively regulates phenotypic activation of mouse pulmonary macrophages

https://doi.org/10.1016/j.cellimm.2018.07.001Get rights and content

Highlights

  • Both GABAARs and GAD, a GABA synthetic enzyme, are expressed in mouse LMϕs.

  • GABAARs in LMϕs are down- and up-regulated by “M1” and “M2” stimulants, respectively.

  • GABAAR signaling restrains “M1” activation but fosters “M2” polarization.

Abstract

In response to micro-environmental cues such as microbial infections or T-helper 1 and 2 (TH1 and TH2) cytokines, macrophages (Mϕs) develop into M1- or M2-like phenotypes. Phenotypic polarization/activation of Mϕs are also essentially regulated by autocrine signals. Type-A γ-aminobutyric acid receptor (GABAAR)-mediated autocrine signaling is critical for phenotypic differentiation and transformation of various cell types. The present study explored whether GABAAR signaling regulates lung Mϕ (LMϕ) phenotypic activation under M1/TH1 and M2/TH2 environments. Results showed that GABAAR subunits were expressed by primary LMϕ of mice and the mouse Mϕ cell line RAW264.7. The expression levels of GABAAR subunits in mouse LMϕs and RAW264.7 cells decreased or increased concurrently with classical (M1) or alternative (M2) activation, respectively. Moreover, activation or blockade of GABAARs distinctively influenced the phenotypic characteristics of Mϕ. These results suggested that microenvironments leading to LMϕ phenotypic polarization concurrently modulates autocrine GABA signaling and its role in Mϕ activation.

Introduction

Macrophages (Mϕs) exist in almost all tissues of the body where they play critical roles in mediating tissue homeostasis as well as primary innate immune responses [1]. Mϕs in the lung (LMϕs) including airway/alveolar and interstitial Mϕs (AMϕs and IMϕs respectively) are the predominant immune cells in pulmonary tissues under normal conditions [2]. Previous studies indicated that LMϕs reacting to environmental cues, such as inhaled pathogens and the cytokine milieu, develop into either classically-activated (M1) or alternatively-activated (M2) Mϕs [3]. For example, LMϕs reacting to the Gram-negative bacterial endotoxin lipopolysaccharide (LPS) and/or the T helper 1 (TH1) cytokine interferon-gamma (INFγ) polarize to the pro-inflammatory M1 phenotype characterized by upregulated inducible nitric oxide synthase (iNOS) expression and tumor necrosis factor alpha (TNFα) secretion. Alternatively, following exposure to allergen or microbes inducing T helper 2 (TH2) cytokines, such as interleukin-4/13 (IL-4/13), LMϕs shift to “anti-inflammatory” and “pro-wound healing” M2a-d phenotypes, which are generally characterized by increased expression of arginase-1 and secretion of IL-10 [4]. LMϕs strategically coordinate innate immune responses by adopting different functional phenotypes. They do so through phagocytosis of invading bacteria and injured tissue cells during pulmonary inflammation. Concurrently with these activities, Mϕ secret specific cytokines to assist in tissue regeneration/repair after lung injury. Recent analyses showed that tissue Mϕs constantly evolve their phenotype in response to their dynamic micro-environment during disease states. In contrast, under typical in vitro conditions, Mϕs exhibit either M1 or M2 phenotypic characteristics of activation, when cultivated with specific immunomodulatory Th1 or Th2 cytokines [5].

The phenotypic activation of Mϕs is also modified by intrinsic autocrine signals [6], [7]. Resident Mϕs in several tissues reportedly produce γ-aminobutyric acid (GABA) from glutamate via glutamic acid decarboxylase (GAD) and express A-type GABA receptors (GABAAR) [8], [9], [10], [11]. This suggests that it is feasible to regulate Mϕs by an autocrine GABA signaling mechanism. Indeed, a recent study by Sanders et al. reported that activating GABAARs using benzodiazepine inhibits AMϕ’s phagocytic activity in Streptococcus pneumoniae-infected mouse lungs, resulting in increased mortality of mice due to worsened pneumonia [12]. On the other hand, we previously showed that airway and alveolar epithelial cells increase GABA secretion in the lung of mice experiencing an asthmatic reaction [13], a typical Th2/M2 pulmonary condition. Taken together, all the available data suggest that pulmonary environments may influence GABA signaling in LMϕs and lung epithelial cells, which in turn further regulates the pulmonary immune responses. Despite these findings, the cellular and molecular mechanisms by which auto- and paracrine GABA regulates the phenotypical activation of LMϕs remain to be determined. This study sought to explore whether autocrine GABA signaling in LMϕ adjusts to M1/TH1 and M2/TH2 environments, and, if so, whether it contributes to the LMϕs’ phenotypic activation state under in vivo and in vitro conditions.

Section snippets

Animals and treatments

C57BL/6 mice (8–10 weeks old, Charles River Laboratories, Senneville, QC, Canada), BALB/c mice (8–10 weeks, Harlan, Indianapolis, IN, USA) and lys-EGFP-ki transgenic mice [14] (totally 106 mice) were used in the present study. The lys-EGFP-ki transgenic mice were originally obtained from Dr. Thomas Graf and bred by homozygous mating in the University of Western Ontario barrier facility. All experimental procedures conform to the Canadian Council on Animal Care guidelines for the care and

Expression of GABA signaling proteins in lung structure cells and LMϕs

Previous studies, including ours, showed that GABAAR subunit proteins are expressed in airway epithelial cells [13], airway smooth muscle cells [27], type II alveolar epithelial cells [28] and AMϕs [12] of rodent lungs. Indeed, here our immunohistochemical assays revealed intense immunofluorescence of α1GABAAR subunit (red) (Fig. 1A) in both airway epithelial cells and type II alveolar epithelial cells that was specifically distinguished by immunofluorescence (blue) of surfactant protein C

Discussion

GABA is the primary inhibitory neurotransmitter in the central nervous system (CNS). GABAARs are pentameric anionic/chloride channels assembled from 19 different subunits (α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3) [32]. The majority of GABAA receptors in neurons are composed of two α-, two β-, and one γ2-subunits [33]. Cumulative evidence from recent studies indicates that GABAAR activation not only regulates neuronal cell maturity in the developmental brain [34] but also modifies phenotypic

Authorship

R.A.W.V., G.A.D., and W-Y.L., designed the experiments and L.J., J.W.P., M.J.E.M., Y-Y.X. participated in experiments, data acquisition and analyses. All authors involved in the interpretation of results. W-Y.L., drafted the manuscript and S.P.C., S.E.G., R.A.V., G.A.D., critically revised the manuscript. G.A.D., and W-Y.L., approved the version to be submitted.

Acknowledgements

Authors thanks Christy R Barreira, Lynda McCaig, and Cynthia Pape for technical assistances in some experiments. Authors also thank Dr. Lisa Cameron for her insightful comments on this manuscript. This study was supported by a CIHR grant (MOP-133504) to W-Y.L., and by a CHIR grant (FRN-114936) to R.A.W.V. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Disclosures

The authors declare no conflicts of interest.

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