Page 57 - M. Immunology

GABAergic activation enhances autophagy and phagosomal

maturation in macrophages during mycobacterial infection

Seul Gi Shin 1,2,3 , Jin Kyung Kim 1,2,3 , Yi Sak Kim 1,2,3 , Hyun-Woo Suh 1,2,3 and Eun-Kyeong Jo 1,2,3*

1 Department of Microbiology, Department of Medical Science, Infection Control Convergence Research Center, Chungnam National University School
3
2
of Medicine, Daejeon 35015, Republic of Korea.

ABSTRACT

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter within the central nervous system and has been extensively studied in neurological disorders,

such as epilepsy, anxiety disorders, and schizophrenia. However, the roles of GABA in antimicrobial host defenses are largely unknown. To define the mechanism by

which GABA triggers the antimicrobial host defense system during intracellular bacterial infection, we used RNA sequencing to analyze the GABA-mediated, genome-

wide transcriptional changes in bone marrow-derived macrophages (BMDMs). GABA treatment led to an upregulation of numerous genes involved in autophagy in

BMDMs. When we next explored whether GABA activated autophagy, we found that GABA significantly increased the mRNA expression levels of autophagy-related

genes involved in autophagosome formation and autolysosome maturation in BMDMs. We further showed that treatment of BMDMs with GABA or GABAAR agonists

robustly increased the autophagosomal membrane-associated LC3-II fractions and autophagic flux. In addition, we showed that GABA-induced autophagy activation

was required for phagosomal maturation during Mtb infection. Taken together, both GABA and GABAergic activation enhanced activation of autophagy and

phagosomal maturation during Mtb infection.

INTRODUCTION

• Gamma-aminobutyric acid (GABA) is the major inhibitory

neurotransmitter within the central nervous system and has

been extensively studied in neurological disorders.

• Preclinical studies suggest the potential application of GABA

or GABAergic (agonistic) drugs as new therapeutics against

autoimmune and inflammatory diseases.

• As a crucial innate immune arm, the autophagy pathway

provides a protective role against a variety of intracellular

bacterial pathogens, including Mycobacterium tuberculosis

(Mtb).

• Nonetheless, it is poorly understood whether and how

GABAergic signaling regulates antimicrobial host defenses in

the context of bacterial infections.

RESULT

GABAergic activation enhances autophagy in macrophages. GABAergic autophagy promotes Mtb phagosomal maturation.

a b

a b

(a–d) BMDMs were infected

with Mtb-ERFP (MOI of 5) for

4 h, incubated with GABA

(100 μM), muscimol (Mus;

100 μM), or isoguvacine

hydrochloride (Iso; 100 μM)

for 18 h, then stained with

LC3 (green; a, b) or LAMP2

(green; c, d), and DAPI (for

nuclei; blue).

d e (a) Cells were visualized by

c d confocal microscopy. Scale

bars, 5 μm. (b) Quantitative

data of colocalization of Mtb-

ERFP and LC3 per cell.

(c) Cells were visualized by

confocal microscopy. Scale
c bars, 5 μm. (d) Quantitative

data of colocalization of Mtb-

ERFP and LAMP2 per cell.

f e–f Mice were injected i.v.

with Mtb (1 × 10 6 CFU),

treated with PBS or GABA

(daily i.p. 200 mg/kg), and

monitored at 14 days post-

e f infection (dpi).

(e) Representative low- (top)

and high-magnification

(bottom) transmission
(a) Heatmap analysis of the RNAseq data (representative of duplicate determinations). BMDMs were electron micrographs of lung

treated with GABA (100 μM) for the indicated times and then subjected to RNAseq analysis. (b) tissues. Scale bars, 1 μm

BMDMs were treated with GABA (100 μM) for 6 h. Cells were subjected to qRT-PCR to determine (top) and 200 nm (bottom).

mRNA levels of autophagy genes. (c) BMDMs were treated with GABA (10, 50, or 100 μM) for 18 h (f) Quantitative data of

and the expression of LC3B was analyzed by flow cytometry (horizontal axis, fluorescence (FITC) phagosomes and

intensity). (d, e) BMDMs were incubated with GABA (100 μM), muscimol (Mus; 100 μM), or autophagosomes containing

isoguvacine hydrochloride (Iso; 100 μM) and then stained with LC3 (green) and DAPI (nuclei; blue). Mtb.

(d) Confocal microscopic analysis. Scale bars, 5 μm. (e) Quantitative data of LC3 punctate analysis.

(f) BMDMs were pretreated with or without bafilomycin A1 (BafA, 200 nM; 2 h) and incubated with

GABA (10, 50, or 100 μM), Mus (100 μM), or Iso (100 μM) for 18 h. LC3 and β-tubulin levels were

evaluated by immunoblotting.

CONCLUSION REFERENCE

 These data reveal that either exogenous GABA treatment or GABAergic activation • Wong, C. G., Bottiglieri, T. & Snead, O. C. 3rd (2003). GABA, gamma-hydroxybutyric acid,

triggers the activation of autophagy in macrophages. and neurological disease. Ann. Neurol. 54, S3–S12.

 GABA and GABAergic activation enhanced phagosomal maturation during Mtb • Prud’homme, G. J., Glinka, Y. & Wang, Q. (2015). Immunological GABAergic interactions

infection. and therapeutic applications in autoimmune diseases. Autoimmun. Rev. 14, 1048–1056.

 our data show that GABA or GABAergic activation induces autophagy, which in

turn promotes phagosomal maturation and host antimicrobial responses to • Gutierrez, M. G. et al. (2004). Autophagy is a defense mechanism inhibiting BCG and

intracellular bacterial infections including Mtb and BCG. Mycobacterium tuberculosis survival in infected macrophages. Cell 119, 753–766.