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Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

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Metabotropic glutamate receptor 5 modulates calcium oscillation and innate immune response induced by

F Liu¹, R Zhou², H Yan³

¹State Key Laboratory of Trauma, Burn and Combined Injury, Chongqing, China; Department of Infection & Immunity, Research Institute of Surgery/Daping Hospital, Third Military Medical University, Chongqing, China.

|September 25, 2014

Abstract:

Microglia, the primary immune cells in the brain, have been implicated as the predominant cells governing inflammation-mediated neuronal damage. In response to immunological challenges such as lipopolysaccharide (LPS), microglia are activated and subsequently inflammatory process is initiated as evidenced by the release of pro-inflammatory chemokines and cytokines. Here we show that Group I metabotropic glutamate receptor 5 (mGluR5) is involved in LPS-induced microglia activation. LPS triggered a similar pattern of [Ca2+]i oscillation in N9, Toll-like receptor 4 (TLR4)-mutant EOC 20, TLR4-wild-type and TLR4-deficient primary mouse microglia, suggesting that LPS-induced [Ca2+]i oscillation is independent of TLR4. The characteristics of [Ca2+]i oscillation induced by LPS are consistent with those observed in mGluR5 activation. In addition, mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) abolished LPS-induced [Ca2+]i oscillation. Immunocytochemistry demonstrated that LPS colocalizes with mGluR5 in microglia and the direct binding of LPS and mGluR5 was further validated by antibody-based fluorescence resonance energy transfer (FRET) technology. Activation of mGluR5 using a selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) significantly expanded LPS-induced nuclear factor-kappa B (NF-κB) activity and CHPG alone increased NF-κB activity as well. But, mGluR5 antagonist MTEP attenuated the actions of LPS, CHPG and the additive effect of LPS and CHPG in microglia. LPS induced tumor necrosis factor-α (TNF-α) secretion in N9 microglia, but not in TLR4-mutant EOC 20 and TLR4-deficient primary mouse microglia. CHPG reduced LPS-caused TNF-α production, but MTEP increased LPS-induced TNF-α production and blocked the effect of CHPG in N9 microglia. These data demonstrate that mGluR5 and TLR4 are two critical receptors that mediate microglia activation in response to LPS, suggesting that mGluR5 may represent a novel target for modulating microglia-dependent neuroinflammation.

Insights

Group I metabotropic glutamate receptor 5 (mGluR5) mediates lipopolysaccharide (LPS)-induced microglia activation, independent of Toll-like receptor 4 (TLR4). Targeting mGluR5 offers a novel strategy for neuroinflammation.

Area of Science:

Neuroimmunology
Cellular Neuroscience
Molecular Biology

Background:

Microglia are key brain immune cells involved in inflammation-mediated neuronal damage.
Lipopolysaccharide (LPS) activates microglia, initiating inflammatory responses via pro-inflammatory cytokines.
The precise molecular mechanisms of LPS-induced microglia activation require further elucidation.

Purpose of the Study:

To investigate the role of Group I metabotropic glutamate receptor 5 (mGluR5) in lipopolysaccharide (LPS)-induced microglia activation.
To determine the relationship between mGluR5, Toll-like receptor 4 (TLR4), and LPS-mediated inflammatory signaling in microglia.
To explore the potential of mGluR5 as a therapeutic target for neuroinflammation.

Main Methods:

Assessed intracellular calcium ([Ca2+]i) oscillations in various microglia cell lines and primary cultures upon LPS stimulation.
Utilized mGluR5 antagonist MTEP and agonist CHPG to investigate receptor involvement.
Employed immunocytochemistry and fluorescence resonance energy transfer (FRET) to confirm LPS and mGluR5 colocalization and binding.
Measured nuclear factor-kappa B (NF-κB) activity and tumor necrosis factor-alpha (TNF-α) secretion under different experimental conditions.

Main Results:

LPS-induced [Ca2+]i oscillations in microglia were independent of TLR4 but consistent with mGluR5 activation.
MTEP abolished LPS-induced [Ca2+]i oscillations, and LPS was found to colocalize and bind with mGluR5.
mGluR5 activation by CHPG enhanced LPS-induced NF-κB activity and TNF-α secretion, while MTEP modulated these responses, indicating a complex interplay between mGluR5 and TLR4 signaling.

Conclusions:

Group I mGluR5 plays a significant role in mediating LPS-induced microglia activation, acting independently of TLR4 in some aspects.
The interaction between mGluR5 and TLR4 pathways influences key inflammatory mediators like NF-κB and TNF-α.
mGluR5 represents a promising novel target for therapeutic intervention in neuroinflammatory conditions.

Read full analysis

Keywords:

Group I metabotropic glutamate receptor 5 NF-κB Toll-like receptors [Ca2+]i oscillation lipopolysaccharide microglia

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