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The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression
Published on: July 24, 2018
The intrinsic microglial clock system regulates interleukin-6 expression
Ryota Nakazato1,2, Shogo Hotta1, Daisuke Yamada1,3
1Division of Pharmaceutical Sciences, Laboratory of Molecular Pharmacology, Kanazawa University Graduate School, Kanazawa, Ishikawa, 920-1192, Japan.
Abstract:
Similar to neurons, microglia have an intrinsic molecular clock. The master clock oscillator Bmal1 modulates interleukin-6 upregulation in microglial cells exposed to lipopolysaccharide. Bmal1 can play a role in microglial inflammatory responses. We previously demonstrated that gliotransmitter ATP induces transient expression of the clock gene Period1 via P2X7 purinergic receptors in cultured microglia. In this study, we further investigated mechanisms underlying the regulation of pro-inflammatory cytokine production by clock molecules in microglial cells. Several clock gene transcripts exhibited oscillatory diurnal rhythmicity in microglial BV-2 cells. Real-time luciferase monitoring also showed diurnal oscillatory luciferase activity in cultured microglia from Per1::Luciferase transgenic mice. Lipopolysaccharide (LPS) strongly induced the expression of pro-inflammatory cytokines in BV-2 cells, whereas an siRNA targeting Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), a core positive component of the microglial molecular clock, selectively inhibited LPS-induced interleukin-6 (IL-6) expression. In addition, LPS-induced IL-6 expression was attenuated in microglia from Bmal1-deficient mice. This phenotype was recapitulated by pharmacological disruption of oscillatory diurnal rhythmicity using the synthetic Rev-Erb agonist SR9011. Promoter analysis of the Il6 gene revealed that Bmal1 is required for LPS-induced IL-6 expression in microglia. Mice conditionally Bmal1 deficient in cells expressing CD11b, including microglia, exhibited less potent upregulation of Il6 expression following middle cerebral artery occlusion compared with that in control mice, with a significant attenuation of neuronal damage. These results suggest that the intrinsic microglial clock modulates the inflammatory response, including the positive regulation of IL-6 expression in a particular pathological situation in the brain, GLIA 2016. GLIA 2017;65:198-208.
Insights
Microglia possess an internal molecular clock that regulates inflammatory responses. The Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) clock gene is crucial for controlling interleukin-6 (IL-6) production in microglia during inflammation and brain injury.
Area of Science:
- Neuroimmunology
- Chronobiology
- Molecular Biology
Background:
- Microglia, the brain's resident immune cells, possess intrinsic molecular clocks, similar to neurons.
- The master clock gene Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) influences microglial inflammatory responses, including interleukin-6 (IL-6) production.
Purpose of the Study:
- To investigate the mechanisms by which microglial molecular clock components regulate pro-inflammatory cytokine production.
- To determine the role of Bmal1 in lipopolysaccharide (LPS)-induced IL-6 expression and its impact on brain injury.
Main Methods:
- Utilized microglial cell lines (BV-2) and primary microglia from Per1::Luciferase transgenic and Bmal1-deficient mice.
- Employed siRNA targeting Bmal1, pharmacological inhibition with SR9011, and promoter analysis of the Il6 gene.
- Investigated Bmal1's role in a mouse model of middle cerebral artery occlusion (MCAO).
Main Results:
- Clock gene transcripts and luciferase activity exhibited diurnal rhythmicity in microglia.
- siRNA-mediated Bmal1 knockdown and Bmal1 deficiency selectively inhibited LPS-induced IL-6 expression.
- Pharmacological disruption of circadian rhythmicity and Bmal1 deficiency in microglia attenuated IL-6 upregulation and neuronal damage following MCAO.
Conclusions:
- The intrinsic microglial molecular clock, particularly Bmal1, positively regulates IL-6 expression.
- Bmal1 plays a critical role in modulating microglial inflammatory responses during pathological conditions like stroke.
- Targeting the microglial clock may offer therapeutic strategies for neuroinflammatory diseases.
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