Abstract
BackgroundOverproduction of proinflammatory cytokines from activated microglia has been implicated as an important contributor to pathophysiology progression in both acute and chronic neurodegenerative diseases. Therefore, it is critical to elucidate intracellular signaling pathways that are significant contributors to cytokine overproduction in microglia exposed to specific stressors, especially pathways amenable to drug interventions. The serine/threonine protein kinase p38α MAPK is a key enzyme in the parallel and convergent intracellular signaling pathways involved in stressor-induced production of IL-1β and TNFα in peripheral tissues, and is a drug development target for peripheral inflammatory diseases. However, much less is known about the quantitative importance of microglial p38α MAPK in stressor-induced cytokine overproduction, or the potential of microglial p38α MAPK to be a druggable target for CNS disorders. Therefore, we examined the contribution of microglial p38αMAPK to cytokine up-regulation, with a focus on the potential to suppress the cytokine increase by inhibition of the kinase with pharmacological or genetic approaches.MethodsThe microglial cytokine response to TLR ligands 2/3/4/7/8/9 or to Aβ1-42 was tested in the presence of a CNS-penetrant p38α MAPK inhibitor, MW01-2-069A-SRM. Primary microglia from mice genetically deficient in p38α MAPK were used to further establish a linkage between microglia p38α MAPK and cytokine overproduction. The in vivo significance was determined by p38α MAPK inhibitor treatment in a LPS-induced model of acute neuroinflammation.ResultsIncreased IL-1β and TNFα production by the BV-2 microglial cell line and by primary microglia cultures was inhibited in a concentration-dependent manner by the p38α MAPK-targeted inhibitor. Cellular target engagement was demonstrated by the accompanying decrease in the phosphorylation state of two p38α MAPK protein substrates, MK2 and MSK1. Consistent with the pharmacological findings, microglia from p38α-deficient mice showed a diminished cytokine response to LPS. Further, oral administration of the inhibitor blocked the increase of IL-1β in the cerebral cortex of mice stressed by intraperitoneal injection of LPS.ConclusionThe p38α MAPK pathway is an important contributor to the increased microglial production of proinflammatory cytokines induced by diverse stressors. The results also indicate the feasibility of targeting p38α MAPK to modulate CNS proinflammatory cytokine overproduction.
Highlights
Overproduction of proinflammatory cytokines from activated microglia has been implicated as an important contributor to pathophysiology progression in both acute and chronic neurodegenerative diseases
The p38a mitogen activated protein kinase (MAPK) inhibitor, 069A, suppresses LPS-induced cytokine up-regulation in the BV-2 microglial cell line The ability of the small molecule p38a MAPK inhibitor, compound 069A, to inhibit proinflammatory cytokine up-regulation was tested in the mouse microglial BV-2 cell line
We found that phosphorylation of the p38a MAPK substrates mitogenactivated protein kinase-activated protein kinase 2 (MK2) (Figure 4C) and mitogen- and stress-activated kinase 1 (MSK1) (Figure 4D) was increased with ligand activation of primary microglia, and that 069A treatment significantly decreased the levels of p-MK2 and p-MSK1
Summary
Overproduction of proinflammatory cytokines from activated microglia has been implicated as an important contributor to pathophysiology progression in both acute and chronic neurodegenerative diseases. It is critical to elucidate intracellular signaling pathways that are significant contributors to cytokine overproduction in microglia exposed to specific stressors, especially pathways amenable to drug interventions. The serine/threonine protein kinase p38a MAPK is a key enzyme in the parallel and convergent intracellular signaling pathways involved in stressor-induced production of IL-1b and TNFa in peripheral tissues, and is a drug development target for peripheral inflammatory diseases. Activation of TLRs on microglia leads to the production of inflammatory mediators, such as IL-1b, IL-6, TNFa, and nitric oxide. TLR engagement and signaling in the CNS provide an important defense mechanism by which microglia respond to external pathogens or host-derived ligands. Tight regulation of microglial activation pathways is essential for appropriate responses to stressor stimuli and maintenance of CNS homeostasis, because uncontrolled or dysregulated inflammatory responses can lead to propagation of detrimental and neurotoxic responses
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