Abstract
The endogenous neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone) has protective activity in animal models of alcoholism, depression, traumatic brain injury, schizophrenia, multiple sclerosis, and Alzheimer’s disease that is poorly understood. Because these conditions involve proinflammatory signaling through toll-like receptors (TLRs), we examined the effects of 3α,5α-THP, and pregnenolone on TLR4 activation in both the periphery and the central nervous system (CNS). We used monocytes/macrophages (RAW264.7) as a model of peripheral immune signaling and studied innately activated TLR4 in the ventral tegmental area (VTA) of selectively bred alcohol-preferring (P) rats. LPS activated the TLR4 pathway in RAW264.7 cells as evidenced by increased levels of p-TAK1, TRAF6, NF-κB p50, phospho-NF-κB- p65, pCREB, HMGB1, and inflammatory mediators, including MCP-1 and TNFα. Both 3α,5α-THP and pregnenolone (0.5–1.0μM) substantially (~80%) inhibited these effects, indicating pronounced inhibition of TLR4 signaling. The mechanism of inhibition appears to involve blockade of TLR4/MD-2 protein interactions in RAW246.7 cells. In VTA, 3α,5α-THP (15 mg/kg, IP) administration reduced TRAF6 (~20%), CRF (~30%), and MCP-1 (~20%) levels, as well as TLR4 binding to GABAA receptor α2 subunits (~60%) and MyD88 (~40%). The data suggest that inhibition of proinflammatory neuroimmune signaling underlies protective effects of 3α,5α-THP in immune cells and brain, apparently involving blocking of protein-protein interactions that initiate TLR4-dependent signaling. Inhibition of pro-inflammatory TLR4 activation represents a new mechanism of 3α,5α-THP action in the periphery and the brain.
Highlights
Neuroimmune signaling in the brain elevates proinflammatory cytokines, chemokines, and their associated receptors to promote central nervous system (CNS) disease in a progressive feed-forward manner[21]
To examine the possibility that 3α,5α-THP inhibits proinflammatory neuroimmune signaling in the periphery, we studied the effects of 3α,5α-THP and pregnenolone on LPS-induced TLR4 activation and pro-inflammatory signaling in mouse monocyte/macrophage RAW264.7 cells
To examine the neurosteroids’ effect on TLR4 signal activation, RAW264.7 cells were treated with LPS (1 μg/ml; 24 hrs) in the absence or presence of 3α,5α-THP (0.5 μM, 1 μM) or pregnenolone (0.5 μM, 1 μM), and cell extracts were assayed for expression of myeloid differentiation primary response 88 (MyD88)-dependent pathway members, by immunoblotting with antibodies to p-TAK1, monocyte chemotactic protein (MCP-1), tumor necrosis factor receptor associated factor 6 (TRAF6), TLR4, and transcription factor NF-κB p5031–33
Summary
Neuroimmune signaling in the brain elevates proinflammatory cytokines, chemokines, and their associated receptors to promote CNS disease in a progressive feed-forward manner[21]. To examine the possibility that 3α,5α-THP inhibits proinflammatory neuroimmune signaling in the periphery, we studied the effects of 3α,5α-THP and pregnenolone on LPS-induced TLR4 activation and pro-inflammatory signaling in mouse monocyte/macrophage RAW264.7 cells. To examine the neurosteroids’ effect on TLR4 signal activation, RAW264.7 cells were treated with LPS (1 μg/ml; 24 hrs) in the absence or presence of 3α,5α-THP (0.5 μM, 1 μM) or pregnenolone (0.5 μM, 1 μM), and cell extracts were assayed for expression of MyD88-dependent pathway members, by immunoblotting with antibodies to p-TAK1, monocyte chemotactic protein (MCP-1), TRAF6, TLR4, and transcription factor NF-κB p5031–33. Cell viability was assessed by the trypan blue exclusion assay, as described in Materials and Methods
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