Endogenous Neurosteroid (3α,5α)3-Hydroxypregnan-20-one Inhibits Toll-like-4 Receptor Activation and Pro-inflammatory Signaling in Macrophages and Brain

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.

Is There a Future for PPARs in the Treatment of Neuropsychiatric Disorders?

“…Allopregnanolone enhances γ-aminobutyric acid action at GABA A receptors [9,10] and improves emotional behavior. Allopregnanolone may also exert an important anti-inflammatory action by binding at α2-containing GABA A receptor subtypes located in glial cells, through inhibition of toll-like 4 receptor/NF-κB pathway [11]. PPAR-γ agonists potentiate the PPAR-γ-induced inhibitory action on NF-κB, which is responsible for microglial activated status, neuroinflammation and neurodegeneration.…”

A Rationale for Allopregnanolone Treatment of Alcohol Use Disorders: Basic and Clinical Studies

“…The mechanism of neuroactive steroid inhibition appears to involve blockade of TLR4/MD‐2 protein interactions in RAW246.7 cells, as both neuroactive steroids blocked the coimmunoprecipitation of TLR4 with MD‐2, a requisite step in the activation of the TLR4‐MyD88‐dependent signaling pathway. These data suggest a novel role for both pregnenolone and allopregnanolone to inhibit TLR4 signaling in the innate immune system (Balan et al, 2019a,b). Similar inhibition of TLR4 signaling by pregnenolone, progesterone, and allopregnanolone was found via degradation of TLR2‐ and TLR4‐associated proteins (Murugan et al, ).…”

“…However, clinical trials in humans have so far been unsuccessful. Thus, alternative ways to target CRF signaling in humans may be beneficial, and neuroactive steroids may represent one way to achieve this goal, given their modulatory action on HPA homeostasis (see below) and extrahypothalamic CRF levels (Balan et al, 2019a,b).…”

“…Recent investigations suggest that neuroactive steroids, pregnenolone, and allopregnanolone inhibit TLR4 signaling in an immortalized macrophage cell line (RAW264.7) as well as innate TLR4 and CRF activation in VTA of alcohol‐preferring P male rats (Balan et al, 2019a,b). In the mouse macrophage cell line, the TLR4 agonist LPS increased all the canonical markers of TLR4 activation, as well as the transcription factor pNFκB, the proinflammatory endogenous TLR4 agonist HMGB1, the chemokine monocyte chemoattractant protein 1 (MCP‐1), and the cytokine tumor necrosis factor‐α (TNF‐α).…”

“…In the P rat VTA, allopregnanolone administration reduced both CRF expression (30%) and innate TLR4 activation via TLR4 binding to the GABA A receptor α2 subunit protein (60%) and/or MyD88 (40%), measured by coimmunoprecipitation (Balan et al, 2019a,b). The data suggest that inhibition of proinflammatory neuroimmune signaling contributes to the protective effects of allopregnanolone in the brain, apparently involving blockade of protein–protein interactions that initiate TLR4‐dependent signaling.…”