Neurosteroid [3α,5α]-3-hydroxy-pregnan-20-one enhances IL-10 production via endosomal TRIF-dependent TLR4 signaling pathway

Front Endocrinol (Lausanne). 2023 Dec 21:14:1299420. doi: 10.3389/fendo.2023.1299420. eCollection 2023.

Abstract

Background: Previous studies demonstrated the inhibitory effect of allopregnanolone (3α,5α-THP) on the activation of inflammatory toll-like receptor 4 (TLR4) signals in RAW264.7 macrophages and the brains of selectively bred alcohol-preferring (P) rats. In the current study, we investigated the impact of 3α,5α-THP on the levels of IL-10 and activation of the TRIF-dependent endosomal TLR4 pathway.

Methods: The amygdala and nucleus accumbens (NAc) of P rats, which exhibit innately activated TLR4 pathways as well as RAW264.7 cells, were used. Enzyme-linked immunosorbent assays (ELISA) and immunoblotting assays were used to ascertain the effects of 3α,5α-THP on the TRIF-dependent endosomal TLR4 pathway and endosomes were isolated to examine translocation of TLR4 and TRIF. Additionally, we investigated the effects of 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) on the levels of IL-10 in RAW264.7 macrophages. Finally, we examined whether inhibiting TRIF (using TRIF siRNA) in RAW264.7 cells altered the levels of IL-10.

Results: 3α,5α-THP administration facilitated activation of the endosomal TRIF-dependent TLR4 pathway in males, but not female P rats. 3α,5α-THP increased IL-10 levels (+13.2 ± 6.5%) and BDNF levels (+21.1 ± 11.5%) in the male amygdala. These effects were associated with increases in pTRAM (+86.4 ± 28.4%), SP1 (+122.2 ± 74.9%), and PI(3)K-p110δ (+61.6 ± 21.6%), and a reduction of TIRAP (-13.7 ± 6.0%), indicating the activation of the endosomal TRIF-dependent TLR4 signaling pathway. Comparable effects were observed in NAc of these animals. Furthermore, 3α,5α-THP enhanced the accumulation of TLR4 (+43.9 ± 11.3%) and TRIF (+64.8 ± 32.8%) in endosomes, with no significant effect on TLR3 accumulation. Additionally, 3α,5α-THP facilitated the transition from early endosomes to late endosomes (increasing Rab7 levels: +35.8 ± 18.4%). In RAW264.7 cells, imiquimod (30 µg/mL) reduced IL-10 while 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) restored IL-10 levels. To determine the role of the TRIF-dependent TLR4 signaling pathway in IL-10 production, the downregulation of TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%). TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%) and 3α,5α-THP (1.0 µM) no longer restored the reduced IL-10 levels.

Conclusion: The results demonstrate 3α,5α-THP enhancement of the endosomal TLR4-TRIF anti-inflammatory signals and elevations of IL-10 in male P rat brain that were not detected in female P rat brain. These effects hold significant implications for controlling inflammatory responses in both the brain and peripheral immune cells.

Keywords: [3α,5α]-3,21-dihydroxypregnan-20-one (3α,5α-THDOC); [3α,5α]-3-hydroxy-pregnan-20-one (3α,5α-THP); neuroinflammation; toll-like receptor 4; toll/interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport
  • Animals
  • Endosomes / metabolism
  • Female
  • Interleukin-10
  • Male
  • Mice
  • Neurosteroids*
  • Pregnanolone*
  • RAW 264.7 Cells
  • Rats
  • Signal Transduction*
  • Toll-Like Receptor 4* / metabolism

Substances

  • Adaptor Proteins, Vesicular Transport
  • Interleukin-10
  • Neurosteroids
  • Pregnanolone
  • Toll-Like Receptor 4

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by Sage Therapeutics Inc. and the Bowles Center for Alcohol Studies at UNC School of Medicine. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.