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

Background: Previous studies demonstrated the inhibitory effect of allopregnanolone (3 alpha,5 alpha-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 3 alpha,5 alpha-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 3 alpha,5 alpha-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 3 alpha,5 alpha-THP and 3 alpha,5 alpha-THDOC (0.1, 0.3, and 1.0 mu M) 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: 3 alpha,5 alpha-THP administration facilitated activation of the endosomal TRIF-dependent TLR4 pathway in males, but not female P rats. 3 alpha,5 alpha-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-p110 delta (+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, 3 alpha,5 alpha-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, 3 alpha,5 alpha-THP facilitated the transition from early endosomes to late endosomes (increasing Rab7 levels: +35.8 +/- 18.4%). In RAW264.7 cells, imiquimod (30 mu g/mL) reduced IL-10 while 3 alpha,5 alpha-THP and 3 alpha,5 alpha-THDOC (0.1, 0.3, and 1.0 mu M) 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 3 alpha,5 alpha-THP (1.0 mu M) no longer restored the reduced IL-10 levels.Conclusion: The results demonstrate 3 alpha,5 alpha-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.