AUTHOR=Balan Irina , Grusca Adelina , O’Buckley Todd K. , Morrow A. Leslie 

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

JOURNAL=Frontiers in Endocrinology

VOLUME=Volume 14 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2023.1299420

DOI=10.3389/fendo.2023.1299420

ISSN=1664-2392

ABSTRACT=Background: Previous studies demonstrated the inhibitory effect of  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 and RAW264.7 macrophage cells were used. 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%). 3α,5α-THP (1.0 µM) did not alter 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.