Inhibition of human macrophage activation via pregnane neurosteroid interactions with toll-like receptors: Sex differences and structural requirements

We recently discovered that (3α,5α)3-hydroxypregnan-20-one (allopregnanolone) inhibits pro-inflammatory toll-like receptor (TLR) activation and cytokine/chemokine production in mouse macrophage RAW264.7 cells. The present studies evaluate neurosteroid actions upon TLR activation in human macrophages from male and female healthy donors. Buffy coat leukocytes were obtained from donors at the New York Blood Center (http://nybloodcenter.org/), and peripheral blood mononuclear cells were isolated and cultured to achieve macrophage differentiation. TLR4 and TLR7 were activated by lipopolysaccharide (LPS) or imiquimod in the presence/absence of allopregnanolone or related neurosteroids and pro-inflammatory markers were detected by ELISA or western blotting. Cultured human monocyte-derived-macrophages exhibited typical morphology, a mixed immune profile of both inflammatory and anti-inflammatory markers, with no sex difference at baseline. Allopregnanolone inhibited TLR4 activation in male and female donors, preventing LPS-induced elevations of TNF-α, MCP-1, pCREB and pSTAT1. In contrast, 3α,5α-THDOC and SGE-516 inhibited the TLR4 pathway activation in female, but not male donors. Allopregnanolone completely inhibited TLR7 activation by imiquimod, blocking IL-1-β, IL-6, pSTAT1 and pIRF7 elevations in females only. 3α,5α-THDOC and SGE-516 partially inhibited TLR7 activation, only in female donors. The results indicate that allopregnanolone inhibits TLR4 and TLR7 activation in cultured human macrophages resulting in diminished cytokine/chemokine production. Allopregnanolone inhibition of TLR4 activation was found in males and females, but inhibition of TLR7 signals exhibited specificity for female donors. 3α,5α-THDOC and SGE-516 inhibited TLR4 and TLR7 pathways only in females. These studies demonstrate anti-inflammatory effects of allopregnanolone in human macrophages for the first time and suggest that inhibition of pro-inflammatory cytokines/chemokines may contribute to its therapeutic actions.


Supplementary Results
A representative example of results from the analysis of the human monocyte-derived macrophage (hMDM) secretome on the RayBiotech L-507 cytokine array (RayBiotech Life, Inc., Peachtree, GA) is shown in Fig. S1. Figure S1. Example of results from the analysis of the hMDM secretome on the RayBiotech L-507 cytokine array. Medium was collected from hMDM cultured overnight in serum free DMEM and centrifuged at 1000 x g. The cell free medium was added to a RayBiotech human antibody array L-507 and processed according to the RayBiotech human antibody array protocol. Slide arrays were scanned using an Agilent technologies DNA microarray scanner.

Supplementary
Female macrophages had a reduced sensitivity to lipopolysaccharide (LPS) as defined by the level of MCP-1 compared to male macrophages. This factor may explain the greater inhibition of LPS-induced elevation of MCP-1 by allopregnanolone (3α,5α-THP) in female macrophages when compared with male macrophages (Fig. S2).
Supplementary Figure S3. Lipopolysaccharide (LPS) and/or allopregnanolone (3α,5α-THP) did not affect TLR4 expression in human monocyte-derived macrophages (hMDM). hMDM cultures (n≥9 cultures from 3 female or 3 male donors/grp) were treated with LPS (1 µg/ml) with or without allopregnanolone (3α,5α-THP; 1 µM). Cells were harvested at 24 h after treatment initiation and examined for the expression of TLR4. Treatments with lipopolysaccharide (LPS) and/or allopregnanolone (3α,5α-THP) did not change TLR4 expression in hMDM from both female and male donors when compared with vehicle control (CTL). There were no differences in the levels of TLR4 between females and males at baseline or after treatments with LPS with or without allopregnanolone (3α,5α-THP). Three-way ANOVA, Tukey's post hoc test, p>0.05.
Imiquimod (IMQ) and/or allopregnanolone (3α,5α-THP) did not affect TLR7 expression in hMDM from both male and female donors and there are no sex differences in the expression of TLR7 at baseline, after IMQ and/or allopregnanolone treatments (Fig. S5).
Supplementary Figure S5. Imiquimod (IMQ) and/or allopregnanolone (3α,5α-THP) did not affect TLR7 expression in human monocyte-derived macrophages (hMDM). hMDM cultures (n≥9 cultures from 3 female or 3 male donors/grp) were treated with IMQ (30 µg/ml) with or without allopregnanolone (3α,5α-THP; 1 µM). Cells were harvested at 24 h after treatment initiation and examined for the expression of both full length (FL; ~121 kDa) and cleaved fragment (~70 kDa) of TLR7. Treatments with IMQ and/or allopregnanolone (3α,5α-THP) did not change TLR7 (full length and cleaved fragment) expression in hMDM from both female and male donors when compared with vehicle control (CTL). There were no differences in the levels of TLR7 (full length and cleaved fragment) between females and males at baseline or after treatments with IMQ with or without allopregnanolone (3α,5α-THP). Three-way ANOVA, Tukey's post hoc test, p>0.05.