Fig 2: Characteristics of cultured human monocyte-derived macrophages. (A) Example of a monolayer of human monocyte-derived macrophages (hMDM) grown on ultralow adhesion plastic. (B) Basal hMDM secretome from 5 independent mixed sex cultures detected by RayBiotech human antibody array L-507 illustrating a mixed inflammatory and anti-inflammatory cytokine profile. (C) hMDM cell lysates analyzed by ELISA express both inflammatory (TNF-a, IL-6, IL-1ß, MCP-1, IFN-?) and anti-inflammatory (IL-13, TGF-ß1, IL-1ra) mediators with no evidence of sex differences at baseline. Each hMDM culture is shown as a single symbol (n = 18), obtained from at least 3 male or 3 female donors. (D) hMDM surface markers determined by flow cytometry using direct immunofluorescence in 4-7 independent mixed sex cultures. (E) Western blot image shows that hMDM from both male and female donors (n=9 cultures from 3 donors/sex) lack ?-aminobutyric acid type A (GABAA) receptor subunits a1, a2, a4, and d at baseline (CTL) or after treatments with lipopolysaccharide (LPS) and/or allopregnanolone (3a,5a-THP). As a positive control, the amygdala from male and female alcohol preferring P rats intraperitoneally injected with vehicle (45% w/v 2-hydroxypropyl-ß-cyclodextrin; 30 min) or allopregnanolone (15 mg/kg; 30 min) was used. (F) Chemical structures of endogenous neurosteroids allopregnanolone and 3a,5a-THDOC (tetrahydrodeoxycorticosterone) and the synthetic 1,2,5-triazole analog of the allopregnanolone, SGE-516. 3a,5a-THDOC differs from the allopregnanolone by a C-21-hydroxyl group at the D-ring. SGE-516 differs from the allopregnanolone by a C-3 cis-methyl and cis-hydrogens at C-5 and C-19, and a C-21-1,2,5-triazole group at the D-ring.

Fig 3: 3a,5a-THDOC and SGE-516 inhibit LPS-induced increases of TNF-a and MCP-1 in human monocyte-derived macrophages (hMDM) from female, but not male donors. Each hMDM culture (n=12 from 3 female (A) or 3 male (B) donors/grp) is shown as a single symbol. Cells were treated with LPS (1 µg/ml; 24h) with or without 3a,5a-THDOC (1 µM; 24h) or SGE-516 (1 µM; 24h). LPS caused a significant increase in the levels of TNF-a (~80% in hMDM from both female (A) and male (B) donors) and MCP-1 (~65% in hMDM from both female (A) and male (B) donors) relative to vehicle control (CTL). The increases of TNF-a were partially inhibited by 3a,5a-THDOC and SGE-516 (~30% and ~35% inhibition, respectively) in hMDM derived from female (A) but not male (B) donors. The increases of MCP-1 were completely inhibited by 3a,5a-THDOC or SGE-516 in hMDM from female (A) but not male (B) donors. Two-way ANOVA, Tukey’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.