Fig 2: LRH-1 inhibition reduces the pro-inflammatory cytokine production in LPS-stimulated RAW 264.7 cells.mRNA expression levels of a Tnf, b Il6, and c Il1b in RAW 264.7 cells pre-treated for 2 h with vehicle (PBS) or 3d2 (40 µM) and subsequently stimulated with control buffer (CTL) or LPS at indicated concentrations for 18 h. Results are shown as relative to murine Gapdh mRNA expression. Mean values of triplicates ± SD of three independent experiments are shown (t test; **p < 0.01, ***p < 0.001). d TNF and e IL-6 secreted levels in the supernatant of RAW 264.7 cells pre-treated with indicated concentrations of 3d2 for 2 h and subsequently stimulated with control buffer (CTL) or LPS at indicated concentrations for 18 h. f TNF secreted levels in the supernatant of RAW 264.7 cells pre-treated for 2 h with SR1848 at indicated concentrations, prior to stimulation with control buffer (CTL) or LPS at indicated concentrations for 18 h. Mean values of triplicates ± SD of a representative experiment (n = 3) are shown (one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001). g Quantification of mRNA levels of Nr5a2 in RAW 264.7 cells transduced with shRNA against Nr5a2 (shNr5a2) or shRNA control (shCtrl). Data are shown as relative to murine Actb mRNA and expressed as fold change to shCtrl cells. h TNF produced in RAW 264.7 cells transduced with shNr5a2 or shCtrl control and subsequent treated with control buffer (CTL) or LPS (30 ng/mL) for 18 h. Dots show technical replicates and bars means ± SD of data pooled from two independent experiment (two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant).

Fig 3: LRH-1 regulates the pro-inflammatory cytokine production in LPS-stimulated bone marrow-derived macrophages and Kupffer cells.a TNF, b IL-6, and c IL-1ß secreted protein levels in the supernatant of bone marrow-derived macrophages (BMDMs) pre-treated for 2 h with vehicle (PBS) or 3d2 (40 µM) and subsequently stimulated with control buffer (CTL) or LPS at indicated concentrations for 18 h. Mean values of triplicates ± SD of a representative experiment (n = 3) are shown (one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001). d TNF protein levels in the supernatant of isolated Kupffer cells from wild-type C57BL/6 mice after 2 h pre-treatment with vehicle (PBS) or 3d2 (40 µM) and subsequently stimulated for 18 h with LPS at indicated concentrations. Mean values of triplicates ± SD of one representative experiment (n = 3) are shown (t test; *p < 0.05, ***p < 0.001). e TNF protein levels in the supernatant of BMDMs generated from wild-type C57BL/6 (WT) or SHP-deficient (SHP-/-) mice and stimulated for 18 h with LPS at indicated concentrations. Mean values of triplicates ± SD of a representative experiment (n = 3) are shown (t test; *p < 0.05, ***p < 0.001).

Fig 4: LRH-1 inhibition limits glycolysis and glutaminolysis in RAW 264.7 cells.a Lactate produced in RAW 264.7 cells supernatant after 2 h pre-treatment with 3d2 (40 µM) or control buffer (PBS), and subsequently stimulated with control buffer (CTL) or LPS (30 ng/ml) for 18 h. Mean values of triplicates ± SD of a representative experiment (n = 2) are shown (t test; *p < 0.05). b ATP levels of RAW 264.7 cells after pre-treatment for 2 h with control buffer (CTL) or 3d2 at indicated concentrations in the medium at normal conditions, or medium supplemented with 8 mM pyruvate (Pyr) or 4 mM l-glutamine (Gln), following 18 h stimulation with LPS (30 ng/ml). The percentage of ATP was normalized to the untreated control. Mean values of triplicates ± SD of a representative experiment (n = 3) are shown. c TNF levels in the supernatant of RAW 264.7 cells pre-treated for 2 h with vehicle (PBS) or 3d2 (40 µM), following 18 h stimulation with control buffer (CTL) or LPS (30 ng/ml) in normal culture medium, or medium supplemented with 8 mM pyruvate (Pyr) or 4 mM Gln. Pooled values (three to four technical replicates) of three independent experiments ± SD are shown (t test or two-way ANOVA; *p < 0.05, **p < 0.01, n.s. = not significant).