Fig 2: LRH-1 inhibition protects from TNF-dependent hepatitis.Wild-type C57BL/6 mice (n = 6–9 per group) were pre-treated with vehicle (PBS) or 3d2 (50 mg/kg body weight) i.p. injections for 1 h and then challenged by i.p. injections of LPS (0.2 mg) and GalN (40 mg). Serum was harvested for the analysis of a TNF protein concentrations and b transaminase (ALT) levels (t test; ***p < 0.001, n.s. = not significant). c Immunohistochemical detection of cleaved caspase-3-positive, apoptotic hepatocytes in liver sections from untreated (CTL) or LPS/GalN i.p. injected mice pre-treated with PBS or 3d2 as described above (scale bar: 150 µm).

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 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).