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 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).

Fig 4: 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 5: Extracellular vesicles favor macrophage reprogramming during the resolution of inflammation. THP-1 monocytes were differentiated under the PMA conditions with extracellular vesicles treatment over 72 h. Culture supernatants were then collected and proinflammatory cytokine secretion were determined by selective ELISA. Levels of IL-6 (a), IL-12p40 (b), IL-12 (c), IL33 (d), IFN-a2 (e) are significantly increased in the diseased mesentery extracellular vesicles group compared to the other groups while TNF-a (f), TGF-b (g), and IL-10 (h) are significantly decreased in the diseased mesentery group. Results are mean ± SEM from four independent experiments. *p< 0.05. ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin 6; PMA, phorbol-12-myristate-13-acetate; TNF-a, tumor necrosis factor-a.