Journal: EBioMedicine

Article Title: Ketogenesis acts as an endogenous protective programme to restrain inflammatory macrophage activation during acute pancreatitis

doi: 10.1016/j.ebiom.2022.103959

Figure Lengend Snippet: βOHB limits proinflammatory macrophage activation via class I HDACs, (a, b) Gene set enrichment analysis (GSEA) of gene expression in murine BMDMs are depicted. (a) Enrichment of TSA-upregulated signature genes (GSE22049) in βOHB-treated BMDMs. (b) Enrichment of HDAC3 depletion related signature genes (GES33162) in βOHB-treated BMDMs. (c, d) BMDMs treated with LPS (100 ng/ml) together with βOHB (10 mM), TSA (1 μΜ), or MS-275 (20 μM) for 4 h. (c) Flow cytometry analysis of intracellular TNFα levels. (d) Quantitative PCR analysis of Nos2, Il6, Il12b , and Tnfa mRNA expression. (e) ChIP-qPCR analysis of H3Ac and H4Ac occupancy in Edn1, Il12a , and Nos2 loci in the indicated group, with IgG as the control. (f) Ingenuity Pathway Analysis (IPA) of genes in cluster A. (g) BMDMs were pretreated with βOHB (10 mM) or TSA (1 μΜ) 1 h prior to LPS (100 ng/ml) stimulation for 15 min. Acylated p65 levels were determined by immunoprecipitation, while p-p65 and total p65 were determined by immunoblotting. (h) Mice were i.p. injected with TSA (0.1 mg/kg), MS-275 (20 mg/kg) or DMSO as a control 1 h prior to the CER-AP (Cer*7) induction and were harvested at 24 h thereafter (n = 5 / group). Frequency of TNFα + macrophagess (F4/80 + CD11b + ) in pancreatic leukocytes from the indicated groups. Data are presented as the mean ± SD in bar graphs (c–e) or the mean ± SEM in dot plots (h). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: On day 6 of culturing, BMDMs were stimulated with LPS (100 ng/ml) for 15 min or 4 h, or injured acinar cell culture medium for further assays. βOHB (10 mM; Sigma-Aldrich), HDAC pan-inhibitor TSA (1 μM; MedChem Express, Shanghai, China) or HDAC1/3 inhibitor MS-275 (20 μM; MedChem Express) was administered 1 h before LPS stimulation.

Techniques: Activation Assay, Gene Expression, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, ChIP-qPCR, Control, Immunoprecipitation, Western Blot, Injection

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Effects of epigenetic inhibitors on IL-1β–induced NUB1 expression in RA and OA fibroblast-like synoviocytes (FLS). RA and OA FLS were pretreated with epigenetic inhibitors and subsequently stimulated with IL-1β (2 ng/mL). NUB1 mRNA expression was quantified by qRT-PCR. Fold change was calculated as the ratio of NUB1 mRNA expression in IL-1β–stimulated cells to the corresponding unstimulated control; specifically, DMSO alone for the IL-1/DMSO condition and the inhibitor-alone control for inhibitor-treated conditions. ( a ) 5-aza-deoxycytidine (5-aza-dC; DNA methyltransferase inhibitor). RA and OA FLS were treated with 5-aza-dC for 14 days prior to stimulation with IL-1β (n = 7 each). 5-aza-dC partially reduced the difference in IL-1β–induced NUB1 expression between RA and OA. Under control conditions, IL-1β–induced NUB1 expression was significantly higher in OA than in RA ( p = 0.024). Following 5-aza-dC treatment, the magnitude of the OA–RA difference was no longer statistically significant ( p = 0.488), indicating partial attenuation of the baseline RA–OA difference. ( b ) EPZ6438 (EZH2 inhibitor; histone methylation inhibitor). RA and OA FLS were treated with EPZ6438 for 12 or 24 hours before IL-1β stimulation (n = 5 each). treatment with EPZ6438 partially reversed the difference in IL-1β–induced NUB1 expression between RA and OA. Under control conditions, IL-1β–induced NUB1 expression was significantly higher in OA than in RA. This difference was no longer significant after EPZ6438 treatment at 12 h ( p = 0.329) or 24 h ( p = 0.512). ( c ) Left panel: ITF2357 (pan-HDAC inhibitor); Right panel: MS275 (HDAC1/3 selective inhibitor). RA and OA FLS were treated with each HDAC inhibitor for 12 or 24 hours, followed by IL-1β stimulation (ITF2357; n = 5 each, MS275; n = 6 each). Under control conditions, IL-1β–induced NUB1 expression was significantly higher in OA than in RA in the ITF2357 panel. Following ITF2357 treatment, the OA–RA difference was no longer statistically significant at 12 h ( p = 0.596) or 24 h ( p = 0.072). Under control conditions, IL-1β–induced NUB1 expression was also significantly higher in OA than in RA in the MS275 panel. Following MS275 treatment, the OA–RA difference was no longer statistically significant at either 12 h ( p = 0.944) or 24 h ( p = 0.846). These data indicate that histone modifications are required for differential induction of NUB1 in OA compared with RA. Circles and squares represent mean fold change for OA and RA FLS, respectively. Statistical analysis was performed at each time point and condition performed by comparing the fold change values between RA and OA FLS using unpaired t test with Welch’s correction (* p < 0.05, ** p < 0.01).

Article Snippet: For epigenetic modulation experiments, we used the EZH2 inhibitor EPZ6438 (Tazemetostat; Selleck Chemicals), the pan-HDAC inhibitor ITF-2357 (givinostat; Selleck Chemicals), and the selective HDAC1/3 inhibitor MS-275 (entinostat; Selleck Chemicals), as previously described in synovial fibroblast studies .

Techniques: Expressing, Quantitative RT-PCR, Control, Methylation