Journal: FASEB BioAdvances

Article Title: Human inborn errors of long‐chain fatty acid oxidation show impaired inflammatory responses to TLR4 ‐ligand LPS

doi: 10.1096/fba.2024-00060

Figure Lengend Snippet: Inborn errors in ETFDH cause impaired responses to LPS. Primary dermal fibroblasts from healthy controls (C1‐2) and primary dermal fibroblasts derived from MADD patients (P1‐2) were (A) blotted for basal expression level of ETF‐QO. The cells were stimulated with LPS 400 ng/mL, and analyzed for (B, C) IL‐6, IL‐8 secretion at 24 h by ELISA or (D–H) cytokines mRNA (IL‐6, CCL2, CXCL10, IL‐1β, and IL‐1α) expression by RT‐QPCR at 6 h. Control dermal fibroblasts (C2) transfected with scrambled siRNA or ETFDH siRNA for 72 h were stimulated with LPS 400 ng/mL for (I–K) 24 h, blotted and quantified for ETF‐QO, and analyzed for IL‐6 secretion or (L–P) 6 h, and analyzed for cytokines mRNA (IL‐6, CCL2, CXCL10, IL‐1β, and TNF) expression by RT‐QPCR. Blots in figures (A and I) are representative of three independent experiments. Data in figures (B–D) and (K–P) are presented as mean ± SEM of four cell culture experiments, while data in figures (E–H) represent mean ± SEM of two cell culture experiments. * p < 0.05, ** p < 0.01 *** p < 0.001, and **** p < 0.0001; compared to untreated control cells (unpaired t test). MADD, Multiple Acyl‐CoA Dehydrogenase Deficiency; ETFDH, Electron Transfer Flavoprotein Dehydrogenase.

Article Snippet: Cytokines in the patient and control dermal fibroblast culture supernatants were measured using enzyme‐linked immunosorbent assay (ELISA) for quantitative detection of IL‐1β (R&D Systems Cat. No. DY201), tumor necrosis factor TNF‐α (BioLegend® Cat. No. 430204), IL‐6 (BioLegend® Cat. No. 430504 and R&D Systems Cat. No. DY206), IL‐8 (Thermo Scientific 88–8086), IL‐10 (BioLegend® Cat. No. 430604 and R&D Systems Cat. No. DY217B), and CXCL10 (R&D Systems Cat. No. DY266), all according to the manufacturer's instructions.

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Transfection, Cell Culture

Journal: FASEB BioAdvances

Article Title: Human inborn errors of long‐chain fatty acid oxidation show impaired inflammatory responses to TLR4 ‐ligand LPS

doi: 10.1096/fba.2024-00060

Figure Lengend Snippet: Reduced LPS responsiveness in severe VLCADD patient cells. Primary dermal fibroblasts from healthy controls (C4‐6) and primary dermal fibroblasts derived from severe VLCADD patients (P5‐8) stimulated with LPS 400 ng/mL were analyzed for (A–D) cytokines mRNA (IL‐6, CCL2, IL‐1β, and IL‐1α) expression by RT‐QPCR at 6 h and (E, F) IL‐6, IL‐8 secretion at 24 h. (G, H) Primary dermal fibroblasts from a healthy control (C4) transfected with scrambled siRNA or ACADVL siRNA for 72 h were stimulated with LPS 400 ng/mL, blotted and quantified for VLCAD expression. Then the cells were analyzed for (I, J) cytokines secretion (IL‐6, IL‐8) at 24 h, (K‐P) cytokines mRNA ( IL‐6 , CCL2 , IL‐1α , IL‐1β , CXCL10 , TNF ), and (Q) TLR4 mRNA expression by RT‐QPCR at 6 h. (R, S) Primary dermal fibroblasts from healthy controls (C4‐6) and primary dermal fibroblasts derived from severe VLCADD patients (P5‐8) were analyzed for basal level TLR4 mRNA expression. (T, U) The cells were stimulated with LPS 400 ng/mL for 24 h, blotted and quantified for JunB. Data in figures (A–F) represent mean ± SEM of two cell culture experiments, while data in figures (I–Q) are presented as mean ± SEM of four cell culture experiments. Blots in figures (G and T) were performed twice in independent experiments. *Represents significance compared to healthy controls or untreated control cells (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, unpaired t test).

Article Snippet: Cytokines in the patient and control dermal fibroblast culture supernatants were measured using enzyme‐linked immunosorbent assay (ELISA) for quantitative detection of IL‐1β (R&D Systems Cat. No. DY201), tumor necrosis factor TNF‐α (BioLegend® Cat. No. 430204), IL‐6 (BioLegend® Cat. No. 430504 and R&D Systems Cat. No. DY206), IL‐8 (Thermo Scientific 88–8086), IL‐10 (BioLegend® Cat. No. 430604 and R&D Systems Cat. No. DY217B), and CXCL10 (R&D Systems Cat. No. DY266), all according to the manufacturer's instructions.

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Control, Transfection, Cell Culture