Journal: The Journal of Experimental Medicine

Article Title: A biallelic mutation in IL6ST encoding the GP130 co-receptor causes immunodeficiency and craniosynostosis

doi: 10.1084/jem.20161810

Figure Lengend Snippet: The GP130 p.N404Y variant causes a defective acute phase response, and P1 primary fibroblasts can be rescued by lentiviral transduction of WT GP130. (A) Cellular and biochemical response to recurrent chest infections and cellulitis in patient GP130 p.N404Y. White blood cell count (WBC), neutrophils, CRP, and fibrinogen measurements are shown at different time points. Black/red lines indicate 3rd/97th percentile or lower/upper limit of the normal range, respectively. (B) CRISPR/Cas9-mediated KO of GP130 in human hepatoma Hep3B cells results in absent GP130 surface expression. (C) IL-6–mediated production of fibrinogen is GP130 dependent. Hep3B GP130-KO cells were stimulated with IL-6 for 24 h, and expression of the fibrinogen α chain ( FGA ; left) and fibrinogen β chain ( FGB ; right) was determined by quantitative PCR. Gene expression was determined relative to the housekeeping gene RPLP0 and expressed as fold-induction compared with unstimulated cells. Data represent summary results from four independent experiments (mean ± SEM). (D) Hep3B GP130-KO cells were transiently transfected with GP130 WT, GP130 p.N404Y-mutant plasmid, and FGA (left) and FGB (right) gene expression was analyzed after 24 h of IL-6 stimulation. Gene expression was determined relative to the housekeeping gene RPLP0 and expressed as fold-induction compared with the unstimulated vector control. Data represent pooled results from four independent experiments (mean ± SEM). (E) Fibroblasts of a healthy donor and P1 were stimulated with the indicated concentrations (ng/ml) of IL-6, IL-11, IL-27, OSM, and LIF. The levels of phospho-STAT3 (p-STAT3) were determined after 15-min stimulation by Phosflow. Titration curves are representative of two independent experiments. Curves are fitted by nonlinear/linear regression analysis. (F) Healthy donor and patient GP130 p.N404Y fibroblasts were treated as in E, and STAT1 phosphorylation (p-STAT1) was analyzed using flow cytometry. Titration curves are representative of two independent experiements. MFI, mean fluorescence intensity. (G) Lentiviral transduction of GP130 WT reconstitutes STAT3 phosphorylation in primary fibroblasts with p.N404Y variant. Fibroblasts were stimulated with 30 ng/ml IL-6 and 50 ng/ml IL-11 for 15 min. Quantification is based on four independent experiments. HD, healthy donor; LV, lentiviral. Differences were determined by Mann-Whitney U test. *, P < 0.05.

Article Snippet: Hep3B cells were obtained from the European Collection of Authenticated Cell Cultures and maintained in DMEM (Sigma-Aldrich) supplemented with 10% FCS.

Techniques: Variant Assay, Transduction, Cell Counting, CRISPR, Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Transfection, Mutagenesis, Plasmid Preparation, Control, Titration, Phospho-proteomics, Flow Cytometry, Fluorescence, MANN-WHITNEY