Journal: The Journal of Experimental Medicine

Article Title: Gain-of-function human UNC93B1 variants cause systemic lupus erythematosus and chilblain lupus

doi: 10.1084/jem.20232066

Figure Lengend Snippet: C haracterization of TLR8 signaling in THP-1 cells. (A) ISG15 expression, assessed by intracellular staining and flow cytometry, in THP-1 cells stimulated for 16 h with CL264 (10 µg/ml), CL307 (TLR7 agonist) (1 µg/ml), R848 (10 µg/ml), TL8-506 (1 µg/ml), CpG-A (5 µM), and CpG-B (5 µM). Relative MFI: mean fluorescence intensity of stimulated condition over NS condition. Mean ± SEM of n = 3 experiments. (B) Relative mRNA levels of indicated genes in THP-1 cells stimulated for 16 h with CL307 (1 µg/ml) or TL8-506 (1 µg/ml). mRNA was assessed by qPCR, normalized to HPRT mRNA, and expressed as a fold induction over NS condition. Mean ± SEM of n = 4 experiments. (C) V5-tagged UNC93B1 expression, assessed by intracellular staining of V5 and flow cytometry, in THP-1 cells stably expressing EV, WT, and variant UNC93B1 (pTrip-SFFV-GFP-2A construct), or non-transduced (NT). Mean ± SEM of n = 3 experiments. (D) V5-tagged UNC93B1 expression, assessed by intracellular staining of V5 (PE) and flow cytometry, in THP-1 cells stably expressing WT, L330R, or R525P UNC93B1, or an EV (pTrip-CMV-Puro-2A construct). Overlayed histogram of PE intensity is shown. Representative experiment of n = 2. (E) Western blot of non-transduced (NT), EV- and UNC93B1-V5-transduced THP-1 protein lysates (pTrip-SFFV-GFP-2A construct), using a conformational anti-UNC93B1 antibodies. Endogenous UNC93B1 migrates at 65 kDa, while the overexpressed, V5-tagged UNC93B1 migrates at a higher molecular weight. (F) Relative mRNA levels of indicated genes in THP-1 cells transduced with EV, WT, and variant UNC93B1 (pTrip-SFFV-GFP-2A construct) and stimulated for 16 h with R848 (1 µg/ml), assessed by qPCR, normalized to HPRT mRNA, and expressed as fold induction over WT NS condition. Mean ± SEM of n = 3 experiments. Two-way ANOVA with Dunnett’s post-hoc test, except for IFNB1 : one-way ANOVA with Holm–Sidak post-hoc test. (G) Syntenin-1 expression level in THP-1 cells stably transduced with EVs (EV1, EV2) or vectors carrying two single sgRNA targeting syntenin-1/ SDCBP gene (sgSyn-1a and sgSyn-1b), harvested 7 days after transduction and selection, and assessed by western blot. Vinculin is a loading control. Representative experiment of n = 3. (H) Baseline ISG score (median of the relative mRNA levels of the ISGs IFI27 , IFI44L , OAS1 , and IFIT1 ), ISGs, IFNB1 , and inflammatory cytokine ( IL6 , IL8 , and TNF ) expression in THP-1 cells stably transduced with EVs (EV1, EV2) or vectors carrying two single sgRNA targeting syntenin-1/ SDCBP gene (sgSyn-1a and sgSyn-1b), at baseline, harvested 7–10 days after transduction and selection. mRNA was assessed by qPCR, normalized to HPRT mRNA, and expressed as a fold induction over EV1. Mean ± SEM of n = 6–10 experiments. (I) ISGs and inflammatory cytokine ( IL6 , IL8 , and TNF ) expression in THP-1 cells stably transduced with EVs (EV1, EV2) or vectors carrying sgRNA targeting syntenin-1/ SDCBP gene, stimulated for 24 h with TL8-506 (1 µg/ml). mRNA was assessed by qPCR, normalized to HPRT mRNA, and expressed as a percentage of the averaged EV1-EV2 fold stimulation over the NS condition. Mean ± SEM of n = 6–10 experiments. (H and I) Kruskal–Wallis test with Dunnett’s post-hoc analysis (for ISG score and IFNB1 ); mixed-effects analysis (REML; restricted maximum likelihood) with uncorrected Fisher’s test (for ISGs and inflammatory cytokines). (J and K) NF-κB SEAP reporter assay in control (sgNTgt) or syntenin-1 KO (sgSyn-1b) THP-1 Dual cell pools stably transduced with EV, WT, and variant UNC93B1 (pTrip-SFFV-GFP-2A construct) stimulated for 16 h with (J) TL8-506 (0.1 µg/ml) and (K) CL307 (5 µg/ml). Data are expressed as fold induction over WT NS. Mean ± SEM of n = 7–8 experiments. (L) ISG score (median of the relative mRNA levels of the ISGs IFI27 , IFI44L , OAS1 , and IFIT1 ) in control (sgNTgt) or TLR8 KO (sgTLR8) THP-1 cell pools stimulated for 24 h with TL8-506 (1 µg/ml). mRNA was assessed by qPCR, normalized to HPRT mRNA, and expressed as a fold induction over control (sgNtgt) in the NS condition. Mean ± SEM of n = 2 experiments. (M) PLA assessing UNC93B1-TLR8 association (yellow dots) with anti-V5 (for UNC93B1) and anti-TLR8 specific antibodies, as in , in unstimulated TLR8 KO THP-1 cell pools stably transduced with WT UNC93B1-V5. No PLA signal is detected. Nuclei (blue) were stained with DAPI, scale bar: 5 μm. Two-way ANOVA with Dunnett’s post-hoc test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Single-guide RNAs (sgRNA) targeting syntenin-1/SDCBP , TLR8 , or non-targeting controls (sgNtgt) ( ) were either designed using the CRISPOR tool ( http://crispor.tefor.net ) (sgSyn-1a), taken from (sgSyn-1b) or taken from , and cloned into lentiviral construct lentiCRISPRv2-hygro, a gift from Brett Stringer (Flinders University, Adelaide, Australia) (plasmid #98291; Addgene ) , following the protocol provided on the plasmid website.

Techniques: Expressing, Staining, Flow Cytometry, Fluorescence, Stable Transfection, Variant Assay, Construct, Western Blot, Molecular Weight, Transduction, Selection, Control, Reporter Assay