Journal: medRxiv

Article Title: IRAK2 deficiency causes a new immune dysregulation disorder

doi: 10.1101/2024.10.21.24315796

Figure Lengend Snippet: (A) Co-immunoprecipitation of whole cell lysates from HEK293T cells transiently expressing Myc-tagged IRAK2-WT or IRAK2-Δex2 and equal levels of IRAK4-Flag or TRAF6-Flag using anti-Flag antibody (IP: Flag). The experiment was repeated three times. (B) Immunoblot showing deficiency of NF- κ B and MAPK signaling in the presence of IRAK2-Δex2 mutation compared with IRAK2-WT, using indicated antibodies in the stable HEK293T-TLR4 cell line. The experiment was repeated three times. (C) NF- κ B signaling was detected by luciferase assay in HEK293T- IRAK2 −/− cells overexpressing either IRAK2-WT or IRAK2-Δex2. Cells were co-transfected with NF- κ B-luciferase reporter and renilla plasmids (n=3). Bars represent the mean ± SD. An unpaired Student’s t -test was performed between the IRAK2-WT and IRAK2-Δex2 groups, ** P < 0.01. The experiment was repeated three times. (D) RNA sequencing analysis of NF- κ B pathway in PBMCs treated with 1 μg/ml LPS, 1 μg/ml R848, respectively, or left untreated for 12 hours. (E) PBMCs from P1, P2, and six unaffected controls were treated with 1 μg/ml LPS, 1 μg/ml R848, respectively, or left untreated for 12 hours. Supernatants were collected for CBA. Bars represent the mean ± SD. (F) RNA sequencing analysis of NF- κ B pathway in BMDMs treated with 100 ng/ml LPS or left untreated for 8 hours (n=3). (G) Quantitative PCR (qPCR) analysis of the expression of the genes related to NF- κ B signaling in BMDMs treated with 100 ng/ml LPS, 25 ng/ml R848, or 50 ng/ml FSL-1, respectively, or left untreated for 8 hours (n=5). Bars represent the mean ± SEM. Unpaired Student’s t -tests were performed between the Irak2 WT/WT and Irak2 Δ ex2/ Δ ex2 groups, * P < 0.05, ** P < 0.01, *** P < 0.001. UNS, unstimulated; ns, not significant.

Article Snippet: BMDMs were stimulated with 100 ng/ml LPS, 25 ng/ml R848 (HY-13740, MCE), and 50 ng/ml FSL-1 (HY-P2036, MCE), respectively for 8 hours prior to RNA extraction.

Techniques: Immunoprecipitation, Expressing, Western Blot, Mutagenesis, Luciferase, Transfection, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: medRxiv

Article Title: IRAK2 deficiency causes a new immune dysregulation disorder

doi: 10.1101/2024.10.21.24315796

Figure Lengend Snippet: (A) The gene set enrichment analysis (GSEA) of RNA sequencing data for PBMCs. NES, normalized enrichment score. (B) Quantification of the 28-gene IFN score of RNA sequencing data of total RNA extracted from PBMCs. Data are presented as the mean ± SD. Significance assessed by unpaired Student’s t -test, *** P < 0.001, ** P < 0.01. (C) Uniform Manifold Approximation and Projection (UMAP) visualization and marker-based annotation of 13 cell subtypes from P1, P2, and three unaffected controls. ‘Macro-like’ cell subset is circled in red. NK, natural killer cells; Treg, regulatory T cells; pDC, plasmacytoid dendritic cells; mDC, myeloid dendritic cells; Macro-like: macrophage-like. (D) Identification of differentially expressed genes between patients (P1 and P2) and three unaffected controls across different cell types. Each point denotes a gene with an adjusted p-value (method= “BH”) less than 0.05. The dashed line represents a Log2FC value of 1, marking the number of differentially expressed genes exceeding this threshold across cell clusters. (E) The bubble graph shows the expression of marker genes for annotating monocytes and ‘Macro-like’ cell subset by scRNA-seq. The size of the bubble indicates the proportion of cells expressing the gene, and the color scale indicates the average expression level. (F) The box plot represents the type I interferon response scores calculated by scRNA-seq for ‘Macro-like’ cell subset. Statistical analysis was performed using Wilcoxon test (*** P < 0.001). (G) The violin plots show the upregulation of IFN-stimulated genes in the ‘Macro-like’ cell subset of P1 and P2 compared to three unaffected controls. (H) The phosphorylation level of STAT1 of CD16+ monocytes within PBMCs from P3 and three unaffected controls, as determined by flow cytometry analysis. (I) qPCR analysis of Ifnβ expression in BMDMs treated with 100 ng/ml LPS, 25 ng/ml R848, or 50 ng/ml FSL-1, respectively, or left untreated for 8 hours (n=5). Bars represent the mean ± SEM. An unpaired Student’s t -test was conducted between Irak2 WT/WT and Irak2 Δ ex2/ Δ ex2 groups, *** P < 0.001.

Article Snippet: BMDMs were stimulated with 100 ng/ml LPS, 25 ng/ml R848 (HY-13740, MCE), and 50 ng/ml FSL-1 (HY-P2036, MCE), respectively for 8 hours prior to RNA extraction.

Techniques: RNA Sequencing, Marker, Expressing, Phospho-proteomics, Flow Cytometry