Journal: The Journal of Biological Chemistry

Article Title: RNA-binding protein tristetraprolin inhibits Th2 cell activation and differentiation in allergic rhinitis by promoting TRIM18 mRNA decay

doi: 10.1016/j.jbc.2026.111240

Figure Lengend Snippet: TTP suppressed T helper 2 (Th2)-type inflammation in AR mice. A , schematics of splenocytes isolation from AR-mice and the differentiation of splenic naive CD4+ T cells model in vitro . B , the levels of Interleukin (IL)-4, IL-5, and IL-13, which belong to the Th2 cytokine family, were measured by ELISA assays in the cell supernatant. The cell supernatant was collected from mononuclear cells of the spleen that had been treated with ionomycin and phorbol 12-myristate 13-acetate (PMA) for 6 h. C , after treatment with ionomycin, PMA, and GolgiPlug, the cells with the double stain of anti-CD4 and anti-IL-4-APC were detected by flow cytometry to analyze the percentage of Th2 cells. Data are shown as means ± SD (N = 3 or 6). One-way analysis of variance (ANOVA) was used for statistical analysis of control mice, AR mice, and AR mice transduced with control vectors or TTP overexpression vectors.

Article Snippet: For flow cytometry, the primary splenocytes were stimulated with 50 ng/ml PMA (Shanghai Yuanye Bio-Technology, Cat No. R32414 ), 1 μg/ml ionomycin (Macklin, Cat No.I838446), and 1 μl/ml BD Golgi Plug for 6 h. Centrifugal collection of cells was resuspended in 100 μl of staining buffer, and surface markers were stained with 5 μl anti-mouse CD4 (RM4-5; Liankebio, Cat No.F21004A02) and 5 μl APC anti-mouse IL-4 (Elabsciencem, Cat No.E-AB-F1204E) for 30 min. After centrifugation, the samples were analyzed using a Flow Cytometer.

Techniques: Isolation, In Vitro, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Control, Transduction, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: RNA-binding protein tristetraprolin inhibits Th2 cell activation and differentiation in allergic rhinitis by promoting TRIM18 mRNA decay

doi: 10.1016/j.jbc.2026.111240

Figure Lengend Snippet: TTP knockdown exacerbated allergic phenotypes and promoted Th2-type inflammation in AR mice. A , experimental scheme of RW-induced AR mice with or without TTP knockdown via intravenous injection of lentiviral vectors (Lv-shNC or Lv-shTTP). B , expression of TTP mRNA in AR mice with or without TTP knockdown. C , immunofluorescence staining for TTP protein ( red ) and CD4 protein ( green ) in normal and RW-exposed nasal tissues. DAPI ( blue ) was used for nuclear staining (Scale bar = 50 μm). D , quantitative analysis of TTP fluorescence intensity in nasal mucosal tissues from lentivirus-injected mice. E , hematoxylin-eosin (H&E)-stained nasal mucosal tissue sections, showing nasal mucosa thickness (Scale bar = 100 μm). Dashed lines indicate the boundary between the epithelial layer and lamina propria. F and G , changes in the number of sneezes and nasal rubbings after TTP knockdown in AR mice. H , schematic of splenocyte isolation from AR mice and in vitro treatment. I – K , Levels of IL-4, IL-5, and IL-13 in the cell culture supernatant of splenocytes treated with ionomycin and PMA for 6 h. Data are presented as means ± SD (N = 3 or 6). Student's t test was used for statistical analysis of AR mice transduced with shNC or shTTP.

Article Snippet: For flow cytometry, the primary splenocytes were stimulated with 50 ng/ml PMA (Shanghai Yuanye Bio-Technology, Cat No. R32414 ), 1 μg/ml ionomycin (Macklin, Cat No.I838446), and 1 μl/ml BD Golgi Plug for 6 h. Centrifugal collection of cells was resuspended in 100 μl of staining buffer, and surface markers were stained with 5 μl anti-mouse CD4 (RM4-5; Liankebio, Cat No.F21004A02) and 5 μl APC anti-mouse IL-4 (Elabsciencem, Cat No.E-AB-F1204E) for 30 min. After centrifugation, the samples were analyzed using a Flow Cytometer.

Techniques: Knockdown, Injection, Expressing, Immunofluorescence, Staining, Fluorescence, Isolation, In Vitro, Cell Culture, Transduction

Journal: The Journal of Biological Chemistry

Article Title: RNA-binding protein tristetraprolin inhibits Th2 cell activation and differentiation in allergic rhinitis by promoting TRIM18 mRNA decay

doi: 10.1016/j.jbc.2026.111240

Figure Lengend Snippet: The TTP protein acted as a negative regulator of TRIM18-mediated Th2-type inflammation by reducing TRIM18 mRNA stability. A , protein–protein interaction network of 16 potential substrates of E3 ubiquitin ligase TRIM18, constructed using the STRING database and supplemented with functional annotations via GO (Gene Ontology) pathway enrichment analyses, highlighting key biological processes and signaling pathways associated with these substrates. B and C , the qRT-PCR and ELISA were performed to detect the mRNA level and secreted protein concentration of IL-5, respectively, in naive CD4+ T cells or in vitro differentiated Th2 cells transfected with TRIM18 overexpression vector or empty vector. D , schematic diagram of the experimental workflow: Naive CD4+ T cells were induced to differentiate into Th2 cells under in vitro and were co-transfected with combinations of TTP overexpression vector, TRIM18 overexpression vector, or corresponding empty vectors. E , the mRNA level of TRIM18 in differentiated Th2 cells from the three groups (Control, TTP-OE, TTP+TRIM18-OE) was detected by qRT-PCR assays. F and G , The mRNA level and secreted protein concentration of IL-5 were measured by qRT-PCR and ELISA, respectively, in differentiated Th2 cells from the three groups (Control, TTP-OE, TTP+TRIM18-OE). H , flow cytometric analysis of the percentage of CD4+IL-4+ Th2 cells among the three groups after in vitro differentiation, and the proportion of double-positive cells was quantified to assess the impact of TTP and/or TRIM18 overexpression on Th2 cell differentiation. Data are shown as means ± SD (N = 3), One-way ANOVA was used for statistical analysis.

Article Snippet: For flow cytometry, the primary splenocytes were stimulated with 50 ng/ml PMA (Shanghai Yuanye Bio-Technology, Cat No. R32414 ), 1 μg/ml ionomycin (Macklin, Cat No.I838446), and 1 μl/ml BD Golgi Plug for 6 h. Centrifugal collection of cells was resuspended in 100 μl of staining buffer, and surface markers were stained with 5 μl anti-mouse CD4 (RM4-5; Liankebio, Cat No.F21004A02) and 5 μl APC anti-mouse IL-4 (Elabsciencem, Cat No.E-AB-F1204E) for 30 min. After centrifugation, the samples were analyzed using a Flow Cytometer.

Techniques: Ubiquitin Proteomics, Construct, Functional Assay, Protein-Protein interactions, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Protein Concentration, In Vitro, Transfection, Over Expression, Plasmid Preparation, Control, Cell Differentiation