Journal: Frontiers in Immunology

Article Title: CRIF1 gene therapy ameliorates inflammatory bowel disease by suppressing TH17 cells and fibrosis through mitochondrial function regulation

doi: 10.3389/fimmu.2025.1618012

Figure Lengend Snippet: Transplantation of CRIF1-overexpressing mitochondria alleviates DSS-induced colitis. (A) Chronic DSS-induced colitis was induced through oral administration of 3% DSS in distilled water for 5 days, followed by regular drinking water three times as shown in the schematic. Mitochondria were isolated from C2C12 cells transfected with p3XFLAG-CMV-10-CRIF1 or mock vector and mitochondria (10 μg in 200 μL saline) were intraperitoneally injected 1 day before and on days 2, 4, 6, 9, 12, and 14 from the start of treatment (n = 5/group). (B) Percentage change in body weight during the disease process. (C) On day 38 after the first DSS administration, colon sections were stained with hematoxylin and eosin (H&E) and antibodies against CRIF1, α-smooth muscle actin (α-SMA), and collagen-I (Col-I). Representative images are shown (original magnification: H&E 200×; IHC 400×, scale bar: 100 µm). Graphs show the mean histological scores for H&E and the numbers of antibody-positive cells for IHC. (D) On day 38 after the first DSS administration, the frequencies of IL-17+CD4+ and CD5+CD1d+IL-10+CD19+ cells in ex vivo spleens of mice were analyzed via flow cytometry. (E) CCD-18Co cells were transfected with pCMV6-Myc-DDK hCRIF1 vector or mock vector; 3 days later, mitochondria were isolated from these cells. Mitochondria (0.3 μg/mL) were cocultured with human PBMCs under the stimulation of anti-CD3 antibody (2 μg/mL); 3 days later, the supernatant was harvested and the amount of IL-17 was measured by ELISA. Values are presented as means ± SDs. *P < 0.05, **P < 0.01. Data are representative of two independent experiments.

Article Snippet: C2C12 cells cells (Korean cell line bank, Seoul, Korea), which are myoblasts from mouse muscle, were transfected with p3XFLAG-CMV-10-CRIF1 or mock vector using Lipofectamine, in accordance with the manufacturer’s recommendation.

Techniques: Transplantation Assay, Isolation, Transfection, Plasmid Preparation, Saline, Injection, Staining, Ex Vivo, Flow Cytometry, Enzyme-linked Immunosorbent Assay