Journal: Journal of Advanced Research

Article Title: Developmentally endothelial locus-1 facilitates intestinal inflammation resolution by suppressing the Cmpk2-cGAS-STING pathway and promoting reparatory macrophage transition

doi: 10.1016/j.jare.2025.04.030

Figure Lengend Snippet: DEL-1 knockdown inhibits inflammation resolution of DSS-induced colitis in the recovery phase. (A) Diagram of the modeling and treatment strategy for the induced repair model. Briefly, mice were treated with AAV-DEL-1 for 4 weeks, followed by DSS feeding for 7 days. Subsequently, DSS was withdrawn and replaced with sterile water for 6 days (n = 5). (B) Body weight loss was calculated as the percent change relative to day 0. (C) Disease activity index (DAI) scores. (D) Representative images of colons. (E) Colonic length. (F) Representative images of hematoxylin and eosin (H&E) staining. (G) Histological score. (H) RT-qPCR of cytokines ( Il1β , Il6 , Tnfα , Il10 , Arg1 , Ifnα , and Ifnβ ), normalized to β-actin . (I, J) Western blot of SPI1, CMPK2 and cGAS-STING pathway related protein expression. The intensity ratio of the target protein to corresponding controls quantified using densitometric analysis, including SPI1/GAPDH, CMPK2/GAPDH, CGAS/GAPDH, p-STING/STING, p-TBK1/TBK1, and p-IRF3/IRF3. Statistical analysis was calculated by student’s t tests. ns (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The adeno-associated virus (AAV) specific for DEL-1 knockdown was obtained from Cyagen Biosciences.

Techniques: Knockdown, Sterility, Activity Assay, Staining, Quantitative RT-PCR, Western Blot, Expressing

Journal: Journal of Advanced Research

Article Title: Developmentally endothelial locus-1 facilitates intestinal inflammation resolution by suppressing the Cmpk2-cGAS-STING pathway and promoting reparatory macrophage transition

doi: 10.1016/j.jare.2025.04.030

Figure Lengend Snippet: DEL-1 promotes intestinal inflammation resolution dependent on cGAS-STING pathway in the repair model. Mice were treated with 2.5 % DSS for 7 days, and then DSS was withdrawn and replaced with sterile water and intraperitoneally injected with DEL-1-Fc (1 ug/mouse) for 6 days (n = 4–6) (A-E). (A, B) Flow cytometry analysis of CD11b + Ly-6G + neutrophils and their percentage in colonic tissues. (C) Assessment of F4/80 (green) and ARG1 (red) expression and colocalization in the colon tissues of the repair model by immunofluorescence. (D, E) Western blot of iNOS, CD206, and ARG1 in colonic tissues, and densitometric analysis quantified the intensity ratio of the target protein to GAPDH. (F-M) Mice were treated with 2.5 % DSS for 7 days, and then DSS was withdrawn and replaced with sterile water and intraperitoneally injected with DEL-1-Fc (1 ug/mouse) and DMXAA (70 ug/mouse) for 6 days (n = 10). (F) Diagram of the modeling and treatment strategy for the induced repair model. (G) Survival rates. (H) Representative images of colons. (I) Colonic length. (J) Representative images of H&E staining. (K) Histological score. (L) RT-qPCR of cytokines ( Il1β , Il6 , Tnfα , Il10 , Arg1 , Ifnα , and Ifnβ ), normalized to β-actin . (N, M) Western blot of iNOS, CD206, and ARG1 in colonic tissues, and densitometric analysis quantified the intensity ratio of the target protein to GAPDH. Statistical analysis was calculated by one-way-analysis of variance (ANOVA). ns (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The adeno-associated virus (AAV) specific for DEL-1 knockdown was obtained from Cyagen Biosciences.

Techniques: Sterility, Injection, Flow Cytometry, Expressing, Immunofluorescence, Western Blot, Staining, Quantitative RT-PCR

Journal: Journal of Advanced Research

Article Title: Developmentally endothelial locus-1 facilitates intestinal inflammation resolution by suppressing the Cmpk2-cGAS-STING pathway and promoting reparatory macrophage transition

doi: 10.1016/j.jare.2025.04.030

Figure Lengend Snippet: DEL-1 attenuates macrophages inflammation and promotes inflammation resolution. (A) The intervention methods of LPS induced the acute and repair model. Macrophages were pulsed with LPS (1 μg/ml) for 4 h (the acute inflammation model), and then replaced with fresh media after washing away the LPS for 24 h (the repair model) (n = 3–4). (B, E) Del-1 mRNA expression was measured by RT-qPCR after pulsed with LPS and hours post LPS withdrawal in RAW264.7 macrophages (B) and BMDMs (E). Results were normalized by β-actin gene. (C, D, F, G) Expression of DEL-1 protein levels were detected using western blot after pulsed with LPS and 24 h post LPS withdrawal in RAW264.7 cells (C, D) and BMDMs (F, G). Densitometry analysis quantified the intensity ratio of DEL-1 to GAPDH. (H) RAW264.7 macrophages were transfected with DEL-1 overexpression plasmids and corresponding controls for 24–36 h, and then subjected to LPS stimulation detailed in (A). The mRNA expression of Il1β , Il6 , Tnfα , Il10 , and Arg1 were measured by RT-qPCR in the acute and repair model, normalized to β-actin . (I) BMDMs were pulsed with LPS (1 μg/ml) and simultaneously treated with DEL-1-Fc (1 μg/ml) for 4 h (the acute inflammation model), or withdrawn LPS stimulation and replaced with fresh media and treated with DEL-1-Fc (1 μg/ml) for 24 h (the repair model). The mRNA expression of Il1β , Il6 , Tnfα , Il10 , and Arg1 were measured by RT-qPCR in the acute and repair model. Results were normalized by β-actin gene. (J-M) RNA-seq analysis of the NC + LPS and OE-DEL-1 + LPS groups of RAW264.7 macrophages 24 h post LPS withdrawal. (J) Heatmaps of DEGs with adjusted p value < 0.05 and |fold change| > 1.2. (K) Gene set enrichment analysis (GSEA). (L) Gene ontology (GO) enrichment analysis. (N) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. (M) Volcano plot of DEGs. Statistical analysis was calculated by student’s t tests or one-way-analysis of variance (ANOVA). ns (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The adeno-associated virus (AAV) specific for DEL-1 knockdown was obtained from Cyagen Biosciences.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Over Expression, RNA Sequencing

Journal: Journal of Advanced Research

Article Title: Developmentally endothelial locus-1 facilitates intestinal inflammation resolution by suppressing the Cmpk2-cGAS-STING pathway and promoting reparatory macrophage transition

doi: 10.1016/j.jare.2025.04.030

Figure Lengend Snippet: DEL-1 down-regulated in the acute phase and up-regulated in the recovery phase in patients with IBD and DSS-induced colitis mice. (A) Comparison of DEL-1 expression in the colon tissues between controls (Ctrl), UC, and CD ( GSE179285 , GSE75214 ). (B) DEL-1 mRNA expression in the colon tissues from controls and IBD patients were measured by RT-qPCR, normalized to GAPDH. (C) Serum DEL-1 concentrations of controls and IBD patients were determined by ELISA. (D) Receiver operating characteristic (ROC) curve of serum DEL-1 in identifying patients with active IBD from the control group. (E) Correlation between serum DEL-1 and CRP or ESR levels were determined by Spearman’s correlation analysis. (F) Immunohistochemical staining of colonic tissues from controls, UC, and CD patients. (G) Comparison of DEL-1 expression in the colon tissues between controls (Ctrl), and colitis in the acute and recovery phase ( GSE42768 ). (H) Relative mRNA levels of DEL-1 in different organs of wild-type mice were detected by RT-qPCR. The Y axis indicate the ratio of the cycle threshold (CT) value of β-actin to DEL-1 (n = 5). (I and J) DEL-1 mRNA (I) and protein (J) expression in the colon tissues of control mice and colitis mice in the acute and recovery phase was measured by RT-qPCR and western blot. (K) The intensity ratio of DEL-1 to GAPDH was quantified by densitometry analysis. (L, M) Assessment of F4/80 (green) and DEL-1 (red) expression and localization in the colon tissues from control mice and colitis in the acute and repair model by immunofluorescence. (N) The DEL-1 fluorescence intensity of control mice and colitis mice in the acute and recovery phase. Statistical analysis was calculated by student’s t tests or one-way-analysis of variance (ANOVA). ns (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The adeno-associated virus (AAV) specific for DEL-1 knockdown was obtained from Cyagen Biosciences.

Techniques: Comparison, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control, Immunohistochemical staining, Staining, Western Blot, Immunofluorescence, Fluorescence

Journal: Journal of Advanced Research

Article Title: Developmentally endothelial locus-1 facilitates intestinal inflammation resolution by suppressing the Cmpk2-cGAS-STING pathway and promoting reparatory macrophage transition

doi: 10.1016/j.jare.2025.04.030

Figure Lengend Snippet: DEL-1 inhibits the Cmpk2-dependent mtDNA synthesis and the activation of cGAS-STING pathway in macrophages. RAW264.7 macrophages were transfected with DEL-1 overexpression plasmids and corresponding controls for 24–36 h, and then pulsed with LPS (1 μg/ml) for 4 h or withdrawal of LPS stimulation for 24 h to induce acute and repair models (n = 3–4). BMDMs were pulsed with LPS (1 μg/ml) and simultaneously treated with DEL-1-Fc (1 μg/ml) for 4 h (the acute inflammation model), or withdrawn LPS stimulation and replaced with fresh media and treated with DEL-1-Fc (1 μg/ml) for 24 h (the repair model) (n = 3–4). (A, D) The mRNA expression of Cmpk2 , Ifnα , and Ifnβ were measured by RT-qPCR in the acute and repair model in RAW264.7 macrophages (A) and BMDMs (D), normalized to β-actin . (B, C, E, F) The expression of CMPK2 and cGAS-STING pathway related protein were determined by western blot in RAW264.7 macrophages (B, C) and BMDMs (E, F), and densitometric analysis quantified the intensity ratio of the target protein to corresponding controls: CMPK2/GAPDH, CGAS/GAPDH, p-STING/STING, p-TBK1/TBK1, and p-IRF3/IRF3. (G, H) Relative total mtDNA amount in macrophages pulsed with LPS and 24 h post LPS withdrawal was detected using RT-qPCR in RAW264.7 macrophages (G) and BMDMs (H). Results were standardized by Tert nuclear (n) DNA. (I, J) Representative images of immunofluorescence staining for dsDNA (green) and mitochondria (red) in the acute and repair model in RAW264.7 cells. Statistical analysis was calculated by one-way-analysis of variance (ANOVA). ns (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The adeno-associated virus (AAV) specific for DEL-1 knockdown was obtained from Cyagen Biosciences.

Techniques: Activation Assay, Transfection, Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Journal of Advanced Research

Article Title: Developmentally endothelial locus-1 facilitates intestinal inflammation resolution by suppressing the Cmpk2-cGAS-STING pathway and promoting reparatory macrophage transition

doi: 10.1016/j.jare.2025.04.030

Figure Lengend Snippet: DEL-1 regulates transcription of Cmpk2 and reparative gene Il10 though transcription factor Spi1. (A, B) The expression of nuclear SPI1 and cytoplasm SPI1 were measured by western blot in DEL-1 overexpressed RAW264.7 macrophages pulsed with LPS and 24 h post LPS withdrawal, and the intensity ratio of the target protein to HISTONE 3 or GAPDH quantified using densitometric analysis (n = 3–4). (C, D) The expression of SPI1 were measured by western blot in BMDMs in the acute and repair model, and densitometric analysis quantified the intensity ratio of the SPI1 to GAPDH (n = 3–4). (E, F) The mRNA expression of Cmpk2 (E) and Il10 (F) were measured by RT-qPCR in RAW264.7 macrophages with Spi1 overexpression, normalized to β-actin . (G-N) Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays analyzed the regulatory role of the transcription factor Spi1 on target genes in RAW264.7 and HEK293T cells. (G) ChIP analyzed the association between Spi1 and the promoter of Cmpk2 gene without intervention. Agarose gel electrophoresis of products (top), and %input calculated from CT values of RT-qPCR (bottom). (H, I) ChIP analyzed the association between Spi1 and the promoter of Cmpk2 gene in the acute and repair model. (J) ChIP analyzed the association between Spi1 and the promoter of Il10 gene without intervention. Agarose gel electrophoresis of products (top), and %input calculated from CT values of RT-qPCR (bottom). (K, L) ChIP analyzed the association between Spi1 and the promoter of Il10 gene in the acute and repair model. (M) The luciferase activity of the Cmpk2 promoter with Spi1 binding sites in HEK293T cells. (N) The luciferase activity of the Il10 promoter with Spi1 binding sites in HEK293T cells. Statistical analysis was calculated by student’s t tests or one-way-analysis of variance (ANOVA). ns (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The adeno-associated virus (AAV) specific for DEL-1 knockdown was obtained from Cyagen Biosciences.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Chromatin Immunoprecipitation, Luciferase, Agarose Gel Electrophoresis, Activity Assay, Binding Assay

Journal: Journal of Advanced Research

Article Title: Developmentally endothelial locus-1 facilitates intestinal inflammation resolution by suppressing the Cmpk2-cGAS-STING pathway and promoting reparatory macrophage transition

doi: 10.1016/j.jare.2025.04.030

Figure Lengend Snippet: DEL-1 induces the ubiquitin – proteasome-dependent degradation of transcription factor Spi1, and further inhibits the Cmpk2-cGAS-STING pathway in macrophages. (A-C) Cmpk2 mRNA (A) and protein (B, C) expression in RAW264.7 macrophages with transfection Cmpk2 overexpression plasmid was determined using RT-qPCR and western blot (n = 4). (D-F) RAW264.7 macrophages were transfected with DEL-1, Spi1, and Cmpk2 overexpression plasmids and corresponding controls for 24–36 h. Cells were pulsed with LPS (1 μg/ml) and the STING pathway agonist DMXAA (1 μg/ml) for 4 h in the acute phase, or withdrawn LPS stimulation and treated with DMXAA for 24 h in the recovery phase (n = 4). The expression of CMPK2 and cGAS-STING pathway related protein were measured by western blot, and densitometric analysis quantified the intensity ratio of the target protein to relevant controls: CMPK2/GAPDH, CGAS/GAPDH, p-STING/STING, p-TBK1/TBK1, and p-IRF3/IRF3. (G) The mRNA expression of Spi1 were determined by RT-qPCR in DEL-1 overexpressed macrophages pulsed with LPS and 24 h post LPS withdrawal. (H, I) DEL-1 overexpressed macrophages treated with cycloheximide (CHX, 60 μg/ml), MG132 (20 uM), and chloroquine (50 uM) in the acute and repair model, and the expression of SPI1 was measured using western blot. Statistical analysis was calculated by student’s t tests or one-way-analysis of variance (ANOVA). ns (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The adeno-associated virus (AAV) specific for DEL-1 knockdown was obtained from Cyagen Biosciences.

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot