Journal: Frontiers in Immunology
Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis
doi: 10.3389/fimmu.2025.1524304
Figure Lengend Snippet: Expression of CD47, its two ligands and several integrin subunits in synovial tissue and peripheral blood samples of human RA patients. (A–J) Violin Plots for increased expression of CD47, SIRP-α, TSP-1, integrin subunits (α4, αM, αv, αL, β1, β2, β3) in synovial tissues from RA patients compared with healthy people. Data were based on RA patient synovial tissue samples (RA=230, Normal=59) published on GPL96, GPL570, GPL11154, GPL1708, GPL10558, and GPL91 platforms from NCBI-GEO. (K–O) . Violin Plots for increased expression of CD47, SIRP-α, TSP-1, and integrin subunits (α4 and αL) in peripheral blood samples from RA patients compared with healthy people. Data were based on RA patient peripheral blood samples (RA=1238, Normal=120) published on GPL6947, GPL570, GPL20171, and GPL13158 platforms from NCBI-GEO.
Article Snippet: Cell lysate was incubated with an anti-CD47 antibody (SANTA CRUZ, SC-12730), an anti-integrin subunit α4 antibody (Novus, NBP2-50445) or an anti-integrin subunit β1 antibody (Abcom, ab24693) that have been properly diluted according to the instructions of the products.
Techniques: Expressing
Figure 2 , rats were sacrificed and synovial tissues of the hind paws were taken for western-blot, quantitative real-time PCR, and RNAseq analysis. (A) Photo of western-blot for the expression of CD47, SIRP-α, TSP-1, integrin subunit α4, αM, αv, αL, β1, β2 and β3. (B–K) Expression of CD47 (B) , SIRP-α (C) , TSP-1 (D) , integrin subunit α4 (E) , αM (F) , αv (G) , αL (H) , β1 (I) , β2 (J) and β3 (K) on an mRNA level in synovial membranes of rats in the four groups were shown and compared. For (B–K) , n=3 (three replicates for detection of each molecule). All of the data show mean ± SD. **P < 0.01. (L) Differential expression of CD47, its two ligands (SIRP-α and TSP-1), and several integrin subunits in synovial membranes of rats in the four groups by RNAseq analysis (n=3, samples of three animals for each group). Gene expression heatmap was generated by Cluster 3.0 with the hierarchical method and R 4.0.3 with the heatmap method. " width="100%" height="100%">
Journal: Frontiers in Immunology
Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis
doi: 10.3389/fimmu.2025.1524304
Figure Lengend Snippet: Expression of CD47, its two ligands and several integrin subunits in rat synovial tissues. On day 28 of the animal experiment shown in Figure 2 , rats were sacrificed and synovial tissues of the hind paws were taken for western-blot, quantitative real-time PCR, and RNAseq analysis. (A) Photo of western-blot for the expression of CD47, SIRP-α, TSP-1, integrin subunit α4, αM, αv, αL, β1, β2 and β3. (B–K) Expression of CD47 (B) , SIRP-α (C) , TSP-1 (D) , integrin subunit α4 (E) , αM (F) , αv (G) , αL (H) , β1 (I) , β2 (J) and β3 (K) on an mRNA level in synovial membranes of rats in the four groups were shown and compared. For (B–K) , n=3 (three replicates for detection of each molecule). All of the data show mean ± SD. **P < 0.01. (L) Differential expression of CD47, its two ligands (SIRP-α and TSP-1), and several integrin subunits in synovial membranes of rats in the four groups by RNAseq analysis (n=3, samples of three animals for each group). Gene expression heatmap was generated by Cluster 3.0 with the hierarchical method and R 4.0.3 with the heatmap method.
Article Snippet: Cell lysate was incubated with an anti-CD47 antibody (SANTA CRUZ, SC-12730), an anti-integrin subunit α4 antibody (Novus, NBP2-50445) or an anti-integrin subunit β1 antibody (Abcom, ab24693) that have been properly diluted according to the instructions of the products.
Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Gene Expression, Generated
Journal: Frontiers in Immunology
Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis
doi: 10.3389/fimmu.2025.1524304
Figure Lengend Snippet: Peptide 4N1K engagement on CD47 increased integrin α4β1 activation and function on human Jurkat cells. (A) CD47, α4 integrin, and cell nucleus were probed and stained by an APC conjugated anti-CD47 antibody, a FITC conjugated anti-α4 antibody or Hoechst staining and their merge signal confirmed the interaction between CD47 and α4 integrin on the cell surface (400×). (B–D) . Western-blot analysis of the bands for CD47, α4, and β1 integrin subunit after immunoprecipitation with an anti-CD47 (B) , anti-α4 (C) , or anti-β1 (D) antibody. Cell lysate which was negatively labeled on the left was precipitated with an isotype control antibody. The whole-cell lysate on the right was named input. (E) Peptide 4N1K treatment increased the adhesion of human Jurkat cells to immobilized VCAM-1 (n=3, three replicate samples for each experimental condition). Cells in the control sample were not incubated with peptides. Typical photos of adhesion cells under various treatment conditions (×100) were shown on the left. (F) Peptide 4N1K treatment increased the migration of human Jurkat cells to immobilized VCAM-1 with SDF-1 as a chemoattractant (n=3, three replicate samples for each experimental condition). Typical photos of migrated cells under various treatment conditions (×200) were shown on the left. (G–J) Peptide 4N1K increased the percent of “extended form” integrin α4β1 with detection by an anti-active form α4β1 antibody. Cells were treated with peptide 4KGG (I) , 4N1K (J) , or without peptide treatment (H) and then were probed with an anti-active form integrin α4β1 antibody and thereafter a FITC-labeled secondary antibody. Human Jurkat cells incubated with an isotype antibody were used as an isotype control sample (G) . Statistical analysis of up-regulation of integrin α4β1 active form was shown in (K) (n=3, three detections for the same experimental condition). Data show mean ± SEM. **P < 0.01. Total expression of integrin α4β1 on Jurkat cells was confirmed with the use of a FITC conjugated rabbit anti-human integrin α4β1 monoclonal antibody (M) . Jurkat cells incubated with an isotype antibody were used as an isotype control sample (L) .
Article Snippet: Cell lysate was incubated with an anti-CD47 antibody (SANTA CRUZ, SC-12730), an anti-integrin subunit α4 antibody (Novus, NBP2-50445) or an anti-integrin subunit β1 antibody (Abcom, ab24693) that have been properly diluted according to the instructions of the products.
Techniques: Activation Assay, Staining, Western Blot, Immunoprecipitation, Labeling, Control, Incubation, Migration, Expressing
Journal: Frontiers in Immunology
Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis
doi: 10.3389/fimmu.2025.1524304
Figure Lengend Snippet: Inside-out signaling pathways lead to integrin α4β1 activation. A monoclonal antibody α-PSα4 was used for the detection of the Ser988-phosphorylated form of the α4 cytoplasmic domain. (A) Peptide 4N1K and TSP increased the amount of phosphorylated α4 subunit in Jurkat cells whereas peptide 4KGG had no effect. (B) PKAI and PP2 inhibited 4N1K effect on α4 subunit phosphorylation in Jurkat cells whereas PTX had no effect. Peptide 4N1K and 4KGG treatment did not change the amount of intracellular cAMP (C) or IP3 (D) in human Jurkat cells (n=3, three replicate samples for each experimental condition). Results are presented as the mean ± SD. For CD3+ T cells isolated from wild-type rats, peptide 4N1K and TSP increased the amount of phosphorylated α4 subunit whereas peptide 4KGG had no effect (E) . PKAI and PP2 inhibited 4N1K effect on α4 subunit phosphorylation whereas PTX had no effect (F) . The amount of intracellular cAMP (H) or IP3 (I) was not significantly changed in wild-type rat CD3+ T cells under treatment with peptide 4N1K, 4KGG, or without peptide treatment (n=3, three replicate samples for each experimental condition). Results are presented as the mean ± SD. For CD3+ T cells isolated from Cd47 knockout rats, the amount of phosphorylated α4 subunit was undetectable whether the cells were treated with peptide 4N1K, TSP (J) , or 4N1K in combination with PKAI, PTX, or PP2 (K) . The total amount of integrin α4β1 was not changed under peptide 4N1K or its combination with PKAI, PTX, or PP2 treatment for CD3+T cells isolated from wild-type rats (G) and Cd47 knockout rats (L) .
Article Snippet: Cell lysate was incubated with an anti-CD47 antibody (SANTA CRUZ, SC-12730), an anti-integrin subunit α4 antibody (Novus, NBP2-50445) or an anti-integrin subunit β1 antibody (Abcom, ab24693) that have been properly diluted according to the instructions of the products.
Techniques: Protein-Protein interactions, Activation Assay, Phospho-proteomics, Isolation, Knock-Out
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