Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: Increased frequency and proinflammatory signature of CD48 high S100A12 + macrophages in rheumatoid arthritis synovium. A UMAP visualization of synovial macrophages clustered into nine subpopulations on the basis of scRNA-seq data. B Disease-stratified analysis showing an increased proportion of CD48 high S100A12 + macrophages in the RA synovium compared with those in the UA, OA, and HC groups. C UMAP plots depicting S100A12 expression intensity across macrophage subclusters in different disease states. D Gene Ontology (GO) enrichment of biological processes in CD48 high S100A12 + marker genes, highlighting enrichment for defense response activation, cytokine production, and leukocyte migration. E Representative images of CD68 and S100A12 immunofluorescence staining in knee synovial tissues from RA and OA patients. Scale bar: 100 μm

Article Snippet: Double immunofluorescence staining was performed as follows: sections were incubated overnight at 4 °C with primary antibodies against CD68 (mouse monoclonal, 1:100 dilution; 66231-2-Ig, Proteintech, China) with S100A12 (rabbit polyclonal, 1:100 dilution; 16630-1-AP, Proteintech, China) or CD68 (1:100 dilution; 66231-2-Ig) with IRF7 (rabbit polyclonal, 1:150 dilution; 22392-1-AP, Proteintech, China).

Techniques: Expressing, Marker, Activation Assay, Migration, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: IRF7 is a specific transcriptional regulator of CD48 high S100A12 + macrophages. A Venn diagram showing overlapping transcription factors (TFs) identified by triplicate SCENIC analyses, with the CD48 high S100A12 + subcluster enriched for NFIL3, TGIF1, FOSL2, IRF7, and STAT1. B Heatmap of regulon activity scores (RASs) for TFs across macrophage subclusters. C Ranking of TFs in CD48 high S100A12 + macrophages by the regulon specificity score (RSS, calculated via Jensen‒Shannon divergence). D UMAP dimensionality reduction of TF activity profiles across subclusters. E – F UMAP plots highlighting spatial overlap between the CD48 high S100A12 + subcluster. ( E ) and cells with elevated IRF7 regulon activity ( F ). G Representative images of immunofluorescence staining for CD68 and IRF7 in knee synovial tissues from RA and OA patients. Scale bar: 100 μm

Article Snippet: Double immunofluorescence staining was performed as follows: sections were incubated overnight at 4 °C with primary antibodies against CD68 (mouse monoclonal, 1:100 dilution; 66231-2-Ig, Proteintech, China) with S100A12 (rabbit polyclonal, 1:100 dilution; 16630-1-AP, Proteintech, China) or CD68 (1:100 dilution; 66231-2-Ig) with IRF7 (rabbit polyclonal, 1:150 dilution; 22392-1-AP, Proteintech, China).

Techniques: Activity Assay, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: IRF7 directly regulates downstream inflammatory genes in M1 macrophages. A ChIP-seq peak heatmaps showing increased IRF7 binding to promoter/enhancer regions in LPS-stimulated M1 macrophages. B Venn diagram of 108 overlapping genes from the IRF7 ChIP-seq data and the SCENIC-predicted target genes. C Reactome pathway enrichment of IRF7-regulated genes, highlighting the involvement of NF-κB, TNF, and Toll-like receptor signalling (key genes: IL-1β, FOS, NF-κB1, PTGS2, and CXCL10). D Bulk RNA-seq heatmap showing the upregulation of IRF7 and target genes in M1-polarized macrophages ( GSE130011 , GSE154346 ). E UMAP plots of NFKB1, PTGS2, IL1B, and CXCL10 expression in the CD48 high S100A12 + subcluster. F RT‒qPCR analysis of IRF7 and M1 marker genes in siRNA-treated macrophages (performed in triplicate, with 3 distinct patient sources used for each repetition). G – H Western blot validation of IRF7 and downstream protein expression following IRF7 knockdown in M1-polarized macrophages (performed in triplicate, with 3 distinct patient sources used for each repetition). Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way ANOVA with the Bonferroni post hoc correction)

Article Snippet: Double immunofluorescence staining was performed as follows: sections were incubated overnight at 4 °C with primary antibodies against CD68 (mouse monoclonal, 1:100 dilution; 66231-2-Ig, Proteintech, China) with S100A12 (rabbit polyclonal, 1:100 dilution; 16630-1-AP, Proteintech, China) or CD68 (1:100 dilution; 66231-2-Ig) with IRF7 (rabbit polyclonal, 1:150 dilution; 22392-1-AP, Proteintech, China).

Techniques: ChIP-sequencing, Binding Assay, RNA Sequencing, Expressing, Marker, Western Blot, Biomarker Discovery, Knockdown

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: Local IRF7 knockdown alters the immune cell composition in CIA mice. A Schematic of intra-articular IRF7 siRNA treatment in collagen-induced arthritis (CIA) model mice. B – C Flow cytometry analysis of the CD86 and CD206 mean fluorescence intensities (MFIs) in F4/80 + macrophages from ankle joints ( n = 6). CD86: NC: 1241 ± 265.4, si-IRF7: 2469 ± 390.3, positive: 3489 ± 570.9, si-mock: 3689 ± 370.1. CD206: NC: 2225 ± 225.9, si-IRF7: 3395 ± 369.4, positive: 978.2 ± 147.9, si-mock: 2022 ± 170.6. D – E Frequencies of Foxp3 + Tregs among CD3 + CD4 + T cells ( n = 6). NC: 1.66% ± 0.15%, si-IRF7: 3.27% ± 0.28%, positive: 0.47% ± 0.17%, si-mock: 1.10% ± 0.22%. F Immunofluorescence staining for S100A12 + inflammatory macrophages in the ankle synovium of different groups. Scale bar: 100 μm. The data are presented as the means ± SDs. Statistical significance: *** P < 0.001, **** P < 0.0001 (one-way ANOVA with Bonferroni post hoc correction)

Article Snippet: Double immunofluorescence staining was performed as follows: sections were incubated overnight at 4 °C with primary antibodies against CD68 (mouse monoclonal, 1:100 dilution; 66231-2-Ig, Proteintech, China) with S100A12 (rabbit polyclonal, 1:100 dilution; 16630-1-AP, Proteintech, China) or CD68 (1:100 dilution; 66231-2-Ig) with IRF7 (rabbit polyclonal, 1:150 dilution; 22392-1-AP, Proteintech, China).

Techniques: Knockdown, Flow Cytometry, Fluorescence, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: Local IRF7 inhibition attenuates joint inflammation and bone erosion in CIA mice. A Representative ankle joint images on day 42 postimmunization. B H&E staining and histological staining. C - E IHC staining for CD68, S100A12, and IRF7 in the ankle synovium. Scale bar: 100 μm. F Quantification of paw thickness at the ankle joint ( n = 6 per group). 42 Days after the first immunization: NC: 8.33 ± 0.02, si-IRF7: 10.39 ± 0.54, positive: 12.08 ± 0.80, si-mock: 12.65 ± 0.57, Statistical significance: **** P < 0.0001 (one-way ANOVA with Bonferroni post hoc correction). G H&E staining and histological scoring of synovial hyperplasia and inflammation ( n = 6). NC: 0.00 (0.00–0.00), si-IRF7: 1.50 (1.00–2.25), positive: 2.50 (1.75–3.00), and si-mock: 3.00 (2.75–3.00). Data are shown as medians with 25% − 75% percentiles. Statistical significance: * P < 0.05 (Kruskal‒Wallis test, followed by post hoc Dunn’s test with Bonferroni correction for multiple comparisons). H - J Semiquantitative analysis analysis of CD68, S100A12, and IRF7 expression via IHC staining via ImageJ ( n = 6). Statistical significance: **** P < 0.0001 (one-way ANOVA with Bonferroni post hoc correction)

Article Snippet: Double immunofluorescence staining was performed as follows: sections were incubated overnight at 4 °C with primary antibodies against CD68 (mouse monoclonal, 1:100 dilution; 66231-2-Ig, Proteintech, China) with S100A12 (rabbit polyclonal, 1:100 dilution; 16630-1-AP, Proteintech, China) or CD68 (1:100 dilution; 66231-2-Ig) with IRF7 (rabbit polyclonal, 1:150 dilution; 22392-1-AP, Proteintech, China).

Techniques: Inhibition, Staining, Immunohistochemistry, Expressing

Journal: Molecular metabolism

Article Title: Dietary sugars, not lipids, drive hypothalamic inflammation.

doi: 10.1016/j.molmet.2017.06.008

Figure Lengend Snippet: Figure 3: RAGE and ALCAM are expressed on non-neuronal cell populations. (A) CML binds to proteins of RAGE (molecular weight z 75 KDa) and ALCAM (molecular weight z 105 KDa); the vehicle does not bind to protein of either receptor, as illustrated by no band detected in binding of ALCAM. (B) RAGE and ALCAM gene expression in mediobasal hypothalami of chow or HCHF mice (n ¼ 6 for chow or for HCHF, P ¼ 0.019 for RAGE, P ¼ 0.006 for ALCAM). (C) RAGE is intensely expressed by microglia (iba1-ir, indicated by white arrowheads). Higher magnifications of the areas framed by dashed lines are presented in D. (E) RAGE is intensely expressed on endothelial cells (laminin-ir, indicated by white arrows). Higher magnifications of the areas framed by dashed lines are presented in F. (G) CML stimulates TNFa, but not PDGF-B, gene expression in cultured primary microglia (n ¼ 6 wells of cells for vehicle, n ¼ 5 for TNFa treatments, P ¼ 0.04 for TNFa). (H & I) CML stimulates microglial reactivity in the mediobasal hypothalamic area, arrowheads point to the areas where the tip of the infusion probes located. (J) Iba1-ir cell number and cell coverage in H & I (n ¼ 4 mice for vehicle, n ¼ 5 for CML). (K) ALCAM is expressed on part of the vasculature (laminin-ir, indicated by white arrows, two pericytes are indicated by white arrowheads); higher magnifications of the areas framed by dashed lines are presented in L. (M) ALCAM is expressed on pericytes (PDGFRb-ir, white arrowheads). Higher magnifications of the areas framed by dashed lines are presented in N. Scale bar: 30 mm in C, E, K and M, 7.5um in D, F, L and N. Data are presented as means s.e.m. *P < 0.05, **P < 0.01. P values for unpaired comparisons were analyzed by two-tailed Student’s t test.

Article Snippet: Briefly, 2 mg recombinant CD166/ALCAM protein (Recombinant Mouse ALCAM/CD166 Fc Chimera; R&D Systems) and RAGE protein (Recombinant Mouse RAGE Fc Chimera R&D Systems) were separated on 10% SDS gels (Bio-rad cat.567-1033) and transferred to PVDF membranes.

Techniques: Molecular Weight, Binding Assay, Gene Expression, Cell Culture, Two Tailed Test

Journal: Molecular metabolism

Article Title: Dietary sugars, not lipids, drive hypothalamic inflammation.

doi: 10.1016/j.molmet.2017.06.008

Figure Lengend Snippet: Figure 4: Deletions of RAGE or ALCAM genes improve metabolic symptoms induced by a HCHF diet and exert diverse impacts on microglia, pericytes, and vasculature in the arcuate nucleus. (A & B) Daily caloric intake (in wk10) and weekly BW gain of chow or HCHF diet-fed WT versus RAGE/ mice (n ¼ 5e8 per group); For weekly BW gain, in all time points, WT and RAGE/ mice have less BW gain on chow diet than on HCHF diet (P < 0.0001); from wk14 to wk16, BW gain on HCHF of RAGE/ mice is significantly less than WT mice. (C & D) Daily caloric intake (in wk10) and weekly BW gain of chow or HCHF diet fed WT mice versus ALCAM/ mice (n ¼ 5e8 per group). For weekly BW gain, from wk2 on, WT and ALCAM/ mice have less BW gain in chow than in HCHF; from wk12, wk14 to wk16, BW-gain on HCHF of ALCAM/ mice is significantly less than WT mice. (E & F) Quantification of the number of iba1-ir microglia and the PDGFRb-ir pericytes in the ARC in chow or HCHF diet fed WT mice versus RAGE/ mice (n ¼ 5e9 per group). (G & H) Quantification of vessel length and vascular density in the ARC from chow or HCHF diet fed WT mice versus RAGE/ mice (n ¼ 5e7 per group). (I & J) Quantification of the number of iba1-ir microglia and the PDGFRb-ir pericytes in the ARC in chow or HCHF diet fed WT mice versus ALCAM/ mice (n ¼ 5e6 per group). (K & L) Quantification of vessel length and vascular density in the ARC from chow or HCHF diet fed WT mice versus ALCAM/ mice (n ¼ 6 per group). Data are presented as means s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Asterisks in B and D indicate significance between WT and RAGE/ or ALCAM/ mice on HCHF diet. Two-way ANOVA followed by Bonferroni multiple comparisons for post-hoc analysis was performed to detect significant interaction between genotype and diet on each parameter.

Article Snippet: Briefly, 2 mg recombinant CD166/ALCAM protein (Recombinant Mouse ALCAM/CD166 Fc Chimera; R&D Systems) and RAGE protein (Recombinant Mouse RAGE Fc Chimera R&D Systems) were separated on 10% SDS gels (Bio-rad cat.567-1033) and transferred to PVDF membranes.

Techniques:

Journal: Molecular metabolism

Article Title: Dietary sugars, not lipids, drive hypothalamic inflammation.

doi: 10.1016/j.molmet.2017.06.008

Figure Lengend Snippet: Figure 5: Deletion of RAGE and ALCAM genes improves metabolic symptoms induced by a HCHF diet. (A & B) Daily caloric intake (in wk10) and weekly body weight gain of chow or HCHF diet-fed WT versus RAGE-ALCAM/ mice (n ¼ 6e11 per group); for weekly BW gain, from wk5 on, WT and RAGE-ALCAM/ mice have less BW gain in chow than in HCHF; From wk1 to wk4 and from wk9 to wk16, there are significant effect of genotype on BW gain on HCHF. (C) Body composition of WT versus RAGE-ALCAM/ mice fed HCHF diet (n ¼ 4e8 per group). (D) Glucose tolerance of WT versus RAGE-ALCAM/ mice fed chow or HCHF diet (n ¼ 5e7 per group). (E) Glucose tolerance of RAGE-ALCAM/

Article Snippet: Briefly, 2 mg recombinant CD166/ALCAM protein (Recombinant Mouse ALCAM/CD166 Fc Chimera; R&D Systems) and RAGE protein (Recombinant Mouse RAGE Fc Chimera R&D Systems) were separated on 10% SDS gels (Bio-rad cat.567-1033) and transferred to PVDF membranes.

Techniques:

Journal: Molecular metabolism

Article Title: Dietary sugars, not lipids, drive hypothalamic inflammation.

doi: 10.1016/j.molmet.2017.06.008

Figure Lengend Snippet: Figure 6: Deletion of RAGE and ALCAM genes reduces microglial reactivity and neovasculature formation in the arcuate nucleus (A, B & C) Quantification of iba1-ir microglial number, coverage and the PDGFRb-ir pericytes number in the ARC from chow or HCHF diet fed WT (n ¼ 5e8 per group) versus RAGE-ALCAM/ mice (n ¼ 7e 10 per group). (D & E) Quantification of vessel length and vascular density in the ARC from chow or HCHF diet fed WT (n ¼ 5e7 per group) versus RAGE-ALCAM/ mice (n ¼ 5e 7 per group). (FeH) Illustrations of the iba1-ir microglia, PDGFRb-ir pericytes and FITC-albumin labeled vessels in WT versus RAGE-ALCAM/ mice fed chow or HCHF diet, with a frame of 0.2 mm 0.2 mm for quantifications in the ARC. (I) Illustration of the skeletonization of vessel in H for vascular density analysis. III: third cerebral ventricle. Scale bar: 50um in F and G, 100um in F. Data are presented as means s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA followed by Bonferroni multiple comparisons for post-hoc analysis was performed to detect significant interaction between genotype and diet on each parameter.

Article Snippet: Briefly, 2 mg recombinant CD166/ALCAM protein (Recombinant Mouse ALCAM/CD166 Fc Chimera; R&D Systems) and RAGE protein (Recombinant Mouse RAGE Fc Chimera R&D Systems) were separated on 10% SDS gels (Bio-rad cat.567-1033) and transferred to PVDF membranes.

Techniques: Labeling

Journal: Biochimica et biophysica acta

Article Title: RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line.

doi: 10.1016/j.bbadis.2014.02.013

Figure Lengend Snippet: Fig. 1. Q2 A) Levels of RAGE in RAGE transfected WM115 cells as determined by ELISA. Levels are expressed in pg RAGE protein per mg of total protein. WM115-RAGE and WM115-RAGE-I expressed 94 fold and 7 fold higher RAGE protein than the MOCK control cells, respectively. B) Binding of the anti-RAGE antibody MAB1145 to WM115-RAGE (filled circles) and WM115- MOCK (filled squares) measured by flow cytometry. The binding curve of MAB1145 to WM115-RAGE was fitted using a 1:1 binding model and showed an affinity of 1.5 (±0.3) nM. RAGE overexpressed in the melanoma cells is properly processed and translocated to the cell-surface, as demonstrated by their recognition by specific antibodies. The experiment was performed in triplicate and the standard deviation is indicated. C–F) Morphology of WM115-MOCK (C), WM115-RAGE-I (D), WM115-RAGE (E) and WM266-MOCK (F) by bright field microscopy. G–H) Differences in morphology between WM115-MOCK (G) and WM115-RAGE (H) transfected cells, as shown by actin staining. Actin was stained with PE conjugated phalloidin and the nuclei were stained with Hoechst 33342. (20× magnification).

Article Snippet: RAGE protein levels in the cell extracts were determined using the Quantikine human RAGE Immunoassay kit (R&D Systems) according to the manufacturer's procedure, and expressed in picogram of RAGE per mg of total protein.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Control, Binding Assay, Cytometry, Standard Deviation, Microscopy, Staining

Journal: Disease markers

Article Title: Fecal S100A12 in Healthy Infants and Children

doi: 10.1155/2013/873582

Figure Lengend Snippet: Faecal S100A12 concentrations in 56 healthy infants and children. Serial stools collected from the first day of life (meconium) to 6 months of age from 7 healthy infants (Population 1) and single stools collected from 49 children (Population 2) were utilised to measure faecal S100A12 concentrations by immunoassay.

Article Snippet: Dilutions of recombinant S100A12 (Recombinant Human EN-RAGE/S100A12, R&D Systems, Minneapolis, MN, USA) were added (100 μ L/well) in order to create a standard curve.

Techniques:

Journal: Disease markers

Article Title: Fecal S100A12 in Healthy Infants and Children

doi: 10.1155/2013/873582

Figure Lengend Snippet: Measurement of fecal S100A12 infants and children. Repeated fecal samples were collected from seven term infants over the first six months of life (a). Single stool samples were collected from 49 healthy infants and children (b). S100A12 concentrations were measured by immunoassay. Only five samples (all in infants) were above the cut-off of 10 mg/kg.

Article Snippet: Dilutions of recombinant S100A12 (Recombinant Human EN-RAGE/S100A12, R&D Systems, Minneapolis, MN, USA) were added (100 μ L/well) in order to create a standard curve.

Techniques:

Journal: Molecules and Cells

Article Title: Characterization of αX I-Domain Binding to Receptors for Advanced Glycation End Products (RAGE)

doi: 10.14348/molcells.2017.0021

Figure Lengend Snippet: Binding of αX and αM I-domains to RAGE and the V-domain of RAGE. (A) A schematic representation of recombinant RAGE and RAGE derived soluble domains. All soluble proteins are fused with a His-tag for purification and detection. (B) SDS-PAGE analysis of purified sRAGE, sRAGEC1/2 and sRAGEV. (C) SPR sensorgram of sRAGE and RAGE-derived soluble domains binding to immobilized GST-αX-I. RAGE-derived proteins (1 μM) were injected to flow over immobilized GST-αX-I on a CM5 sensor chip (1800 RU). (D) Binding of sRAGEV and sRAGEC1/2 to GST-αX-I on microtiter plates. sRAGEV and sRAGEC1/2 (0.5 μM or 1.0 μM) were loaded on microtiter plates coated with GST-αX-I. Data are means ± S. E. (n = 3). (E, F) Binding of the I-domains to the sRAGE (E) and sRAGEV (F) on microtiter plates. GST and αX and αM I-domains (0.5 μM or 1.0 μM) were loaded on microtiter plates coated with sRAGE and sRAGEV. Data are means ± S. E. (n = 3).

Article Snippet: Recombinant RAGE fused with the human IgG Fc region produced from mammalian cells was purchased from R&D Systems (USA).

Techniques: Binding Assay, Recombinant, Derivative Assay, Purification, SDS Page, Injection