cxc motif chemokine ligand 6  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: A stage specific NETs-related signature in alcoholic steatohepatitis: from molecular subtyping to therapeutic vulnerabilities

doi: 10.3389/fimmu.2025.1711388

Figure Lengend Snippet: Construction and performance evaluation of a three-gene diagnostic signature for ASH. (A) Expression levels of FOS, MMP7, and CXCL6 within the training cohorts (integrated from microarray datasets GSE103580 and GSE28619 following batch effect correction). (B) ROC analysis was used to evaluate the diagnostic performance of three-gene signature in the training cohort. (C) A nomogram integrating the three biomarkers to predict the probability of ASH. (D) Calibration curve assessing the predictive accuracy of the nomogram and a decision curve analysis evaluating its clinical utility. (E) Architecture and weights of a neural network model, illustrating the contribution of each gene to ASH prediction. (F) Expression levels of the three genes in the external validation cohort (integrated from RNA-seq datasets GSE142530 and GSE155907 after batch correction). (G) ROC analysis of the individual genes and the combined model in the external validation cohort (AUC = 0.983). *p < 0.05, ***p < 0.001.

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against MPO (1:8000, Abcam, ab188211), F4/80 (1:500, CST, 70076S), MMP7 (1:200, Proteintech, 10374-2-AP), or CXCL6 (1:200, Abclonal, TD13470).

Techniques: Diagnostic Assay, Expressing, Microarray, Biomarker Discovery, RNA Sequencing

Journal: Frontiers in Immunology

Article Title: A stage specific NETs-related signature in alcoholic steatohepatitis: from molecular subtyping to therapeutic vulnerabilities

doi: 10.3389/fimmu.2025.1711388

Figure Lengend Snippet: Identification and characterization of NETs-related molecular subtypes in ASH. (A–C) The optimal cluster number was identified as k=2 through unsupervised consensus clustering. This conclusion is supported by evidence from the consensus matrix at k=2 (A) , the CDF plots for k values from 2 to 9 (B) , and the delta area plot (C) . (D) Box plots showing the expression levels of FOS, MMP7, and CXCL6 in clusters C1 and C2. (E) Heatmap showing distinct expression profiles of representative genes between the two subtypes. (F) PCA plot visualizing the separation of samples belonging to the two molecular subtypes. (G) Differential immune cell infiltration profiles between the two clusters. (H, I) GSVA results revealing significantly enriched GO biological processes (H) and KEGG pathways (I) for each subtype.

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against MPO (1:8000, Abcam, ab188211), F4/80 (1:500, CST, 70076S), MMP7 (1:200, Proteintech, 10374-2-AP), or CXCL6 (1:200, Abclonal, TD13470).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: A stage specific NETs-related signature in alcoholic steatohepatitis: from molecular subtyping to therapeutic vulnerabilities

doi: 10.3389/fimmu.2025.1711388

Figure Lengend Snippet: Validation of the NETs signature in a short-term NIAAA mouse model of ASH. (A) Schematic representation of the experimental design for the short-term NIAAA model. (B) Representative liver histology showing H&E, Oil Red O, Masson’s trichrome, and Sirius red staining (200× magnification; scale bar = 100 μm). (C) Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). (D) Quantification of hepatic triglyceride (TG) content. (E) Assessment of hepatic oxidative stress levels indicated by glutathione (GSH), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content. (F) ELISA analysis of hepatic pro-inflammatory cytokines TNF-α and IL-6 levels. (G) Western blot analysis of lipid metabolism-related proteins (PPAR-γ, PPAR-α) and the antioxidant enzyme HO-1 in whole liver lysates. (H) Western blot analysis of oxidative stress and inflammation markers (MDA, Arg1, MCP-1, and IL-10) in whole liver lysates. (I) Immunofluorescence staining of FOS (H) (400×; scale bar = 50 μm) and quantification of the relative fluorescence intensity of FOS. (J) Immunohistochemical staining of MMP7 and CXCL6 (G) (200×; scale bar = 100 μm) and quantification of the positive area. (K) Western blot analysis of FOS, MMP7, and CXCL6 protein expression in whole liver lysates. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. .

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against MPO (1:8000, Abcam, ab188211), F4/80 (1:500, CST, 70076S), MMP7 (1:200, Proteintech, 10374-2-AP), or CXCL6 (1:200, Abclonal, TD13470).

Techniques: Biomarker Discovery, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Fluorescence, Immunohistochemical staining, Expressing

Journal: Frontiers in Immunology

Article Title: A stage specific NETs-related signature in alcoholic steatohepatitis: from molecular subtyping to therapeutic vulnerabilities

doi: 10.3389/fimmu.2025.1711388

Figure Lengend Snippet: Validation of the NETs signature in a chronic ASH mouse model and in ASH patients. (A) Flowchart illustrating the protocol for the chronic-plus-binge ethanol feeding model. (B) Representative liver histopathological images stained with H&E, Oil Red O, Masson’s trichrome, and Sirius Red (200× magnification; scale bar = 100 μm). (C) Serum AST and ALT levels. (D) Hepatic TG and TC content. (E) Analysis of hepatic oxidative stress markers (GSH, SOD, MDA). (F) Hepatic levels of pro-inflammatory cytokines TNF-α, IL-6. (G) Western blot analysis of PPAR-γ, PPAR-α, and HO-1 protein expression in whole liver lysates. (H) Western blot analysis of MDA, Arg1, MCP-1, and IL-10 protein levels in whole liver lysates. (I) Immunofluorescence detection of FOS and quantification of FOS relative fluorescence intensity (400×; scale bar =50 μm). (J) Immunohistochemical staining of MMP7 and CXCL6 alongside quantitative analysis of the positive staining area (200×; scale bar = 100 μm). (K) Western blot analysis of FOS, MMP7, and CXCL6 protein expression in the chronic mouse model using whole liver lysates. (L) Western blot analysis of FOS, MMP7, and CXCL6 protein expression in whole liver lysates from healthy and ASH patients. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 .

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against MPO (1:8000, Abcam, ab188211), F4/80 (1:500, CST, 70076S), MMP7 (1:200, Proteintech, 10374-2-AP), or CXCL6 (1:200, Abclonal, TD13470).

Techniques: Biomarker Discovery, Staining, Western Blot, Expressing, Immunofluorescence, Fluorescence, Immunohistochemical staining

Journal: Kidney Diseases

Article Title: CXCL6 Orchestrates Macrophage-Driven Inflammation in Diabetic Kidney Disease and Represents a Druggable Target

doi: 10.1159/000548806

Figure Lengend Snippet: Flow diagram of the study design. A comprehensive overview of the study workflow, illustrating the integration of bioinformatics analysis, experimental validation, and therapeutic exploration to investigate the pathogenesis of diabetic kidney disease (DKD). Gene expression datasets ( GSE30529 , GSE47184 , GSE175759 , and GSE142025 ) were analyzed to identify differentially expressed genes (DEGs) and key modules associated with DKD progression using weighted gene correlation network analysis (WGCNA). KEGG pathway enrichment analysis highlighted significant signaling pathways, and machine-learning approaches (LASSO, SVM, and RF) identified CXCL6 as the hub gene. Single-cell RNA sequencing (scRNA-seq) data further localized CXCL6 expression to proximal tubular epithelial cells in the kidney. Experimental validation was conducted using renal tissues from DKD patients and mice, as well as in vitro studies with HG-stimulated HK-2 cells and THP-1 macrophages to confirm CXCL6’s role in macrophage recruitment and inflammatory cytokine release. Virtual screening and molecular docking identified salvianolic acid B as a CXCL6-targeting inhibitor, which was shown to mitigate inflammation and macrophage migration in in vitro studies. This integrative approach bridges bioinformatics and experimental data, highlighting CXCL6’s role in DKD and its therapeutic potential.

Article Snippet: CXCL6 antibody (DF13470), aquaporin-1 (AQP1) antibody (sc-25287), and F4/80 antibody (70076) were purchased from Affinity Biosciences (Jiangsu, China), Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Cell Signaling Technology (Boston, MA, USA), respectively.

Techniques: Biomarker Discovery, Gene Expression, Protein-Protein interactions, RNA Sequencing, Expressing, In Vitro, Migration

Journal: Kidney Diseases

Article Title: CXCL6 Orchestrates Macrophage-Driven Inflammation in Diabetic Kidney Disease and Represents a Druggable Target

doi: 10.1159/000548806

Figure Lengend Snippet: Screening DEGs from three datasets and using machine learning to screen hub gene. Volcano plots of DEGs from GSE30529 ( a ), GSE47184 ( b ), and GSE175759 ( c ) are presented. Venn diagrams show the intersection of DEGs across the three datasets ( d ), identifying 41 upregulated ( e ) and 5 downregulated ( f ) DEGs. g The top significant KEGG pathways in common DEGs. h The Venn diagram shows CXCL6, a hub gene that screens common DEGs using three machine-learning approaches. i The hub gene CXCL6 was significantly increased in patients with DKD compared to healthy controls (* p < 0.05).

Article Snippet: CXCL6 antibody (DF13470), aquaporin-1 (AQP1) antibody (sc-25287), and F4/80 antibody (70076) were purchased from Affinity Biosciences (Jiangsu, China), Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Cell Signaling Technology (Boston, MA, USA), respectively.

Techniques:

Journal: Kidney Diseases

Article Title: CXCL6 Orchestrates Macrophage-Driven Inflammation in Diabetic Kidney Disease and Represents a Druggable Target

doi: 10.1159/000548806

Figure Lengend Snippet: GSE142025 dataset identifies key modules associated with advanced DKD via WGCNA. a Correlation between module eigengenes and clinical traits of DKD. The turquoise module was closely related to advanced DKD. b The top 20 of significant KEGG pathways in turquoise module. c The expression level of CXCL6 in advanced DKD was significantly increased in GSE142025 database (*** p < 0.001). The mRNA expression level of CXCL6 was negatively correlated with eGFR in both the GSE30529 ( d ) and GSE175759 ( e ) databases. f Relative expression level of CXCL6 in the various cell types between healthy controls and DKD from the KIT website. g The protein expression of CXCL6 and proximal tubular epithelial marker (AQP1) in human kidney tissues of DKD and healthy control kidney using double immunofluorescence staining. h Representative CXCL6 immunohistochemical staining in healthy control kidney and renal biopsies of human DKD. Data were represented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. healthy control.

Article Snippet: CXCL6 antibody (DF13470), aquaporin-1 (AQP1) antibody (sc-25287), and F4/80 antibody (70076) were purchased from Affinity Biosciences (Jiangsu, China), Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Cell Signaling Technology (Boston, MA, USA), respectively.

Techniques: Expressing, Marker, Control, Double Immunofluorescence Staining, Immunohistochemical staining, Staining

Journal: Kidney Diseases

Article Title: CXCL6 Orchestrates Macrophage-Driven Inflammation in Diabetic Kidney Disease and Represents a Druggable Target

doi: 10.1159/000548806

Figure Lengend Snippet: CXCL6 induction in DKD and screening of potential regulatory transcription factors (TFs). a CXCL6 was detected in the urine of healthy controls ( n = 12) and DKD patients ( n = 24), and the urinary CXCL6 protein concentration (µg/mg creatinine) was corrected with urinary creatinine. Linear regression shows that urinary CXCL6 levels in DKD patients are correlated with renal function (estimated glomerular filtration rate [eGFR]) ( b ), serum creatinine (Scr) ( c ), and urinary albumin-to-creatinine ratio (UACR) ( d ) compared to healthy controls. e Real-time PCR show that CXCL6 mRNA levels increased in HG-induced HK-2 cell injury. f ELISA analysis of CXCL6 in extracellular fluid of HK-2 cells revealed elevated levels following HG treatment. g Immunofluorescence staining of CXCL6 (red) in HK-2 cells with or without HG-treated. h t-SNE visualization of regulon activity in PCT cells, comparing diabetic kidney disease (DKD) and control (CTRL) groups. i Heatmap analysis delineated differential activation patterns of TFs and regulons in DKD compared to CTRL. j Area under the curve scores of the FOXP2 (18 g) target gene set show elevated activity in DKD, suggesting enhanced transcriptional regulation (upper). t-SNE visualization of binary regulon activity reveals distinct clustering, confirming disease-specific FOXP2 activation in DKD compared to CTRL (bottom). k A schematic illustration depicts the FOXP2 motif within the promoter region of the CXCL6 locus. Data were represented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. healthy control or NC. PCT, proximal tubule cell.

Article Snippet: CXCL6 antibody (DF13470), aquaporin-1 (AQP1) antibody (sc-25287), and F4/80 antibody (70076) were purchased from Affinity Biosciences (Jiangsu, China), Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Cell Signaling Technology (Boston, MA, USA), respectively.

Techniques: Protein Concentration, Filtration, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Activity Assay, Control, Activation Assay

Journal: Kidney Diseases

Article Title: CXCL6 Orchestrates Macrophage-Driven Inflammation in Diabetic Kidney Disease and Represents a Druggable Target

doi: 10.1159/000548806

Figure Lengend Snippet: Levels of CXCL6 were increased in db/db mice. a Analysis of blood glucose levels. b Analysis of serum creatinine (Scr) levels. c Analysis of urinary albumin-to-creatinine ratio (UACR). d CXCL6 was detected in the urine of db/m and db/db mice ( n = 6), with the urinary CXCL6 protein concentration corrected by urinary creatinine (µg/mg creatinine). e , f Linear regression analysis reveals a correlation between urinary CXCL6 and Scr and UACR in db/db mice. g Histological observations of kidney sections stained with PAS. IHC and quantification of CXCL6 ( h ) and F4/80 ( i ) in db/db renal tissue. The levels of mRNA for CXCL6, a-SMA, and FN were analyzed by real-time PCR ( j ), and linear regression analysis showed that the mRNA level of CXCL6 is also correlated with a-SMA ( k ) and FN ( l ) in db/db mice. Data represent the mean ± SEM of 6 mice in vivo. Statistically significant differences were determined by independent sample t test and one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. db/m.

Article Snippet: CXCL6 antibody (DF13470), aquaporin-1 (AQP1) antibody (sc-25287), and F4/80 antibody (70076) were purchased from Affinity Biosciences (Jiangsu, China), Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Cell Signaling Technology (Boston, MA, USA), respectively.

Techniques: Protein Concentration, Staining, Real-time Polymerase Chain Reaction, In Vivo

Journal: Kidney Diseases

Article Title: CXCL6 Orchestrates Macrophage-Driven Inflammation in Diabetic Kidney Disease and Represents a Druggable Target

doi: 10.1159/000548806

Figure Lengend Snippet: CXCL6 was chemotactic to macrophages and increased the release of inflammatory cytokines in macrophages. a Uniform manifold approximation and projection (UMAP) plot showing cell cluster identities and the cell cluster abundance in control and DKD. ENDO, endothelial cells; MES, mesangial cells; PODO, podocytes; CFH+, complement factor H-positive cells; LOH, loop of Henle; CD, collecting duct; PC, principal cell; IC, intercalated cell; DCT, distal convoluted tubule; PCT, proximal tubular cells; LEUK, leukocyte. b t-SNE plot showing CXCR2 in macrophages. c The protein levels of CXCR2 in THP-1 cells were significantly upregulated following stimulation with CXCL6. d Flow cytometry analysis showed that CXCL6 cytokines could polarize THP-1 cells into pro-inflammatory macrophages. e The protein expression of CXCR2 and pro-inflammatory macrophage marker (iNOS) in human kidney tissues of DKD and healthy control kidney using double immunofluorescence staining. f The mRNA expression levels of TNF-a, IL-6, and IL-23 in THP-1 cells after CXCL6 stimulation were detected by real-time PCR. g Transwell migration assays showed that the addition of CXCL6 cytokines to extracellular fluid increased the migration of THP-1 cells. Data represent the mean ± SEM. of at least three independent experiments in vitro. Statistically significant differences were determined by independent sample t test and one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MΦ.

Article Snippet: CXCL6 antibody (DF13470), aquaporin-1 (AQP1) antibody (sc-25287), and F4/80 antibody (70076) were purchased from Affinity Biosciences (Jiangsu, China), Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Cell Signaling Technology (Boston, MA, USA), respectively.

Techniques: Control, Flow Cytometry, Expressing, Marker, Double Immunofluorescence Staining, Real-time Polymerase Chain Reaction, Migration, In Vitro

Journal: Kidney Diseases

Article Title: CXCL6 Orchestrates Macrophage-Driven Inflammation in Diabetic Kidney Disease and Represents a Druggable Target

doi: 10.1159/000548806

Figure Lengend Snippet: The potential pathways by which CXCL6 regulates the polarization and migration of THP-1 cells. a Volcano plot of differential gene expression between MΦ and MΦ+CXCL6 THP-1 cells. b Results of KEGG pathway enrichment analysis based on RNA-seq of THP-1 cells. c GSEA results based on the RNA-Seq data. d Representative Western blot of CXCR2 protein expression in coculture models in vitro. e The mRNA expressions of TNF-a, IL-6, and IL-23 in THP-1 cells were analyzed by RT-qPCR in HG-induced HK-2 cells with or without Si-CXCL6. f The migration of THP-1 cells in HG-induced HK-2 cells with or without Si-CXCL6 was analyzed by Transwell migration assay. g Schematic diagram of the virtual screening-based strategy for exploring anti-renal inflammatory TCM monomers targeting CXCL6. h Molecular docking of salvianolic acid B to the catalytic core of CXCL6. Data represent the mean ± SEM. of at least three independent experiments in vitro. Statistically significant differences were determined by independent sample t test and one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. HG. NC, normal control; HG, high glucose.

Article Snippet: CXCL6 antibody (DF13470), aquaporin-1 (AQP1) antibody (sc-25287), and F4/80 antibody (70076) were purchased from Affinity Biosciences (Jiangsu, China), Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Cell Signaling Technology (Boston, MA, USA), respectively.

Techniques: Migration, Gene Expression, RNA Sequencing, Western Blot, Expressing, In Vitro, Quantitative RT-PCR, Transwell Migration Assay, Control

Journal: Kidney Diseases

Article Title: CXCL6 Orchestrates Macrophage-Driven Inflammation in Diabetic Kidney Disease and Represents a Druggable Target

doi: 10.1159/000548806

Figure Lengend Snippet: In vitro experiments showed that inhibition of CXCL6 secretion by Sal-B can alleviate the release of inflammatory cytokines in macrophages and the chemotaxis of macrophages. a CCK-8 measured drug toxicity and optimal concentration of salvianolic acid B. b Representative immunofluorescence staining showed that salvianolic acid B treatment reduced the CXCL6 abundance in HG-treated HK-2 cells ( n = 3). Scale bar = 20 μm. c Schematic diagram of coculture of HK-2 cells and THP-1 cells. d Representative Western blot and quantification of CXCR2 protein expression in coculture models in vitro. e The mRNA expressions of TNF-a, IL-6, and IL-23 in THP-1 cells were analyzed by RT-qPCR in HG-induced HK-2 cells with or without Sal-B. f The migration of THP-1 cells in HG-induced HK-2 cells with or without Sal-B was analyzed by Transwell migration assay. Data represent the mean ± SEM. of at least three independent experiments in vitro. Statistically significant differences were determined by independent sample t test and one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC and Sal-B. p < 0.05, p < 0.01, p < 0.001, vs. HG. NC, normal control; HG, high glucose; Sal-B, salvianolic acid B.

Article Snippet: CXCL6 antibody (DF13470), aquaporin-1 (AQP1) antibody (sc-25287), and F4/80 antibody (70076) were purchased from Affinity Biosciences (Jiangsu, China), Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Cell Signaling Technology (Boston, MA, USA), respectively.

Techniques: In Vitro, Inhibition, Chemotaxis Assay, CCK-8 Assay, Concentration Assay, Immunofluorescence, Staining, Western Blot, Expressing, Quantitative RT-PCR, Migration, Transwell Migration Assay, Control