Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) CXCL9, 10 and 11 can all bind and signal through the chemokine receptor CXCR3 that is typically found on T cells. (B) The EMBL-ELI expression atlas (human) was analysed for relatedness in expression of CXCR3 and its’ ligands CXCL9, 10 and 11 across all tissues or (C) in distinct tissues and diseases.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Expressing

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) Schematic of the in vivo air pouch leukocyte recruitment model. (B) Analysis of chemokine concentration in the carrageenan inflamed air pouch. (C) Representative tSNE of all murine cells gated on live, single, CD45 + and built on CD4, CD8, F4/80, Ly6C, Ter119, CD3, TCRβ, CXCR3, Ly6G, CD11c, B220, CD11b, CD64, Siglec F, NK1.1 and TCRγδ. FlowSOM clusters are illustrated by gates. (D) tSNE analysis of air pouches injected with equimolar amounts of CXCL9, 10 and 11. (E) Quantification of all leukocytes (CD45 + ) and T cells within the air pouch following injection of CXCL9, 10 or 11. E analysed using a one-way ANOVA.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: In Vivo, Concentration Assay, Injection

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) Schematic of HS GAG structure, including sulphation points and the enzymes that produce them. (B) Normalised (relative to wild type) binding of labelled CXCL9, 10 or 11 to genetically modified CHO cells. (C) Normalised and absolute binding of CXCL9, 10 and 11 to CHO cells in which KS^ST1/2/3 have been genetically removed. (D and E) Normalised binding of CXCL9, 10 and 11 to CHO cells genetically engineered to express the enzymes regulating 3-O GAG sulphation. (F) EMBL-ELI expression atlas analysis of relatednessCXCL9, 10 and 11 and GAG sulphation gene expression. B and D, data plotted as mean from three separate pooled experiments. C and E data plotted as mean ± SEM from three separate pooled experiments and analysed using a one-way ANOVA.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Binding Assay, Genetically Modified, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) CXCL9, 10 and 11 all bind to the same receptor with different affinities and biased signalling outcomes and are found in over-lapping expression patterns during inflammation and disease. (B) Differential GAG interactions means that CXCL9 is more likely to be retained on GAGs on the cell surface or within the ECM, with CXCL10 and CXCL11 being more likely to be present in their soluble state.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Expressing

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 1 RT-PCR analysis of CXCL10, CXCL9, and CXCL11 gene expression in freshly isolated Langerhans cells (fLC), 48 h cultured LC (cLC), interferon-c (IFN-c)-stimulated cLC (c-cLC), freshly isolated splenic dendritic cells (fDC), 48 h cultured splenic DC (cDC), and IFN-c- stimulated cDC (c-cDC). LC and splenic DC were purified and cul- tured for 48 h in the absence or presence of 100 ng per mL of IFN-g. Samples from fresh and cultured LC and splenic DC were collected and mRNA expression was analyzed using specific primers for each chemokine. CXCL10, CXCL9, and CXCL11 mRNA expression was hardly detectable in fLC. In cLC, mRNA expression of CXCL10 and CXCL11, but not CXCL9, was induced. CXCL9 mRNA was strongly expressed only in g-cLC. In addition, mRNA for CXCL10 and CXCL11 was also strongly expressed in g-cLC. In splenic DC, mRNA for these T helper 1(Th1)-type chemokines was almost undetectable both in fDC and cDC. When stimulated with IFN-g, CXCL10, CXCL9, and CXCL11 mRNA expression was induced. Data are representative of three inde- pendent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Isolation, Cell Culture, Expressing

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 2 Production of T helper 1-type chemokines during culture of Langerhans cells (LC) and splenic dendritic cells (DC). Purified LC and splenic DC were cultured with or without interferon-g (IFN-g), and the concentration of CXCL10, CXCL9, and CXCL11 was measured at different time points (0, 12, 24, 36, 48 h) in the supernatants by ELISA. Representative data of three independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 3 Chemotaxis of mCXCR3-transfected 2B4 T cells. Langerhans cells (LC) and splenic dendritic cells (DC) were cultured for 48 h with 100 ng per mL of interferon-g (IFN-g) and the supernatants were collected. The culture supernatants were preincubated with or without neutralizing anti-chemokine monoclonal antibody (mAb) indicated in the figure for 30 min, and assessed for chemotactic activity to CXCR3 transfectant. RPMI 10 medium alone served as a negative control. RPMI 10 medium containing recombinant chemokine served as a positive control. The supernatants of IFN-g-stimulated LC and splenic DC exhibited chemo- tactic activity to CXCR3 transfectant, which is mediated at least by CXCL10 and CXCL9. Mean (SD) (n ¼ 3). Data are representative of three independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Chemotaxis Assay, Transfection, Cell Culture, Activity Assay, Negative Control, Recombinant, Positive Control

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 7 Regulation of T helper 1-type chemokines produced by La- ngerhans cells (LC) and splenic dendritic cells (DC). LC (&) and splenic DC (’) were purified and cultured (1.5 106 cells per mL per 200 mL in each well) for 48 h with or without various stimuli. The supe- rnatants were collected and the concentration of CXCL10 (a), CXCL9 (b), and CXCL11 (c) was measured by ELISA. CXCL10 production by LC was induced by interferon-g (IFN-g), interleukin (IL)-12, lipopoly- saccharide (LPS), Staphylococcus aureus Cowen 1 (SAC), and poly- inosinic–polycytidylic acid (Poly(I:C)). In the case of CXCL9 and CXCL11, only IFN-g induced their production by LC. In the case of splenic DC, the production of CXCL10, CXCL9, and CXCL11 was in- duced by IFN-g, IL-18, LPS, and Poly(I:C). Results are the mean (SD) (n ¼ 4). Significant increase (po0.05) compared with the unstimulated group. Data are representative of four independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Produced, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Cyclin G2 in macrophages triggers CTL-mediated antitumor immunity and antiangiogenesis via interferon-gamma.

doi: 10.1186/s13046-022-02564-2

Figure Lengend Snippet: Fig. 7 Cyclin G2 knockout in macrophages attenuates the inhibitory effects of IFN-γ on colon cancer cell growth. A–C MC38 cells were mixed with BMDMs from WT and Ccng2−/− C57BL/6 mice at a ratio of 5:1 and injected subcutaneously into C57BL/6 mice, which were then treated with IFN-γ at specific times. Gross tumors (A), tumor weights (B), and tumor volumes (C) were measured at the endpoint. Data were analyzed with the unpaired Student’s t-test. Data are presented as the mean ± SEM (n = 5). D A schematic model depicting the role of cyclin G2 in macrophages after IFN-γ treatment. Upregulated cyclin G2 after IFN-γ treatment inhibited the interaction between PP2Ac and STAT1, thereby increasing the nuclear import of STAT1 and promoting CXCL9 transcription. Increased CXCL9 secretion can promote CTL chemotaxis and inhibit vascular endothelial cell angiogenesis, ultimately inhibiting tumor progression. **p < 0.01; ****p < 0.0001

Article Snippet: A total of 10 × 104 cells/well were suspended in RPMI 1640 containing 0.2% FBS in the upper chamber, and the lower chamber was filled with the conditioned medium of BMDMs from C57BL/6 mice with or without recombinant mouse CXCL9 protein (492-MM-010/CF, R&D Systems).

Techniques: Knock-Out, Injection, Chemotaxis Assay

Journal:

Article Title: Antifibrotic Effects of CXCL9 and Its Receptor CXCR3 in Livers of Mice and Humans

doi: 10.1053/j.gastro.2009.03.053

Figure Lengend Snippet: Murine quantitative trait locus and gene expression analysis. (A) Schematic diagram of the Cxc chemokine cluster on chromosome 5 (90,207,953 to 94,974,241 base pairs). The SNPs rs4225368, rs4225374, and rs3023048 tag the proximal, middle, and distal part of the gene cluster. Regression analysis in the F2 progeny showed that the SNPs tagging the middle and distal part of the cluster are significantly associated with the histologic stage of liver fibrosis induced by CCl4 (P = .026 and P = .036, respectively). (B –F) Quantitative expression of IFN-inducible chemokines and their receptor Cxcr3 in BALB/cJ and A/J mice after challenge with CCl4. Whereas the mRNA expression levels of (D) Cxcl10, E ( ) Cxcl11, and (F) Cxcr3 do not differ between the strains, the expression of Cxcl9 was 2.3-fold increased in the resistant strain (P = .002, B). (C) The increased mRNA expression of CXCL9 was confirmed by CXCL9 protein analysis (C).

Article Snippet: Immunohistochemistry of CXCL9 in Human Liver Tissue Immunohistochemical staining was performed with a mouse monoclonal anti-human CXCL9 antibody (R&D Systems).

Techniques: Gene Expression, Expressing

Journal:

Article Title: Antifibrotic Effects of CXCL9 and Its Receptor CXCR3 in Livers of Mice and Humans

doi: 10.1053/j.gastro.2009.03.053

Figure Lengend Snippet: Human genetic analyses. (A) Schematic diagram of the distal part of the human CXC chemokine cluster on chromosome 4q21 (77,380 –77,420 kilobases). Eight SNPs cover the genes for the chemokines CXCL9, CXCL10, and CXCL11. (B ) These SNPs were genotyped in 404 patients with mild (stage F0/F1, 2n = 474) or advanced (stage F2–F4, 2n = 334) liver fibrosis. The overall haplotype distribution is different between patients with mild fibrosis and subjects with severe fibrosis (P = .01). Specifically, the fourth most common haplotype, dCXC_4, is significantly more prevalent in individuals with advanced fibrosis (15.0%) compared with patients with mild fibrosis (6.4%; *P = .001). (C) dCXC_4 is tagged by rs3733236 in the 3′-untranslated region of CXCL9. The minor (risk) allele of this SNP is functionally associated with reduced serum concentrations in patients with HCV infection and mild fibrosis (n = 60, P = .02).

Article Snippet: Immunohistochemistry of CXCL9 in Human Liver Tissue Immunohistochemical staining was performed with a mouse monoclonal anti-human CXCL9 antibody (R&D Systems).

Techniques: Infection

Journal:

Article Title: Antifibrotic Effects of CXCL9 and Its Receptor CXCR3 in Livers of Mice and Humans

doi: 10.1053/j.gastro.2009.03.053

Figure Lengend Snippet: Analyses of CXCL9 in human liver fibrosis. (A) CXCL9 mRNA expression is significantly associated with the degree of liver fibrosis in HCV (P = .04). (B) Association of CXCL9 mRNA expression with liver fibrosis is confirmed in patients with fibrosis due to nonalcoholic steatohepatitis (P = .002). (C) Immunohistochemistry of CXCL9 confirms a strong up-regulation in chronic liver damage (normal liver, left; HCV-induced cirrhosis, right). (D ) The mRNA of both splice variants of the CXCL9 receptor CXCR3 are present in HCV-infected liver. (E) CXCL9 and (F) CXCL10 serum concentrations are also associated with the severity of liver fibrosis in HCV infection (P = .01 and P = .001, respectively).

Article Snippet: Immunohistochemistry of CXCL9 in Human Liver Tissue Immunohistochemical staining was performed with a mouse monoclonal anti-human CXCL9 antibody (R&D Systems).

Techniques: Expressing, Immunohistochemistry, Infection

Journal:

Article Title: Antifibrotic Effects of CXCL9 and Its Receptor CXCR3 in Livers of Mice and Humans

doi: 10.1053/j.gastro.2009.03.053

Figure Lengend Snippet: Functional analysis of CXCL9 in stellate cells. (A) CXCL9 mRNA is expressed in HSCs and is significantly increased (P = .02) after treatment of the cells with IFN-γ. (B and C) Stimulation of LX-2 cells with CXCL9 leads to a dose-dependent down-regulation of TGFB (P = .01) and COLA1A mRNA expression (P = .001), respectively. The columns represent mean values ± SEM of 3 independent experiments. (D) The down-regulation of collagen by CXCL9 in HSCs is confirmed by Western blot with an antibody detecting the immature (175 kilodaltons) and mature (120 kilodaltons) chain of collagen. (E) Both splice variants of the CXCL9 receptor CXCR3 are present in LX-2 cells. (F) However, stimulation of the CXCR3-B isoform with CXCL4 leads only to a modest increase in COLA1A mRNA.

Article Snippet: Immunohistochemistry of CXCL9 in Human Liver Tissue Immunohistochemical staining was performed with a mouse monoclonal anti-human CXCL9 antibody (R&D Systems).

Techniques: Functional Assay, Expressing, Western Blot