cxcl16 neutralization  (Bio X Cell)

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) CXCL16 mRNA expression in the kidney during MuPyV infection. Expression is shown as fold change relative to sham infected samples. Data are from three independent experiments (n = 10-11). (B) Expression of NKCC2 [marks the ascending loop of Henle ], CD8, and CXCL16 in epithelia in sham-infected and 8 dpi kidneys; bottom right photomicrographs are merged images. Representative of two independent experiments. (C) CXCL16 expression in sham-infected, 4 dpi, and 8 dpi kidney lysates (left). Western blot image is representative of two independent experiments with each lane indicating protein lysate from kidneys of individual mice. Protein band intensity quantification for sCXCL16 was normalized to β-actin and analyzed by ImageLab and normalized to the loading control (right). Data are combined from two independent experiments (n = 3-5). (D) Experimental design of in vivo CXCL16 mAb administration. Mice were administered 250 µg of CXCL16 mAb or control rat IgG every two days from days 4-14 post infection and euthanized at 15 dpi. Numbers of CD45 mAb i.v.-negative, CD8 + CD44 + D b -LT359 tetramer + T cells and CD4 + CD44 + T cells in kidneys and spleens of infected mice given anti-CXCL16 or control rat IgG. Data were analyzed by one-way ANOVA (A and C) and by multiple Mann-Whitney tests (D).

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Infection, Western Blot, Control, In Vivo, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) Dense lymphoplasmacytic infiltrate surrounding and infiltrating cortical tubules (H&E, x400). (B) Immunohistochemical staining for Large T antigen in nuclei of tubular epithelium (x250). (C) Expression of CXCR6 on infiltrating CD8 + cells. Photomicrographs (right) are enlarged images in the white squares (left); bottom right are merged images. No 1 o , no primary antibody. (D) Log-fold change of CXCL16 and CXCR6 across four independent studies, each comparing KTx biopsies from patients with PVAN and stable graft function. The error bars indicate the 95% confidence interval of log-fold change, with horizontal dashed grey line indicating log-fold change = 0, or no difference. GSE120495 is an RNA-seq study, while remaining studies are microarray based. Given the heterogeneity of the data, a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. The combined p-values for CXCL16 = 0.000337 and CXCR6 = 2.72x10 -05 . ns, non-significant at adjusted p-value > 0.05; *, statistical significance at adjusted p-value ≤ 0.05; **, statistical significance at adjusted p-value ≤ 0.01; ***, statistical significance at adjusted p-value ≤ 0.001.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Immunohistochemical staining, Staining, Expressing, RNA Sequencing, Microarray

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: Summary of differential gene expression of CXCL16 and CXCR6 in four studies of KTx biopsies with PVAN. Because of the heterogeneity of the data (i.e., one RNA-seq, three microarray), a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. P-values significant after false discover rate (FDR) correction are bolded and italicized in the Adj. P-value column. Cauchy combined p-values < 0.05 are bolded and italicized.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Gene Expression, Microarray

Journal: Heliyon

Article Title: Sodium butyrate restricts neutrophils migration and NETs formation through reducing macrophage-derived CXCL16 in calculous cholecystitis

doi: 10.1016/j.heliyon.2024.e25189

Figure Lengend Snippet: The increased NETs formation is associated with pathological classification of calculous cholecystitis. (a) Blood neutrophil percentage count of normal control, chronic cholecystitis and acute cholecystitis (n = 20). (b) Proportion of neutrophils forming NETs in patients with control, chronic cholecystitis and acute cholecystitis (n = 20). (c–d) Quantification of plasmatic dsDNA and MPO-DNA in healthy controls, patients with chronic cholecystitis and acute cholecystitis (n = 20). (e) Concentration of serum CXCL16 in healthy controls, patients with chronic cholecystitis and acute cholecystitis (n = 20). (f) Correlation analysis between CXCL16 and neutrophil amount. (g) Immunohistochemical staining for Neutrophil Elastase in cholecyst sections from patients with chronic cholecystitis (n = 20) and acute cholecystitis (n = 20). (g) Immunohistochemical staining for CXCL16 in cholecyst sections from patients with chronic cholecystitis (n = 20) and acute cholecystitis (n = 20).

Article Snippet: Briefly, the neutrophils were stained with calcein-AM (2 μg·mL-1, C3099, Invitrogen) for 15 min at 37 °C and seeded on the upper chamber of the transwell inserts with 3.0 μm pore-size transparent PET membranes (FALCON-353096) and co-cultured with RAW264.7 cells or exosomes derived from treatment RAW264.7 cells or CXCL16 neutralizing antibody (0.25 μg; R&D Systems, USA)/recombinant CXCL16 (100 ng/mL; MedChemExpress, USA) in the lower chamber for 1 h at 37 °C according to the instructions.

Techniques: Concentration Assay, Immunohistochemical staining, Staining

Journal: Heliyon

Article Title: Sodium butyrate restricts neutrophils migration and NETs formation through reducing macrophage-derived CXCL16 in calculous cholecystitis

doi: 10.1016/j.heliyon.2024.e25189

Figure Lengend Snippet: The release of exosomes containing CXCL16 in plasma was elevated in calculous cholecystitis. (a) EVs obtained from plasma of patients with calculous cholecystitis were observed by TEM. Scale bar 200 nm. (b–c) The distribution and concentration of EVs were obtained by NanoSight tracking analysis. (d) Correlation between total exosomal protein concentration and MPO-DNA complexes obtained from plasma of patients with calculous cholecystitis (e–f) CD63, TSG101, HSP70 as markers of EVs and CXCL16 were detected by western blotting. Levels of EVs markers and CXCL16 were compared with the normal group. (g) The expression of CXCR6 in gallbladder tissue was evaluated by immunohistochemical staining. *, **and ***denote P < 0.05, P < 0.01 and P < 0.001 compared to the normal group, respectively.

Article Snippet: Briefly, the neutrophils were stained with calcein-AM (2 μg·mL-1, C3099, Invitrogen) for 15 min at 37 °C and seeded on the upper chamber of the transwell inserts with 3.0 μm pore-size transparent PET membranes (FALCON-353096) and co-cultured with RAW264.7 cells or exosomes derived from treatment RAW264.7 cells or CXCL16 neutralizing antibody (0.25 μg; R&D Systems, USA)/recombinant CXCL16 (100 ng/mL; MedChemExpress, USA) in the lower chamber for 1 h at 37 °C according to the instructions.

Techniques: Concentration Assay, Protein Concentration, Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: Heliyon

Article Title: Sodium butyrate restricts neutrophils migration and NETs formation through reducing macrophage-derived CXCL16 in calculous cholecystitis

doi: 10.1016/j.heliyon.2024.e25189

Figure Lengend Snippet: Sodium butyrate reduced exosomal CXCL16 secreted by LPS-induced RAW264.7 cells. (a) Schematic of the protocol for exosome collection. (b) EVs obtained from medium were observed by TEM. Scale bar 200 nm. (c) The distribution of EVs were obtained by NanoSight tracking analysis.(d–e) Westernblot analysis (d) of exosomal proteins (CD63, TSG101, HSP70 and CXCL16) collected from the same volume RAW264.7 cells culture supernatant and quantitative analysis (e). **and***denote P < 0.01 and P < 0.001 compared to the control group, & and && denote P < 0.05 and P < 0.01 compared to the NaB (0) group, respectively.

Article Snippet: Briefly, the neutrophils were stained with calcein-AM (2 μg·mL-1, C3099, Invitrogen) for 15 min at 37 °C and seeded on the upper chamber of the transwell inserts with 3.0 μm pore-size transparent PET membranes (FALCON-353096) and co-cultured with RAW264.7 cells or exosomes derived from treatment RAW264.7 cells or CXCL16 neutralizing antibody (0.25 μg; R&D Systems, USA)/recombinant CXCL16 (100 ng/mL; MedChemExpress, USA) in the lower chamber for 1 h at 37 °C according to the instructions.

Techniques:

Journal: Heliyon

Article Title: Sodium butyrate restricts neutrophils migration and NETs formation through reducing macrophage-derived CXCL16 in calculous cholecystitis

doi: 10.1016/j.heliyon.2024.e25189

Figure Lengend Snippet: Sodium butyrate reduced neutrophil migration and NETs formation by inhibiting exosomal CXCL16 secretion. (a) Representative fluorescence images of neutrophil migration assay. Neutrophils were stained with calcein-AM and co-cultured with RAW264.7 cells exosomes in the presence of LPS, Sodium butyrate or CXCL16 antagonist/recombinant CXCL16, quantitative assessment of neutrophil migration across a permeable transwell chamber. Data represent mean ± SD (n = 5), ***p < 0.001 compared to control group, && and &&& denote p < 0.01 and p < 0.001 compared to LPS group. (c) CXCR6 protein levels were detected by Western blot. (d–e) Neutrophilic extracellular traps (NETs) were identified by MPO staining observed by confocal microscopy (green). Quantification of dsDNA and circulating NET structures in the supernatant of cultured PMNs using PicoGreen fluorescent dye and MPO-DNA-ELISA, respectively.** P < 0.01 compared to control group, & and && denote P < 0.05 and P < 0.01 compared to LPS group. $ denote P < 0.05 compared to LPS + NaB(H) group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Briefly, the neutrophils were stained with calcein-AM (2 μg·mL-1, C3099, Invitrogen) for 15 min at 37 °C and seeded on the upper chamber of the transwell inserts with 3.0 μm pore-size transparent PET membranes (FALCON-353096) and co-cultured with RAW264.7 cells or exosomes derived from treatment RAW264.7 cells or CXCL16 neutralizing antibody (0.25 μg; R&D Systems, USA)/recombinant CXCL16 (100 ng/mL; MedChemExpress, USA) in the lower chamber for 1 h at 37 °C according to the instructions.

Techniques: Migration, Fluorescence, Staining, Cell Culture, Recombinant, Western Blot, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: a B220 + cells and Th17 cells in the BM niche in WT mice and BCR-ABL tTA mice ( n = 3 mice per group) were subjected to immunofluorescence staining. Representative images were shown. b The numbers of Th17 cells migrating to culture medium or to leukemia cells treated with or without 50 ng/ml rhIL-17A were determined by cell counting and FCS analysis. c Real-time PCR analysis of the relative mRNA levels of chemokine genes in SupB15 (left) and BV173 cells (right) treated with rhIL-17A (100 ng/ml) or PBS control. d The human IL-5, CXCL5, CCL5 and CXCL16 levels in the culture supernatant of SupB15 cells treated with or without rhIL-17A (100 ng/ml) were measured by ELISA. e The CXCL16 concentrations in the serum of BCR-ABL tTA mice and WT mice ( n = 5 mice per group) were quantified with a Mouse Chemokine Assay Q1 (Raybiotech). f The CXCL16 concentrations in the serum of HDs ( n = 7 samples) and primary Ph + B-ALL patients ( n = 14 samples) were measured by ELISA. g Schematic strategy for studying the differentiation of Th17 cells with or without rmCXCL16 stimulation in vitro. h Representative FACS plots and quantification of the percentage of Th17 cells in naïve CD4 + T cells with or without rmCXCL16 treatment. i Schematic strategy for studying the migration of Th17 cells cocultured with leukemia cells with or without anti-CXCL16 mAb treatment in vitro. j , k Flow cytometric analysis of the percentage of Th17 cells in the lower chambers of the inserts ( j ) and proliferation activity of leukemia cells ( k ) in a coculture system with or without anti-CXCL16 treatment. The gating strategy for isolating Th17 cells is shown in Supplementary Fig. . (b–d , h , j , k) n = 3 independent experiments. Statistical significance was calculated by ( c – f , j , k ) two-tailed Student’s t test; ( b , h ) one-way ANOVA with Tukey’s multiple comparison tests ; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: For the coculture assay, viable Th17 cells were cocultured with B-ALL cells at a ratio of 1:1, and recombinant human CXCL16 (PeproTech, 300-55), recombinant human IL-17A (PeproTech, 200-17), the anti-human IL-17A neutralizing antibody (Bio X Cell, SIM0013), the anti-human CXCL16 neutralizing antibody (R&D, AF976), anti-goat IgG (R&D, AB-108-C) or recombinant human IgG1 Fc (Bio X Cell, BE0096) was added to the indicated coculture systems.

Techniques: Immunofluorescence, Staining, Cell Counting, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, In Vitro, Migration, Activity Assay, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: Primary Ph + B-ALL cells were treated with or without rhIL-17A for 24 h, and relative CXCL16 mRNA level ( a ) and CXCL16 secretion ( b ) were evaluated by real-time PCR and ELISA, respectively. c Representative immunofluorescence images of B220 (red) and CXCL16 (green) staining in spleen tissue sections of WT mice and BCR-ABL tTA mice treated with or without 50 µg/kg rmIL-17A for a duration of 3 weeks (twice a week) ( n = 3 mice per group). d Flow cytometric analysis and quantification of CXCL16 expression in primary mouse B-ALL cells treated with or without rmIL-17A. e The protein levels of p65 and p-p65 in the cytoplasm and p-p65 in the nucleus of primary mouse leukemia cells treated with or without rmIL-17A were measured by Western blotting. f Immunofluorescence of p-P65 in primary B-ALL cells treated with or without rhIL-17A. g The effect of rhIL-17A treatment on NF-kB transcriptional activity. HEK 293T cells were transfected with a synthetic NF-kB luciferase reporter construct (pNF-kB-Luc) for 12 h and then treated with different concentrations of rhIL-17A for 24 h. NF-kB transcriptional activity was detected by a luciferase assay. h ChIP‒qPCR analyses of the binding of NF-kB to the CXCL16 promoter region in SupB15 cells treated with or without rhIL-17A. i – k The effect of BAY11-7082, rIL-17A or BAY11-7082 in combination with rIL-17A on the cytoplasmic and nuclear protein levels of p65, p-p65, and CXCL16 in primary Ph + B-ALL cells. i The indicated protein band intensities were quantified using ImageJ software. The relative CXCL16 mRNA level ( j ) in primary Ph + B-ALL cells and CXCL16 protein level ( k ) in the supernatant of primary Ph + B-ALL cells were measured by RT‒PCR and ELISA, respectively. (a, b , d – k ) n = 3 independent experiments. Statistical significance was calculated by ( a , b , d ) two-tailed Student’s t-test; ( e , g – k ) one-way ANOVA with Tukey’s multiple comparison tests ; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: For the coculture assay, viable Th17 cells were cocultured with B-ALL cells at a ratio of 1:1, and recombinant human CXCL16 (PeproTech, 300-55), recombinant human IL-17A (PeproTech, 200-17), the anti-human IL-17A neutralizing antibody (Bio X Cell, SIM0013), the anti-human CXCL16 neutralizing antibody (R&D, AF976), anti-goat IgG (R&D, AB-108-C) or recombinant human IgG1 Fc (Bio X Cell, BE0096) was added to the indicated coculture systems.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing, Western Blot, Activity Assay, Transfection, Luciferase, Construct, Binding Assay, Software, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: a Flow cytometric analysis of the percentages of B220 dim CD19 + cells in BM, spleens, LNs and PB of mice with secondary transplantation treated with or without rmCXCL16 ( n = 6 mice per group). b – d Representative images of Wright-Giemsa-stained PB smears ( b , top), H&E staining of spleens ( b , bottom), spleens ( c ) and spleen weights ( d ) from the indicated mice ( n = 6 mice per group). e Representative images of Ki67 staining in the spleen tissues from the indicated mice. n = 4 fields, two different mice per group. f Flow cytometric analysis of the percentages of Th17 cells in the PB, LNs, spleens and BM from the indicated mice ( n = 6 mice per group). g Spleen tissues from the indicated mice were subjected to immunofluorescence staining for IL-17A (red), CD4 (green), and B220 (rose red). Representative images of Th17 cells were shown. n = 4 fields, two different mice per group. h Kaplan–Meier survival curves for the indicated mice ( n = 8 mice per group). i Schematic strategy for investigating the effects of anti-CXCL16 mAb alone or combined with imatinib on Ph + B-ALL progression. j – l Representative images of spleens ( j ), spleen weights ( k ), Wright-Giemsa-stained PB smears ( l, top ), and H&E staining of the spleen ( l, bottom ) from leukemia mice treated with the indicated agents ( n = 3 mice per group). m Flow cytometric analysis of the percentages of B220 dim CD19 + cells in the PB, BM, LNs and spleens from leukemia mice treated with the indicated agents ( n = 3 mice per group). n The percentage of Ki-67 + cells in the spleen was detected by immunofluorescence staining in the indicated mice. Data are presented as means ± S.E.M of eight random fields of view from three different mice per group. o Flow cytometric analysis of the percentages of Th17 cells in the PBMCs, LNs, spleens and BM of leukemia mice treated with the indicated agents ( n = 3 mice per group). (a , m ) The gating strategy for B220 dim CD19 + cells was shown in Supplementary Fig. . ( f , o ) The gating strategy for Th17 cells in the CD4 + T cells was shown in Supplementary Fig. . Statistical significance was calculated by ( a, d – g ) two-tailed Student’s t-test; ( k , m – o) one-way ANOVA with Tukey’s multiple comparison tests ; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: For the coculture assay, viable Th17 cells were cocultured with B-ALL cells at a ratio of 1:1, and recombinant human CXCL16 (PeproTech, 300-55), recombinant human IL-17A (PeproTech, 200-17), the anti-human IL-17A neutralizing antibody (Bio X Cell, SIM0013), the anti-human CXCL16 neutralizing antibody (R&D, AF976), anti-goat IgG (R&D, AB-108-C) or recombinant human IgG1 Fc (Bio X Cell, BE0096) was added to the indicated coculture systems.

Techniques: Transplantation Assay, Staining, Immunofluorescence, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: The Th17 cell population and IL-17A expression are distinctively increased in Ph + B-ALL patients, and high expression of IL-17A promotes the progression of Ph + B-ALL. IL-17A promotes the proliferation and survival of Ph + B-ALL cells by activating the BCR-ABL and IL6/JAK/STAT3 signaling pathways. Moreover, IL-17A can increase the secretion of the chemokine CXCL16 from leukemia cells by activating NF-kB, which in turn mediates the differentiation and recruitment of Th17 cells to the leukemia niche microenvironment. Targeting IL-17A or CXCL16 in the leukemia niche microenvironment attenuates the progression of Ph + B-ALL.

Article Snippet: For the coculture assay, viable Th17 cells were cocultured with B-ALL cells at a ratio of 1:1, and recombinant human CXCL16 (PeproTech, 300-55), recombinant human IL-17A (PeproTech, 200-17), the anti-human IL-17A neutralizing antibody (Bio X Cell, SIM0013), the anti-human CXCL16 neutralizing antibody (R&D, AF976), anti-goat IgG (R&D, AB-108-C) or recombinant human IgG1 Fc (Bio X Cell, BE0096) was added to the indicated coculture systems.

Techniques: Expressing