Journal: Scientific Reports

Article Title: Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer

doi: 10.1038/srep35855

Figure Lengend Snippet: ( a ) Fucoidan modulates CC chemokines transcription of THP-1-derived macrophages during the polarization process. The expression of selected CC chemokines in non-treated and fucoidan-treated M0/M1/M2-like macrophages were assessed by quantitative real-time PCR and expressed as a fold change compared with M0 macrophages. ( b ) Fucoidan modulates CCL22 secretion level of THP-1-derived macrophages during the polarization process. After 48 h polarization, the conditioned media created by non-treated and fucoidan-treated M0/M1/M2-like macrophages were collected. ELISA was then used on the various conditioned media to measure the levels of secreted CCL22. ( c , d ) CCL22 transcription ( c ) and concentration in culture media ( d ) of peripheral blood monocyte-derived macrophages. ( e , f ) CCL22 transcription ( e ) and concentration ( f ) in non-treated and fucoidan-treated polarized M2 macrophages exposure to IL-4 and/or IL-13. The quantitative real-time PCR data expressed as relative fold change compared with control (polarized M2 macrophages). The value represent means ± SD, n = 3: *P < 0.05, **P < 0.01.

Article Snippet: Recombinant human CCL22, M-CSF, IFN-γ, IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer

doi: 10.1038/srep35855

Figure Lengend Snippet: ( a ) Diagram showing the protocol for MHCC-97 migration assay and the PBMC/CD4 + lymphocytes recruitment assay. Upper: Supernatants of THP-1-derived M2 macrophages under non- or fucoidan pretreatment were collected and add into lower chamber of transwell with or without neutralizing anti-CCL22 antibody or recombine human CCL22, respectively. Down left: MHCC-97H were seeded in upper chamber. Transwelled cells were stained after 24 h and counted. Down right: PBMC (I) from healthy human peripheral blood separated by lymphocyte separation liquid or microbeads selected CD4 + lymphocytes (II) were used as upper chamber components of transwell. Transwelled cells were collected after 6h, and flow cytometry was used to detect the cellular components. FCM: flow cytometry. ( b ) Migration assays were carried out in MHCC-97H cells by using transwell chamber assay. Migrating cells located on the lower surface were fixed in methanol and stained with eosin. The scale bar represents 50 μm. The graph represents fold change. ( c ) Flow cytometry settings used to detect human CD3 + CD4 −/+ cells among PBMC recruited to lower chamber contained non-treated and fucoidan-treated THP-1-derived M2 macrophages supernatants from donor human PBMC seeded in the upper chambers (left). %CD3 + CD4 + and %CD3 + CD4 − cells are the percentage of CD3 + CD4 + or CD3 + CD4 − cells among recruited PBMC (right). ( d , e ) Flow cytometry settings used to detect human CD25 + FoxP3 + (Treg) cells among CD4 + lymphocytes recruited to the bottom chamber filled with conditioned M2 supernatants as tumor cell migration assay. Graphs showed the total numbers of CD4 + lymphocytes ( d ) and the flow cytometry (left) and percentage (right) of CD25 + FoxP3 + cells among recruited CD4 + lymphocytes ( e ). Control group: the bottom chamber filled with M2 supernatants. The value represent means ± SD, n = 3: *P < 0.05, **P < 0.01 versus the control.

Article Snippet: Recombinant human CCL22, M-CSF, IFN-γ, IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Derivative Assay, Staining, Flow Cytometry, Transwell Chamber Assay, Cell Migration Assay, Control

Journal: Scientific Reports

Article Title: Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer

doi: 10.1038/srep35855

Figure Lengend Snippet: ( a , b ) THP-1-derived M2 macrophages were treated with the NF-κB inhibitor BAY11-7082, the PI3K inhibitor Wortmannin or the p38-MAPK inhibitor SB203580 for 24 h. The cells and culture supernatants were collected. CCL22 transcription ( a ) and concentration ( b ) were measured by quantitative real-time PCR and ELISA, respectively. ( c ) M2 macrophages derived from THP-1 cells were pretreated with the Wortmannin or the SB203580 for 30 min, followed by incubation with or without Fucoidan for 1 h. Whole-cell lysates were analyzed by western blot using the appropriate antibodies. The original blots are presented in . Densitometry ratios of phospho-p65-NF-κB, AKT and p38-MAPK were normalized to β-actin levels. The cells treated with DMSO were used as control. The value represent means ± SD, n = 3. *P < 0.05, **P < 0.01 versus the control. ( d ) p65-NF-κB immunofluorescence of THP-1-derived M0 and M2 polarized macrophages, which were treated as described in ( c ). Green (anti-p65-NF-κB) indicates p65-NF-κB distribution, and blue indicates the location of nucleus. The scale bar represents 10 μm.

Article Snippet: Recombinant human CCL22, M-CSF, IFN-γ, IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Control, Immunofluorescence

Journal: Scientific Reports

Article Title: Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer

doi: 10.1038/srep35855

Figure Lengend Snippet: Green arrow represents activation; truncated red line, inhibition. The dotted lines represent the non-critical effect of AKT and p38-MAPK on fucoidan-mediated downregulation of CCL22.

Article Snippet: Recombinant human CCL22, M-CSF, IFN-γ, IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Inhibition

Journal: Clinical Cancer Research

Article Title: CCL22 Recruits CD4-positive CD25-positive Regulatory T Cells into Malignant Pleural Effusion

doi: 10.1158/1078-0432.ccr-08-2641

Figure Lengend Snippet: Fig. 3. CCL22 (A) and CCL17 (B) are present in malignant pleural effusions and sera from patient with lung cancer (n = 33). CCL22 and CCL17 concentrations were measured with ELISA. Horizontal bars, medians. Comparisons of CCL22 and CCL17 levels between malignant pleural effusion and sera were made using a Wilcoxon signed-rank test.

Article Snippet: Right after collection of malignant pleural effusion samples, 10 Ag of recombinant human CCL22 (R&D Systems Inc.) in vehicle (0.1% human serum albumin in 0.9% saline) were injected into the pleural space of five www.aacrjournals.org Clin Cancer Res 2009;15(7) April 1, 20092233 Research. on March 24, 2015.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Clinical Cancer Research

Article Title: CCL22 Recruits CD4-positive CD25-positive Regulatory T Cells into Malignant Pleural Effusion

doi: 10.1158/1078-0432.ccr-08-2641

Figure Lengend Snippet: Fig. 4. Expression of CCL22 in malignant cells (top), macrophages (middle), andT lymphocytes (bottom). Malignant cells (A), macrophages (D), andTcells (G) were incubated with mouse antiepithelial membrane antigen, anti-CD163, and anti-CD3 mAbs, respectively, and then were stained with fluorescein-labeled goat anti-mouse IgG (green). CCL22 expression was detected by rabbit polyclonal Ab targeted against CCL22 and then rhodamine-labeled goat antirabbit IgG (red; B, E, H). Immunoflorescence double-staining indicate that all malignant cells (C), most of macrophages (F), and most of Tcells (I) express CCL22. Closed arrows, CD163-positive macrophages that do not express CCL22; open arrows, CD3-positiveTcells that do not express CCL22. Original magnification: 1,000.

Article Snippet: Right after collection of malignant pleural effusion samples, 10 Ag of recombinant human CCL22 (R&D Systems Inc.) in vehicle (0.1% human serum albumin in 0.9% saline) were injected into the pleural space of five www.aacrjournals.org Clin Cancer Res 2009;15(7) April 1, 20092233 Research. on March 24, 2015.

Techniques: Expressing, Incubation, Membrane, Staining, Labeling, Double Staining

Journal: Clinical Cancer Research

Article Title: CCL22 Recruits CD4-positive CD25-positive Regulatory T Cells into Malignant Pleural Effusion

doi: 10.1158/1078-0432.ccr-08-2641

Figure Lengend Snippet: Fig. 5. Malignant pleural effusion is chemotactic for CD4-positive CD25-positive Tcells. Pleural effusions from patients with lung cancer (n = 5) were used to stimulate chemotaxis of peripheral blood CD4-positive CD25-positiveTcells isolated from healthy adults. Data are expressed as percent of control. Open bars, chemotaxis in the absence of anti-CCL22 or anti-CCL17 mAb; hatched bars, irrelevant isotype controls; closed bars, chemotaxis in the presence of anti-CCL22 or anti-CCL17 mAb. * P < 0.05, compared with the corresponding group without anti-CCL22 mAb were determined by Kruskal-Wallis one-wayANOVA on ranks.

Article Snippet: Right after collection of malignant pleural effusion samples, 10 Ag of recombinant human CCL22 (R&D Systems Inc.) in vehicle (0.1% human serum albumin in 0.9% saline) were injected into the pleural space of five www.aacrjournals.org Clin Cancer Res 2009;15(7) April 1, 20092233 Research. on March 24, 2015.

Techniques: Chemotaxis Assay, Isolation, Control

Journal: Clinical Cancer Research

Article Title: CCL22 Recruits CD4-positive CD25-positive Regulatory T Cells into Malignant Pleural Effusion

doi: 10.1158/1078-0432.ccr-08-2641

Figure Lengend Snippet: Fig. 6. Changes of CD4-positive CD25-positiveTcell numbers in malignant pleural effusion from patients with lung cancer, who were intrapleurally injected with vehicle and recombinant human CCL22. n = 5 for each group. Points, mean values; error bars, SE at each time point. *P < 0.05 compared within-group change from baseline measurements determined by one-way repeated-measures ANOVA.

Article Snippet: Right after collection of malignant pleural effusion samples, 10 Ag of recombinant human CCL22 (R&D Systems Inc.) in vehicle (0.1% human serum albumin in 0.9% saline) were injected into the pleural space of five www.aacrjournals.org Clin Cancer Res 2009;15(7) April 1, 20092233 Research. on March 24, 2015.

Techniques: Injection, Recombinant

Journal: bioRxiv

Article Title: Macrophages foster adaptive anti-tumor immunity by ZEB1-dependent cytotoxic T cell chemoattraction

doi: 10.1101/2024.02.26.582102

Figure Lengend Snippet: A . Differentially expressed genes in unstimulated or LPS or IL-4 stimulated LysM ΔZeb1 compared to LysM Ctrl BMDMs as measured by a customized RT2 array and depicted in log 2 fold change of expression. nd marks non-detectable mRNA levels. All transcripts were normalized to Gapdh (n=3). B . Relative mRNA expression of Ccl2 and Ccl22 in LysM Ctrl and LysM ΔZeb1 BMDMs (n=3; means ±SD; 2-way ANOVA). C . Comparison of transcript and secretome alterations of Ccl2 and Ccl22 in LysM ΔZeb1 compared to LysM Ctrl BMDMs (n≥5; means ±SD).

Article Snippet: Final concentrations of cytokines were 1.93 ng/ ml CCL2 (R&D Systems, 479-JE-050) and 0.18 ng/ ml CCL22 (R&D Systems, 439-MD-025) and of antibodies 5 μg/ml IgG (Diagenode, C15410206), anti-CCL2 (Novus Biologicals, NBP1-07035SS) or anti-CCL22 (abcam, ab124768), respectively.

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: Macrophages foster adaptive anti-tumor immunity by ZEB1-dependent cytotoxic T cell chemoattraction

doi: 10.1101/2024.02.26.582102

Figure Lengend Snippet: A-B . Venn diagrams of differentially expressed genes (DEGs) of LysM Ctrl (blue) and LysM ΔZeb1 BMDMs (red) after stimulation with LPS (A) or IL-4 (B) compared to unstimulated in total (left) and divided in up-/downregulated DEGs (right). C . GO term enrichment analysis for DEGs (FDR<0.05) uniquely up- or downregulated by LysM ΔZeb1 BMDMs after LPS stimulation. D . Log 2 fold change of expression of selected trafficking genes after LPS stimulation. X marks no significant deregulation. E . Representative images and quantification of OPP incorporation of LysM Ctrl and LysM ΔZeb1 BMDMs with 0h, 4h and 16h LPS pre-stimulation (n=3; means ±SD; 2-way ANOVA). F-G . Representative arrays of intracellular cytokines of LysM Ctrl and LysM ΔZeb1 BMDMs with LPS stimulation or additional Brefeldin A and Monensin treatment (F) and quantification of intracellular CCL2 and CCL22 after LPS, Brefeldin A and Monensin treatment (G; n≥2).

Article Snippet: Final concentrations of cytokines were 1.93 ng/ ml CCL2 (R&D Systems, 479-JE-050) and 0.18 ng/ ml CCL22 (R&D Systems, 439-MD-025) and of antibodies 5 μg/ml IgG (Diagenode, C15410206), anti-CCL2 (Novus Biologicals, NBP1-07035SS) or anti-CCL22 (abcam, ab124768), respectively.

Techniques: Expressing

Journal: bioRxiv

Article Title: Macrophages foster adaptive anti-tumor immunity by ZEB1-dependent cytotoxic T cell chemoattraction

doi: 10.1101/2024.02.26.582102

Figure Lengend Snippet: A . Confluence of KPC cells alone or co-cultured with LysM Ctrl or LysM ΔZeb1 BMDMs (n=3). B . Representative images at t=28h and quantification over time of KPC cell invasion into a scratch wound without or with co-culture of LysM Ctrl or LysM ΔZeb1 BMDMs (n=3). C . Confluence of KPC cells alone or with LysM Ctrl or LysM ΔZeb1 BMDM conditioned medium (CM) (n=2 KPC CM, n=3 LysM Ctrl and LysM ΔZeb1 CM). D . Transwell migration assay of CD8+ T cells alone or towards LysM Ctrl or LysM ΔZeb1 BMDMs in absence or presence of recombinant CCL2 and CCL22 (left panel, n>3) or absence or presence of anti-CCL2 and anti-CCL22 antibodies (right panel, n=3). Means ±SD; *:p<0.05; **:p<0.01; ns: not significant; 2-way ANOVA.

Article Snippet: Final concentrations of cytokines were 1.93 ng/ ml CCL2 (R&D Systems, 479-JE-050) and 0.18 ng/ ml CCL22 (R&D Systems, 439-MD-025) and of antibodies 5 μg/ml IgG (Diagenode, C15410206), anti-CCL2 (Novus Biologicals, NBP1-07035SS) or anti-CCL22 (abcam, ab124768), respectively.

Techniques: Cell Culture, Co-Culture Assay, Transwell Migration Assay, Recombinant

Journal:

Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity

doi:

Figure Lengend Snippet: Chemotaxis of human and murine T lymphocytes to conditioned media of genetically modified DCs. (a) CEM cells. Human T lymphocyte CEM cells placed in the upper chamber of a transwell chamber were assayed for chemotaxis in response to serial dilution of supernatant from DCs transduced with AdMDC, AdNull, or PBS alone (naive control) in the lower chamber. (b) EL4 cells. The study was similar to that in a, but the murine T lymphocyte EL4 cell line was used. (c) Suppression of AdMDC-mediated chemotaxis. CEM and EL4 cells were assayed for chemotaxis in response to 1:2 dilution of supernatant from AdMDC-modified DCs. Where indicated, anti–MDC neutralizing Ab or mouse IgG control Ab was added to the supernatant in the lower chambers at 10 μg/ml at the initiation of assay or cells were placed to the upper chamber in the presence of recombinant (r) MDC or TARC at 100 ng/ml. For all parts, the number of cells migrating to the lower chamber at 37°C for 4 hours was counted by FACS analysis. Migration index was calculated as the number of cells migrating to the conditioned media over the number of cells migrating to control medium. Results represent the mean ± SE (n = 3 per data point).

Article Snippet: Where indicated, neutralizing anti-human MDC mAb or isotype-matched mouse IgG control Ab (R&D Systems Inc.) was added to the condition medium in the lower chamber at 10 μg/ml, or recombinant human MDC or recombinant human TARC (R&D Systems Inc.) was added to the cell suspension in the upper chamber at 100 ng/ml.

Techniques: Chemotaxis Assay, Genetically Modified, Serial Dilution, Transduction, Modification, Recombinant, Migration