Journal: Cells

Article Title: CCL4 Affects Eosinophil Survival via the Shedding of the MUC1 N-Terminal Domain in Airway Inflammation.

doi: 10.3390/cells14010033

Figure Lengend Snippet: Figure 1. Full-length MUC1 (MUC1-FL) expression in the epithelial cells of nasal polyps. (A) Im- munofluorescence analysis of nasal polyps obtained from patients with chronic rhinosinusitis with nasal polyps (CRSwNP) with high eosinophil count (left panels; i–iii) or those with low eosinophil count (right panels; iv–vi). MUC1-FL (green), C-terminal domain (MUC1-C, red), and the nucleus (blue) are stained. Images were captured by an FV3000 confocal microscope (400× objectives). The scale bars in the bottom-right corner indicate 10 µm. (B) Correlation of MUC1-FL expression with eosinophil count in nasal polyps. MUC1-FL intensity is indicated as a ratio to epithelial cell adhesion molecule (EpCAM) (n = 39).

Article Snippet: Human eosinophils were separated with high purity (>98%) from peripheral blood obtained from healthy individuals with slightly increased eosinophils (approximately 300–500/μL) using an eosinophil isolation kit with a MACS system (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Expressing, Staining, Microscopy

Journal: Cells

Article Title: CCL4 Affects Eosinophil Survival via the Shedding of the MUC1 N-Terminal Domain in Airway Inflammation.

doi: 10.3390/cells14010033

Figure Lengend Snippet: Figure 2. Effect of eosinophils on full-length MUC1 (MUC1-FL) expression in airway epithelial cells. (A–D) BEAS-2B cells were coincubated overnight with purified peripheral blood eosinophils. MUC1- FL mRNA levels (A), MUC1-FL protein levels (B), and MUC1 C-terminal domain (MUC1-C) protein levels (C) were evaluated. (D) Immunofluorescence analysis of MUC1-FL (green), MUC1-C (red), and the nucleus (blue) are shown in the upper (without eosinophils) and lower (with eosinophils) panels. Images were captured by an FV3000 confocal microscope (400× objectives). Scale bars in the bottom-right corner indicate 10 µm. Results were representative of at least three experiments. (E) MUC1-FL protein expression in BEAS-2B coincubated with the supernatants of eosinophilic mucin overnight. Patients underwent endoscopic sinus surgery under general anesthesia. Mucin samples were collected from the sinuses of refractory ECRS subjects. Values in (A–C,E) represent the mean ± SEM of four experiments; # p < 0.05, ## p < 0.01 (vs. vehicle).

Article Snippet: Human eosinophils were separated with high purity (>98%) from peripheral blood obtained from healthy individuals with slightly increased eosinophils (approximately 300–500/μL) using an eosinophil isolation kit with a MACS system (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Expressing, Purification, Immunofluorescence, Microscopy

Journal: Cells

Article Title: CCL4 Affects Eosinophil Survival via the Shedding of the MUC1 N-Terminal Domain in Airway Inflammation.

doi: 10.3390/cells14010033

Figure Lengend Snippet: Figure 3. Relation between full-length MUC1 (MUC1-FL) and CCL4 expression in the epithelial cells of nasal polyps. (A) Immunofluorescence staining of nasal polyps obtained from patients with CRSwNP with high or low eosinophil count. MUC1-FL, CCL4, and epithelial cell adhesion molecule (EpCAM) expression levels were evaluated. MUC1-FL (pink), CCL4 (green), EpCAM (orange), MBP (red), and the nucleus (blue) are stained with hematoxylin and eosin (H&E). Images were captured by an FV3000 confocal microscope (100× objectives). The scale bars in the bottom-right corner indicate 100 µm. (B,C) Correlation of CCL4 expression with the eosinophil count in nasal polyps (B) and MUC1-FL expression (C). MUC1-FL and CCL4 intensities are indicated as a ratio to EpCAM (n = 39).

Article Snippet: Human eosinophils were separated with high purity (>98%) from peripheral blood obtained from healthy individuals with slightly increased eosinophils (approximately 300–500/μL) using an eosinophil isolation kit with a MACS system (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Expressing, Immunofluorescence, Staining, Microscopy

Journal: Cells

Article Title: CCL4 Affects Eosinophil Survival via the Shedding of the MUC1 N-Terminal Domain in Airway Inflammation.

doi: 10.3390/cells14010033

Figure Lengend Snippet: Figure 4. Effect of CCL4-mediated reduction of full-length MUC1 (MUC1-FL) on eosinophil survival. (A,B) BEAS-2B was stimulated overnight with recombinant human CCL4 (10 µg/mL). MUC1-FL expression (A) and matrix metalloproteases (ADAM17 and MMP14) mRNA levels (B) in BEAS-2B. (C–E) BEAS-2B and purified eosinophils were coincubated with or without anti-CCL4 neutralizing antibody (10 µg/mL). MUC1-FL protein levels in BEAS-2B (C), CCL4 concentration in supernatants of cell culture (D), and eosinophil survival (E) were evaluated. (F) Purified eosinophils were incubated overnight on a recombinant human MUC1-coated plate, followed by the evaluation of their survival. Images (MUC1-FL, green; nucleus, blue) in A were captured by an FV3000 confocal microscope (400× objectives) with scale bars (20 µm) in the bottom-right corner, which were representative of at least three experiments. The values in (B–F) represent the mean ± SEM of four experiments. # p < 0.05, ## p < 0.01 (vs. without rhCCL4 in (B), without eosinophils in (C,D), without BEAS-2B in (E), and without rhMUC1 in (F)). ** p < 0.01 (between the two groups).

Article Snippet: Human eosinophils were separated with high purity (>98%) from peripheral blood obtained from healthy individuals with slightly increased eosinophils (approximately 300–500/μL) using an eosinophil isolation kit with a MACS system (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Recombinant, Expressing, Purification, Concentration Assay, Cell Culture, Incubation, Microscopy

Journal: Molecular medicine reports

Article Title: Analysis of murine and human Treg subsets in inflammatory bowel disease.

doi: 10.3892/mmr.2017.6912

Figure Lengend Snippet: Figure 5. Serum inflammatory cytokines in patients with ulcerative colitis. Serum from ulcerative colitis patients and healthy donors were collected and inflammatory cytokines were determined by ELISA. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 as indicated. IL, interleukin; TSLP, thymic stromal lymphopoietin; MMP, matrix metalloproteinase.

Article Snippet: Serum cytokine levels were analyzed using the following commercially available ELISA kits according to the manufacturer's protocol: MMP-2, Total MMP-2 Quantikine ELISA kit (cat. no. MMP200; Quantikine, R&D Systems, Inc., Minneapolis, MD, USA); thymic stromal lymphopoietin, Legend MaxTM Human TSLP ELISA kit with Pre-coated Plates (cat. no. 434208; BioLegend, Inc., San Diego, CA, USA); MMP-9, Legend MaxTM Human MMP-9 ELISA kit with Pre-coated Plates (cat. no. 440707; BioLegend, Inc.); IL-17A, Legend MaxTM Human IL-17A ELISA kit with Pre-coated Plates (cat. no. 433918; BioLegend, Inc.); IL-25, Human IL-17E/IL-25 AccuSignal ELISA kit (cat. no. KOA0468; Rockland Immunochemicals, Inc., Limerick, PA, USA); and eotaxin, Human Eotaxin ELISA kit (cat. no. KOA0159; Rockland Immunochemicals, Inc.).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: Single-cell profiling of PBMCs from RA patients and HC revealed distinct clustering patterns of CD20 + T cell populations. a t-SNE plot of CD20 + T cells from PBMCs of RA patients ( n = 9) and HC ( n = 3), colored by group. b tSNE plot (left panel) and subgroup proportion pie chart (right panel) of CD20 + T cells from all donors. Subgroups were divided based on the expression levels of marker genes CD4 or CD8A . c tSNE plot of CD20 + T cells from PBMCs of all donors, showing 3 clusters based on the unsupervised graph-based clustering. d Dot plot for expression of marker genes for three CD20 + T cell subtypes. The color of the dots shows the relative expression levels of genes, and the size of the dots shows the percentage of expressed cells. e Heatmap showing expression levels of discriminative genes for each cluster. Cluster 0 prominently features high expression of CCL5 , NKG7 and GNLY ; Cluster 1 is defined by combined enrichment of CCR7 , IL6ST and TRABD2A ; Cluster 2 shows distinct expression of GATA3 , ITGB1 and S100A11 . f Feature plots of single-cell expression for identified genes across three CD20 + T cell clusters

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Single Cell, Expressing, Marker

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: The proportion of CCL5 + T cells in CD20 + CD8 + T cells was significantly elevated in naïve RA patients. a Schematic representation of gating strategy for flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) derived from HC and RA patients. b The frequencies of CD20 + CD8 + T cells were analyzed in PBMCs samples collected from HC ( n = 63) and naïve RA patients ( n = 69). Data shown as median with interquartile range (IQR), Mann-Whitney U test, **** P < 0.0001. c The frequencies of CCL5 + cells within CD20 + CD8 + T cells were shown for HC ( n = 32) and naïve RA patients ( n = 35). Data shown as median with interquartile range (IQR), Mann-Whitney U test, **** P < 0.0001. d The proportion of CCL5 + cells was significantly higher in CD20 + CD8 + T cells than in CD20 – CD8 + T cells in both HC and RA patients. Data shown as median with interquartile range (IQR), Wilcoxon matched-pairs signed-rank test, **** P < 0.0001. e The proportion of CCL5 + cells within CD20 + CD8 + T cells positively correlated with inflammation and disease activity of RA. Data shown as individual scatter points, Spearman’s rank correlation, P < 0.05. f KEGG pathway analysis revealed that the top five most significantly enriched pathways for DEGs between CD20 + CD8 + T and CD20 – CD8 + T cells included the chemokine signaling pathway, and CXCR3 and CCR5 were identified as genes significantly enriched in this pathway. g Violin plots (upper panel) demonstrated that the expression levels of CXCR3 and CCR5 genes were significantly upregulated in CD20 + CD8 + T cells compared to CD20 – CD8 + T cells. **** P < 0.0001; * P < 0.05. UMAP plot (lower panel) visualized the distribution of CXCR3 + or CCR5 + cells among CD8 + T cells. ( h Flow cytometric analysis demonstrated significantly higher proportions of CXCR3 + or CCR5 + cells among CD20 + CD8 + T cells compared to CD20 – CD8 + T cells in PBMCs from RA patients ( n = 27 in each group). Wilcoxon matched-pairs signed-rank test, **** P < 0.0001

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Derivative Assay, MANN-WHITNEY, Activity Assay, Expressing

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: CD20 + CD8 + T cells may promote the expansion of invasive FAPα + FLS through CCL5-mediated activation of the JAK-STAT signaling pathway. a Gating strategy for identifying FLS involved sequential selection of CD45-negative (CD45 – ) cells followed by positive selection for PDPN (podoplanin) expression. The frequencies of FAPα + cells in the PDPN + CD45 – CD31 – population were quantified and compared between normal human controls (NH, n = 9) and RA patients ( n = 9). (mean ± SEM, unpaired two-tailed t -test, *** P < 0.001). b Representative flow cytometry plots depict FAPα + cells (gated on PDPN + CD45 – CD31 – populations) with corresponding quantitative analysis following 48-hour treatment under various experimental conditions. (mean ± SEM, n = 7 in each group, one-way ANOVA with Tukey’s post hoc test, * P < 0.05, **** P < 0.0001). c Representative images from the Transwell assay demonstrate the migratory capacity of FLS under the experimental conditions corresponding to panel (b). Scale bar: 50 μm. (mean ± SEM, n = 3 in each group, one-way ANOVA with Tukey’s post hoc test, * P < 0.05; *** P < 0.001). d The frequency of FAPα-expressing FLS was assessed by flow cytometry following 48-hour incubation with CCR1 antagonist BX471 (5 µM) or CCR5 antagonist maraviroc (5 µM) in IL-1β (5 ng/mL) + CD20 + CD8 + T-CM cultures. (mean ± SEM, n = 6 in each group, one-way ANOVA with Tukey’s post hoc test, * P < 0.05; ** P < 0.01). e Representative histogram plots demonstrate FAPα expression in FLS following stimulation with IL-1β (5 ng/mL) in combination with either CD20 + CD8 + T-CM alone, or T-CM + tofacitinib (JAK inhibitor). (mean ± SEM, n = 6 in each group, paired t- test, *** P < 0.001). ( f ) ELISA was performed to quantify CCL5 concentrations in culture supernatants under experimental conditions corresponding to panels (B) and (C). (mean ± SEM, n = 6 in each group, one-way ANOVA with Welch’s correction, followed by Dunnett’s T3 post hoc test, ** P < 0.01). g Heatmap depicting transcriptomic profiling results, showing expression levels of differentially expressed genes across experimental groups. h GO (upper panel) and KEGG pathway (lower panel) analyses of upregulated genes in FLS treated with CD20 – CD8 + T-CM + IL-1β (left), and FLS treated with CD20 + CD8 + T-CM + IL-1β (right), both compared to IL-1β alone control (CTL). q -value < 0.05

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Activation Assay, Selection, Expressing, Two Tailed Test, Flow Cytometry, Transwell Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: Schematic of CCL5 + CD20 + CD8 + T cells driving synoviocyte transformation. In RA, circulating CD20 + CD8 + T cells with high co-expression of chemokine receptors CXCR3 and CCR5 are chemoattracted from peripheral blood into synovial tissues by cognate chemokines (such as CXCL9 and CCL5). CD20 + CD8 + T cells further engage in crosstalk with fibroblast-like synoviocytes (FLS) through CCL5 secretion. This chemokine-dependent signaling engages cognate receptors (i.e., CCR1, CCR5) on FLS, subsequently activating the JAK/STAT signaling cascade, inducing phenotypic transformation of FLS (PDPN + CD45 – CD31 – ) into pathogenic FAPα + subpopulation. The FAPα + subpopulation demonstrates enhanced proliferative capacity, increased invasive migration, and robust secretion of synovial inflammatory cytokines, chemokines, and pro-angiogenic factors, collectively driving pannus formation and progressive joint destruction. ECM: extracellular matrix; MMPs: matrix metalloproteinases; TNFα: tumor necrosis factor α.

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Transformation Assay, Expressing, Migration

Journal: Digestive Diseases and Sciences

Article Title: Acetylcholinesterase Inhibitor Pyridostigmine Bromide Attenuates Gut Pathology and Bacterial Dysbiosis in a Murine Model of Ulcerative Colitis

doi: 10.1007/s10620-019-05838-6

Figure Lengend Snippet: PB ameliorates the DSS-induced eosinophilic infiltration and increased expression of IL-5 and eotaxin in the colon, and the reduction in body weight. a Colonic tissues from mice treated with DSS alone or PB+DSS were analyzed for eosinophils on H&E-stained sections and were compared with controls (magnification 400×). b Western blot analysis of IL-5 in colonic homogenates (70 µg). Blots were probed for IL-5 and β-actin levels and quantitated as the ratio of IL-5/β-actin following densitometric analysis. c Eotaxin expression by Western blots of colonic homogenates (70 µg) (left panel), densitometric quantitation of eotaxin (right panel). d RT-qPCR analysis of eotaxin mRNA expression in total colonic RNA. e PB effects on DSS-induced body weight reduction on day 8 after DSS treatment. Groups are as follows: Control (Cont), DSS: dextran sodium sulfate-treated, and DSS+PB: DSS and pyridostigmine bromide-treated. Figures are representative of two separate experiments ( n = 4–5/group) for A, B, C, and D and one experiment for E ( n = 6–8 animals). *≤ 0.01; **≤ 0.001; ***≤ 0.0001

Article Snippet: The sections were stained with antibodies to ZO-1 (Invitrogen Inc., Carlsbad, CA, USA) or anti-eosinophil antibody (Novus Biologicals, Littleton, CO, USA), or isotype control antibodies.

Techniques: Expressing, Staining, Western Blot, Quantitation Assay, Quantitative RT-PCR, Control