Journal: Signal Transduction and Targeted Therapy

Article Title: Employing epigenetic protein degradation techniques to block CCL5-mediated photodynamic therapy via a programmed delivery platform

doi: 10.1038/s41392-025-02542-y

Figure Lengend Snippet: Mechanistic study of the antitumor effects of ARV/Ce6@RDP micelles. a Venn diagrams of the intersection between upregulated genes in Ce6@RDP (+) vs RDP and downregulated genes in ARV/Ce6@RDP (+) vs Ce6@RDP (+) for 4T1 and B16F10 cells based on RNA-seq analysis. b Venn diagrams of the intersection between upregulated genes in Ce6@RDP (+) vs RDP and upregulated genes in ARV/Ce6@RDP (+) vs Ce6@RDP (+) for 4T1 and B16F10 cells. c Heatmap of the intersection genes from ( a ) and ( b ) expressed in 4T1 and B16F10 cells receiving different treatments. d Quantification of the CCL5 level in the supernatants of treated 4T1 and B16F10 cells ( n = 3 per group, two-tailed unpaired Student’s t -test). Cell viability ( e ) and apoptosis ( f ) analysis of Ccl5 -knockdown 4T1 and B16F10 cells with and without treatment with PDT ( n = 3 per group, two-tailed unpaired Student’s t -test). g – n Effects of Ccl5 knockdown on PDT efficacy in vivo. Tumor growth curves ( g , n = 6 per group, two-way ANOVA with Tukey’s multiple comparisons test), post-treatment tumor photographs ( h ), and tumor weight analysis ( i , n = 6 per group, two-tailed unpaired Student’s t -test) of Ccl5 -knockdown 4T1 cells and control cell xenografts treated with or without PDT; Tumor growth curves ( j , n = 5 per group, two-way ANOVA with Tukey’s multiple comparisons test), post-treatment tumor photographs ( k ) and tumor weight analysis ( l , n = 5 per group, two-tailed unpaired Student’s t -test) of Ccl5 -knockdown B16F10 cells and control cell xenografts treated with or without PDT; Expression levels of the CCL5 protein ( m ) and proteins ( n ) related to proliferation and apoptosis in tumor tissues collected from Ccl5 -knockdown 4T1 and B16F10 xenografts after treatment with or without PDT. o Flow cytometric analyses of M2 polarization in BMDMs after treatment with culture medium from Ccl5 -knockdown 4T1 cells or control cells ( n = 3 per group, two-tailed unpaired Student’s t -test). p M2 polarization analysis of BMDMs treated with culture medium from Ccl5 -overexpressing 4T1 cells and control cells ( n = 3 per group, two-tailed unpaired Student’s t -test). q Mrc1 , Arg1 , Irf4 , Ym1 , and Cd274 gene expression in BMDMs incubated with culture medium from different 4T1 cells determined by qPCR ( n = 3 per group, two-tailed unpaired Student’s t -test). r Recruitment of M2 macrophages by Ccl5 -knockdown or Ccl5 -overexpressing 4T1 cells ( n = 3 per group; two-tailed unpaired Student’s t -test; scale bar: 50 µm). s CFSE staining analysis of 4T1 cell proliferation in BMDMs subjected to different treatments ( n = 3 per group, two-tailed unpaired Student’s t -test). t Effects of nanomedicines on Ccl5 promoter activity detected by dual-luciferase assay ( n = 3 per group, two-tailed unpaired Student’s t -test). u ChIP‒qPCR analysis of BRD4 protein binding to the Ccl5 promoter in B16F10 cells ( n = 3 per group; two-way ANOVA with Sidak’s multiple comparisons test). v Schematic diagram of ARV-825-mediated inhibition of Ccl5 gene transcription to enhance PDT. The figure was created with Figdraw.com. The data are presented as the means ± SDs for in vitro experiments and means ± SEMs for in vivo experiments

Article Snippet: For IHC, the slides were subsequently separately stained with primary antibodies against Ki67 (# GB111141 , Servicebio), CD31 (# GB113151 , Servicebio), PDL1 (#ab213480, Abcam), CCL5 (SC-365826, Santa Cruz), CD206 (# GB113497 , Servicebio), and Foxp3 (# GB112325 , Servicebio), followed by incubation with an HRP-conjugated secondary antibody.

Techniques: RNA Sequencing, Two Tailed Test, Knockdown, In Vivo, Control, Expressing, Gene Expression, Incubation, Staining, Activity Assay, Luciferase, Protein Binding, Inhibition, In Vitro

Journal: Cell

Article Title: NK Cells Stimulate Recruitment of cDC1 into the Tumor Microenvironment Promoting Cancer Immune Control

doi: 10.1016/j.cell.2018.01.004

Figure Lengend Snippet:

Article Snippet: For neutralization of CCL5 and XCL1, 50μg of anti-CCL5 and 50μg of anti-XCL1 antibodies or of isotype-matched control antibodies were injected i.v. at the time of tumor transplantation, followed by a second injection two days later (R&D Systems; CCL5: AF478 and MAB478; XCL1: AF486, MAB486; Isotype controls: AB-108-C, MAB006).

Techniques: Virus, Staining, Control, Recombinant, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: STING/RANTES Pathway in Airway Epithelium Stimulates Sensitization to Der p1 in an Asthma Model

doi: 10.1101/2023.07.30.550251

Figure Lengend Snippet: (A) HBEpC were cultured with cGAMP (14nM), H 2 O 2 , (100μM) or cGAMP+H 2 O 2 for 4 hours. (B-F) qPCR analysis of human IFNα (B) and IFNβ (C) and epithelial cytokines, IL-33 (D), TSLP (E), and RANTES (F). RANTES was stimulated by cGAMP and cGAMP+ H 2 O 2 . IFNβ was also stimulated by cGAMP+H 2 O 2 . Statistical analysis is performed by one-way ANOVA (means ± SEM, n=4). * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: To elucidate the role of RANTES as an adjuvant during sensitization, the mice were sensitized with recombinant RANTES protein (R&D systems, Minneapolis, MN, USA, 478-MR) and 1 μg of HDM on Day 1.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: STING/RANTES Pathway in Airway Epithelium Stimulates Sensitization to Der p1 in an Asthma Model

doi: 10.1101/2023.07.30.550251

Figure Lengend Snippet: (A) 8 weeks mice were treated with 20 ng of RANTES with 1 μg of HDM or 1 μg of HDM intra nasally on Day 1. Mice were challenged with 1 μg of HDM intranasally on Day 7, then lungs were extracted and analyzed on Day 8. (B) Pictures of lung sections of control and RANTES-adjuvanted, HDM-sensitized mice stained with H-E. Scale bar: 200 μm. Scale bar in picture of high magnification view: 40 μm. (C) Number of cells between bronchus and alveoli analyzed by ImageJ. Statistical analysis is performed by ordinary Mann Whitney’s U test (means ± SEM, 3 points/ section, n=5). (D) Pictures of lung sections in PBS and RANES-adjuvanted, HDM-sensitized mouse stained with PAS/Alcian blue. Scale bar: 80 μm. (E) Ratio of PAS/Alcianble area per epithelial cells (%). **** p < 0.0001

Article Snippet: To elucidate the role of RANTES as an adjuvant during sensitization, the mice were sensitized with recombinant RANTES protein (R&D systems, Minneapolis, MN, USA, 478-MR) and 1 μg of HDM on Day 1.

Techniques: Control, Staining

Journal: BMC Cancer

Article Title: CCL5-CCR5 interactions modulate metabolic events during tumor onset to promote tumorigenesis

doi: 10.1186/s12885-017-3817-0

Figure Lengend Snippet: MMTV-PyMT.CCR5 −/− tumor cells harvested from NSG mice exhibit lower glucose uptake, reduced GLUT-1 expression and lower levels of cellular metabolites than MMTV-PyMT.CCR5 +/+ tumor cells. MMTV-PyMT.CCR5 +/+ and MMTV-PyMT.CCR5 −/− tumors were harvested from recipient NSG mice when they were either just palpable (~20mm 3 ), i.e. tumor onset, or ~700mm 3 , endpoint. Following 16 h in culture, a glucose uptake, b GLUT-1 expression, c intracellular lactate and d CCL5 production, were measured. Values are the means +/− SEM of technical triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: CCL5 levels in culture supernatants were measured using anti-mouse CCL5 (DY478) and anti-human CCL5 (DRN00B) ELISA kits from R&D Systems, according to the manufacturer’s protocol.

Techniques: Expressing

Journal: BMC Cancer

Article Title: CCL5-CCR5 interactions modulate metabolic events during tumor onset to promote tumorigenesis

doi: 10.1186/s12885-017-3817-0

Figure Lengend Snippet: In vitro, MDA-MB-231.CCR5 −/− cells are less metabolically active than MDA-MB-231.CCR5 +/+ cells. a Cells were either left untreated (medium alone), treated with 10 nM CCL5, or pre-treated with 2 mM 2-DG for 1 h prior to CCL5 treatment, or maintained in medium containing 5 mM glutamine. For all, medium was changed every other day and the treatment(s) reapplied. Cell proliferation was quantified using an MTT assay as described in Methods. The proliferation index is normalized against untreated conditions (ie medium alone). Values are means ± SEM of triplicate assays and each data point combines the data from 3 independent experiments. Statistical analysis was performed comparing untreated cells with CCL5-treated cells and inhibitor-treated cells with CCL5 + inhibitor treated cells, or comparing CCL5-treated with CCL5 + inhibitor-treated cells. b CCL5 levels were measured from culture supernatants after 16 h incubation. c Glucose uptake, d GLUT-1 expression, e intracellular ATP and f intracellular lactate were measured as described in Methods in cells treated with 10 nM CCL5 or 2 μM oligomycin for 3 h. Data are expressed as percent-change relative to untreated MDA-MB-231.CCR5 +/+ cells. Values are means ± SEM of triplicate assays and each data point combines the data from 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: CCL5 levels in culture supernatants were measured using anti-mouse CCL5 (DY478) and anti-human CCL5 (DRN00B) ELISA kits from R&D Systems, according to the manufacturer’s protocol.

Techniques: In Vitro, Metabolic Labelling, MTT Assay, Incubation, Expressing

Journal: BMC Cancer

Article Title: CCL5-CCR5 interactions modulate metabolic events during tumor onset to promote tumorigenesis

doi: 10.1186/s12885-017-3817-0

Figure Lengend Snippet: MDA-MB-231.CCR5 −/− tumor cells harvested from NSG mice exhibit lower glucose uptake, reduced GLUT-1 expression and lower levels of cellular metabolites than MDA-MB-231.CCR5 +/+ tumor cells. MDA-MB-231.CCR5 +/+ and MDA-MB-231.CCR5 −/− tumors were harvested from recipient NSG mice when they were either just palpable (~20mm 3 ), i.e. tumor onset, or ~700mm 3 , endpoint. Following 16 h in culture, a glucose uptake, b GLUT-1 expression, and c CCL5 production, were measured. Values are the means ± SEM of technical triplicates. ** p < 0.01 and *** p < 0.001

Article Snippet: CCL5 levels in culture supernatants were measured using anti-mouse CCL5 (DY478) and anti-human CCL5 (DRN00B) ELISA kits from R&D Systems, according to the manufacturer’s protocol.

Techniques: Expressing

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 THP-1 macrophages were treated with 15 mM lactate for 24 h, and the mRNA levels of chemokines were measured by quantitative PCR. The growth medium of control macrophages was titrated to pH6.1 using sterile HCl. (B) 3×10 5 THP-1 macrophages were incubated with different concentrations of lactate for 24 h, and CCL5 gene expression was determined with quantitative PCR. (C) 10 6 THP-1 macrophages were exposed to increasing concentrations of lactate for 48 h, and the secretion of CCL5 was measured by ELISA. (D) 10 6 human primary macrophages from breast cancer patients (n=9) were cultured with different concentrations of lactate for 48 h, and CCL5 production was detected. (E) 10 6 MDA-MB-231 cells were pre-treated with 15μM GSK 2837808A for 2 h, then the media were changed, and cells were cultured for another 24 h. The conditional media (MD-231 CM) were collected and applied to 10 6 THP-1 macrophages. CCL5 concentrations were detected with ELISA. (F) Immunohistochemical staining of CD68 and CCL5 in tumor adjacent tissues (control) and breast tumors (n=28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Real-time Polymerase Chain Reaction, Control, Sterility, Incubation, Gene Expression, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 48 h, and the expression of key regulators in Notch, NF-κB, STAT3 and HIF, were detected by western blot. (B) 3×10 5 THP-1 macrophages were cultured with 15 mM lactate for 24 h, and the mRNA levels of Notch ligands and receptors were measured by quantitative PCR. (C) Western blot for Notch ligands and receptors in THP-1 (10 6 ) macrophages after 48 h lactate treatment. (D) Lactate stimulated the expression of NICD in a time and dose-dependent manner. 10 6 THP-1 macrophages were treated with 15 mM lactic acid for 48 h. Data presented were representatives of at least three independent experiments. (E) 10 6 THP-1 macrophages were transfected with 50nM siNotch1, or pretreated with 50μM DAPT for 2 h, and then cultured with 15 mM lactate for 48 h. The secretion of CCL5 was measured by ELISA. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 72 h, and then cells were washed twice and fresh media were added. Macrophages were cultured for another 24 h and the conditional media (lactate CM) was collected. The effect of CM on breast cancer cell migration was measured by double chamber transwell assay. 5μg/ml anti-CCL5 neutralizing antibody significantly decreased lactate CM-induced cell migration. (B) 10 6 MCF-7 cells were co-cultured with 15 mM lactate-activated macrophages in the presence of 5μg/ml anti-CCL5 antibody or not, and protein levels of EMT markers were tested by western blot. (C) 10 6 breast cancer cells were co-cultured with 10 6 lactate-activated THP-1 macrophages (or 10 6 lactate-activated primary macrophages) for different time points, and the expression of CCR5 was monitored by western blot. (D) MDA-MB-231 and MCF-7 cells were transfected with shCCR5 plasmids, or pre-treated with 5μM Maraviroc for 2 h, then cell migration induced by lactate CM was detected by double chamber transwell assay. Lactate CM was described in (A). (E) MCF-7 cells (10 6 ) were transfected with pcDNA3.1-CCR5, and then cultured with 10ng/ml CCL5 for 24 h. The expression of E-cadherin, N-cadherin and vimentin was investigated by western blot. (F) 10 6 Human primary macrophages (No. 4 and No. 9) were treated with 15 mM lactate for 72 h and CM was collected as described in (A). The migration of MDA-MB-231 cells was measured in the presence of primary macrophage CM. 5μg/ml anti-CCL5 neutralizing antibody, shRNAs designed against CCR5, or 5μM Maraviroc, significantly reduced primary macrophage CM-induced cell migration. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Migration, Transwell Assay, Western Blot, Expressing, Transfection

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 MDA-MB-231 and MCF-7 cells were stimulated with 1-5ng/ml TGF-β1 for 24 h, and total RNA was isolated and tested for CCR5 mRNA by quantitative PCR. (B) Western blot for CCR5 protein in breast cancer cells (10 6 ) under TGF-β1 stimulation for 48 h. Data presented were representatives of at least three independent experiments. (C) MDA-MB-231 and MCF-7 cells (3×10 5 ) were co-transfected with pGL3-CCR5 and pRL-TK and exposed to different concentrations of TGF-β1 for 24 h, and luciferase activities were determined. (D) MDA-MB-231 and MCF-7 cells were pre-treated with 5μM SIS3 for 2 h, and cells were subjected to luciferase assay. (E) 10 6 MCF-7 cells were transfected with TGFβRI/ALK5 siRNA, and were then co-cultured with lactate-activated THP-1 macrophages (ratio 1:1) for 24 h. The protein levels of CCR5 were assayed by western blot. (F) The expression of TGF-β1, CCL5 and CCR5 in clinical samples obtained from breast cancer patients. The mRNA levels were measured by quantitative PCR, and the correlation between TGF-β1 and CCL5-CCR5 axis was shown. (G) Representative IHC staining for TGF-β1, CCL5 and CCR5 in breast cancer samples. The sample used was derived from 28 breast cancer cases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Cell Culture, Expressing, Immunohistochemistry, Derivative Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Glucose uptake, lactic acid production and ATP levels in breast cancer cells co-cultured with lactate-activated THP-1 macrophages, with or without 5μg/ml anti-CCL5 neutralizing antibody. The co-culture system was described in Figure . (B) Western blots for glycolytic enzymes in breast cancer cells treated as in (A). (C) MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, and then subjected to cell co-culture. Glucose uptake, lactic acid production and ATP levels were measured after co-culture. The co-culture system was described in Figure . (D) The protein levels of HK2, PKM2 and LDHA in MDA-MB-231 cells cultured as in (C). (E) Recombinant human CCL5 induced aerobic glycolysis in breast cancer cells. MDA-MB-231 and MCF-7/CCR5 cells were treated with increasing concentrations of CCL5 for 12 h, and glucose uptake, lactic acid production and ATP levels were detected. (F) Western blots for glycolytic enzymes in MDA-MB-231 and MCF-7/CCR5 cells after stimulation with CCL5. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Co-Culture Assay, Western Blot, Transfection, Recombinant

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Western blot for AMPK, c-Myc, HIF-1α and Akt in breast cancer cells co-cultured with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 72 h. Results presented were representatives of at least three independent experiments. (B) The expression of AMPK downstream signaling target ACC in breast cancer cells co-cultured as in (A). (C) MDA-MB-231 and MCF-7 cells were transfected with 50 nM AMPKα1 siRNA, or pretreated with 10μM compound C for 4 h, and then incubated with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 48 h. The glucose uptake, lactic acid production and ATP levels were detected. (D) The inhibition of AMPK abrogated macrophage-induced EMT in MCF-7 cells. Cells were treated as described in (C). After co-culture, the expression of EMT markers, E-cadherin and vimentin, was measured by western blot. (E) Recombinant human CCL5 induced the phosphorylation of AMPK in MDA-MB-231 and MCF-7/CCR5 cells. 10 6 cells were treated with 50ng/ml CCL5 for defferent time points as indicated, and phosphorylated AMPK and total AMPK were investigated by western blot. (F) Inhibition of CCR5 in MDA-MB-231 cells significantly attenuated macrophage-induced AMPK phosphorylation. MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, then co-cultured with 15 mM lactate-activated macrophages as described in (A). After co-culture, the phosphorylation of AMPK was detected by western blot. (G) Expressions of CCL5, CCR5 and p-AMPK in samples obtained from breast cancer patients (n =28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Incubation, Inhibition, Co-Culture Assay, Recombinant, Phospho-proteomics

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) MDA-MB-231 cells were co-cultured with 15 mM lactate-activated THP-1 macrophages for 7 days, in the presence of 5μg/ml anti-CCL5 neutralizing antibody or not. MDA-MB-231 cells were then collected and injected into the tail vein of nude mice. After two weeks, animals were sacrificed and metastatic nodules on lung surfaces were counted. (B) CCR5, HK2 and p-AMPK were immunostained in MDA-MB-231 metastases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Injection

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: CCR5 Inhibition Prevents Cardiac Dysfunction in the SIV/Macaque Model of HIV

doi: 10.1161/JAHA.114.000874

Figure Lengend Snippet: CCL5 decreases cardiomyocyte contractility via CCR5. Confocal microscopy showing expression of (A) CCR5 (red) on isolated adult rhesus macaque ventricular cardiomyocyte (RM VCM) merged with (B) sarcomeric actin (green). C, Representative twitch traces for VCM sequentially exposed to human recombinant CCL5 (100 nmol/L), then CCL5 (100 nmol/L) with maraviroc (MVC, 500 nmol/L). Compared to basal conditions (grey, left), CCL5 decreased contractility, shown by the reduced, flattened twitch amplitude (red, middle). The CCL5‐induced decline in contractility was reversed by subsequent addition of MVC with CCL5 (green, right). D, Representative twitch traces for reciprocal experiments showing VCM sequentially exposed to MVC (500 nmol/L) followed by a combination CCL5 (100 nmol/L) and MVC (500 nmol/L). Addition of MVC (blue, middle) did not alter contraction from basal (grey, left). Furthermore, addition of MVC prior to MVC+CCL5 (turquoise, left) prevented CCL5‐induced changes in contractility with twitch traces unchanged from basal. E, Summary data for RM VCM exposed to CCL5 (100 nmol/L) followed by MVC (500 nmol/L) with CCL5 (100 nmol/L). CCL5 significantly decreased fractional shortening compared to basal. Subsequent addition of MVC modulated the CCL5 effect towards basal shortening. F, CCL5 significantly increases the time from basal to 50% peak sarcomere length (t to pk) compared to basal conditions. Subsequent CCL5+MVC modulated t to pk shortening towards basal conditions. G, CCL5 did not significantly change the time from peak shortening to 50% baseline (t to bl) compared to basal conditions although t to bl in cells treated with CCL5+MVC was shorter than both basal and CCL5 treatment. Paired t ‐tests, mean value indicated by the top of bar with bars representing standard error.

Article Snippet: Cells were sequentially exposed to recombinant human CCL5 (278‐RN, R&D Systems) 100 nmol/L alone and with 500 nmol/L of MVC (NIH/AIDS Reagent).

Techniques: Confocal Microscopy, Expressing, Isolation, Recombinant

Journal: Frontiers in Immunology

Article Title: T lymphocyte characteristics and immune repertoires in the epicardial adipose tissue of heart failure patients

doi: 10.3389/fimmu.2023.1126997

Figure Lengend Snippet: Amplified lymphocyte activation features in EAT of HF patients. (A) GO-BP functional enrichment analyses of up-regulated DEGs in EAT of HF patients. (B) “Lymphocyte activation” and “myeloid leucocyte activation” GO term genes in DEGs of HF-EAT. (C) Immune cell infiltration analyses of HF-EAT and control EAT by CIBERSORT. (D) Differentially expressed key genes in HF-EAT. (E) Expression of T cell-inflamed GEPs in HF-EAT and control EAT. (F) Correlation of CCL5 and GZMK expression with T cell-inflamed GEPs score in GSE24425. (G) Top 10 potential key TFs of DEGs in HF-EAT identified by ChEA3 database. (H) PPI network of top 10 potential TFs. * P < 0.05, ** P < 0.01.

Article Snippet: Used antibodies were as follows: human CD3 antibody (Servicebio, China), human CCL5 antibody (R&D systems, USA), and human GZMK antibody (R&D systems, USA).

Techniques: Amplification, Activation Assay, Functional Assay, Control, Expressing

Journal: Frontiers in Immunology

Article Title: T lymphocyte characteristics and immune repertoires in the epicardial adipose tissue of heart failure patients

doi: 10.3389/fimmu.2023.1126997

Figure Lengend Snippet: Verification of T cells infiltration and key molecules in EAT. (A, B) CD3-specific immunohistochemical staining in EAT and SAT. (C, D) Gating strategy and representative flow cytometry results of IFN-γ + T lymphocytes in EAT. (E, F) Representative flow cytometry results for proportion of T lymphocytes memory subtypes in EAT. (G) Representative fluorescent staining images of CCL5 and GZMK with CD3 from EAT of HF patients (scale: 50 μm). ** P < 0.01, *** P < 0.001 and ns refers to no significance.

Article Snippet: Used antibodies were as follows: human CD3 antibody (Servicebio, China), human CCL5 antibody (R&D systems, USA), and human GZMK antibody (R&D systems, USA).

Techniques: Immunohistochemical staining, Staining, Flow Cytometry