Journal: Journal of Advanced Research

Article Title: CCR1 hi /CCL5 hi macrophage-mediated CCL5 hi T cell chemotaxis in salivary gland aggravates Sjögren’s syndrome

doi: 10.1016/j.jare.2025.06.076

Figure Lengend Snippet: CCR1-overexpressing macrophages induce T and B cell migration. (A) Immunofluorescence (IF) detection of CD68 expression in THP-1 cells induced by PMA (100 nM, 24 h). (B) Quantitative analysis of CD68 expression based on IF staining fluorescence intensity. (C) Schematic diagram of the detection of macrophage-induced T and B cell migration. (D, E) Western blot analysis and quantitative evaluation of CCR1 and CCL5 expression in macrophages after transfection with the CCR1 overexpression plasmid. (F) ELISA measurement of CCL5 concentration in supernatants from CCR1-overexpressing macrophages. (G, H) Transwell assay evaluating macrophage-mediated migration of T and B cells, with quantitative analysis of migrated cell numbers. Statistical significance was determined using one-way ANOVA.

Article Snippet: The release level of CCL5 in cell culture supernatants was assessed using a commercial ELISA kit (EK1129, Lianke Biotechnology Co., Ltd., Hangzhou, China) following the manufacturer’s instructions.

Techniques: Migration, Immunofluorescence, Expressing, Staining, Fluorescence, Western Blot, Transfection, Over Expression, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transwell Assay

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: CCL5 enhances LPS-induced PAD4-dependent NETosis in primary neutrophils. (A) Blood samples were collected from each animal group 24 h after CLP, and plasma concentrations of cfDNA/MPO (NETs) were determined ( n = 10 per group). (B) Confocal images of NETs formation in primary neutrophils were stimulated with LPS (1 mg/ml, 2 h) in the presence or absence of CCL5 (50 ng/ml). Blue, 4′,6-diamidino-2-phenylindole (DAPI); green, myeloperoxidase (MPO). Scale bar = 50 mm. (C) Liver was harvested from each group 24 h post-CLP induction and examined by immunoblotting (IB) with the indicated antibodies. (D) The primary neutrophils isolated from each group were stimulated with LPS with or without CCL5 and examined by IB with the indicated antibodies. The quantification of indicated protein ratio was shown in the right. (E-I) The effect of GSK484 on LPS/CCL5-induced NETs formation. The isolated neutrophils from bone marrow were pre-treated with GSK484 (50 nM, 1 h) and further stimulated with either LPS or CCL5 (n = 3). Sytox Green staining (E), superoxide (2-OH-E+) release (F), mitochondrial ROS (G), total cellular ROS (H) and elastase activity (I) were measured. Results are shown as means ± SE. * p < 0.05; ** p < 0.01 (by a one-way ANOVA).

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Clinical Proteomics, Western Blot, Isolation, Staining, Activity Assay

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: Macrophage-derived CCL5 via PP4 drives NETs formation. (A) CCL5 production by isolated BMDMs from WT and PP4 myel−KO mice, stimulated with LPS (1 mg/ml, 24 h), was measured by an ELISA. (B–F) NETs formation (B), mitoROS (C), total cellular ROS (D), elastase activity (E) and immunofluorescence images showing co-localization of myeloperoxidase (MPO, green) and DAPI (blue) (F) in differentiated HL-60 cells treated with conditioned media (CM) from LPS-induced WT and PP4 myel−KO BMDMs, with either IgG control or CCL5-neutralizing antibody (10 ng) for 24 h. All results are presented as the means ± SE. * p < 0.05; ** p < 0.01 (by a one-way ANOVA). Scale bar = 50 mm.

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Isolation, Enzyme-linked Immunosorbent Assay, Activity Assay, Immunofluorescence, Control

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: PP4 suppresses CCL5 expression through the TBK1/IRF3 signaling pathway. (A) Immunoblotting (IB) of the indicated antibodies in LPS-stimulated BMDM cells isolated from WT and PP4 myel−KO mice. (B) BMDMs were stimulated with LPS (1 mg/ml) and assessed by IB using the indicated antibodies after immunoprecipitation (IP) with anti-PP4. (C) IB of TBK1 and β-actin in BMDMs transfected with empty vector (EV, siNT) or siTBK1. (D) The mRNA levels of IL-6, TNF-α, CCL5 and MIP-1a in BMDMs from PP4 myel−KO mice stimulated with LPS in the presence of siNT or siTBK1. Results were normalized to the expression of ACTB (encoding β-actin) and presented relative to those of untreated cells ( n = 3). (E) BMDMs infected with adenoviruses expressing either an EV (Ad-PGK), HA-PP4 WT (Ad-PP4), or HA-PP4 mutant (Ad-PP4mut) were stimulated with LPS and assessed by IB analysis with the indicated antibodies. (F) CCL5 production by BMDMs transduced as in E and stimulated with LPS for 24 h (n = 3). (G) NETs formation by differentiated HL-60 cells was treated with conditioned media (CM) from LPS-induced either Ad-PGK or Ad-PP4 transduced BMDMs (n = 3). Data are presented as mean ± SE for each group. * p < 0.05; ** p < 0.01 (by a one-way ANOVA).

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Isolation, Immunoprecipitation, Transfection, Plasmid Preparation, Infection, Mutagenesis

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: LPS induces CCR5 upregulation in neutrophils via ERK1/2 signaling. (A) Neutrophils from WT and PP4 myel−KO mice were isolated 24 h after CLP and analyzed by IB using CCR5 and b-action antibodies. (B) Expression of CCR5 in differentiated HL-60 (dHL60) cells was stimulated with LPS (1 mg/ml, 24 h), with or without the indicated inhibitors. Results were normalized to the expression of ACTB (encoding β-actin) and presented relative to control cells. (C) dHL60 cells treated siNT or siPP4 were stimulated with LPS and subsequently analyzed by IB using the indicated antibodies. (D) IP of anti-PP4 from LPS-stimulated dHL60 cells, followed by IB with antibodies against pERk1/2 or PP4. Total cell lysate (TCL) IB was performed with the indicated antibodies. (E) Neutrophils isolated from PP4 myel−KO mice were pre-treated with PD98059 (20 mM, 1 h) and further stimulated with LPS (1 mg/ml, 2 h). IB was analyzed by using indicated antibodies. (F) siPP4-treated dHL60 cells were stimulated with LPS in the presence or absence of PD98059, followed by IB with indicated antibodies. (G) Neutrophils isolated from PP4 myel−KO mice bone marrow infected with adenoviruses expressing EV (Ad-PGK) or gene encoding HA-PP4 WT (Ad-PP4) or HA-PP4 mutant (Ad-PP4mut) were stimulated with LPS, followed by IB analysis with the indicated antibodies. The quantification of indicated protein ratio was shown in the right. (H and I) NETs formation (H) and elastase activity (I) in isolated neutrophils was measured following LPS in the presence or absence of CCL5. All results are expressed as the means ± SE.* p < 0.05; ** p < 0.01 (by a one-way ANOVA). (n = 3).

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Expressing, Control, Infection, Mutagenesis, Activity Assay

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: Targeting the CCL5–CCR5 axis attenuates neutrophil hyperactivation. (A) The effect of CCR5 inhibitors on LPS/CCL5-induced NETs formation by using Sytox Green staining. Primary neutrophils isolated from WT and PP4 myel−KO mice were pre-treated with TAK-779 (10 nM, 1hr) or UK-427857 (10 nM, 1hr), and further stimulated with either LPS (1 mg/ml) or CCL5 (50 ng/ml) for 24 h. (B and C) The production of mitoROS (B) and elastase activity (C) by neutrophils as in A, was quantified. (D) Differentiated HL-60 (dHL60) cells treated with siNT or siCCR5 were analyzed by IB using the indicated antibodies. (E-H) NETs formation (E), mitoROS (F), elastase activity (G), and immunofluorescence images of myeloperoxidase (MPO, green) and DAPI (blue) co-localization (H) in dHL60 cells transfected with siNT or siCCR5, followed by LPS stimulation with or without CCL5. All results are presented as the means ± SE.* p < 0.05; ** p < 0.01 (by a one-way ANOVA). Scale bar = 50 mm. (n = 3). (I) Schematic representation of the central role of PP4 in neutrophil recruitment and activation.

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Isolation, Activity Assay, Immunofluorescence, Transfection, Activation Assay

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: CCL5 deficiency reduces NETs formation and improves survival in CLP-induced sepsis. (A) Survival curves of WT and CCL5 KO mice following CLP. (B) Plasma levels of cfDNA/MPO (NETs) in WT or CCL5 KO mice were determined 24 h after CLP. (C-E) NETs formation by Sytox Green staining (C), and the production of mitoROS, total cellular ROS and superoxide release (D), and Elastase activity (E) was measured in differentiated HL-60 cells treated with conditioned media (CM) from LPS-induced WT and CCL5 KO BMDMs in the presence or absence methyl-β-cyclodextrin (MβCD, 5 mM). Data are the mean ± SE for each group. * p < 0.05, ** p < 0.01 (by one-way ANOVA). ( n = 6).

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Clinical Proteomics, Staining, Activity Assay