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: Neutrophils were permeabilized and stained with mouse anti-MPO (SC-52707, Santa Cruz) with an Alexa FluorTM 488, goat anti-mouse IgG (H + L) cross-absorbed secondary antibody (A-11001, Invitrogen).

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: 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: Neutrophils were permeabilized and stained with mouse anti-MPO (SC-52707, Santa Cruz) with an Alexa FluorTM 488, goat anti-mouse IgG (H + L) cross-absorbed secondary antibody (A-11001, Invitrogen).

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: Neutrophils were permeabilized and stained with mouse anti-MPO (SC-52707, Santa Cruz) with an Alexa FluorTM 488, goat anti-mouse IgG (H + L) cross-absorbed secondary antibody (A-11001, Invitrogen).

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