Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: Expression of NOD2 is elevated in the lung tissue of patients with COPD The expression of NOD2 was evaluated in lung tissue samples from healthy subjects and patients with COPD. ( A ) Schematic diagram of the experimental workflow for analyzing human lung tissue. ( B ) Quantitative real-time PCR (qRT-PCR) analysis of NOD2 mRNA. ( C ) Representative images of immunohistochemical (IHC) staining for NOD2 protein. ( D ) Quantification of IHC staining. ( E ) Representative images of fluorescence in situ hybridization (FISH) for NOD2 mRNA (green), with nuclei counterstained with DAPI (blue). ( F ) Quantification of FISH signal. ( G ) Representative multicolor immunofluorescence images showing CD66b-positive neutrophils (pink) and NOD2 (green), with nuclei counterstained with DAPI (blue). Enlarged views of the boxed regions are shown on the right. Scale bars: 20 μm. ( H ) Immunoblotting analysis and quantification of NOD2 protein levels in differentiated human HL-60-derived neutrophil-like cells and primary mouse neutrophils following CSE stimulation for 0, 2, 4, or 8 h, with untreated cells serving as the 0-h controls. ( I) qRT-PCR analysis of NOD2 mRNA expression in the corresponding human and mouse neutrophil models. Data are presented as mean ± SD. Data in B, D, and F were analyzed using an unpaired two-tailed Student’s t-test; data in H and I were analyzed separately for each cell model using one-way ANOVA with Tukey’s test. ** p < 0.01 versus the corresponding healthy-subject or CSE 0-h control group

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Expressing, Tissue, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Analysis, Immunohistochemical staining, Immunohistochemistry, Fluorescence, In Situ Hybridization, Immunofluorescence, Western Blot, Derivative Assay, Two Tailed Test, Control

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: NOD2 deficiency ameliorates cigarette smoke-induced lung inflammation and injury in vivo The functional role of NOD2 was evaluated in a cigarette smoke (CS)-induced mouse model of COPD using wild-type (wt) and NOD2-knockout (NOD2-/-) mice, with lentiviral-mediated NOD2 re-expression (NOD2-OE) in NOD2-/- COPD mice. ( A ) Schematic of the in vivo experimental design. ( B ) qRT-PCR analysis of NOD2 mRNA expression in lung tissue. ( C ) Representative images of hematoxylin and eosin (H&E) staining of lung tissue sections. Scale bars: 200 μm in the upper panels and 100 μm in the enlarged lower panels. ( D ) Representative images of Masson’s trichrome staining for collagen deposition in lung tissue sections. Scale bar: 100 μm. ( E ) Quantification of total cells, neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF), and ELISA measurements of TNF-α, IL-6, and CXCL1 levels in BALF supernatant. ( F ) Representative images of IHC staining for E-cadherin and N-cadherin in lung tissue. Scale bar: 50 μm. ( G ) Quantification of the average optical density (AOD) for E-cadherin and N-cadherin IHC staining. ( H ) qRT-PCR analysis of senescence-associated secretory phenotype (SASP) factors (CSF3, CCL3, and IL1α) in lung tissue. Data are presented as mean ± SD. Data were analyzed using one-way ANOVA followed by Tukey’s test. * p < 0.05 and ** p < 0.01 Control vs. wt-COPD groups; # p < 0.05 and ## p < 0.01 wt-COPD vs. NOD2 −/− -COPD groups; & p < 0.05 and && p < 0.01 NOD2 −/− -COPD + vector vs. NOD2 −/− -COPD + NOD2-OE groups

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: In Vivo, Functional Assay, Knock-Out, Expressing, Quantitative RT-PCR, Analysis, Tissue, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Control, Plasmid Preparation

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: NOD2 activation induced excessive mitophagy in the lungs of CS-exposed mice The role of NOD2 in regulating mitophagy was assessed in lung tissue and primary neutrophils isolated from mice in the CS-induced COPD model. ( A ) Representative transmission electron microscopy (TEM) images showing mitochondrial ultrastructure and mitophagosome-like structures in lung tissue. Insets show magnified views of mitochondrial morphology and mitochondria-containing autophagosomes. Scale bars: 500 nm. ( B ) Representative TEM images showing mitochondrial ultrastructure and mitophagosome-like structures in primary neutrophils isolated from the corresponding mouse groups. Insets show magnified views of mitochondrial morphology and mitochondria-containing autophagosomes. Scale bars: 500 nm. ( C ) Immunoblotting analysis of mitophagy-related proteins (Beclin1, LC3I/II, PINK1, Parkin, NIX, and BNIP3) in whole-lung tissue lysates. Data are presented as mean ± SD. Data in C were analyzed using one-way ANOVA followed by Tukey’s test. ** p < 0.01 Control vs. wt -COPD groups; and ## p < 0.01 wt -COPD vs. NOD2 −/− -COPD groups; & p < 0.05 and && p < 0.01 NOD2 −/− -COPD + vector vs. NOD2 −/− -COPD + NOD2-OE groups

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Activation Assay, Tissue, Isolation, Transmission Assay, Electron Microscopy, Morphology, Western Blot, Analysis, Control, Plasmid Preparation

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: NOD2 promotes Neutrophil Extracellular Trap (NET) formation in the CS-induced COPD mouse model The association between NOD2, autophagy, and Neutrophil Extracellular Trap (NET) formation was investigated in the lung tissue of mice from the CS-induced COPD model. ( A) Representative immunofluorescence images showing co-localization of the neutrophil marker Ly6G (green) and the autophagy marker LC3B (red), with nuclei counterstained with DAPI (blue). Scale bars: 20 μm in the upper panels and 10 μm in the enlarged lower panels. ( B ) Representative immunofluorescence images showing staining for the NET markers citrullinated histone H3 (CitH3, green) and myeloperoxidase (MPO, red), with nuclei counterstained with DAPI (blue). Scale bars: 20 μm in the upper panels and 10 μm in the enlarged lower panels. ( C ) Quantification of the NET biomarkers CitH3-DNA and MPO-DNA complexes in serum and lung tissue homogenates by ELISA. ( D ) Immunoblotting analysis of the neutrophil marker Ly6G and the NET marker CitH3 in whole-lung tissue lysates. ( E ) Representative immunofluorescence images showing colocalization of the neutrophil marker Ly6G (red), the NET marker CitH3 (green), and NOD2 (pink) in lung tissue, with nuclei counterstained with DAPI (blue). Scale bars: 20 μm in the upper panels and 10 μm in the enlarged lower panels. Data are presented as mean ± SD. C and D were analyzed using one-way ANOVA followed by Tukey’s test. ** p < 0.01 Control vs. wt -COPD groups; and ## p < 0.01 wt -COPD vs. NOD2 −/− -COPD groups; && p < 0.01 NOD2 −/− -COPD + vector vs. NOD2 −/− -COPD + NOD2-OE groups

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Tissue, Immunofluorescence, Marker, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Analysis, Control, Plasmid Preparation

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: Neutrophil depletion minimizes genotype-dependent differences in CS-induced lung injury, inflammation, fibrosis, and NET formation The dependence of NOD2-associated COPD pathology on neutrophils was evaluated by depleting neutrophils in CS-exposed wild-type (wt) and NOD2 −/− mice. Mice were randomly assigned to the wt -COPD + IgG, wt -COPD + Anti-Ly6G, NOD2 −/− -COPD + IgG, and NOD2 −/− -COPD + Anti-Ly6G groups ( n = 6 per group). ( A ) Schematic of the experimental design. Mice were exposed to CS for 12 weeks and received 200 µg of anti-Ly6G antibody or an equivalent dose of isotype IgG intraperitoneally three times per week from week 2 until the end of the experiment. ( B ) Representative flow-cytometry plots and quantification of CD11b + Ly6G + neutrophils in lung tissue and peripheral blood. ( C ) Representative H&E-stained images of lung tissue sections. Scale bars: 200 μm in the upper panels and 100 μm in the enlarged lower panels. ( D ) Representative images of Masson’s trichrome staining for pulmonary collagen deposition. Scale bars: 100 μm. ( E ) Quantification of total cells, neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF), together with ELISA measurements of TNF-α, IL-6, and CXCL1 levels in BALF supernatants. ( F ) Quantification of CitH3-DNA and MPO-DNA complexes in serum and lung tissue homogenates by ELISA. Data are presented as mean ± SD. Data were analyzed using two-way ANOVA followed by Tukey’s test. ** p < 0.01 vs. the wt -COPD + IgG group; ## p < 0.01 vs. the wt-COPD + Anti-Ly6G group; & p < 0.05, & p < 0.01 vs. the NOD2 −/− -COPD + IgG group

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Pathology, Flow Cytometry, Tissue, Staining, Enzyme-linked Immunosorbent Assay

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: Knockdown of NOD2 inhibits CSE-induced NET formation, ROS production, and mitophagy in human neutrophils The role of NOD2 in cigarette smoke extract (CSE)-induced Neutrophil Extracellular Trap (NET) formation, reactive oxygen species (ROS) accumulation, and lysosome–mitochondrion spatial relationships were evaluated in differentiated HL-60-derived neutrophil-like cells. ( A ) Schematic of the in vitro experimental design and treatment groups. ( B ) Representative DAPI-stained images of extracellular DNA structures and quantification of NET formation based on DAPI-stained nuclear morphology. Scale bars: 20 μm. ( C ) Extracellular DNA levels in cell culture supernatants determined using a PicoGreen dsDNA assay. ( D ) Myeloperoxidase (MPO) levels in cell culture supernatants determined by ELISA. ( E ) Neutrophil elastase (NE) levels in cell culture supernatants determined by ELISA. ( F ) Representative flow-cytometry histograms and quantitative analysis of intracellular ROS levels measured using DCFH-DA staining. ( G ) Representative immunofluorescence images showing the spatial relationship between LAMP1-positive lysosomes (green) and TOM20-positive mitochondria (red), with nuclei counterstained with DAPI (blue). The colocation efficients for LAMP1 and TOM20 was determined by ImageJ and shown on the right. Scale bars: 20 μm. Data are presented as mean ± SD. Quantitative data were analyzed using one-way ANOVA followed by Tukey’s test. ** p < 0.01 vs. Control group; ## p < 0.01 vs. CSE + sh-NC group

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Knockdown, Derivative Assay, In Vitro, Staining, Morphology, Cell Culture, Picogreen Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Analysis, Immunofluorescence, Control

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: ZDHHC5-mediated S-palmitoylation is essential for NOD2 stability and signaling The association of ZDHHC5 with NOD2 and the contribution of NOD2 S-palmitoylation to its stability, localization, and downstream signaling were investigated in differentiated HL-60-derived neutrophil-like cells. ( A ) Schematic of the proposed mechanism by which ZDHHC5-mediated S-palmitoylation promotes NOD2 membrane localization. ( B ) Representative immunofluorescence images showing NOD2 (red) in cells with or without CSE stimulation. Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm. ( C ) Acyl-biotin exchange (ABE) assay evaluating Flag-NOD2 S-palmitoylation in cells with or without CSE stimulation. HAM, hydroxylamine. ( D ) Immunoblotting analysis and quantification of NOD2 protein levels following treatment with increasing concentrations of the palmitoylation inhibitor 2-bromopalmitate (2BP) or with 100 µM 2BP for the indicated durations. ( E ) Representative immunofluorescence images showing NOD2 (red) in cells treated with vehicle or 2BP. Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm. ( F ) Co-immunoprecipitation analysis of the association between endogenous ZDHHC5 and NOD2. ( G ) Representative immunofluorescence images showing ZDHHC5 (green) and NOD2 (red), with nuclei counterstained with DAPI (blue). Scale bars: 20 μm. ( H ) Schematic of human NOD2 showing its functional domains and the positions of the C395S and C1033S mutations. ( I ) Immunoblotting analysis of wild-type (WT) and mutant Flag-NOD2 in total-cell lysates and membrane fractions during a cycloheximide (CHX) chase, with quantification of the relative NOD2 protein remaining shown on the right. GAPDH and Na + /K + -ATPase were used as loading controls for the total-cell lysates and membrane fractions, respectively. ( J ) ABE assay comparing the S-palmitoylation levels of WT Flag-NOD2, the C395S and C1033S single mutants, and the C395S/C1033S double mutant. ( K ) Immunoblotting analysis and quantification of p65 and p38 phosphorylation in cells expressing wt Flag-NOD2 or the C395S/C1033S double mutant following CSE stimulation. Data are presented as mean ± SD. Data in D and K were analyzed using one-way ANOVA followed by Tukey’s test. Data in I were analyzed using two-way ANOVA followed by Tukey’s test. ** p < 0.01 versus the corresponding 0 µM, 0 h, or wt control group; ## p < 0.01 versus the CSE + Flag-NOD2 wt group

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Stability, Derivative Assay, Membrane, Immunofluorescence, Western Blot, Analysis, Co-Immunoprecipitation Assay, Functional Assay, Mutagenesis, Phospho-proteomics, Expressing, Control

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: ZDHHC5 overexpression partially counteracts the effects of NOD2 knockdown in human neutrophil-like cells The ability of ZDHHC5 overexpression to counteract the effects of NOD2 knockdown was evaluated in CSE-stimulated, differentiated HL-60-derived neutrophil-like cells. ( A ) Schematic of the in vitro experimental design and treatment groups. ( B ) Representative DAPI-stained images of extracellular DNA structures and quantification of NET formation based on DAPI-stained nuclear morphology. Scale bars: 20 μm. ( C ) Extracellular DNA levels in cell culture supernatants determined using a PicoGreen dsDNA assay. ( D ) MPO levels in cell culture supernatants determined by ELISA. ( E ) NE levels in cell culture supernatants determined by ELISA. ( F ) Representative flow cytometry histograms and quantitative analysis of intracellular ROS levels measured using DCFH-DA staining. ( G ) Representative immunofluorescence images showing LAMP1-positive lysosomes (green) and TOM20-positive mitochondria (red), with nuclei counterstained with DAPI (blue). The colocalization coefficients for LAMP1 and TOM20 were determined by ImageJ and shown on the right. Scale bars: 20 μm. Data are presented as mean ± SD. Data were analyzed using two-way ANOVA followed Tukey’s test. ** p < 0.01 versus the CSE + sh-NC + Vector group; ## p < 0.01 versus the CSE + sh-NOD2 + ZDHHC5 group

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Over Expression, Knockdown, Derivative Assay, In Vitro, Staining, Morphology, Cell Culture, Picogreen Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Analysis, Immunofluorescence, Plasmid Preparation

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: Proposed model linking ZDHHC5-associated NOD2 S-palmitoylation to mitophagy and NET formation in COPD. CS exposure increases NOD2 and ZDHHC5 expression in pulmonary neutrophils. ZDHHC5-mediated NOD2 S-palmitoylation supports NOD2 stability and membrane localization, promoting NF-κB/MAPK signaling and mitophagy, intracellular ROS accumulation, and NET formation. Additional NOD2-independent effects of ZDHHC5 may also contribute to COPD-associated inflammation

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Expressing, Stability, Membrane

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Neutrophils develop a lipid‐laden state through free fatty acid uptake. (A–C) Neutrophils uptake free fatty acids (FAs). Human neutrophils were exposed to free fatty acids (PA, palmitic acid, 50 µM; OA, oleic acid, 50 µM; LA, linoleic acid, 50 µM). Lipid droplets (LDs) were visualized by BODIPY staining and immunofluorescence microscopy. SSO, sulfo‐ N ‐succinimidyl esters (CD36 inhibitor, 100 µM); Lipo, lipofermata (FATP2 inhibitor, 5 µM). (A) Schematic illustration of free FAs uptake by neutrophils. Representative immunofluorescence images (B) and quantification of LDs (C) in neutrophils exposed to free FAs. (D–F) Neutrophils acquire lipids from adipocytes. Mouse neutrophils were co‐cultured with differentiated 3T3‐L1 (d3T3‐L1) in the presence of TNF‐α, and LDs within the neutrophils were examined using immunofluorescence microscopy. (D) Schematic illustration of adipocyte‐to‐neutrophil lipid transfer. (E) Representative immunofluorescence images of neutrophils co‐cultured with adipocytes. Adipocytes are delineated by dashed lines, and neutrophils surrounding the adipocytes are indicated by arrowheads. (F) Quantification of LDs in neutrophils co‐cultured with adipocytes. Neutrophils co‐cultured with adipocytes were harvested, and intracellular LDs were measured using BODIPY staining. (G and H) Metabolic pathways in lipid‐laden neutrophils. (G) qPCR analysis of lipid metabolism‐related genes in vehicle‐ and OA‐treated neutrophils. (H) Heatmap showing the relative mRNA expression levels in OA‐treated neutrophils compared with the vehicle‐treated neutrophils. (I) Intracellular concentrations of FAs and TGs in OA‐treated neutrophils. (J–M) Lipidomics analysis in neutrophils exposed to OA. (J) Principal component analysis of glycerolipids in neutrophils. (K) Differential metabolite analysis in OA‐treated neutrophils. Scatter plot showing the log2 fold change of metabolites between OA‐ and vehicle‐treated neutrophils, with upregulated DG and TG species marked. (L and M) Heatmap showing the relative expression levels of selected DG and TG species enriched in OA‐treated neutrophils. Each heatmap annotates lipid species and their fatty acid composition. ABHD5, Abhydrolase domain containing 5; ACAA2, Acetyl‐CoA acyltransferase; ACACA, Acetyl‐CoA carboxylase alpha; ACAT, Acetyl‐CoA: cholesterol acyltransferase; ACLY, ATP citrate lyase; AGPAT, 1‐acylglycerol‐3‐phosphate O‐acyltransferase; ACSL, Acyl‐CoA synthetase long‐chain family member; CD36, Cluster of differentiation 36; CPT, Carnitine palmitoyltransferase; DGAT, (Diacylglycerol O‐acyltransferase); FABP, Fatty acid‐binding protein; FASN, Fatty acid synthase; G0S2, G0/G1 switch gene 2; HADHA, Hydroxyacyl‐CoA dehydrogenase / 3‐ketoacyl‐CoA thiolase / enoyl‐CoA hydratase alpha subunit; HILPDA, Hypoxia‐inducible lipid droplet‐associated protein; LIPE, Lipase E; MGAT, Mannosyl‐glycoprotein beta‐1,2‐N‐acetylglucosaminyltransferase; MGLL, Monoglyceride lipase; PLIN, Perilipin; PNPLA, Patatin‐like phospholipase domain containing; SLC27A4, Solute carrier family 27 member 4. All results are expressed as mean ± SEM. * p < .05; ** p < .01; *** p < .001.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: Staining, Immunofluorescence, Microscopy, Cell Culture, Expressing, Binding Assay

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Lipid‐laden neutrophils transfer lipids to the hepatocytes. (A–C) Lipid transfer from lipid‐laden neutrophils (LNs) to hepatocytes. Mouse neutrophils were co‐cultured with either undifferentiated 3T3‐L1 or differentiated 3T3‐L1 (d3T3‐L1) and subsequently co‐cultured with AML12 cells for 2 h. Neutrophils were removed and intracellular fat levels in AML12 cells were quantified using BODIPY staining. NNs, normal neutrophils; LNs, lipid‐laden neutrophils; AML12+OA, AML12 cells treated with oleic acid (OA, 50 µM); AML12+LA, AML12 cells treated with linoleic acid (LA, 50 µM); AML12+d3T3L1, AML12 cells co‐cultured with d3T3‐L1 cells at the ratio of 1:1; AML12+NNs, AML12 cells co‐cultured with NNs; AML12+LNs, AML12 cells co‐cultured with LNs. (A) Schematic representation of the experiment. (B) Left, representative immunofluorescence images. Right, quantification of BODIPY fluorescence in AML12 cells. (C) qPCR analysis of the metabolic genes in AML12 cells. (D–F) Lipid transfer from neutrophils exposed to free FAs to hepatocytes. Human neutrophils exposed to OA were subsequently co‐cultured with human HepG2 cells for 2 h. Neutrophils were removed and intracellular fat levels were quantified using BODIPY staining. HepG2+Veh; HepG2 cells treated with vehicle, HepG2+OA; HepG2 cells treated with OA (50 µM), HepG2+NNs, HepG2 cells co‐cultured with NNs; HepG2+LNs, HepG2 cells co‐cultured with LNs. (D) Schematic representation of the experiment. (E) Quantification of BODIPY fluorescence in HepG2 cells (F) qPCR analysis of metabolic pathway genes in HepG2 cells. Dgat1, Diacylglycerol O‐Acyltransferase 1; LPIN, Lipin family member; Scd, Stearoyl‐CoA Desaturase. All results are expressed as mean ± SEM. * p < .05; ** p < .01; *** p < .001.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: Cell Culture, Staining, Immunofluorescence, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Lipid‐laden neutrophils transfer lipids to hepatocytes via extracellular vesicles. (A, B) LNs transfer lipids to HepG2 cells in the absence of direct cell to cell contact. (A) HepG2 cells were co‐cultured with either NNs or LNs in a transwell chamber, and intracellular BODIPY fluorescence was quantified. (B) Conditioned media from LNs mediate lipid transfer to HepG2 cells. HepG2 cells were treated with supernatants derived from either NNs or LNs, and intracellular BODIPY fluorescence was quantified. (C) Extracellular vesicles (EVs) mediate lipid transfer from LNs to hepatocytes. HepG2 cells were treated with EVs isolated from either NNs (NNEVs) or LNs (LNEVs), and intracellular BODIPY fluorescence was quantified. Veh, HepG2 cells treated with vehicle; +NNEVs, HepG2 cells treated with NNEVs; +LNEVs, HepG2 cells treated with LNEVs. (D) LNEVs exhibit increased lipid content. Left, representative immunofluorescence images. Right, quantification of BODIPY fluorescence in neutrophil‐derived EVs. (E) Quantification of lipid contents in neutrophil‐derived EVs. (F) In vivo distribution of fluorescence‐labeled neutrophil‐derived EVs. Mice were intravenously injected with fluorescence‐tagged neutrophil‐derived EVs, and bioluminescence was assessed 24 h after administration using IVIS spectrum in vivo imaging system. All results are expressed as mean ± SEM. * p < .05; ** p < .01; *** p < .001.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: Cell Culture, Fluorescence, Derivative Assay, Isolation, Immunofluorescence, In Vivo, Labeling, Injection, In Vivo Imaging

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Transcriptomic profiling of HepG2 cells exposed to extracellular vesicles derived from lipid‐laden neutrophils. HepG2 cells were treated with extracellular vesicles (EVs) derived from NNs (+NNEVs) or LNs (+LNEVs) for 2 h and subsequently subjected to bulk RNA sequencing. (A) Schematic representation of the experimental procedure. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighting enrichment of human disease‐associated pathways in EV‐treated HepG2 cells. (C) KEGG pathway analysis of metabolic processes altered by neutrophil‐derived EVs. (D) Gene Ontology (GO) Biological Process enrichment analysis of differentially expressed genes. (E) Metabolic pathways modulated by LNEVs in HepG2 cells. Upper panel, a heatmap depicting differentially expressed genes (DEGs) in +LNEVs relative to vehicle‐treated HepG2 cells. Lower panel, a schematic summary illustrating alterations in lipid metabolic genes in +LNEVs based on KEGG metabolic pathway mapping.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: Derivative Assay, RNA Sequencing

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Lipid‐laden neutrophils contribute to hepatic fat accumulation in vivo. (A–E) Effect of neutrophil depletion on hepatic fat accumulation in a murine model of diet‐induced obesity. (A) Schematic representation of the experimental design. C57BL/6J mice were fed either a high‐fat diet (HFD) or normal chow (NC). Neutrophils were depleted via intraperitoneal administration of an anti‐Ly6G antibody. (B) Representative immunofluorescence images showing BODIPY staining of peripheral neutrophils isolated from mice. ( C ) Immunofluorescence analysis of liver sections, showing the presence of BODIPY + Ly6G + lipid‐laden neutrophils in livers of HFD‐fed mice. (D and E) Visualization of hepatic lipid accumulation using Oil Red O staining (D) and BODIPY immunofluorescence staining (E). (F–J) Effects of adoptive transfer of LNs on hepatic fat accumulation. (F) Schematic illustration of the experimental design. C57BL/6J mice were intravenously injected with either LNs or NNs at two‐day intervals for 10 days. (G and H) Representative images and quantification of hepatic lipid accumulation assessed by Oil Red O staining. (I and J) Representative images and quantification of hepatic lipid accumulation assessed by BODIPY fluorescence. All results are expressed as mean ± SEM. * p < .05; ** p < .01; *** p < .001.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: In Vivo, Immunofluorescence, Staining, Isolation, Adoptive Transfer Assay, Injection, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Neutrophils from patients with MASLD exhibit a lipid‐laden phenotype. (A) Schematic overview of the experimental workflow for the isolation and analysis of neutrophils and EVs from patients with MASLD and healthy volunteers. (B–E) Phenotypic and metabolic characterization of neutrophils isolated from patients with MASLD. Neutrophils were isolated from healthy volunteers and patients with MASLD and subjected to BODIPY staining, intracellular TG quantification, and qPCR analysis of the lipid metabolic genes. (B) Representative immunofluorescence images of neutrophils isolated from healthy volunteers and patients with MASLD stained with BODIPY to visualize LDs, with LDs indicated by arrows. (C) Quantification of LD counts per neutrophils from healthy volunteers and patients with MASLD. (D) Intracellular TG levels in neutrophils isolated from healthy volunteers and patients with MASLD. (E) qPCR analysis of lipid metabolism‐related genes in neutrophils, comparing expression levels between patients with MASLD and healthy volunteers. (F and G) Characterization of circulating EVs isolated from patients with MASLD. EVs were isolated from serum of healthy volunteers (HV EVs) and patients with MASLD (MASLD EVs) and analyzed for miRNAs expression and TG content. (F) qPCR analysis of selected miRNAs in HV EVs and MASLD EVs. (G) Quantification of TG concentrations in HV EVs and MASLD EVs. (H) qPCR analysis of selected miRNAs in neutrophils isolated from HV (HV Neu) and MASLD patients (MASLD Neu). (I) Spearman correlation analysis between miRNA levels in circulating EVs and neutrophils from patients with MASLD. All results are expressed as mean ± SEM. * p < .05; ** p < .01; *** p < .001.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: Isolation, Staining, Immunofluorescence, Expressing