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