Palmitoylation Enzyme ZDHHC5 Emerges as Driver of Neutrophil Inflammation in COPD

Chronic obstructive pulmonary disease is marked by a stubborn infiltration of neutrophils into the lung, and researchers have now traced a molecular chain of events that appears to sustain that infiltration. The work, conducted in human lung tissue samples, a cigarette smoke-induced mouse model of COPD, and neutrophil-like cells grown in the laboratory, positions the ZDHHC5-NOD2 axis as a previously unrecognized contributor to the neutrophilic inflammation that defines the disease.

The investigation began with a clinical observation. Lung tissue collected from patients with COPD during pulmonary nodule resection showed markedly higher levels of NOD2 messenger RNA and protein than tissue from healthy volunteers with normal lung function, as measured by quantitative PCR, immunohistochemistry and fluorescence in situ hybridization. Multicolor immunofluorescence staining revealed that NOD2 signals overlapped with CD66b-positive neutrophils in COPD lung sections, although NOD2 was also detectable outside the neutrophil compartment. When the researchers exposed differentiated HL-60-derived neutrophil-like cells and primary mouse neutrophils to cigarette smoke extract, NOD2 protein and mRNA rose progressively over two, four and eight hours of stimulation, indicating that the receptor is inducible by smoke exposure in both human and mouse neutrophil models.

To test whether NOD2 actually drives disease, the team turned to a mouse model in which animals breathe cigarette smoke at 350 milligrams of total particulate matter per cubic meter for 75 minutes twice daily, five days a week, for 12 weeks. In wild-type mice, this regimen produced the expected pathology: inflammatory cell infiltration, alveolar wall thickening, collagen deposition, and elevated levels of the inflammatory mediators TNF-alpha, IL-6 and CXCL1 in bronchoalveolar lavage fluid. In NOD2 knockout mice subjected to the same exposure, these changes were substantially attenuated, and staining patterns consistent with epithelial-mesenchymal transition, including reduced E-cadherin and elevated N-cadherin, were partially reversed. Reintroducing NOD2 via lentiviral vector restored the pathological picture, and the expression of senescence-associated secretory phenotype factors CSF3, CCL3 and IL1-alpha followed the same pattern.

The most striking finding concerned mitochondria. Transmission electron microscopy of lung tissue from smoke-exposed wild-type mice revealed severe mitochondrial swelling and vacuolization, along with autophagosomes containing mitochondrial material, hallmarks of excessive mitophagy. These ultrastructural changes were markedly reduced in NOD2-deficient mice and returned upon NOD2 re-expression. Immunoblotting confirmed the picture at the molecular level: the mitophagy-related proteins Beclin1, PINK1, Parkin, NIX and BNIP3, as well as the ratio of LC3II to LC3I, were all elevated in the lungs of smoke-exposed wild-type mice, reduced by NOD2 deficiency, and restored by NOD2 overexpression. Primary neutrophils isolated from the airways of these mice showed the same mitochondrial damage and mitophagosome accumulation, tying the effect directly to the effector cells under study.

NET formation tracked closely with these mitochondrial changes. In the lungs of smoke-exposed wild-type mice, immunofluorescence showed strong staining for the NET markers citrullinated histone H3 and myeloperoxidase, and enzyme-linked immunosorbent assays detected elevated concentrations of CitH3-DNA and MPO-DNA complexes in both serum and lung tissue homogenates. All of these signals diminished in NOD2 knockout mice and rebounded with NOD2 re-expression. Multicolor staining further showed overlap among NOD2, the neutrophil marker Ly6G, and citrullinated histone H3, placing NOD2 within NET-forming neutrophils in the inflamed lung.

To probe whether NOD2-associated pathology depends on neutrophils, the researchers depleted them with an anti-Ly6G antibody beginning at week two of the smoke exposure. Depletion lowered neutrophil abundance in lung tissue and peripheral blood in both genotypes and, crucially, narrowed the differences between wild-type and NOD2-deficient mice: after depletion, both groups showed similarly mild lung injury, reduced collagen deposition, lower BALF inflammatory cell counts and cytokine levels, and reduced NET biomarkers. The authors are careful to note that this experiment supports neutrophil dependence of NOD2-associated pathology but does not, by itself, establish that NOD2 acts exclusively within neutrophils, particularly given the global nature of the knockout.

In vitro experiments fleshed out the cellular mechanism. In CSE-stimulated HL-60-derived neutrophil-like cells, NOD2 knockdown reduced the proportion of cells forming NET-like extracellular DNA structures and lowered the release of extracellular DNA, myeloperoxidase and neutrophil elastase into the culture supernatant. Intracellular reactive oxygen species, measured by DCFH-DA flow cytometry, also fell with NOD2 loss, and the spatial overlap between LAMP1-positive lysosomes and TOM20-positive mitochondria, an indicator of mitophagy, was visibly attenuated. Parallel experiments in primary neutrophils from knockout mice corroborated each of these results. Conversely, when NOD2 was overexpressed in both cell types, treatment with the autophagy inhibitor 3-MA blunted the resulting increases in NET formation, NET-associated protein release and lysosome-mitochondrion colocalization, supporting autophagy and mitophagy as functional contributors to the NET-promoting effects of NOD2.

The study then dissected the chemical modification at the heart of the pathway. Cigarette smoke extract increased NOD2 S-palmitoylation, as shown by an acyl-biotin exchange assay, and treatment with the palmitoylation inhibitor 2-bromopalmitate reduced NOD2 protein levels in a concentration- and time-dependent manner. Co-immunoprecipitation detected an association between endogenous ZDHHC5 and NOD2, and mutating two cysteine residues, C395 and C1033, to serine reduced NOD2 palmitoylation, accelerated its degradation during a cycloheximide chase, diminished its presence in membrane fractions, and weakened CSE-induced phosphorylation of the inflammatory signaling proteins p65 and p38. ZDHHC5 itself was elevated in COPD lung tissue and in CSE-stimulated neutrophil models, and experimentally altering ZDHHC5 expression correspondingly raised or lowered NOD2 protein abundance in human and mouse neutrophils.

Rescue experiments tested how far ZDHHC5 could compensate for NOD2 loss. In smoke-exposed mice receiving NOD2 knockdown, ZDHHC5 overexpression partially restored lung inflammation, collagen deposition, EMT-associated marker changes, neutrophil autophagy-related activity, NET marker staining, and soluble NET biomarkers, even though NOD2 mRNA remained suppressed, pointing to post-transcriptional or NOD2-independent effects. In NOD2 knockout neutrophils, ZDHHC5 overexpression partially restored NET formation, ROS accumulation, mitophagy-associated colocalization and p65 and p38 phosphorylation, but the responses generally remained below those of ZDHHC5-overexpressing wild-type cells. The authors interpret this partial rescue as evidence of additional NOD2-independent ZDHHC5-regulated mechanisms, and they note that ZDHHC5-mediated palmitoylation of other inflammatory proteins, such as NLRP3 and GSDMD, offers plausible routes that remain untested in COPD neutrophils.

The authors are explicit about the limits of their work. The mouse model relies on cigarette smoke exposure, and whether the ZDHHC5-NOD2 axis operates in COPD independent of tobacco exposure remains to be verified. Global NOD2 deletion, systemic lentiviral delivery, and colocalization-based mitophagy assessment do not establish neutrophil-intrinsic causality or direct measurement of mitophagic flux, and future studies will need neutrophil-specific genetic models, direct flux assays, and single-cell analyses to resolve which lung cell populations drive the observed changes. Within those constraints, the study delivers a coherent mechanistic account: cigarette smoke raises ZDHHC5 in neutrophils, ZDHHC5 palmitoylates NOD2 at two specific cysteines, the stabilized membrane-associated receptor promotes mitophagy, ROS accumulation and NET formation, and the resulting neutrophilic inflammation exacerbates lung injury in a cigarette smoke-induced model of COPD. The pathway now stands as a candidate target for future therapeutic exploration.

Subject of Research: The role of ZDHHC5-mediated S-palmitoylation of NOD2 in neutrophil extracellular trap formation and inflammation in COPD

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

Article References: Dai, Z., Chen, X., Peng, Y., Yu, Q., Xie, B., Ma, R., You, S., Hu, C., & Chen, Q. (2026). ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD. Biomarker Research, 14(1), Article 140. https://doi.org/10.1186/s40364-026-01001-2

Image Credits: AI Generated

DOI: 10.1186/s40364-026-01001-2

Keywords: COPD, ZDHHC5, NOD2, S-palmitoylation, neutrophil extracellular traps, mitophagy, reactive oxygen species, cigarette smoke, neutrophils, inflammation, mouse model, HL-60 cells