Abstract
Neutrophils, the first cells to arrive at the site of inflammation, are rather short-lived cells and thus have to be constantly replenished. During neutrophil development, vesicle dynamics need to be fine-tuned and impaired vesicle trafficking has been linked to failure in neutrophil maturation. Here, we characterized the role of VPS18 as a central core component of CORVET & HOPS tethering complexes for neutrophil development. Using CRISPR/Cas9-engineered Hoxb8 cells with heterozygous mutations in Vps18, we found that VPS18 deficiency interfered with neutrophil development due to tethering complex instability. As a result, vesicle dynamics were impaired with a strong increase in LC3B-II and p62 levels, indicating autophagosome accumulation and reduced autophagic flux. With transmission electron microscopy, we verified the increase in autophagosomes and also found irregularly shaped vesicular structures in Vps18 mutants. Subsequently, Vps18 mutant neutrophil progenitors underwent premature apoptosis. We described a novel patient with a heterozygous stop-gain mutation in VPS18 suffering from neutropenia and recurrent infections. To verify our findings in the human system, we used human induced pluripotent stem cells (iPSCs). Upon differentiation into neutrophils, loss of VPS18 resulted in an almost complete absence of iPSC-derived developing neutrophils. Heterozygous VPS18 mutant and patient mutation-harboring iPSCs were characterized by strongly reduced numbers of developing neutrophils. Zebrafish larvae with heterozygous mutations in vps18 were also characterized by significantly reduced neutrophil numbers. This study shows the pivotal impact of VPS18 for adequate vesicle dynamics during neutrophil development which might be relevant in the context of vesicle trafficking during granulopoiesis and congenital neutropenia.
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The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD049111.
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Acknowledgements
The authors thank Jennifer Truong, Tanja Vlaovic, Tanja Weißer, Ulrike Wilhelm-Forster, Sabine Schlink, Azin Rasti, and Hafez Gabara for excellent technical assistance. The authors are grateful to Dr. Steffen Dietzel (Core Facility Bioimaging, Biomedical Center, LMU Munich) for the support with fluorescence microscopy. We acknowledge the Core Facility Flow Cytometry at the Biomedical Center, LMU Munich, for providing equipment and expertise.
Funding
German Research Foundation collaborative research grant CRC914 (projects A02 (DM-B and BW), A08 (CK), A13 (FM)) and Z03 (BW)). German Research Foundation collaborative research grant TRR332 (#449437943; projects A02 (OS), B01 (CK) and C03 (DM-B and BW)). Open Access funding enabled and organized by Projekt DEAL.
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DM-B designed and performed experiments, analyzed data and wrote the manuscript. AB performed experiments, analyzed data and wrote the manuscript. JG, MIL, JC, FM, MRi, RdC, AZ, KM, XW, EdVG, MK, MRo, MT, BP and OS performed experiments and analyzed data. SF-W, BS, CK and BW provided their expertise. IS, JY, OS-S and RS provided critical patient information. DM-B and BW acquired funding and supervised the study.
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All methods were performed in accordance with the relevant guidelines and regulations. All studies involving human samples were conducted in accordance with the Declaration of Helsinki. For isolation of human neutrophils, written informed consent was obtained from all volunteers prior to blood donation. The blood collection was approved by the ethics committee of the LMU Munich (No. 259-14). Written informed consent was obtained from the family of the patient to perform research on the samples of the patient and the father. Collection of blood samples to perform genome analyses at the Dr. von Hauner Children’s Hospital was approved by the ethics committee of the LMU Munich (No. 66-14). The use of iPS cells was authorized by the ethics committee of the LMU Munich (No. 24-0697 OMICS-SE). Adult zebrafish were raised and housed and experiments with zebrafish larvae were performed in accordance with animal protection standards of the LMU Munich and approved by the government of Upper Bavaria (Regierung von Oberbayern, No. 55.2-2532.Vet_02-17-21).
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Gao, J., Bader, A., Linder, M.I. et al. Mutations in VPS18 lead to a neutrophil maturation defect associated with disturbed vesicle homeostasis. Cell Death Dis (2026). https://doi.org/10.1038/s41419-025-08338-w
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DOI: https://doi.org/10.1038/s41419-025-08338-w
