Biology subjects

Cheng, J.

Publications and source records attributed to Cheng, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Spatiotemporal Mapping of Tertiary Lymphoid Structure Heterogeneity Shapes Immune Niches and Clinical Outcomes in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal malignancy with limited therapeutic options. The spatial architecture and functional diversity of tertiary lymphoid structures (TLSs) in iCCA remain unclear. Here, we present a multimodal spatial atlas of TLSs and identified Intra-tumoral TLSs (iTLSs) as independent prognostic markers. Bulk proteomic profiling of 214 discovery and 155 validation cases identified a four-tier TLS-based TME classification system and supported development of a TLS-predictive random forest classifier. Imaging mass cytometry revealed that iTLS tumors harbor structured immune architectures, where M1-like tissue-resident macrophages (RTMs), dendritic cells, and CXCL13 CD4 T cells co-localize to form antigen-presenting neighborhoods (apc-CNs) spatially coupled to TLS core regions (TLScore-CNs). Single-cell spatial transcriptomics further resolved 61 TLSs into 14 spatial niches and defined a pseudo-temporal maturation continuum: aggregated, activated, and post-activated. Intra-niche communication, primarily mediated by ifnCAFs, iCAFs, and CXCL12 macrophages, evolved dynamically with maturation. Single-nucleus RNA-sequencing combined with Tangram-based spatial mapping revealed CXCL12 macrophages and iCAFs forming a peripheral band in aggregated TLSs, while ifnCAFs infiltrated TLS interiors during activation. These findings define TLS heterogeneity and provide insights for stroma-directed immunotherapy. TeaserAn atlas of tertiary lymphoid structures reveals immune-stroma interactions in Intrahepatic cholangiocarcinoma.

immunology↗

bioRxiv2025

Structural Basis of Asymmetric Class C GPCR activation and distinct Gq coupling induced by force stimulation

Mechanosensation is essential for diverse physiological processes. While many G protein-coupled receptors (GPCRs) are known to be mechanosensitive, the underlying mechanism remains largely unknown. Here, we reveal that mechanical force induces Gq activation of mGlu2, a balance modulator residing in kinocilia, but not other 7 mGlu members. Force activates mGlu2 through a conserved N-terminal force transduction motif (FTM) via a unique cis mechanism. We engineered a potent FTM-derived peptide agonist that recapitulates force-induced activation and resolved cryo-EM structures of apo-mGlu2, FTM-mGlu2, LFTM-{Psi}EK-mGlu2 and LFTM-{Psi}EK-mGlu2-Gq. The structures reveal that an atypical FTM binding to a previously uncharacterized pocket induces asymmetric 7TM domain rearrangement, enabling Gq coupling via an ICL1-TM3/6/7 interface, fundamentally distinct from the glutamate-induced Gi coupling mode of mGlu2. Compared with Gi-coupled mGlu2, the 5 helix of Gq rotated by 180{degrees}, penetrating deeper (8 [A]) into a hydrophobic pocket. Disruption of the M7947.32-F7806.57-F7766.53 hydrophobic triad core and a conformational propagation path predominantly comprising TM6-7 residues are identified as key elements mediating force induced mGlu2 activation. Further In vivo rescue experiments support that mGlu2s mechanosensitivity is dependent on FTM and is required for vestibular function. This work establishes a paradigm for class C GPCR mechanotransduction, revealing unprecedented structural mechanisms underlying force-induced Gq coupling and offering a chemical toolset to modulate mechanical signaling of GPCR.

cell biology↗

bioRxiv2025

The functional divergence of two ethylene receptor subfamilies that exhibit Ca2+-permeable channel activity

Ethylene is a gaseous plant hormone crucial for regulating plant growth, development, and stress adaptation, yet the molecular basis underlying ethylene receptor function remains elusive. Here, we show that subfamily I receptors constitute core ethylene-sensing module and function epistatically to subfamily II receptors. Notably, we discover that only subfamily I receptors possess Ca2+-permeable channel activity, which are indispensable for ethylene-induced cytosolic calcium influx. Overall, this work supports a mechanistic framework in which subfamily I receptors integrate ethylene sensing with Ca2+ influx, providing new insight into how plants translate hormonal cues into downstream signaling events.

plant biology↗

bioRxiv2025

ETSAM: Effectively Segmenting Cell Membranes in cryo-Electron Tomograms

Cryogenic Electron Tomography (cryo-ET) is an emerging experimental technique to visualize cell structures and macromolecules in their native cellular environment. Accurate segmentation of cell structures in cryo-ET tomograms, such as cell membranes, is crucial to advance our understanding of cellular organization and function. However, several inherent limitations in cryo-ET tomograms, including the very low signal-to-noise ratio, missing wedge artifacts from limited tilt angles, and other noise artifacts, collectively hinder the reliable identification and delineation of these structures. In this study, we introduce ETSAM - a two-stage Segment Anything Model 2 (SAM2)-based fine-tuned AI method that effectively segments cell membranes in cryo-ET tomograms. It is trained on a diverse dataset comprising 83 experimental tomograms from the CryoET Data Portal (CDP) database and 28 simulated tomograms generated using PolNet. ETSAM achieves state-of-the-art performance on an independent test set comprising 10 experimental tomograms for which ground-truth annotations are available. It robustly segments cell membranes with high sensitivity and precision, significantly outperforming existing deep learning methods.

bioinformatics↗

bioRxiv2025

SpNeigh: spatial neighborhood and differential expression analysis for high-resolution spatial transcriptomics

Spatial transcriptomics technologies such as Xenium, MERFISH, and Visium HD enable high-resolution profiling of gene expression while preserving tissue architecture. However, most computational methods for spatial analysis do not explicitly model local tissue context, such as boundaries, neighborhoods, or gradients. Here we present SpNeigh, an R package for spatial neighborhood analysis and spatially-aware differential expression modeling. SpNeigh includes tools for boundary detection, spatial neighborhood extraction, distance-based weighting, and gradient-based statistical testing. It supports both region-based differential expression and smooth spatial modeling using spline-based regression, along with a spatial enrichment index that identifies genes enriched near defined spatial features. We demonstrate the utility of SpNeigh across multiple platforms and tissues, including mouse brain, human breast cancer, and human liver, revealing intermediate populations at tissue interfaces, immune microenvironment differences, and spatially zonated gene expression patterns. SpNeigh offers a flexible and interpretable framework for dissecting spatial gene expression dynamics in complex tissues.

bioinformatics↗

bioRxiv2025

Infusible Extracellular Matrix Biomaterial Enhances Cell-Specific Pro-Repair Responses Following Acute Myocardial Infarction

To mitigate the pathological effects of myocardial infarction, we developed and investigated pro-reparative decellularized extracellular matrix (ECM) biomaterials: an intravascularly infusible ECM (iECM). However, the cellular and molecular mechanisms by which iECM mediates repair are unknown because investigations have relied on bulk techniques. Here, we leverage single nucleus RNA sequencing (snRNAseq) to measure pro-repair in various cell types across acute timepoints (1, 3 and 7 days post infusion). In iECM, we found pro-reparative macrophage activation, fibroblast remodeling, increased vasculaure development, lymphangiogenesis, cardioprotection, and neurogenesis. These findings were validated through spatial transcriptomics. Thus, we define the pro-reparative nature of decellularized ECM biomaterials on cardiac cell types and elucidate previously undiscovered therapeutic pathways, further demonstrating the potential of iECM as an MI therapy as well as display the wealth of data generated from next-generation sequencing.

bioengineering↗

bioRxiv2025

Modular Noncovalent Functionalization of Electrospun Piezoelectric Scaffolds with Bioactive Nanocarriers

Electrospun scaffolds offer a promising platform for immune-instructive materials, but stable and modular functionalization with bioactive signals remains a technical challenge. Here, we develop a surface coating strategy for electrospun scaffolds that consist of poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE), a piezoelectric polymer, using electrostatic adsorption of charged nanoparticles. We show that under certain conditions, these piezoelectric scaffolds are suitable substrates for electrostatic self-assembly, and that the density of nanoparticle coatings can be tuned by adjusting buffer pH, ionic strength, and nanoparticle concentration. This approach enables robust and uniform coating of both polymeric nanoparticles and soft nanocarriers such as liposomes, without requiring covalent surface modification. Liposome-coated scaffolds are cytocompatible with adherent epithelial and suspension immune cells and support lipid exchange at the cell-material interface. Using a supramolecular tethering strategy, we use liposome coatings to present interleukin-15 (IL-15) from the scaffold surface and demonstrate localized, sustained cytokine signaling. Together, these findings establish a modular approach for post-fabrication, noncovalent scaffold functionalization with bioactive nanocarriers, offering new opportunities for tissue and immune engineering.

bioengineering↗

bioRxiv2025

RNA viruses in water shape the viromes of shrimp and fish in aquaculture systems

Aquatic environments host immense RNA viral diversity, yet the extent to which waterborne viruses influence the virome of aquatic animals remains unclear. Here, we conducted a comprehensive longitudinal metatranscriptomic analysis of water, shrimp, and fish across multiple culture stages in aquaculture systems. We identified 3,211 RNA viruses spanning 21 viral supergroups, with 97.4% representing novel species. Water exhibited the greatest viral diversity (3,093 species), followed by shrimp (1,280) and fish (398), with most viruses detected in shrimp and fish also detected in water. Diversity analyses revealed increasing richness and significant compositional shifts in 92 high-abundance viruses (>1,000 RPM) across culture stages. Viral abundance in aquatic animals was strongly correlated with that in water, and the temporal trajectories of the 16 most abundant viruses were closely synchronized between water and shrimp. Furthermore, 337 virus species were shared among shrimp and two fish species, of which 309 were present in water. These findings demonstrated that RNA viruses in water shape the composition and dynamics of aquatic animal viromes, underscoring the importance of water virome surveillance and management for disease prevention in shrimp aquaculture.

microbiology↗

bioRxiv2025

Using gnotobiotic mice to decipher effects of gut microbiome repair in undernourished children on tuft and goblet cell function

Studies have implicated perturbations in the postnatal development of the gut microbiome as a contributing factor to childhood undernutrition. Compared to a standard ready-to-use supplementary food, a microbiome-directed complementary food (MDCF-2) designed to repair these perturbations produced superior improvements in ponderal and linear growth in clinical trials of Bangladeshi children with moderate acute malnutrition. Here, reverse translation experiments are performed where intact fecal microbiomes collected from trial participants prior to and at the end of treatment are introduced into female gnotobiotic mice just after delivery of their pups. Pups received diets designed to resemble those consumed by children in the trials to recreate "unrepaired" and "repaired" gut ecosystems. Analyses of the abundances of bacterial strains (metagenome-assembled genomes), their expressed genes and metabolic products, combined with assessments of ponderal growth and intestinal epithelial lineage transcriptomes (single-nucleus RNA-Seq with follow-up immunocytochemistry) disclosed effects of MDCF-2 associated microbiome repair that cannot be determined, in part because no treatment control arms cannot be ethically incorporated into these trials. Specifically, microbiome repair in these mice produced significant increases in ponderal growth, changes microbial gene expression consistent with a less virulent gut ecosystem and changes in expression of (i) components of gut epithelial cell junctions in the enterocytic and goblet cell lineages, (ii) pathways for synthesis and secretion of eicosanoid immune effectors in chemosensory tuft cells, and (iii) goblet cell pathways involved in glycosylation and secretion of mucin. Experiments of the type described can help formulate and test hypotheses about how microbiome repair affects host biology. SIGNIFICANCE STATEMENTUndernutrition is a global health problem. Recent clinical trials of a gut microbiome-directed complementary food (MDCF-2) designed to repair the perturbed gut microbiomes of undernourished Bangladesh children produced superior growth outcomes versus a standard nutritional supplement. Given ethical considerations and tissue sampling constraints associated with these types of studies, we colonized gnotobiotic mice postnatally with microbiome samples obtained from trial participants before and after treatment to model "unrepaired" and "repaired" gut ecosystems. Using a multi-omics approach, we uncover heretofore unappreciated changes in expressed chemosensory tuft cell, mucus-producing goblet cell and absorptive enterocytic functions and interactions accompanying microbiome repair. Extending microbiome clinical trials back to preclinical models ( reverse translation) provides mechanistic insights that can inform design/interpretation of future interventions.

microbiology↗

bioRxiv2025

CryoFSL: an annotation-efficient, few-shot learning framework for robust protein particle picking in cryo-EM micrographs

AO_SCPLOWBSTRACTC_SCPLOWAccurate identification of protein particles in cryo-electron microscopy (cryo-EM) micrographs is crucial for high-resolution structure determination, but remains challenging due to the heavy reliance on extensive annotated datasets and the difficulty of ensuring robustness under low signal-to-noise ratio (SNR) conditions. Current approaches require large annotations and exhibit poor generalization to new protein targets. We present CryoFSL (Cryo-EM Few Shot-Learning), a novel few-shot learning framework built upon Segment Anything Model 2 (SAM2) with lightweight adapters, enabling robust particle picking using as few as five labeled micrographs, significantly reducing annotation burden. The frameworks hierarchical adapter design supports dynamic feature modulation for low-SNR and heterogeneous conditions, resolving the trade-off between annotation burden and performance. CryoFSL surpasses both traditional template-based methods and state-of-the-art deep learning models across diverse proteins in the few-short learning setting, achieving superior recall, precision and 3D reconstruction resolution with minimal supervision. It maintains stability across heterogeneous micrographs and consistently detects high-quality particles with fewer false positives. Notably, CryoFSL achieves competitive density map reconstruction resolution with just a fraction of the particles picked by other methods, redefining efficiency and quality in cryo-EM analysis. This work paves the way for scalable, generalizable, and annotation-efficient particle picking pipelines. The code is available at GitHub.

bioinformatics↗

bioRxiv2025

Integrating Protein and DNA Embeddings for Improving Genome-Wide Transcription Factor Binding Site Prediction

Transcription factors (TFs) regulate gene expression by binding to specific DNA sites on genome, making accurate TF binding site prediction critical for understanding gene regulation and downstream phenotypes. Current deep learning methods use only DNA-related information to predict TF binding sites, ignoring the fact that different TF protein sequences and structures recognize distinct DNA patterns. Not leveraging TF information not only limits prediction accuracy but also makes the methods not generalizable to predicting binding sites of new TFs that do not exist in the traning data. Here, we present TransBind, a protein-aware deep learning architecture that integrates DNA sequence information with protein embeddings containing both sequence and structural information derived from a protein language model pretrained on DNA-binding proteins, to improve TF binding site prediction. Through the cross-attention, a TF embedding selectively attends to genomic regions according to its unique binding properties. Evaluated on the data of 690 ChIP-seq experiments spanning 161 TFs across 91 human cell types, TransBind achieves an AUROC of 0.950 and AUPR of 0.371--representing a [≥]11.3% relative AUPR improvement over state-of-the-art methods including TBiNet, DanQ, and DeepSEA. The model outperformed existing methods in [≥]97.1% of TF-cell type combinations. It also recovered 160 known TF binding motifs in the JASPAR database, providing the biological interpretability of the model. Moreover, the approach enables zero-shot prediction for unseen TFs, demonstrating its potential of generalizing to new, poorly characterized TFs. The source code of TransBind is available at https://github.com/jianlin-cheng/TransBind.

bioinformatics↗

bioRxiv2025

Systematic evaluation of metatranscriptomic differential gene expression in silico, in vitro, and in vivo enables elucidation of inter-species cross-feeding

Metatranscriptomic (MTX) sequencing quantifies gene expression from the collective genomes of microbial communities (microbiomes), enabling assessment of functional activity rather than functional potential. While differential expression testing is essential for RNA-sequencing analysis, current metatranscriptomic approaches have only been benchmarked on simulated data, resulting in a lack of standard practices for analysis of real datasets. Here, we use mock communities (defined mixtures of microbial cells with known properties) to quantitatively assess robustness and susceptibility of current approaches to various confounders including organisms low relative abundance, differential abundance, low prevalence, global transcriptional output changes, and compositional effects. We show that no current method is robust to all confounders and method performance on simulated data does not generalize to real datasets. We then apply the same approaches to MTX datasets generated from gnotobiotic mice colonized with defined consortia of human bacterial strains and show that the method nominated by the mock community comparisons successfully inferred cross-feeding dynamics that were subsequently validated in vitro. Finally, using metagenome-assembled genomes from a human clinical study, we leverage genome-level sequencing depth and detection of genes to exclude low information samples on a per-organism basis to overcome confounding low prevalence and enhance differential expression inference.

genomics↗

bioRxiv2025

JointMap identified SERPINA3+ chondrocytes as a therapeutic target for osteoarthritis

BackgroundOsteoarthritis (OA) is a prevalent degenerative joint disease that affects nearly half of the population aged over 65. Recent single-cell transcriptional studies have identified heterogeneous OA-specific chondrocyte subtypes, yet a unified subtype-classification remains incomplete. MethodsIn this study, we integrated a total of 545,946 cells to develop JointMap, an interspecies atlas. The chondrocyte subtypes were analyzed using machine learning algorithms were validated via human samples, animal models, and in vitro cell culture. We performed KAS-seq and analyzed the spatial characters for JointMap. ResultsBased on clinical diagnosis and cross-omic indexes, we identified a novel OA-elevated chondrocyte subtype, SERPINA3+ chondrocytes. Notably, we uncovered their upregulated chondrogenesis function and intercellular communication patterns as potential therapeutic targets. ConclusionsIn summary, JointMap provides a unified framework for the systematic annotation of chondral tissues across species and helps in the identification of druggable networks for articular diseases.

bioinformatics↗

bioRxiv2025

CenSegNet: a generalist high-throughput deep learning framework for centrosome phenotyping at spatial and single-cell resolution in heterogeneous tissues

Centrosome abnormalities (CA) are a hallmark of epithelial cancers, yet their spatial complexity and phenotypic heterogeneity remain poorly resolved due to limitations in conventional image analysis. We present CenSegNet (Centrosome Segmentation Network), a modular deep learning framework for high-resolution, context-aware segmentation of centrosomes and epithelial architecture across diverse tissue types. Integrating a dual-branch architecture with uncertainty-guided refinement, CenSegNet achieves state-of-the-art performance and generalisability across both immunofluorescence and immunohistochemistry modalities, outperforming existing models in accuracy and morphological fidelity. Applied to tissue microarrays (TMAs) containing 911 breast cancer sample cores from 127 patients, CenSegNet enables the first large-scale, spatially resolved quantification of numerical and structural CA at single-cell resolution. These CA subtypes are mechanistically uncoupled, exhibiting distinct spatial distributions, age-dependent dynamics, and associations with histological tumour grade, hormone receptor status, genomic alterations, and nodal involvement. Structural CA levels are additionally associated with overall survival, supporting the clinical relevance of spatially resolved CA patterns. Discordant CA profiles at tumour margins are linked to local aggressiveness and stromal remodelling. To support broad adoption and reproducibility, CenSegNet is released as an open-source Python library. Together, our findings establish CenSegNet as a scalable, generalisable platform for spatially resolved centrosome phenotyping in intact tissues, enabling systematic dissection of the biology of this organelle and its dysregulation in cancer and other epithelial diseases.

cell biology↗

bioRxiv2025

High-Fat Diet-Induced Obesity Impairs Endothelium-Dependent Relaxation via MLCK Activation: Reversal by ML-7 in Rabbits

ObjectiveObesity is an independent risk factor for CVD(cardiovascular diseases) and elevated cardiovascular mortality, though the precise mechanisms remain incompletely elucidated. Impaired endothelium-dependent relaxation is an early manifestation of cardiovascular diseases. This study investigated the impact of HFD(high-fat diet)-induced obesity on endothelium-dependent relaxation function and explored the potential role of MLCK(myosin light chain kinase) signaling. MethodsForty-five New Zealand White rabbits were randomized into three groups: a control group (normal diet), HFD(high-fat diet) group, and an ML-7 group (HFD plus the MLCK inhibitor ML-7). After 8 weeks of feeding, vascular endothelial function was assessed in vivo by measuring FMD(flow-mediated dilation). Serum lipids and blood glucose levels were determined. Endothelium-dependent vasodilation was evaluated ex vivo using aortic ring assays. The protein expression levels of MLCK and eNOS(endothelial nitric oxide synthase), as well as the phosphorylation level of MLC(myosin light chain), in rabbit arteries were analyzed by Western blot. ResultsHFD-fed rabbits developed obesity, dyslipidemia (elevated TC[total cholesterol], LDL-C[low-density lipoprotein cholesterol], and ox-LDL[oxidized low-density lipoprotein]), perirenal adipose tissue deposition, and significant endothelial dysfunction, evidenced by impaired endothelium-dependent relaxation function and morphological endothelial damage. Western blot analysis revealed upregulated MLCK and eNOS expression, along with increased phosphorylation of MLC in the arterial walls of obese rabbits, despite no significant change in NO(nitric oxide) levels compared to controls. Treatment with ML-7 ameliorated endothelium-dependent relaxation function impairment. Furthermore, the inherent limitations of FMD as a non-invasive technique in terms of accuracy and sensitivity for evaluating endothelium-dependent dilation. ConclusionsObesity-induced endothelial dysfunction arise from visceral fat accumulation, dyslipidemia, oxidative stress, and MLCK overexpression. Notably, MLCK inhibition via ML-7 restores vascular function, highlighting its therapeutic potential in obesity-related vascular injury.

physiology↗

bioRxiv2025

High-resolution spatial analysis reveals pregnenolone metabolism as a potential target for immuno-suppressive microenvironment of BRAF mutant colorectal cancer

BRAF mutant colorectal cancer (CRC) is highly aggressive and often resistant to traditional therapies, presenting a significant challenge in cancer immunotherapy. Understanding the tumor microenvironment (TME) and identifying novel immune checkpoints are crucial for improving treatment outcomes. Here, we employed high-resolution spatial analysis and single-cell RNA sequencing to map the TME of BRAF mutant CRC at single-cell resolution, focusing on the interactions between exhausted CD8+ T cells and C1QC+ macrophages. Mechanistically, we discovered that C1QC+ macrophages drive CD8+ T cell exhaustion through pregnenolone synthesis via the steroidogenic enzyme CYP11A1. Our findings identify pregnenolone metabolism as a previously unrecognized immunosuppressive pathway in BRAF mutant CRC, and suggest that steroid biosynthesis is a generalizable mechanism of immune evasion beyond endocrine-related malignancies. Targeting this pathway offers a promising therapeutic strategy for enhancing anti-tumor immunity in BRAF mutant CRC and potentially other cancers.

cancer biology↗

bioRxiv2025

Time-Resolved Mapping in Calves Reveals BoHV-1 Shift From Mucosal Replication to Trigeminal Neuroinvasion and IFN-α-bPML Antagonism

Bovine herpesvirus 1 (BoHV-1) causes huge cattle losses, yet the transition from mucosal replication to neuroinvasion is poorly resolved. Using a controlled calf model, we integrated quantitative virology and transcriptomics to map pathogenesis and define the role of promyelocytic leukemia protein (PML). Calves inoculated intranasally/ocularly (1.4x10^6 pfu/head) were sampled daily (1-14 dpi) for gB-qPCR; tissues at 4 and 14 dpi were analyzed for viral DNA and trigeminal ganglia (TG) mRNA-seq. Shedding peaked at 3-6 dpi (nasal > ocular >> rectal) and declined by 10-14 dpi. Tonsils bore the highest burden at 4 dpi; TG was low-positive at 4 dpi but remained positive at 14 dpi, indicating neuroinvasion. TG programs shifted from early proteostasis priming (4 dpi) to immune/ECM activation with synaptic repression (14 dpi). In MDBK/Vero cells, IFN- increased bovine PML (bPML) and enlarged PML nuclear bodies (PML-NBs), reducing very-early viral DNA, whereas BoHV-1 disrupted PML-NB integrity. bPML isoforms diverged (bPML1/6 pro-viral; bPML3/4/5 antiviral). Cross-species STRING mapped a conserved PML-SUMO1-UBE2I-DAXX-SP100 core. These data delineate the mucosal-to-neuronal trajectory, establish PML as both effector and viral target, and nominate SUMO/ubiquitin-linked proteostasis as a tractable antiviral leverage point.

microbiology↗

bioRxiv2025

Mutations in VPS18 lead to a neutrophil maturation defect associated with disturbed vesicle homeostasis

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 LC3-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.

physiology↗