Biology subjects

Everts, B.

Publications and source records attributed to Everts, B..

15 recordsLinked to original sources

bioRxiv2026

Physiological fatty acid uptake reveals spatial and systemic constraints on nutrient accessibility in vivo

Immune cells rely on exogenous fatty acids (FA) for membrane synthesis, bioenergetics and signalling, yet current approaches cannot accurately quantify physiological FA uptake in vivo. Here, we use cyclopropene-tagged fatty acids (cpFA) that, unlike existing FA-uptake tools, are taken up by physiologically relevant mechanisms. We measure FA uptake at single-cell resolution in vivo and uncover a previously unappreciated distinction between nutrient uptake capacity and nutrient accessibility. Although arachidonic acid exhibits the highest uptake capacity ex vivo across immune populations, it displays limited tissue accessibility in vivo, whereas palmitate is broadly accessible. In vivo nutrient-uptake measurements reveal that tissue architecture shapes nutrient accessibility, with spatial constraints in the spleen and exclusion of circulating FA, but not amino acids, from the thymus. Together, these findings identify nutrient accessibility as a distinct layer of metabolic regulation and reveal that immune-cell metabolism is shaped by spatial and systemic constraints on nutrient access HighlightsO_LINutrient accessibility is a distinct layer of metabolic regulation C_LIO_LIPhysiological FA uptake differs from ex vivo uptake capacity C_LIO_LISpatial and systemic factors govern fatty-acid accessibility C_LIO_LITissue context shapes immune-cell metabolism in vivo C_LI In briefUsing bioorthogonal FA to quantify physiological nutrient uptake in vivo, Wang et al. show that nutrient accessibility is distinct from nutrient uptake capacity. Tissue architecture and systemic FA distribution create spatial constraints on nutrient access, revealing an underappreciated layer of metabolic regulation in immune cells.

immunology↗

bioRxiv2026

O-GlcNAcylation and low glycolysis underpin Th2 polarization by dendritic cells

Activation of dendritic cells (DCs) is dependent on rewiring of their cellular metabolism. However, the metabolic requirements for DCs to prime T helper 2 (Th2) responses are still poorly understood. Using unbiased transcriptomics and non-targeted metabolomics we find that helminth antigen-conditioned human DCs suppress glycolysis while increasing hexosamine biosynthesis to fuel protein O-GlcNAcylation. Functionally, glycolytic inhibition of DCs selectively enhanced, while blocking O-GlcNAcylation impaired, Th2-priming capacity. In helminth infection and allergic challenge, Th2 responses were also attenuated in vivo in mice with specific deletion of O-GlcNAc Transferase (OGT) in CD11c-expressing cells. Mechanistically, through proteomic analysis and functional validation, we identified O-GlcNAcylation as a critical negative regulator of immune synapse formation by controlling cytoskeletal organization via Fascin-1 and Zyxin, thereby dampening TCR signalling to promote Th2 polarization. Altogether we reveal a novel metabolic program in DCs that governs Th2 polarization, that could potentially be harnessed to treat type 2 mediated inflammatory diseases.

immunology↗

bioRxiv2026

Supervised machine learning versus expert assessment of ultrastructural changes in wild-type and OGT knockout macrophages

Automated transmission electron microscopy (TEM) generates large datasets that challenge traditional qualitative analysis of cellular ultrastructure. Quantitative assessment of structural differences between different samples remains difficult due to structural variability in thin sections of organelles. Here we applied supervised machine learning (sML) to segment, quantify and compare cellular structures -including nuclei, chromatin, mitochondria, rough endoplasmic reticulum, and endocytic vesicles- in large TEM images of wild-type macrophages versus those with altered cellular physiology due to deficiency in O-GlcNAc Transferase (OGT). sML revealed that OGT knockout macrophages are larger and more oval, with increased euchromatin, nucleoli size, and relative mitochondrial and rER surface areas. Comparison with six TEM experts showed sML provides more objective and sensitive quantification of subtle differences, while expert consensus is only achieved for larger structural variations. These findings demonstrate that sML enhances quantitative TEM analysis and complements human expertise in ultrastructural studies.

cell biology↗

bioRxiv2026

O-GlcNAcylation drives macrophage IL-4 responsiveness and tissue residency through metabolic and cell cycle calibration

The metabolic requirements for macrophage IL-4 polarization remain contentious, while immunometabolic studies of tissue resident macrophages are still sparse. Hexosamine biosynthesis has gained attention regarding its immune regulatory potential via downstream O-GlcNAcylation. Here we identify protein O-GlcNAcylation as a requirement for IL-4 polarization in vitro and proliferative expansion in vivo during cytokine challenge or infection. We further show that O-GlcNAcylation is critical for controlling tissue residency. By enforcing metabolic and cell cycle quiescence during differentiation, O-GlcNAcylation is needed for adult monocytes to establish a long-live residency program. In this context, its absence leads to perpetual DNA vulnerability and damage via reactive oxygen species, resulting in a senescent-like state poised for cell death. Conversely, long-lived populations instead require O-GlcNAcylation for self-renewal and inflammatory expansion. Our findings altogether suggest O-GlcNAcylation, fueled by hexosamine biosynthesis, serves as a central metabolic rheostat for resident macrophage formation and maintenance during homeostasis and disease.

immunology↗

bioRxiv2025

IRF4 is a master regulator of multinucleated giant cell formation

Multinucleated giant cells (MGCs) are a hallmark pathological feature of a wide range of diseases, yet the mechanisms underlying their formation and function remain poorly understood. Although it is recognized that MGCs arise from a heterogeneous pool of myeloid precursors, how a committed MGC fate develops therein remains unknown. Here, combining temporal in vitro and in vivo differentiation of bone marrow-derived myeloid precursors with single cell and bulk RNA sequencing as well as CRISPR/Cas9-mediated gene editing, we shed insight into how MGCs emerge. Our findings reveal that coordinated upregulation of cell fusion genes and cellular metabolism, particularly oxidative phosphorylation, regulate the transition from macrophage progenitors to fusion competent cells. In vivo fate-mapping unveils Ms4a3-, and Cd11c-but not Cx3cr1-traced cells as the predominant precursor populations for MGCs in a lung granuloma formation model. Notably, transcription factor profiling in the progression from early myeloid precursors to pre-MGCs identifies IRF4 as a key molecular switch driving MGC generation. IRF4+ MGCs are present in different pathologies, including Schistosoma mansoni egg induced granulomas, Aspergillus fumigatus conidia mediated allergic airway inflammation and human head and neck squamous cell carcinomas. Mechanistically, IRF4 controls critical fusion related genes such as Dcstamp and Ocstamp. Consequently, Irf4 deficient cells are unable to develop into MGCs. Collectively, our work delineates the trajectory of MGC differentiation, establishing IRF4 as a defining transcription factor required for the generation of fusion-competent progenitors that ultimately give rise to MGCs.

immunology↗

bioRxiv2025

A Novel Cyclopropenyl Fatty Acid Library Reveals Tissue-Specific Preferences for Regulatory T Cell Uptake Through Click-Chemistry

The activation of T-cells is heavily shaped by the nutrients that are available during activation. Fatty acids can have highly pleiotropic effects in this process. On the one hand, they are essential for driving T-cell activation, yet on the other hand they can affect curtailed T-cell activation. These differences are likely dependent on the nature and absolute uptake of the fatty acids. Quantifying the uptake of specific FAs by individual cells and studying the effect on the phenotype of the cell is currently not possible with existing tools. Here we therefore use the live-cell compatible Inverse Electron-Demand Diels-Alder reaction combined with the synthesis of saturated, unsaturated and polyunsaturated fatty acids carrying the 1-carbon cyclopropene click group. Single cell uptake studies of these various clickable FAs in primary immune cell mixtures show highly divergent uptake behaviour between different immune cells, with polyunsaturated fatty acids markedly preferred by all immune cells tested.

immunology↗

bioRxiv2024

Oleic acid triggers CD4+ T cells to be metabolically rewired and poised to differentiate into proinflammatory T cell subsets upon activation

T cells are the most common immune cells in atherosclerotic plaques and the function of T cells can be altered by fatty acids. Here, we show that pre-exposure of CD4+ T cells to oleic acid, an abundant fatty acid linked to cardiovascular events, results in a preferential differentiation into pro-inflammatory subsets upon activation by upregulating core metabolic pathways. RNA-sequencing of non-activated CD4+ T cells revealed that oleic acid upregulates genes encoding enzymes responsible for cholesterol and fatty acid biosynthesis. Transcription footprint analysis linked this rewiring to the differentiation of pro-inflammatory subsets. Indeed, spectral flow cytometry showed that pre-exposure to oleic acid results in a skew toward IL-9, IL-17A, IL-5 and IL-13 producing T cells upon activation. Importantly, inhibition of either cholesterol or fatty acid biosynthesis abolishes this effect, suggesting a beneficial role for statins beyond cholesterol lowering. Taken together, fatty acids may affect inflammatory diseases by influencing T cell metabolism.

genomics↗

bioRxiv2023

Integration of Mass Cytometry and Mass Spectrometry Imaging for Spatially Resolved Single Cell Metabolic Profiling

Integration of spatial omics technologies can provide important insights into the biology of tissues. We combined mass spectrometry imaging-based metabolomics and imaging mass cytometry-based immunophenotyping on the same single tissue section to reveal metabolic heterogeneity within tissues and its association with specific cell populations like cancer cells or immune cells. This approach has the potential to greatly increase our understanding of tissue-level interplay between metabolic processes and their cellular components.

cancer biology↗

bioRxiv2023

A PD-1-ST2 axis controls Th2 effector function in tissue via a metabolic checkpoint

Type 2 immune responses characterise both helminth infections and atopic disease such as allergy or asthma, but a better understanding of the mechanisms that regulate these responses is key to improving therapeutic and vaccination strategies. Immuno-metabolic studies over the last two decades have suggested T cell activation broadly requires rapid increases in glycolysis and oxidative phosphorylation. In contrast, we show that CD4+ T helper 2 (Th2) cells activated in vivo, using models of helminth infection, do not acquire a glycolytic metabolism. Instead, we show that Th2 cells solely increase their oxidative metabolism, associated with increased fatty acid uptake. Rather than contributing directly to effector function, our data reveal that Th2 cells switch to fatty acid oxidation downstream of PD-1 signalling to promote expression of the IL-33 receptor (ST2). These data provide insight into the spatial regulation of T cell metabolism, and suggest that PD-1 blockade therapies may be effective in Th2 disorders.

immunology↗

bioRxiv2023

AMPK activation induces RALDHhigh tolerogenic dendritic cells through rewiring of glucose and lipid metabolism

It is well known that dendritic cell (DC) activation and function are underpinned by profound changes in cellular metabolism. Several studies indicate that the ability of DCs to promote tolerance is dependent on catabolic metabolism. The AMP-activated kinase (AMPK) is a central nutrient and energy sensor whose activation promotes catabolism while inhibiting ATP-consuming anabolic pathways. Yet the contribution of AMPK activation to DC tolerogenicity remains unknown. Here, we show that AMPK activation renders human monocyte-derived DCs tolerogenic as evidenced by an enhanced ability to drive differentiation of regulatory T cells, a process dependent on increased RALDH activity. This is accompanied by a number of distinct metabolic changes, in particular increased breakdown of glycerophospholipids, enhanced mitochondrial fission-dependent fatty acid oxidation, and upregulated glucose catabolism. This metabolic rewiring is functionally important as we found interference with these metabolic processes to reduce to various degrees AMPK-induced RALDH activity as well as the tolerogenic capacity of moDCs. Altogether, our findings reveal a key role for AMPK signaling in shaping DC tolerogenicity, and suggest that AMPK may serve as new target to direct DC-driven immune responses in therapeutic settings.

immunology↗

bioRxiv2023

Metabolic sensor AMPK licenses CD103+ dendritic cells to induce Treg responses

Dendritic cells (DCs) play a crucial role in promoting tolerance through priming of regulatory T cells (Treg). Several studies indicate DC tolerogenicity is dependent on catabolic metabolism. However, the role of AMP-activated Kinase (AMPK), a key energy and nutrient sensor driving catabolic metabolism, in this process is unclear. We found that human retinoic acid-induced tolerogenic CD103+ DCs (RA-DCs) display increased AMPK signaling. Interestingly, RA-DCs, but not vitamin-D3- or dexamethasone-induced tolerogenic DCs, required AMPK for Treg induction. Mechanistically, AMPK underpinned RA-driven tolerogenicity by promoting RALDH activity in a FoxO3-dependent manner. Correspondingly, mice deficient for AMPK in DCs (CD11c{Delta}AMPK1) harbored reduced frequencies of intestinal CD103+CD11b+ DCs with impaired RALDH activity. Importantly, upon infection with parasitic worm Schistosoma mansoni, that elicits strong Th2 and Treg responses, CD11c{Delta}AMPK1 mice showed a defect in Treg accumulation and concomitantly, displayed an impaired ability to control Type 2 immunity-driven granulomatous inflammation against the parasite eggs. Together, our findings identify AMPK as a key regulator of tolerance by CD103+ DCs. SummaryDendritic cells (DCs) are critical for inducing tolerance. However, how metabolic cues control their tolerogenicity is still poorly understood. Patente et al demonstrate that AMPK is crucial for Treg induction by retinoic acid-primed tolerogenic CD103+ DCs.

immunology↗

bioRxiv2022

Spectral flow cytometry reveals metabolic heterogeneity in tissue macrophages

Tissue-macrophage populations are constituted by a mosaic of phenotypes, yet new methods are needed to link metabolic status to the range of phenotypes in vivo. We therefore designed a high-dimensional panel for spectral flow cytometry to investigate the heterogeneity of tissue macrophage metabolism at steady-state, and their metabolic adaptation in response to infection. Distinct metabolic profiles were observed between tissue macrophages from different peripheral organs, as well as within populations from a specific site. As such, our data show multiple metabolic states in macrophages corresponding to relative stages of maturity in both the peritoneal cavity and small intestine. Immune perturbation with helminth infection further showed that peritoneal macrophages acquire an overall highly metabolically active profile, whereas responding intestinal macrophages displayed minimal changes in their metabolic phenotype. Thus, we demonstrate that high-dimensional, flow-based analysis is an exciting method to interrogate the metabolic heterogeneity and dynamics of tissue-macrophage populations.

immunology↗

bioRxiv2021

LKB1 signalling in dendritic cells controls whole-body metabolic homeostasis by limiting T helper 17 priming

Obesity-associated metaflammation drives the development of insulin resistance and type 2 diabetes, notably through modulating innate and adaptive immune cells in metabolic organs. The nutrient sensor liver kinase B1 (LKB1) has recently been shown to control cellular metabolism and T cell priming functions of dendritic cells (DCs). Here, we report that hepatic DCs from high-fat diet (HFD)-fed obese mice display increased LKB1 phosphorylation and that LKB1 deficiency in DCs (CD11c{Delta}LKB1) worsened HFD-driven hepatic steatosis, systemic insulin resistance and glucose intolerance. Loss of LKB1 in DCs was associated with increased cellular expression of Th17-polarizing cytokines and increased hepatic CD4+ IL-17A+ Th17 cells in HFD-fed mice. Importantly, IL-17A neutralization rescued metabolic perturbations in HFD-fed CD11c{Delta}LKB1 mice. Mechanistically, disrupted metabolic homeostasis was independent of the canonical LKB1-AMPK axis. Instead, we provide evidence for involvement of the AMPK-related salt-inducible kinase(s) in controlling Th17-polarizing cytokine expression in LKB1-deficient DCs. Altogether, our data reveal a key role for LKB1 signalling in DCs in protection against obesity-induced metabolic dysfunctions by limiting hepatic Th17 differentiation.

immunology↗

bioRxiv2020

Soluble mannose receptor induces pro-inflammatory macrophage activation andobesity-associated metaflammation

Pro-inflammatory activation of macrophages in metabolic tissues is critically important in induction of obesity-induced metaflammation. Here, we demonstrate that the soluble mannose receptor (sMR) plays a direct, functional role in both macrophage activation and metaflammation. We show that sMR binds CD45 on macrophages and inhibits its phosphatase activity, leading to a Src/Akt/NF-{kappa}B-mediated cellular reprogramming towards an inflammatory phenotype both in vitro and in vivo. Remarkably, increased serum sMR levels were observed in obese mice and humans and directly correlated with body weight. Additionally, MR deficiency lowers pro-inflammatory macrophages in metabolic tissues and protects against hepatic steatosis and whole-body metabolic dysfunctions in high-fat diet-induced obese mice. Conversely, administration of sMR in lean mice increases serum pro-inflammatory cytokines, activates tissue macrophages and promotes insulin resistance. Altogether, our results reveal sMR as novel regulator of pro-inflammatory macrophage activation which could constitute a new therapeutic target for metaflammation and other hyperinflammatory diseases.

immunology↗

bioRxiv2018

Functional genomics and programmed genome editing of omega-1 of the blood fluke Schistosoma mansoni

CRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 ({omega}1) of Schistosoma mansoni as a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of {omega}1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5-and 3-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within {omega}1. CRISPResso analysis of amplicons spanning the DSB revealed [~]4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding {omega}1 were reduced >80% and lysates of {omega}1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the {omega}1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of {omega}1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding {omega}1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of {omega}1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of {omega}1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

molecular biology↗