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Ferroptosis plays an important role in the occurrence and development of many diseases, including neurodegenerative diseases. Thus, ferroptosis inhibitors able to cross the blood-brain barrier may have therapeutic potential. The best ferroptosis inhibitors so far are lipophilic radical trapping antioxidants (RTAs) that block lipid peroxidation in membranes. Several generations of ferrostatins have been synthesized, among which UAMC-3203 showed high potency in animal models with improved properties compared to ferrostatin-1. To further improve its pharmacokinetics properties, drug-likeness, and permeability, we modified UAMC-3203 by decreasing the size of the molecule and reducing its polarity by replacing the sulfonamide first by amide groups and subsequently by isosteric oxazoles. Herein, we present the design, synthesis, and biological evaluation of a novel series of oxazole RTAs with high potency, excellent oral bioavailability, and high concentrations in brain tissue.

The discovery of diverse molecular mechanisms of regulated cell death has opened new avenues for cancer therapy. Ferroptosis, a unique form of cell death driven by iron-catalyzed peroxidation of membrane phospholipids, holds particular promise for targeting resistant cancer types. This review critically examines current literature on ferroptosis, focusing on its defining features and therapeutic potential. We discuss how molecular profiling of tumors and liquid biopsies can generate extensive multi-omics datasets, which can be leveraged through machine learning-based analytical approaches for patient stratification. Addressing these challenges is essential for advancing the clinical integration of ferroptosis-driven treatments in cancer care., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Regeneration of small intestinal mucosal tissue could offer a promising strategy for Crohn's disease patients suffering from chronic inflammatory damage. Here, we aimed to develop hydrogels that mirror the villi and crypts of the small intestine and exhibit a physiological stiffness of G' ~ 1.52 kPa. For this purpose, we developed gelatin-methacryloyl-aminoethyl-methacrylate (gel-MA-AEMA)-, and gelatin-methacryloyl-norbornene (gel-MA-NB)-based biomaterial inks to fabricate 3D hydrogels ("villi only" versus "crypts and villi") with digital light processing (DLP) and co-cultured Caco-2/HT29-MTX cells. Gel-MA-AEMA was selected for its higher amount of methacrylates which was hypothesized to provide superior photo-crosslinking kinetics and hence superior DLP fabrication potential while gel-MA-NB was evaluated for its selective functionalization potential with thiolated bioactive compounds following DLP processing, resulting from its incorporated NB moieties which remain unreacted during the DLP process. Both gel-MA-AEMA-, and gel-MA-NB-based hydrogels exhibited a physiologically relevant stiffness, but only the gel-MA-AEMA-based biomaterial ink could be successfully utilized for printing hydrogels encompassing villi and crypts. Paracellular permeability of small sized marker molecules in combination with transepithelial electrical resistance measurements showed the formation of a functional barrier over time on all hydrogel constructs. Transmission electron microscopy and enterocyte differentiation marker genes' expression levels revealed the superior differentiation of Caco-2 on the 3D constructs compared to 2D hydrogel sheets. In summary, while both hydrogels enhanced functional barrier formation and enterocyte differentiation, gel-MA-AEMA proved more conducive to DLP compared to gel-MA-NB. Furthermore, our study underscored the benefits of cultivating intestinal cells on soft 3D constructs, enhancing cell barrier properties and differentiation, thus providing added value over traditional 2D supports., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sandra Van Vlierberghe is a co-founder of the companies BIO INX BV and 4Tissue BV, but the author declares to have no known financial interest that could have appeared to influence the work reported in this paper. The other authors declare to have no competing financial or commercial interest or personal relationship that could have appeared to influence the work reported in this manuscript., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Human inborn errors of immunity (IEI) represent a diverse group of genetic disorders affecting the innate and/or adaptive immune system. Some IEI entities comprise defects in DNA repair factors, resulting in (severe) combined immunodeficiencies, bone marrow failure, predisposition to malignancies, and potentially resulting in radiosensitivity (RS). While other IEI subcategories such as common variable immunodeficiency (CVID) and immune dysregulation disorders also associate with lymphoproliferative and malignant complications, the occurrence of RS phenotypes in the broader IEI population is not well characterized. Nonetheless, identifying RS in IEI patients through functional testing is crucial to reconsider radiation-related therapeutic protocols and to improve overall patient management. This study aimed to investigate chromosomal RS in a diverse cohort of 107 IEI patients using the G0 cytokinesis-block micronucleus (MN) assay. Our findings indicate significant variability in RS across specific genetic and phenotypical subgroups. Severe RS was detected in all ataxia-telangiectasia (AT) patients, a FANCI deficient and ERCC6L2 deficient patient, but not in any other IEI patient included in this cohort. Age emerged as an influencing factor for both spontaneous and radiation-induced MN yields, while the manifestation of additional clinical features, including infection susceptibility, immune dysregulation, or malignancies did not associate with increased MN levels. Our extensive analysis of RS in the IEI population underscores the clinical importance of RS assessment in AT patients and supports RS testing in all IEI patients suspected of having a DNA repair disorder associated with RS., Competing Interests: Declarations. Ethics Approval: This study was reviewed and approved by the Ethics Committee of the Ghent University Hospital (reference no. 2012/593, 2019/0461 and 2019/1565). Written informed consent was obtained from all participants in this study, in accordance with the 1975 Helsinki Declaration. Conflicts of Interest: The authors declare no competing interests., (© 2025. The Author(s).)

Our understanding of the functional heterogeneity of resident versus recruited macrophages in the diseased liver is limited. A population of recruited lipid-associated macrophages (LAMs) has been reported to populate the diseased liver alongside resident Kupffer cells (KCs). However, the precise roles of these distinct macrophage subsets remain elusive. Here, using proteogenomics, we have identified LAMs in multiple models of liver injury. Moreover, we found that this phenotype is not specific to recruited macrophages, as a subset of resident KCs can also adopt a LAM-like phenotype in the mouse and human liver. By combining genetic mouse models targeting the distinct populations, we determined that both recruited LAMs and resident LAM-like KCs play crucial roles in tissue repair. Specifically, triggering receptor expressed on myeloid cells 2 (TREM2) expression on either resident or recruited macrophages is required for the efficient clearance of dying cells, enhancing repair and preventing exacerbated fibrosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

Parasitic helminths induce the production of interleukin-4 (IL-4), which causes the expansion of virtual memory CD8 + T cells (T VM cells), a cell subset that contributes to the control of coinfection with intracellular pathogens. However, the mechanisms regulating IL-4-dependent T VM cell activation and expansion remain ill defined. Here, we used single-cell RNA sequencing of CD8 + T cells to identify pathways that control IL-4-dependent T VM cell responses. Gene signature analysis of CD8 + T cells identified a cell cluster marked by CD22, a canonical regulator of B cell activation, as a selective surface marker of IL-4-induced T VM cells. CD22 + T VM cells were enriched for interferon-γ and granzyme A and retained a diverse TCR repertoire while enriched in self-reactive CDR3 sequences. CD22 intrinsically regulated the IL-4-induced CD8 + T cell effector program, resulting in reduced responsiveness of CD22 + T VM cells and regulatory functions to infection and inflammation. Thus, helminth-induced IL-4 drives the expansion and activation of T VM cells that is counterinhibited by CD22.

Introduction: In mammals, Paneth cells, located in the crypts of the small intestine, produceantimicrobial peptides that serve to keep the intestinal microbiome under control. a-Defensins are the primary antimicrobial peptides produced by these cells., Methods: We used 148 publicly available bulk RNA-seq samples on purified PCs, proteomics on enriched purified PC proteins and Defa peptide activity assays to detect all Defa transcrips, including potential chimeric transcrips., Results: We identified 28 expressed Defa genes in mice, with up to 85% of Paneth cell RNA reads mapping to these genes. Chimeric mRNAs, involving sequences from two different Defa genes, were detected in most experiments. Despite their low abundance (less than 0.3%), mass spectrometry confirmed the presence of chimeric peptides. Synthetic versions of these peptides demonstrated antibacterial activity against multiple bacterial species., Conclusion: We show the existence of chimeric Defa transcripts and peptides in mice that are biologically active. We propose a possible stochatic mechanism or that the activation of the UPR patway may play a role in their production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Timmermans, Wallaeys, De Beul, Garcia-Gonzales and Libert.)

Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward proteome analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain abundant proteins at concentrations that vastly exceed the concentrations of proteins found in the EV proteome. Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion, and precipitation methods. Here, we describe filter-aided extracellular vesicle enrichment (FAEVEr) as an approach based on 300 kDa molecular weight cutoff filtration that allows the processing of multiple samples in parallel within a reasonable time frame and at moderate cost. We demonstrate that FAEVEr is capable of quantitatively retaining EV particles on filters, while allowing extensive washing with the mild detergent Tween-20 to remove interfering non-EV proteins. The retained particles are directly lysed on the filter for a complete recovery of the EV protein cargo toward proteome analysis. Here, we validate and optimize FAEVEr on recombinant EV material and apply it on conditioned medium as well as on complex bovine serum, human plasma, and urine. Our results indicate that EVs isolated from MCF7 cells cultured with or without serum have a drastic different proteome because of nutrient deprivation., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

Prenatal radiation-induced DNA damage poses a significant threat to neurodevelopment, resulting in microcephaly which primarily affects the cerebral cortex. So far, mechanistic studies were done in rodents. Here, we leveraged human cortical organoids to model fetal corticogenesis. Organoids were X-irradiated with moderate or high doses at different time points. Irradiation caused a dose- and time-dependent reduction in organoid size, which was more prominent in younger organoids. This coincided with a delayed and attenuated DNA damage response (DDR) in older organoids. Besides the DDR, radiation induced premature differentiation of neural progenitor cells (NPCs). Our transcriptomic analysis demonstrated a concerted p53-E2F4/DREAM-dependent repression of primary microcephaly genes, which was independently confirmed in cultured human NPCs and neurons. This was a human-specific feature, as it was not observed in mouse embryonic brains or primary NPCs. Thus, human cortical organoids are an excellent model for DNA damage-induced microcephaly and to uncover potentially targetable human-specific pathways., Competing Interests: The authors declare no competing interests., (© 2025 The Author(s).)

Ataxia telangiectasia and Rad3-related (ATR) kinase and its interacting protein ATRIP orchestrate the replication stress response. Homozygous splice variants in the ATRIP gene, resulting in ATRIP deficiency, were identified in two patients of independent ancestry with microcephaly, primordial dwarfism, and recurrent infections. The c.829+5G>T patient exhibited lymphopenia, poor vaccine responses, autoimmune features with hemolytic anemia, and neutropenia. Immunophenotyping revealed reduced CD16+/CD56dim NK cells and absent naïve T cells, MAIT cells, and iNKT cells. Lymphocytic defects were characterized by TCR oligoclonality, abnormal class switch recombination, and impaired T cell proliferation. ATRIP deficiency resulted in low-grade ATR activation but impaired CHK1 phosphorylation under genotoxic stress. ATRIP-deficient cells inadequately regulated DNA replication, leading to chromosomal instability, compromised cell cycle control, and impaired cell viability. CRISPR-SelectTIME confirmed reduced cell fitness for both variants. This study establishes ATRIP deficiency as a monogenic cause of microcephalic primordial dwarfism, highlights ATRIP's critical role in protecting immune cells from replication stress, and offers new insights into its canonical functions., (© 2025 Duthoo et al.)

The purpose of this review is to describe the immune function of the liver, guiding the reader from the homeostatic tolerogenic status to the aberrant activation demonstrated in chronic liver disease. An extensive description of the pathways behind the inflammatory modulation of the healthy liver will be provided focusing on the complex immune cell network residing within the liver. The limit of tolerance will be presented in the context of organ transplantation, seizing the limits of homeostatic mechanisms that fail in accepting the graft, progressing eventually toward rejection. The triggers and mechanisms behind chronic activation in metabolic liver conditions and viral hepatitis will be discussed. The last part of the review will be dedicated to one of the greatest paradoxes for a tolerogenic organ, developing autoimmunity. Through the description of the three most common autoimmune liver diseases, the autoimmune reaction against hepatocytes and biliary epithelial cells will be dissected.

Extracellular vesicles (EVs) are small membranous carriers of protein, lipid, and nucleic acid cargoes and play a key role in intercellular communication. Recent work has revealed the previously under-recognized participation of endoplasmic reticulum (ER)-associated proteins (ERAPs) during EV secretion, using pathways reminiscent of viral replication and secretion. Here, we present highlights of the literature involving ER/ERAPs in EV biogenesis and propose mechanistic parallels with ERAPs exploited during viral infections. We propose that ERAPs play an active role in the release of EVs and viral particles, and we present views on whether viruses hijack or enhance pre-existing ERAP-dependent secretory machineries or whether they repurpose ERAPs to create new secretory pathways., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: Over the past few decades, there has been a sudden rise in the incidence of Multiple Sclerosis (MS) in Western countries. However, current treatments often show limited efficacy in certain patients and are associated with adverse effects, which highlights the need for safer and more effective therapeutic approaches. Environmental factors, particularly dietary habits, have been observed to play a substantial role in the development of MS. In this study, we are the first to investigate the potential protective effect of the phytohormone abscisic acid (ABA) in MS. ABA, which is abundant in fruits such as figs, apricots and bilberries, is known to cross the blood-brain barrier and has demonstrated neuroprotective effects in conditions like depression and Alzheimer's disease., Methods: In this study, we investigated whether ABA supplementation enhances remyelination in both ex vivo and in vivo mouse models., Results: Our results indicated that ABA enhanced remyelination and that this enhanced remyelination is associated with increased lipid droplet load, reduced levels of degraded myelin, and a higher abundance of F4/80+ cells in the demyelinated brain of mice treated with ABA. In in vitro models, we further demonstrated that ABA treatment elevates lipid droplet formation by enhancing the phagocytic capacity of macrophages. Additionally, in a mouse model of microglial activation, we showed that ABA-treated mice maintain a less inflammatory microglial phenotype., Conclusion: Our findings highlight a crucial role for macrophages and microglia in enabling ABA to enhance the remyelination process. Furthermore, ABA's ability to improve remyelination together with its ability to reduce microglial activation, make ABA a promising candidate for modulating macrophage phenotype and reducing neuroinflammation in MS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Van Gaever, Mingneau, Vanherle, Driege, Haegman, Van Wonterghem, Xie, Vandenbroucke, Hendriks, Beyaert and Staal.)

Hepatic steatosis is a central phenotype in multi-system metabolic dysfunction and is increasing in parallel with the obesity pandemic. We use a translational approach integrating clinical phenotyping and outcomes, circulating proteomics, and tissue transcriptomics to identify dynamic, functional biomarkers of hepatic steatosis. Using multi-modality imaging and broad proteomic profiling, we identify proteins implicated in the progression of hepatic steatosis that are largely encoded by genes enriched at the transcriptional level in the human liver. These transcripts are differentially expressed across areas of steatosis in spatial transcriptomics, and several are dynamic during stages of steatosis. Circulating multi-protein signatures of steatosis strongly associate with fatty liver disease and multi-system metabolic outcomes. Using a humanized "liver-on-a-chip" model, we induce hepatic steatosis, confirming cell-specific expression of prioritized targets. These results underscore the utility of this approach to identify a prognostic, functional, dynamic "liquid biopsy" of human liver, relevant to biomarker discovery and mechanistic research applications., Competing Interests: Declaration of interests R.S., J.E.B., and A.S.P. have filed for a patent relevant to the findings in this manuscript. R.S. is supported in part by grants from the National Institutes of Health (NIH) and the American Heart Association (AHA). R.S. has equity ownership in Thryv Therapeutics and has served as a consultant for Amgen and Cytokinetics. R.S. is a co-inventor on a patent for ex-RNA signatures of cardiac remodeling (not relevant to the current work), and other patents on proteomic signatures of fitness and lung disease. A.S.P. and E.C. are supported by the AHA Strategically Focused Research Network in Cardiometabolic Disease. J.F.K.S. and G.M. are employees of Emulate, Inc. (a maker of the liver-on-a-chip) and may hold equity interest in Emulate, Inc. S.D. holds a research grant from Bristol Myers Squibb, is a founder and holds equity in Switch Therapeutics, and is a founder and consultant and holds equity for Thryv Therapeutics. J.K. has served as a consultant to Gilead, Merck, ViiV Healthcare, and Janssen and also received research support from Gilead Sciences and Merck. R.K. is supported in part by grants from the NIH; has received grants from AstraZeneca, PneumRx/BTG, and Spiration; has received consulting fees from CVS Caremark, AstraZeneca, GlaxoSmithKline, and CSA Medical; and has received speaking fees from GlaxoSmithKline, AstraZeneca, and Boehringer Ingelheim. K.A. is supported by an AHA Career Development Award (#929347). J.A.F. serves as a consultant or advisory board member for Kynos Therapeutics, Resolution Therapeutics, Ipsen, River 2 Renal Corp., Stimuliver, Global Clinical Trial Partners, and Guidepoint and has received speaker’s fees from HistoIndex and research grant funding from GlaxoSmithKline, Intercept Pharmaceuticals, and Genentech. T.J.K. undertakes consultancy work for Perspectum, Clinnovate Health, Kynos Therapeutics, Fibrofind, HistoIndex, Concept Life Sciences, and Resolution Therapeutics and has received speaker’s fees from Incyte Corporation and Servier Laboratories. K.V.K.-J. is a member of the scientific advisory board at Dyrnamix. J.J.C. receives project funding from GE Healthcare, Siemens Healthineers, TheraTech, and the NIH. M.N. has received speaking honoraria from Cytokinetics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abstract: Hepatic encephalopathy, defined as neuropsychiatric dysfunction secondary to liver disease, is a frequent decompensating event in cirrhosis. Its clinical impact is highlighted by a notable increase in patient mortality rates and a concomitant reduction in overall quality of life. Systemically, liver disease, liver function failure, portosystemic shunting, and associated multi-organ dysfunction result in the increase of disease-causing neurotoxins in the circulation, which impairs cerebral homeostasis. Key circulating neurotoxins are ammonia and inflammatory mediators. In the brain, pathophysiology is less well understood, but is thought to be driven by glial cell dysfunction. Astrocytes are the only brain resident cells that have ammonia-metabolizing machinery and are therefore putatively most susceptible to ammonia elevation. Based on a large body of mostly in vitro evidence, ammonia-induced cellular and molecular disturbances include astrocyte swelling and oxidative stress. Microglia, the brain resident macrophages, have been linked to the translation of systemic inflammation to the brain microenvironment. Recent evidence from animal studies has provided novel insights into old and new downstream effects of astrocyte and microglial dysfunction such as toxin clearance disruption and myeloid cell attraction to the central nervous system parenchyma. Furthermore, state of the art research increasingly implicates neuronal dysfunction and possibly even irreversible neuronal cell death. Cell-type specific investigation in animal models highlights the need for critical revision of the contribution of astrocytes and microglia to well- established and novel cellular and molecular alterations in hepatic encephalopathy. In this review, we therefore give a current and comprehensive overview of causes, features, and consequences of astrocyte and microglial dysfunction in hepatic encephalopathy, including areas of interest for future investigation., (Copyright © 2025 Neural Regeneration Research.)

Background : Small vulnerable newborns (SVNs), including those born preterm, small for gestational age, or with low birth weight, are at higher risk of neonatal mortality and long-term health complications. Early exposure to maternal vaginal microbiota and breastfeeding plays a critical role in the development of the neonatal microbiota and immune system, especially in low-resource settings like Burkina Faso, where neonatal mortality rates remain high. Objectives : The DenBalo study aims to investigate the role of maternal and neonatal factors, such as vaginal and gut microbiota, immune development, and early nutrition, in shaping health outcomes in SVNs and healthy infants. Methods : This prospective cohort observational study will recruit 141 mother-infant pairs (70 SVNs and 71 healthy controls) from four health centers in Bobo-Dioulasso, Burkina Faso. The mother-infant pairs will be followed for six months with anthropometric measurements and biospecimen collections, including blood, breast milk, saliva, stool, vaginal swabs, and placental biopsies. Multi-omics approaches, encompassing metagenomics, metabolomics, proteomics, and immune profiling, will be used to assess vaginal and gut microbiota composition and functionality, immune cell maturation, and cytokine levels at critical developmental stages. Conclusions : This study will generate comprehensive data on how microbiota, metabolomic, and proteomic profiles, along with immune system development, differ between SVNs and healthy infants. These findings will guide targeted interventions to improve neonatal health outcomes and reduce mortality, particularly in vulnerable populations.

To use phages in a personalized therapy and industrial applications, an accurate quantification is needed. The gold standard method, namely the culture-based double agar overlay (DAO) method, provides an accurate estimate of the number of infectious phages but is laborious and time-intensive. Quantitative polymerase chain reaction (qPCR) can be used as a fast alternative but tends to overestimate the number of infectious phage particles. Here we describe the use of a DNase treatment before quantification of the Staphylococcus aureus phage ISP with qPCR to obtain a more accurate estimate of the number of infectious phage particles. We showed that DNase treatment results in a significant decrease of the concentration when measured with qPCR although for two out of three tested ISP phage stocks, there was still a significant difference with the DAO method. We also showed that the discrepancy between quantification with qPCR and the DAO method is dependent on the storage period of the phage stock, with a larger discrepancy for older stocks. Additionally, we used negative contrast immune electron microscopy to confirm the presence of DNA in the medium of the phage stock and the impact of the DNase treatment on the free DNA., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Van Overfelt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Asthma is characterized by lung eosinophilia, remodeling, and mucus plugging, controlled by adaptive Th2 effector cells secreting IL-4, IL-5, and IL-13. Inhaled house dust mite (HDM) causes the release of barrier epithelial cytokines that activate various innate immune cells like DCs and basophils that can promote Th2 adaptive immunity directly or indirectly. Here, we show that basophils play a crucial role in the development of type 2 immunity and eosinophilic inflammation, mucus production, and bronchial hyperreactivity in response to HDM inhalation in C57Bl/6 mice. Interestingly, conditional depletion of basophils during sensitization did not reduce Th2 priming or asthma inception, whereas depletion during allergen challenge did. During the challenge of sensitized mice, basophil-intrinsic IL-33/ST2 signaling, and not FcεRI engagement, promoted basophil IL-4 production and subsequent Th2 cell recruitment to the lungs via vascular integrin expression. Basophil-intrinsic loss of the ubiquitin modifying molecule Tnfaip3, involved in dampening IL-33 signaling, enhanced key asthma features. Thus, IL-33-activated basophils are gatekeepers that boost allergic airway inflammation by controlling Th2 tissue entry., (© 2024 Schuijs et al.)

Background & Aims: Liver macrophages fulfill various homeostatic functions and represent an essential line of defense against pathogenic insults. However, it remains unclear whether a history of infectious disease in the liver leads to long-term alterations to the liver macrophage compartment., Methods: We utilized a curable model of parasitic infection invoked by the protozoan parasite Trypanosoma brucei brucei to investigate whether infection history can durably reshape hepatic macrophage identity and function. Employing a combination of fate mapping, single-cell CITE-sequencing, single-nuclei multiome analysis, epigenomic analysis, and functional assays, we studied the alterations to the liver macrophage compartment during and after the resolution of infection., Results: We show that T. brucei brucei infection alters the composition of liver-resident macrophages, leading to the infiltration of monocytes that differentiate into various infection-associated macrophage populations with divergent transcriptomic profiles. Whereas infection-associated macrophages disappear post-resolution of infection, monocyte-derived macrophages engraft in the liver, assume a Kupffer cell (KC)-like profile and co-exist with embryonic KCs in the long-term. Remarkably, the prior exposure to infection imprinted an altered transcriptional program on post-resolution KCs that was underpinned by an epigenetic remodeling of KC chromatin landscapes and a shift in KC ontogeny, along with transcriptional and epigenetic alterations in their niche cells. This reprogramming altered KC functions and was associated with increased resilience to a subsequent bacterial infection., Conclusion: Our study demonstrates that a prior exposure to a parasitic infection induces trained immunity in KCs, reshaping their identity and function in the long-term., Impact and Implications: Although the liver is frequently affected during infections, and despite housing a major population of resident macrophages known as Kupffer cells (KCs), it is currently unclear whether infections can durably alter KCs and their niche cells. Our study provides a comprehensive investigation into the long-term impact of a prior, cured parasitic infection, unveiling long-lasting ontogenic, epigenetic, transcriptomic and functional changes to KCs as well as KC niche cells, which may contribute to KC remodeling. Our data suggest that infection history may continuously reprogram KCs throughout life with potential implications for subsequent disease susceptibility in the liver, influencing preventive and therapeutic approaches., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4-expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T-cell differentiation. The mature T cells generated from primary human hematopoietic precursor cells were extensively characterized using single-cell RNA and combined T-cell receptor (TCR) sequencing. These predominantly CD8α T cells exhibit transcriptional and TCR CDR3 characteristics similar to the recently described human polyclonal αβ unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as IKZF2 (Helios), and the expression of various natural killer receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3-associated autoreactive features and early rearrangements of the TCR-α chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Regulated cell death is a fate of cells in (patho)physiological conditions during which extrinsic or intrinsic signals or redox equilibrium pathways following infection, cellular stress or injury are coupled to cell death modalities like apoptosis, necroptosis, pyroptosis or ferroptosis. An immediate survival response to cellular stress is often induction of autophagy, a process that deals with removal of aggregated proteins and damaged organelles by a lysosomal recycling process. These cellular processes and their regulation are crucial in several human diseases. Exploiting high-throughput assays which discriminate distinct cell death modalities and autophagy are critical to identify potential therapeutic agents that modulate these cellular responses. In the past few years, luciferase-based assays have been widely developed for assessing regulated cell death and autophagy pathways due to their simplicity, sensitivity, known chemistry, different spectral properties and high-throughput potential. Here, we review basic principles of bioluminescent reactions from a mechanistic perspective, along with their implication in vitro and in vivo for probing cell death and autophagy pathways. These include applying luciferase-, luciferin-, and ATP-based biosensors for investigating regulated cell death modalities. We discuss multiplex bioluminescence platforms which simultaneously distinguish between the various cell death phenomena and cellular stress recovery processes such as autophagy. We also highlight the recent technological achievements of bioluminescent tools for the prediction of drug effectiveness in pathways associated with regulated cell death.

Autoinflammatory diseases (AIDs) are a group of immunodysregulatory disorders resulting in the increased release or signaling of pro-inflammatory cytokines. Patients with AIDs present systemic inflammation in sterile conditions, which are mainly caused by defects in the innate immune system. Fever is one of the typical symptoms of this derailed immune signaling. In addition, autoinflammatory diseases manifest with varying other symptoms during flare-ups and interphasic periods. The diagnosis of these rare diseases poses numerous challenges. This paper provides an overview of AIDs that arise in childhood and in which fever commonly presents as a symptom. It outlines clinical signs, pathophysiology, diagnosis, and management for each syndrome. Additionally, we discuss a comprehensive diagnostic approach for children where an AID is suspected., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Meertens, Hoste, Tavernier and Haerynck.)

Patients with non-infectious systemic inflammation may suffer from one of many diseases, including hyperinflammation (HI), autoinflammatory disorders (AID), and systemic autoimmune disease (AI). Despite their clinical overlap, the pathophysiology and patient management differ between these disorders. We aimed to investigate blood biomarkers able to discriminate between patient groups. We included 44 patients with active clinical and/or genetic systemic inflammatory disease (9 HI, 27 AID, 8 systemic AI) and 16 healthy controls. We quantified 55 serum proteins and combined multiple machine learning algorithms to identify five proteins (CCL26, CXCL10, ICAM-1, IL-27, and SAA) that maximally separated patient groups. High ICAM-1 was associated with HI. AID was characterized by an increase in SAA and decrease in CXCL10 levels. A trend for higher CXCL10 and statistically lower SAA was observed in patients with systemic AI. Principal component analysis and unsupervised hierarchical clustering confirmed separation of disease groups. Logistic regression modelling revealed a high statistical significance for HI (P = 0.001), AID, and systemic AI (P < 0.0001). Predictive accuracy was excellent for systemic AI (AUC 0.94) and AID (0.91) and good for HI (0.81). Further research is needed to validate findings in a larger prospective cohort. Results will contribute to a better understanding of the pathophysiology of systemic inflammatory disorders and can improve diagnosis and patient management., Competing Interests: Declarations Competing Interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Extracellular vesicles (EVs) are pivotal in intercellular communication, impacting diverse physiological and pathological processes. Current in vitro EV biogenesis studies often utilize pharmacological inhibitors, inducing off-target effects and overlooking cell-specific production nuances. Addressing these limitations, we utilized CRISPR/Cas9 to generate heterozygous full-body and conditional sphingomyelin phosphodiesterase 3 (Smpd3) knockout (KO) transgenic mice. Smpd3 , also known as neutral sphingomyelinase 2 (nSMase2), triggers membrane curvature through sphingomyelin hydrolysis to ceramide, thereby influencing exosome release. Intriguingly, Smpd3 deficiency demonstrated no impact on EV release both in vitro and in vivo, underscoring its potential cell-type-specific role in EV biogenesis. Notably, bone marrow derived macrophages (BMDMs) did exhibit reduced EV release upon Alix deletion. Our findings open avenues for subsequent inquiries, enriching our knowledge of EV biogenesis and illuminating intercellular communication in health and disease., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Background: The long-term effectiveness of immunotherapies against Multiple Myeloma (MM) remains elusive, demonstrated by the inevitable relapse in patients. This underscores the urgent need for an in-depth analysis of the MM tumor-immune microenvironment (TME). Hereto, a representative immunocompetent MM mouse model can offer a valuable approach to study the dynamic changes within the MM-TME and to uncover potential resistance mechanisms hampering effective and durable therapeutic strategies in MM., Methods: We generated a comprehensive single-cell RNA-sequencing atlas of the MM-TME in bone marrow and spleen encompassing different stages of disease, using the immunocompetent 5T33MM mouse model. Through comparative analysis, we correlated our murine dataset with the pathogenesis in MM patients by reanalyzing publicly available datasets of human bone marrow samples across various disease stages. Using flow cytometry, we validated the dynamic changes upon disease progression in the 5T33MM model. Furthermore, interesting target populations, as well as the immune-boosting anti-CD40 agonist (αCD40) therapy were tested ex vivo on murine and human primary samples and in vivo using the 5T33MM model., Results: In this study, we identified the heterogenous and dynamic changes within the TME of murine and human MM. We found that the MM-TME was characterized by an increase in T cells, accompanied with an exhausted phenotype. Although neutrophils appeared to be rather innocuous at early disease stages, they acquired a pro-tumorigenic phenotype during MM progression. Moreover, conventional dendritic cells (cDCs) showed a less activated phenotype in MM, underscoring the potential of immune-boosting therapies such as αCD40 therapy. Importantly, we provided the first pre-clinical evaluation of αCD40 therapy and demonstrated successful induction of cDC- and T-cell activation, accompanied by a significant short-term anti-tumor response., Conclusions: This resource provides a comprehensive and detailed immune atlas of the evolution in human and murine MM disease progression. Our findings can contribute to immune-based patient stratification and facilitate the development of novel and durable (immune) therapeutic strategies in MM., (© 2024. The Author(s).)

Environmental enteric dysfunction (EED), a small intestinal disorder found at a high prevalence in stunted children, is associated with gut mucosal barrier disruption and decreased absorptive capacity due to reduced intact small intestinal villi 1-4 . To test the hypothesis that intergenerational transmission of a perturbed small intestinal microbiota contributes to undernutrition by inducing EED 5 , we characterized two consortia of bacterial strains cultured from duodenal aspirates from stunted Bangladeshi children with EED - one of which induced local and systemic inflammation in gnotobiotic female mice. Offspring of dams that received this inflammatory consortium exhibited immunologic changes along their gut that phenocopied features of EED in children. Single nucleus plus bulk RNA-sequencing revealed alterations in inter-cellular signaling pathways related to intestinal epithelial cell renewal, barrier integrity and immune function while analyses of cerebral cortex disclosed alterations in glial- and endothelial-neuronal signaling pathways that regulate neural growth/axonal guidance, angiogenesis and inflammation. Analysis of ultrasonic vocalization calls in gnotobiotic P5-P9 pups indicated increased arousal and perturbed neurodevelopment in the offspring of dams harboring the inflammation-inducing consortium. Cohousing experiments and follow-up screening of candidate disease-promoting bacterial isolates identified a strain typically found in the oral microbiota ( Campylobacter concisus ) as a contributor to enteropathy. Given that fetal growth was also impaired in the dams with the consortium that induced enteropathy, this preclinical model allows the effects of the human small intestinal microbiota on both pre- and postnatal development to be ascertained, setting the stage for identification of small intestinal microbiota-targeted therapeutics for (intergenerational) undernutrition., Competing Interests: Competing Interests - D.R. and A.O. are co-founders of Phenobiome Inc., a company pursuing development of computational tools for predictive phenotype profiling of microbial communities.

Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs. Abbreviations : BAF: bafilomycin A 1 ; CASP3: caspase 3; Caspases: cysteine-aspartic proteases; cCASP3: cleaved CASP3; CQ: chloroquine; CHX: cycloheximide; cPARP: cleaved poly (ADP-ribose) polymerase; DEPs: differential expressed proteins; ETO: etoposide; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain-like; MSC: mesenchymal stem cell; MTORC1: mechanistic target of rapamycin kinase complex 1; Nec1s: necrostatin 1s; NFKB/NF-kB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PLA: proximity ligation assay; RCD: regulated cell death; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RUBCNL/PACER: RUN and cysteine rich domain containing beclin 1 interacting protein like; si Ctrl : small interfering RNA nonsense; siRNA: small interfering RNA; TdT: terminal deoxynucleotidyl transferase; Tm: tunicamycin; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a.