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Systemic inflammatory conditions are classically characterized by an acute hyperinflammatory phase,followed by a late immunosuppressive phase that elevates the susceptibility to secondary infections. Comprehensive mechanistic understanding of these phases is largely lacking. To address this gap,we leveraged a controlled,human in vivo model of lipopolysaccharide (LPS)-induced systemic inflammation encompassing both phases. Single-cell RNA sequencing during the acute hyperinflammatory phase identified an inflammatory CD163+SLC39A8+CALR+ monocyte-like subset (infMono) at 4 h post-LPS administration. The late immunosuppressive phase was characterized by diminished expression of type I interferon (IFN)-responsive genes in monocytes,impaired myelopoiesis and a pronounced attenuation of the immune response on a secondary LPS challenge 1 week after the first. The infMono gene program and impaired myelopoiesis were also detected in patient cohorts with bacterial sepsis and coronavirus disease. IFNβ treatment restored type-I IFN responses and proinflammatory cytokine production and induced monocyte maturation,suggesting a potential treatment option for immunosuppression. Stunnenberg et al. use a model of lipopolysaccharide injection in humans to characterize the transcriptomic landscape of bone marrow and blood immune cells during the hyperinflammatory and immunosuppressed phases of systemic inflammation. View Publication

The prognosis for adult B-cell acute lymphoblastic leukemia remains unfavorable,especially in the context of relapsed and refractory disease. Exploring the molecular mechanisms underlying disease progression holds significant promise for improving clinical outcomes. In this investigation,utilizing single-cell transcriptome sequencing technology,we discerned a correlation between Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) and the progression of B-cell acute lymphoblastic leukemia. Our findings reveal a significant upregulation of UHRF1 in cases of relapsed and refractory B-cell acute lymphoblastic leukemia,thereby serving as a prognostic indicator for poor outcomes. Both deletion of UHRF1 or overexpression of its downstream target secreted frizzled-related protein 5 (SFRP5) resulted in the inhibition of leukemia cell proliferation,promoting cellular apoptosis and induction of cell cycle arrest. Our results showed that UHRF1 employs methylation modifications to repress the expression of SFRP5,consequently inducing the WNT5A-P38 MAPK-HK2 signaling axis,resulting in the augmentation of lactate,the critical metabolic product of aerobic glycolysis. Furthermore,we identified UM164 as a targeted inhibitor of UHRF1 that substantially inhibits P38 protein phosphorylation,downregulates HK2 expression,and reduces lactate production. UM164 also demonstrated antileukemic activity both in vitro and in vivo. In summary,our investigation revealed the molecular mechanisms of epigenetic and metabolic reprogramming in relapsed and refractory B-cell acute lymphoblastic leukemia and provides potential targeted therapeutic strategies to improve its inadequate prognosis. The schematic model showed the regulator network of UHRF1-SFRP5-WNT5A-P38 MAPK-HK2 in B-ALL. View Publication

The development of hematopoietic cell lineages is a highly complex process governed by a delicate interplay of various transcription factors. The expression of these factors is influenced,in part,by epigenetic signatures that define each stage of cell differentiation. In particular,the formation of B lymphocytes depends on the sequential silencing of stemness genes and the balanced expression of interdependent transcription factors,along with DNA rearrangement. We have investigated the impact of Dido3 deficiency,a protein involved in chromatin status readout,on B cell differentiation within the hematopoietic compartment of mice. Our findings revealed significant impairments in the successive stages of B cell development. The absence of Dido3 resulted in remarkable alterations in the expression of essential transcription factors and differentiation markers,which are crucial for orchestrating the differentiation process. Additionally,the somatic recombination process,responsible for generation of antigen receptor diversity,was also adversely affected. These observations highlight the vital role of epigenetic regulation,particularly the involvement of Dido3,in ensuring proper B cell differentiation. This study reveals new mechanisms underlying disruptive alterations,deepening our understanding of hematopoiesis and may potentially lead to insights that aid in the development of therapeutic interventions for disorders involving aberrant B cell development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-025-01394-x. View Publication

Squalene-based emulsion (SE) adjuvants like MF59 and AS03 are used in protein subunit vaccines against influenza virus (e.g.,Fluad,Pandemrix,Arepanrix) and SARS-CoV-2 (e.g.,Covifenz,SKYCovione). We demonstrate the critical role of uric acid (UA),a damage-associated molecular pattern (DAMP),in triggering immunogenicity by SE adjuvants. In mice,SE adjuvants elevated DAMP levels in draining lymph nodes. Strikingly,inhibition of UA synthesis reduced vaccine-induced innate immunity,subsequently impairing optimal antibody and T cell responses. In vivo treatment with UA crystals elicited partial adjuvant effects. In vitro stimulation with UA crystals augmented the activation of dendritic cells (DCs) and B cells and altered multiple pathways in these cells,including inflammation and antigen presentation in DCs and cell proliferation in B cells. In an influenza vaccine model,UA contributed to protection against influenza viral infection. These results demonstrate the importance of DAMPs,specifically the versatile role of UA in the immunogenicity of SE adjuvants,by regulating DCs and B cells. View Publication

Extracellular DNA (eDNA) is crucial for the structural integrity of bacterial biofilms as they undergo transformation from B-DNA to Z-DNA as the biofilm matures. This transition to Z-DNA increases biofilm rigidity and prevents binding by canonical B-DNA-binding proteins,including nucleases. One of the primary defenses against bacterial infections are Neutrophil Extracellular Traps (NETs),wherein neutrophils release their own eDNA to trap and kill bacteria. Here we show that H-NS,a bacterial nucleoid associated protein (NAP) that is also released during biofilm development,is able to incapacitate NETs. Indeed,when exposed to human derived neutrophils,H-NS prevented the formation of NETs and lead to NET eDNA retraction in previously formed NETs. NETs that were exposed to H-NS also lost their ability to kill free-living bacteria which made H-NS an attractive therapeutic candidate for the control of NET-related human diseases. A model of H-NS release from biofilms and NET incapacitation is discussed. View Publication

Most cancer patients diagnosed with late-stage head and neck squamous cell carcinoma are treated with chemoradiotherapy,which can lead to toxicity. One potential alternative is tumor-limited conversion of a prodrug into its cytotoxic form. We reason this could be achieved by transient and tumor-specific expression of purine nucleoside phosphorylase (PNP),an Escherichia coli enzyme that converts fludarabine into 2-fluoroadenine,a potent cytotoxic drug. To efficiently express bacterial PNP in tumors,we evaluate 44 chemically distinct lipid nanoparticles (LNPs) using species-agnostic DNA barcoding in tumor-bearing mice. Our lead LNP,designated LNP intratumoral (LNPIT),delivers mRNA that leads to PNP expression in vivo. Additionally,in tumor cells transfected with LNPIT,we observe upregulated pathways related to RNA and protein metabolism,providing insight into the tumor cell response to LNPs in vivo. When mice are treated with LNPIT-PNP,then subsequently given fludarabine phosphate,we observe anti-tumor responses. These data are consistent with an approach in which LNP-mRNA expression of a bacterial enzyme activates a prodrug in solid tumors. Lipid nanoparticles (LNPs) delivering mRNA after intratumoral administration could be a promising cancer treatment strategy. Here this group reports the intratumoral delivery of mRNA with LNPs inducing the expression of purine nucleoside phosphorylase and inhibiting the progression of head and neck squamous cell carcinoma in vivo. View Publication

Myocardial infarction (MI) is a major global health concern. Although myeloid cells are crucial for tissue repair in emergency haematopoiesis after MI,excessive myelopoiesis can exacerbate scarring and impair cardiac function. Bone marrow (BM) haematopoietic stem cells (HSCs) have the unique capability to replenish the haematopoietic system,but their role in emergency haematopoiesis after MI has not yet been established. Here we collected human sternal BM samples from over 150 cardiac surgery patients,selecting 49 with preserved cardiac function. We show that MI causes detrimental transcriptional and functional changes in human BM HSCs. Lineage tracing experiments suggest that HSCs are contributors of pro-inflammatory myeloid cells infiltrating cardiac tissue after MI. Therapeutically,enforcing HSC quiescence with the vitamin A metabolite 4-oxo-retinoic acid dampens inflammatory myelopoiesis,thereby modulating tissue remodelling and preserving long-term cardiac function after MI. Rettkowski,Romero-Mulero et al. show that myocardial infarction impacts bone marrow haematopoietic stem cells and leads to inflammatory myelopoiesis,which can be dampened by treatment with 4-oxo-retinoic acid,promoting cardiac recovery. View Publication

AbstractBackgroundChimeric antigen receptor (CAR) T-cell therapy depends on T cells that are genetically modified to recognize and attack cancer cells. Their effectiveness thus hinges on the functionality of a patient’s own T cells. Since CAR T-cell therapy is currently only approved for advanced cancers after at least one line of chemotherapy,we evaluated the potential negative effects of prior exposure to chemotherapy on T-cell functionality.MethodsWe studied T cells of two B-cell non-Hodgkin’s lymphoma patient cohorts,one collected before treatment (pre-therapy) and the other after one or more (median 3) lines of chemotherapy (post-therapy). Leveraging advanced multiparameter flow cytometry,single-cell RNA sequencing (scRNA-seq),whole-genome DNA methylation arrays and in vitro functionality testing of generated CAR T cells,we compared patient samples in their suitability for effective CAR T-cell therapy.ResultsWe discovered significant modifications in T-cell subsets and their transcriptional profiles secondary to chemotherapy exposure. Our analysis revealed a discernible shift towards phenotypically more differentiated T cells and an upregulation of markers indicative of T-cell exhaustion. Additionally,scRNA-seq and DNA methylation analyses revealed gene expression and epigenetic changes associated with diminished functionality in post-therapy T cells. Cytotoxicity assays demonstrated superior killing efficacy of CAR T cells derived from treatment-naïve patients compared with those with chemotherapy history.ConclusionsThese findings corroborate that employing T cells collected prior to frontline chemotherapy could enhance the effectiveness of CAR T-cell therapy and improve patient outcomes. View Publication

AbstractObjectivesWe performed multiparameter phenotyping of peripheral B cells in anti-Jo-1 antibody positive idiopathic inflammatory myopathies (IIM) to delineate disease-associated immunological profiles and the influence of B cells on disease activity.MethodsPurified B cells from peripheral blood mononuclear cells from 16 patients with anti-Jo-1 antibody positive IIM (7 with untreated active IIM,4 with active and treated IIM and 5 with inactive IIM) were analysed by multiparameter spectral flow cytometry. Dimensionality reduction and clustering analysis were applied to pre-gated CD19+B cells. Serum levels of 21 cytokines and anti-Jo-1 IgG autoantibodies were determined. All patients with IIM in this study were positive for anti-Jo-1 antibody.ResultsAnti-Jo-1 antibody levels correlated positively to disease activity. Flow cytometry demonstrated B-cell dysregulation with significantly lower CD73 expression on naïve,switched memory and double negative B cells in patients with active IIM. Clustering analysis further revealed expansions of CD73− IgM+naïve B cells and CD73− CD95+ switched memory B cells in active IIM. In unswitched memory B cells,CD73+CD21+ cells were decreased in active IIM. Patients with active IIM had significantly higher serum levels of B-cell activating factor,inducible protein-10,interleukin-6 and sCD40L which correlated with changes in B-cell populations.ConclusionsSince CD73 has an immunoregulatory function by modulating the ATP/adenosine pathway,which is also targeted by methotrexate,the low CD73 B-cell expression in anti-Jo-1 antibody-positive IIM may lead to B-cell hyperactivation. These novel findings further highlight B cells as central in the pathogenesis of IIM and important therapeutic targets. View Publication

BackgroundGraft-versus-host disease (GVHD) and relapse are major complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Metabolites play crucial roles in immune regulation,but their causal relationships with GVHD and relapse remain unclear.MethodsWe utilized genetic variants from genome-wide association studies (GWAS) of 309 known metabolites as instrumental variables to evaluate their causal effects on acute GVHD (aGVHD),gut GVHD,chronic GVHD (cGVHD),and relapse in different populations. Multiple causal inference methods,heterogeneity assessments,and pleiotropy tests were conducted to ensure result robustness. Multivariable MR analysis was performed to adjust for potential confounders,and validation MR analysis further confirmed key findings. Mediation MR analysis was employed to explore indirect causal pathways.ResultsAfter correction for multiple testing,we identified elevated pyridoxate and proline levels as protective factors against grade 3–4 aGVHD (aGVHD3) and relapse,respectively. Conversely,glycochenodeoxycholate increased the risk of aGVHD3,whereas 1-stearoylglycerophosphoethanolamine had a protective effect. The robustness and stability of these findings were confirmed by multiple causal inference approaches,heterogeneity,and horizontal pleiotropy analyses. Multivariable MR analysis further excluded potential confounding pleiotropic effects. Validation MR analyses supported the causal roles of pyridoxate and 1-stearoylglycerophosphoethanolamine,while mediation MR revealed that pyridoxate influences GVHD directly and indirectly via CD39 + Tregs. Pathway analyses highlighted critical biochemical alterations,including disruptions in bile acid metabolism and the regulatory roles of vitamin B6 derivatives. Finally,clinical metabolic analyses,including direct fecal metabolite measurements,confirmed the protective role of pyridoxate against aGVHD.ConclusionsOur findings provide novel insights into the metabolic mechanisms underlying GVHD and relapse after allo-HSCT. Identified metabolites,particularly pyridoxate,may serve as potential therapeutic targets for GVHD prevention and management.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12916-025-04026-w. Key points1. Strong evidence has found the protective effect of pyridoxate against aGVHD and has been validated in patients.2. Mediated MR analysis suggests that pyridoxate may reduce aGVHD risk by increasing CD39+ Tregs level.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12916-025-04026-w. View Publication

T cells have been reported to play critical roles in preventing of microsporidia dissemination. However,there roles and functions of each subset remain unclear. Here in the study,we performed a thorough analysis of murine splenic T-cell response analysis via single-cell RNA sequencing during microsporidia E. cuniculi infection. We demonstrated that Type I T helper (Th1) cells,T follicular helper (Tfh) cells,effector CD8 + T cells and proliferating CD8 + T cells were activated and expanded after infection. Activated Th1 cells and Tfh cells presented significantly upregulated gene expression of Ifng and Il21,respectively. A subcluster of Th1 cells with high Csf1 expression was detected after infection. Subsets of activated CD8 + T cells were markedly enriched with high expression of cytotoxic-function related genes such as Gzma and Gzmb,whereas some active CD8 T cells were enriched with proliferation-function related genes Mki67 and Stmn1. Other subsets of T cells including NK T cells,Myb+ T cells,γδ T cells and Cxcr6+ T cells,were also analyzed in this study yet no expansion was observed. In summary,our findings provide in-depth and comprehensive insights into T-cell responses during microsporidia infection,which will be valuable for further investigations. This study provides a comprehensive landscape of mouse T cells responses during microsporidia infection at a single-cell resolution reporting that Th1,Tfh,effector and proliferating CD8 + T cell subsets were activated and expanded upon infection. View Publication

Objective: Hypoxia–glycolysis–lactylation (HGL) may play a crucial role in neonatal sepsis (NS). This study aims to identify HGL marker genes in NS and explore immune microenvironment among NS subtypes. Materials and Methods: The gene expression dataset GSE69686,comprising 64 NS cases and 85 controls,was selected for analysis. Based on the screened HGL-related marker genes,diagnostic prediction models were constructed using nine machine learning algorithms,and molecular subtypes of NS were identified through consensus clustering. Subsequently,the heterogeneity of biological functions and immune cell infiltration among the different subtypes was analyzed. Finally,the marker genes and lactylation were validated using the GSE25504 dataset,clinical samples,and mouse neutrophil,respectively. Results: MERTK,HK3,PGK1,and STAT3 were identified and validated as marker genes,and the diagnostic prediction model for NS constructed using the support vector machine (SVM) algorithm exhibited optimal predictive performance. Based on gene expression patterns,two distinct NS subtypes were identified. Functional enrichment analysis highlighted significant immune-related pathways,while immune infiltration analysis revealed differences in neutrophil proportions between the subtypes. Furthermore,the expression levels of marker genes were positively correlated with neutrophil infiltration. Importantly,the experimental validation results were consistent with the findings from the bioinformatics analysis. Conclusion: This study identified the distinct NS subtypes and their associated marker genes. These findings will contribute to elucidating the disease's heterogeneity and establishing appropriate personalized therapeutic approaches. View Publication