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IntroductionCurrent antiretroviral therapy (ART) for HIV infection reduces plasma viral loads to undetectable levels and has increased the life expectancy of people with HIV (PWH). However,this increased lifespan is accompanied by signs of accelerated aging and a higher prevalence of age-related comorbidities. Tat (Trans-Activator of Transcription) is a key protein for viral replication and pathogenesis. Tat is encoded by 2 exons,with the full-length Tat ranging from 86 to 101 aa (Tat101). Introducing a stop codon in position 73 generates a 1 exon,synthetic 72aa Tat (Tat72). Intracellular,full-length Tat activates the NF-κB pro-inflammatory pathway and increases antiapoptotic signals and ROS generation. These effects may initiate a cellular senescence program,characterized by cell cycle arrest,altered cell metabolism,and increased senescence-associated secretory phenotype (SASP) mediator release However,the precise role of HIV-Tat in inducing a cellular senescence program in CD4+ T-cells is currently unknown.MethodsJurkat Tetoff cell lines stably transfected with Tat72,Tat101,or an empty vector were used. Flow cytometry and RT-qPCR were used to address senescence biomarkers,and 105 mediators were assessed in cell supernatants with an antibody-based membrane array. Key results obtained in Jurkat-Tat cells were addressed in primary,resting CD4+ T-cells by transient electroporation of HIV-Tat-FLAG plasmid DNA.ResultsIn the Jurkat cell model,expression of Tat101 increased the levels of the senescence biomarkers BCL-2,CD87,and p21,and increased the release of sCD30,PDGF-AA,and sCD31,among other factors. Tat101 upregulated CD30 and CD31 co-expression in the Jurkat cell surface,distinguishing these cells from Tat72 and Tetoff Jurkats. The percentage of p21+,p16+,and γ-H2AX+ cells were higher in Tat-expressing CD4+ T-cells,detected as a FLAG+ population compared to their FLAG- (Tat negative) counterparts. Increased levels of sCD31 and sCD26 were also detected in electroporated CD4+ T-cell supernatants.DiscussionIntracellular,full-length HIV-Tat expression increases several senescence biomarkers in Jurkat and CD4+ T-cells,and SASP/Aging mediators in cell supernatants. Intracellular HIV-Tat may initiate a cellular senescence program,contributing to the premature aging phenotype observed in PWH. Graphical Abstract View Publication

Tetracyclines are essential bacterial protein synthesis inhibitors under continual development to combat antibiotic resistance yet suffer from unwanted side effects. Mitoribosomes - responsible for generating oxidative phosphorylation (OXPHOS) subunits - share structural similarities with bacterial machinery and may suffer from cross-reactivity. Since lymphocytes rely upon OXPHOS upregulation to establish immunity,we set out to assess the impact of ribosome-targeting antibiotics on human T cells. We find tigecycline,a third-generation tetracycline,to be the most cytotoxic compound tested. In vitro,5–10 μM tigecycline inhibits mitochondrial but not cytosolic translation,mitochondrial complex I,III and IV expression,and curtails the activation and expansion of unique T cell subsets. By cryo-EM,we find tigecycline to occupy three sites on T cell mitoribosomes. In addition to the conserved A-site found in bacteria,tigecycline also attaches to the peptidyl transferase center of the large subunit. Furthermore,a third,distinct binding site on the large subunit,aligns with helices analogous to those in bacteria,albeit lacking methylation in humans. The data provide a mechanism to explain part of the anti-inflammatory effects of these drugs and inform antibiotic design. Tetracyclines impair cellular function by targeting ribosomes. Here,the authors demonstrate that tigecycline impairs T cell function by selectively inhibiting mitochondrial protein synthesis and uncover the structural basis for mitoribosome inhibition and its role in immunosuppression. View Publication

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