技术资料

AbstractNeurodegeneration in the autoimmune disease multiple sclerosis still poses a major therapeutic challenge. Effective drugs that target the inflammation can only partially reduce accumulation of neurological deficits and conversion to progressive disease forms. Diet and the associated gut microbiome are currently being discussed as crucial environmental risk factors that determine disease onset and subsequent progression. In people with multiple sclerosis,supplementation of the short-chain fatty acid propionic acid,as a microbial metabolite derived from the fermentation of a high-fiber diet,has previously been shown to regulate inflammation accompanied by neuroprotective properties. We set out to determine whether the neuroprotective impact of propionic acid is a direct mode of action of short-chain fatty acids on CNS neurons. We analysed neurite recovery in the presence of the short-chain fatty acid propionic acid and butyric acid in a reverse-translational disease-in-a-dish model of human-induced primary neurons differentiated from people with multiple sclerosis-derived induced pluripotent stem cells. We found that recovery of damaged neurites is induced by propionic acid and butyric acid. We could also show that administration of butyric acid is able to enhance propionic acid-associated neurite recovery. Whole-cell proteome analysis of induced primary neurons following recovery in the presence of propionic acid revealed abundant changes of protein groups that are associated with the chromatin assembly,translational,and metabolic processes. We further present evidence that these alterations in the chromatin assembly were associated with inhibition of histone deacetylase class I/II following both propionic acid and butyric acid treatment,mediated by free fatty acid receptor signalling. While neurite recovery in the presence of propionic acid is promoted by activation of the anti-oxidative response,administration of butyric acid increases neuronal ATP synthesis in people with multiple sclerosis-specific induced primary neurons. In human multiple sclerosis-specific neurons,differentiated via induced pluripotent stem cells,Gisevius et al. display neuroregeneration mediated by the short-chain fatty acids propionic and butyric acid. Intracellularly,free fatty acid receptor signalling leads to inhibition of histone deacetylase activity,thereby altering the oxidative stress response and cellular protein biosynthesis. Graphical Abstract Graphical Abstract View Publication

This study investigates the impact of sodium channel protein type 1 subunit alpha (SCN1A) gene knockout (SCN1A KO) on brain development and function using cerebral organoids coupled with a multiomics approach. From comprehensive omics analyses,we found that SCN1A KO organoids exhibit decreased growth,dysregulated neurotransmitter levels,and altered lipidomic,proteomic,and transcriptomic profiles compared to controls under matrix-free differentiation conditions. Neurochemical analysis reveals reduced levels of key neurotransmitters,and lipidomic analysis highlights changes in ether phospholipids and sphingomyelin. Furthermore,quantitative profiling of the SCN1A KO organoid proteome shows perturbations in cholesterol metabolism and sodium ion transportation,potentially affecting synaptic transmission. These findings suggest dysregulation of cholesterol metabolism and sodium ion transport,with implications for synaptic transmission. Overall,these insights shed light on the molecular mechanisms underlying SCN1A-associated disorders,such as Dravet syndrome,and offer potential avenues for therapeutic intervention. View Publication

Using live-cell microscopy,we find that loss of VPS13C in human neurons disrupts lysosomal morphology and dynamics with increased inter-lysosomal tethers,leading to impaired lysosomal motility and defective lysosomal function as well as a decreased phospho-Rab10-mediated lysosomal stress response. Loss-of-function mutations in VPS13C are linked to early-onset Parkinson’s disease (PD). While VPS13C has been previously studied in non-neuronal cells,the neuronal role of VPS13C in disease-relevant human dopaminergic neurons has not been elucidated. Using live-cell microscopy,we investigated the role of VPS13C in regulating lysosomal dynamics and function in human iPSC-derived dopaminergic neurons. Loss of VPS13C in dopaminergic neurons disrupts lysosomal morphology and dynamics with increased inter-lysosomal contacts,leading to impaired lysosomal motility and cellular distribution,as well as defective lysosomal hydrolytic activity and acidification. We identified Rab10 as a phospho-dependent interactor of VPS13C on lysosomes and observed a decreased phospho-Rab10-mediated lysosomal stress response upon loss of VPS13C. These findings highlight an important role of VPS13C in regulating lysosomal homeostasis in human dopaminergic neurons and suggest that disruptions in Rab10-mediated lysosomal stress response contribute to disease pathogenesis in VPS13C-linked PD. View Publication

Human induced pluripotent stem cells (hiPSCs) generate multiple clones with inherent heterogeneity,leading to variations in their differentiation capacity. Previous studies have primarily addressed line-to-line variations in differentiation capacity,leaving a gap in the comprehensive understanding of clonal heterogeneity. Here,we aimed to profile the heterogeneity of hiPSC clones and identify predictive biomarkers for cardiomyocyte (CM) differentiation capacity by integrating transcriptomic,epigenomic,endogenous retroelement,and protein kinase phosphorylation profiles. We generated multiple clones from a single donor and validated that these clones exhibited comparable levels of pluripotency markers. The clones were classified into two groups based on their differentiation efficiency to CMs—productive clone (PC) and non-productive clone (NPC). We performed RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq). NPC was enriched in vasculogenesis and cell adhesion,accompanied by elevated levels of phosphorylated ERK1/2. Conversely,PC exhibited enrichment in embryonic organ development and transcription factor activation,accompanied by increased chromatin accessibility near transcription start site (TSS) regions. Integrative analysis of RNA-seq and ATAC-seq revealed 14 candidate genes correlated with cardiac differentiation potential. Notably,TEK and SDR42E1 were upregulated in NPC. Our integrative profiles enhance the understanding of clonal heterogeneity and highlight two novel biomarkers associated with CM differentiation. This insight may facilitate the identification of suboptimal hiPSC clones,thereby mitigating adverse outcomes in clinical applications. View Publication

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart,lungs,kidney,and brain are all hallmarks of the disease,the most severe symptoms of TSC are often neurological,including seizures,autism,psychiatric disorders,and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented,it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes,we observed aberrant early neurodevelopment in vitro,including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions,including loci associated with key genes in neurodevelopment. Collectively,these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated,with implications for whether and how prenatal treatment should be pursued. View Publication

Regulated cell death is a key component of the innate immune response,which provides the first line of defense against infection and homeostatic perturbations. However,cell death can also drive pathogenesis. The most well-defined cell death pathways can be categorized as nonlytic (apoptosis) and lytic (pyroptosis,necroptosis,and PANoptosis). While specific triggers are known to induce each of these cell death pathways,it is unclear whether all cell types express the cell death proteins required to activate these pathways. Here,we assessed the protein expression and compared the responses of immune and non-immune cells of human and mouse origin to canonical pyroptotic (LPS plus ATP),apoptotic (staurosporine),necroptotic (TNF-α plus z-VAD),and PANoptotic (influenza A virus infection) stimuli. When compared to fibroblasts,both mouse and human innate immune cells,macrophages,expressed higher levels of cell death proteins and activated cell death effectors more robustly,including caspase-1,gasdermins,caspase-8,and RIPKs,in response to specific stimuli. Our findings highlight the importance of considering the cell type when examining the mechanisms regulating inflammation and cell death. Improved understanding of the cell types that contain the machinery to execute different forms of cell death and their link to innate immune responses is critical to identify new strategies to target these pathways in specific cellular populations for the treatment of infectious diseases,inflammatory disorders,and cancer. View Publication

Background:Throughout HIV infection,productively infected cells generate billions of viral particles and are thus responsible for body-wide HIV dissemination,but their phenotype during AIDS is unknown. As AIDS is associated with immunological changes,analyzing the phenotype of productively infected cells can help understand HIV production during this terminal stage.Methods:Blood samples from 15 untreated viremic participants (recent infection,n=5; long-term infection,n=5; active opportunistic AIDS-defining disease,n=5) and 5 participants virologically controlled on antiretroviral therapy (ART) enrolled in the Analysis of the Persistence,Reservoir and HIV Latency (APRIL) study (NCT05752318) were analyzed. Cells expressing the capsid protein p24 (p24+ cells) after 18 hours of resting or 24 hours of stimulation (HIV-Flow) revealed productively infected cells from viremic participants or translation-competent reservoir cells from treated participants,respectively.Results:The frequency of productively infected cells tended to be higher during AIDS in comparison with recent and long-term infections (median,340,72,and 32/million CD4+ T cells,respectively) and correlated with the plasma viral load at all stages of infection. Altogether,these cells were more frequently CD4low,HLA-ABClow,CD45RA-,Ki67+,PD-1+,with a non-negligible contribution from pTfh (CXCR5+PD-1+) cells,and were not significantly enriched in HIV coreceptors CCR5 nor CXCR4 expression. The comparison markers expression between stages showed that productively infected cells during AIDS were enriched in memory and exhausted cells. In contrast,the frequencies of infected pTfh were lower during AIDS compared to non-AIDS stages. A UMAP analysis revealed that total CD4+ T cells were grouped in 7 clusters and that productive p24+ cells were skewed to given clusters throughout the course of infection. Overall,the preferential targets of HIV during the latest stages seemed to be more frequently highly differentiated (memory,TTD-like) and exhausted cells and less frequently pTfh-like cells. In contrast,translation-competent reservoir cells were less frequent (5/million CD4+ T cells) and expressed more frequently HLA-ABC and less frequently PD-1.Conclusions:In long-term infection and AIDS,productively infected cells were differentiated and exhausted. This could indicate that cells with these given features are responsible for HIV production and dissemination in an immune dysfunction environment occurring during the last stages of infection. View Publication

SummaryMesenchymal stromal cells (MSCs) have been modified via genetic or pharmacological engineering into potent antigen-presenting cells-like capable of priming responding CD8 T cells. In this study,our screening of a variant library of Accum molecule revealed a molecule (A1) capable of eliciting antigen cross-presentation properties in MSCs. A1-reprogrammed MSCs (ARM) exhibited improved soluble antigen uptake and processing. Our comprehensive analysis,encompassing cross-presentation assays and molecular profiling,among other cellular investigations,elucidated A1’s impact on endosomal escape,reactive oxygen species production,and cytokine secretion. By evaluating ARM-based cellular vaccine in mouse models of lymphoma and melanoma,we observe significant therapeutic potency,particularly in allogeneic setting and in combination with anti-PD-1 immune checkpoint inhibitor. Overall,this study introduces a strong target for developing an antigen-adaptable vaccination platform,capable of synergizing with immune checkpoint blockers to trigger tumor regression,supporting further investigation of ARMs as an effective and versatile anti-cancer vaccine. Graphical abstract Highlights•Treatment with A1/antigen mix reprograms MSCs into antigen-presenting cells•The antigen cross-presenting ability of ARM cells require ROS and UPR•ARMs synergize with immune-checkpoint inhibitors in priming potent antitumoral activity Classification Description: Immunology; Pharmaceutical engineering; Cancer View Publication

Human milk oligosaccharides (HMOs) can modulate the intestinal barrier and regulate immune cells to favor the maturation of the infant intestinal tract and immune system,but the precise functions of individual HMOs are unclear. To determine the structure-dependent effects of individual HMOs (representing different structural classes) on the intestinal epithelium as well as innate and adaptive immune cells,we assessed fucosylated (2′FL and 3FL),sialylated (3′SL and 6′SL) and neutral non-fucosylated (LNT and LNT2) HMOs for their ability to support intestinal barrier integrity,to stimulate the secretion of chemokines from intestinal epithelial cells,and to modulate cytokine release from LPS-activated dendritic cells (DCs),M1 macrophages (MØs),and co-cultures with naïve CD4+ T cells. The fucosylated and neutral non-fucosylated HMOs increased barrier integrity and protected the barrier following an inflammatory insult but exerted minimal immunomodulatory activity. The sialylated HMOs enhanced the secretion of CXCL10,CCL20 and CXCL8 from intestinal epithelial cells,promoted the secretion of several cytokines (including IL-10,IL-12p70 and IL-23) from LPS-activated DCs and M1 MØs,and increased the secretion of IFN-γ and IL-17A from CD4+ T cells primed by LPS-activated DCs and MØs while reducing the secretion of IL-13. Thus,3′SL and 6′SL supported Th1 and Th17 responses while reducing Th2 responses. Collectively,our data show that HMOs exert structure-dependent effects on the intestinal epithelium and possess immunomodulatory properties that confer benefits to infants and possibly also later in life. View Publication

SummaryHispanic/Latino children have the highest risk of acute lymphoblastic leukemia (ALL) in the US compared to other racial/ethnic groups,yet the basis of this remains incompletely understood. Through genetic fine-mapping analyses,we identified a new independent childhood ALL risk signal near IKZF1 in self-reported Hispanic/Latino individuals,but not in non-Hispanic White individuals,with an effect size of ∼1.44 (95% confidence interval = 1.33–1.55) and a risk allele frequency of ∼18% in Hispanic/Latino populations and <0.5% in European populations. This risk allele was positively associated with Indigenous American ancestry,showed evidence of selection in human history,and was associated with reduced IKZF1 expression. We identified a putative causal variant in a downstream enhancer that is most active in pro-B cells and interacts with the IKZF1 promoter. This variant disrupts IKZF1 autoregulation at this enhancer and results in reduced enhancer activity in B cell progenitors. Our study reveals a genetic basis for the increased ALL risk in Hispanic/Latino children. Graphical abstract Highlights•IKZF1 variants contribute to the increased risk of ALL in Hispanic/Latino children•Risk allele is associated with Indigenous American ancestry and underwent selection•Risk variant impacts IKZF1 enhancer that is selectively active in B cell development•Risk allele reduces enhancer activity,chromatin accessibility,and IKZF1 expression Genetic fine-mapping across the IKZF1 gene revealed a variant associated with childhood ALL that contributes to the increased risk of this disease in Hispanic/Latino individuals. The ALL risk allele reduces enhancer activity and IKZF1 expression specifically in B cell progenitors,likely resulting in stalled B cell development and an increased risk of ALL. View Publication

SummaryMonocytes (Mos) are crucial in the evolution of metabolic dysfunction-associated steatotic liver disease (MASLD) to metabolic dysfunction-associated steatohepatitis (MASH),and immunometabolism studies have recently suggested targeting leukocyte bioenergetics in inflammatory diseases. Here,we reveal a peculiar bioenergetic phenotype in circulating Mos of patients with MASH,characterized by high levels of glycolysis and mitochondrial (mt) respiration. The enhancement of mt respiratory chain activity,especially complex II (succinate dehydrogenase [SDH]),is unbalanced toward the production of reactive oxygen species (ROS) and is sustained at the transcriptional level with the involvement of the AMPK-mTOR-PGC-1α axis. The modulation of mt activity with dimethyl malonate (DMM),an SDH inhibitor,restores the metabolic profile and almost abrogates cytokine production. Analysis of a public single-cell RNA sequencing (scRNA-seq) dataset confirms that in murine models of MASH,liver Mo-derived macrophages exhibit an upregulation of mt and glycolytic energy pathways. Accordingly,the DMM injection in MASH mice contrasts Mo infiltration and macrophagic enrichment,suggesting immunometabolism as a potential target in MASH. Graphical abstract Highlights•Circulating monocytes (Mos) in patients with MASH show a bioenergetic reprogramming•SDH inhibition in vitro restores MASH Mo bioenergetics,abolishing cytokine production•In mice,energy pathways are upregulated in liver Mo-derived macrophages during MASH•SDH inhibition in vivo reduces Mo infiltration and differentiation in MASH Sangineto et al. investigate the bioenergetics and mitochondrial activity of circulating monocytes in patients with MASH,revealing a hypermetabolic state also identified in liver monocyte-derived macrophages through transcriptomic analysis. Immunometabolic modulation via SDH inhibition attenuates inflammation both in vitro and in vivo,ameliorating MASH. View Publication

T lymphocyte activation plays a pivotal role in adaptive immune response and alters the spatial organization of nuclear architecture that subsequently impacts transcription activities. Here,using stochastic optical reconstruction microscopy (STORM),we observe dramatic de-condensation of chromatin and the disruption of nuclear envelope at a nanoscale resolution upon T lymphocyte activation. Super-resolution imaging reveals that such alterations in nuclear architecture are accompanied by the release of nuclear DNA into the cytoplasm,correlating with the degree of chromatin decompaction within the nucleus. The authors show that under the influence of metabolism,T lymphocyte activation de-condenses chromatin,disrupts the nuclear envelope,and releases DNA into the cytoplasm. Taken together,this result provides a direct,molecular-scale insight into the alteration in nuclear architecture. It suggests the release of nuclear DNA into the cytoplasm as a general consequence of chromatin decompaction after lymphocyte activation. The authors show that under the influence of metabolism,T lymphocyte activation de-condenses chromatin,disrupts the nuclear envelope,and releases DNA into the cytoplasm. View Publication