技术资料

Hepatitis E virus (HEV) is a major cause of viral hepatitis worldwide. Pigs are the natural host of HEV genotype 3 and the main reservoir of HEV. As the host range of HEV genotype 3 expands,the possibility that HEV from various species can be transmitted to humans via pigs is increasing. We investigated the potential cross-species transmission of HEV by infecting minipigs with swine HEV (swHEV),rabbit HEV (rbHEV),and human HEV (huHEV) and examining their histopathological characteristics and distribution in various organs. Fifteen specific-pathogen-free Yucatan minipigs were infected with swHEV,rbHEV,huHEV,or a mock control. In the present study,we analysed faecal shedding,viremia,and serological parameters over a seven-week period. Our results indicated that swHEV exhibited more robust shedding and viremia than non-swHEVs. Only swHEV affected the serological parameters,suggesting strain-specific differences. Histopathological examination revealed distinct patterns in the liver,pancreas,intestine,and lymphoid tissues after infection with each HEV strain. Notably,all three HEVs induced histopathological changes in the pancreas,supporting the association of HEVs with acute pancreatitis. Our results also identified skeletal muscle as a site of HEV antigen presence,suggesting a potential link to myositis. In conclusion,this study provides valuable insights into the infection dynamics of different HEV strains in minipigs,emphasizing the strain-specific variations in virological,serological,and histological parameters. The observed differences in infection kinetics and tissue tropism will contribute to our understanding of HEV pathogenesis and the potential for cross-species transmission. View Publication

Humanized mouse models are valuable tools for investigating the human immune system in response to infection and injury. We have previously described the human immune system (HIS)-DRAGA mice (HLA-A2.HLA-DR4.Rag1KO.IL-2RgKO.NOD) generated by infusion of Human Leukocyte Antigen (HLA)-matched,human hematopoietic stem cells from umbilical cord blood. By reconstituting human cells,the HIS-DRAGA mouse model has been utilized as a “surrogate in vivo human model” for infectious diseases such as Human Immunodeficiency Virus (HIV),Influenza,Coronavirus Disease 2019 (COVID-19),scrub typhus,and malaria. This humanized mouse model bypasses ethical concerns about the use of fetal tissues for the humanization of laboratory animals. Here in,we demonstrate the presence of human microglia and T cells in the brain of HIS-DRAGA mice. Microglia are brain-resident macrophages that play pivotal roles against pathogens and cerebral damage,whereas the brain-resident T cells provide surveillance and defense against infections. Our findings suggest that the HIS-DRAGA mouse model offers unique advantages for studying the functions of human microglia and T cells in the brain during infections,degenerative disorders,tumors,and trauma,as well as for testing therapeutics in these pathological conditions. View Publication

BackgroundThe encoding of cell intrinsic drug resistance states in breast cancer reflects the contributions of genomic and non-genomic variations and requires accurate estimation of clonal fitness from co-measurement of transcriptomic and genomic data. Somatic copy number (CN) variation is the dominant mutational mechanism leading to transcriptional variation and notably contributes to platinum chemotherapy resistance cell states. Here,we deploy time series measurements of triple negative breast cancer (TNBC) single-cell transcriptomes,along with co-measured single-cell CN fitness,identifying genomic and transcriptomic mechanisms in drug-associated transcriptional cell states.ResultsWe present scRNA-seq data (53,641 filtered cells) from serial passaging TNBC patient-derived xenograft (PDX) experiments spanning 2.5 years,matched with genomic single-cell CN data from the same samples. Our findings reveal distinct clonal responses within TNBC tumors exposed to platinum. Clones with high drug fitness undergo clonal sweeps and show subtle transcriptional reversion,while those with weak fitness exhibit dynamic transcription upon drug withdrawal. Pathway analysis highlights convergence on epithelial-mesenchymal transition and cytokine signaling,associated with resistance. Furthermore,pseudotime analysis demonstrates hysteresis in transcriptional reversion,indicating generation of new intermediate transcriptional states upon platinum exposure.ConclusionsWithin a polyclonal tumor,clones with strong genotype-associated fitness under platinum remained fixed,minimizing transcriptional reversion upon drug withdrawal. Conversely,clones with weaker fitness display non-genomic transcriptional plasticity. This suggests CN-associated and CN-independent transcriptional states could both contribute to platinum resistance. The dominance of genomic or non-genomic mechanisms within polyclonal tumors has implications for drug sensitivity,restoration,and re-treatment strategies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-024-03318-3. View Publication

BackgroundHuman bone marrow mesenchymal stem cell (MSC) administration reduces inflammation in pre-clinical models of sepsis and sepsis-related lung injury,however clinical efficacy in patients has not yet been demonstrated. We previously showed that Alveolar Macrophage (AM) 11β-hydroxysteroid dehydrogenase type-1 (HSD-1) autocrine signalling is impaired in critically ill sepsis patients,which promotes inflammatory injury. Administration of transgenic MSCs (tMSCs) which overexpress HSD-1 may enhance the anti-inflammatory effects of local glucocorticoids and be more effective at reducing inflammation in sepsis than cellular therapy alone.MethodsMSCs were transfected using a recombinant lentiviral vector containing the HSD-1 and GPF transgenes under the control of a tetracycline promoter. Thin layer chromatography assessed HSD-1 reductase activity in tMSCs. Mesenchymal stem cell phenotype was assessed by flow cytometry and bi-lineage differentiation. HSD-1 tMSCs were co-cultured with LPS-stimulated monocyte-derived macrophages (MDMs) from healthy volunteers prior to assessment of pro-inflammatory cytokine release. HSD-1 tMSCs were administered intravenously to mice undergoing caecal ligation and puncture (CLP).ResultsMSCs were transfected with an efficiency of 91.1%,and maintained an MSC phenotype. Functional HSD-1 activity was demonstrated in tMSCs,with predominant reductase cortisol activation (peak 8.23 pM/hour/100,000 cells). HSD-1 tMSC co-culture with LPS-stimulated MDMs suppressed TNFα and IL-6 release. Administration of transgene activated HSD-1 tMSCs in a murine model of CLP attenuated neutrophilic inflammation more effectively than transgene inactive tMSCs (medians 0.403 v 1.36 × 106/ml,p = 0.033).ConclusionThe synergistic impact of HSD-1 transgene expression and MSC therapy attenuated neutrophilic inflammation in a mouse model of peritoneal sepsis more effectively than MSC therapy alone. Future studies investigating the anti-inflammatory capacity of HSD-1 tMSCs in models of sepsis-related direct lung injury and inflammatory diseases are required. View Publication

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the gut. There is growing evidence in Crohn’s disease (CD) of the existence of a preclinical period characterized by immunological changes preceding symptom onset that starts years before diagnosis. Gaining insight into this preclinical phase will allow disease prediction and prevention. Analysis of preclinical serum samples,up to 6 years before IBD diagnosis (from the PREDICTS cohort),revealed the identification of a unique glycosylation signature on circulating antibodies (IgGs) characterized by lower galactosylation levels of the IgG fragment crystallizable (Fc) domain that remained stable until disease diagnosis. This specific IgG2 Fc glycan trait correlated with increased levels of antimicrobial antibodies,specifically anti-Saccharomyces cerevisiae (ASCA),pinpointing a glycome–ASCA hub detected in serum that predates by years the development of CD. Mechanistically,we demonstrated that this agalactosylated glycoform of ASCA IgG,detected in the preclinical phase,elicits a proinflammatory immune pathway through the activation and reprogramming of innate immune cells,such as dendritic cells and natural killer cells,via an FcγR-dependent mechanism,triggering NF-κB and CARD9 signaling and leading to inflammasome activation. This proinflammatory role of ASCA was demonstrated to be dependent on mannose glycan recognition and galactosylation levels in the IgG Fc domain. The pathogenic properties of (anti-mannose) ASCA IgG were validated in vivo. Adoptive transfer of antibodies to mannan (ASCA) to recipient wild-type mice resulted in increased susceptibility to intestinal inflammation that was recovered in recipient FcγR-deficient mice. Here we identify a glycosylation signature in circulating IgGs that precedes CD onset and pinpoint a specific glycome–ASCA pathway as a central player in the initiation of inflammation many years before CD diagnosis. This pathogenic glyco-hub may constitute a promising new serum biomarker for CD prediction and a potential target for disease prevention. Here the authors show that there are alterations in the glycome profile of serum IgG Fc regions that precede the diagnosis of Crohn’s disease by many years and are associated with complications during disease. This altered glycome triggers innate immune cell activation at a preclinical phase,which is the basis for the transition from healthy tissue to intestinal inflammation. View Publication

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a poor innate immune response following infection. This study evaluates the effects of transforming growth factor beta 1 (TGFβ1) up-regulated by PRRSV on gene expressions of co-stimulatory molecules,type I interferon (IFN),type I IFN-regulated genes (IRGs),pattern recognition receptors,and pro-inflammatory cytokines in PRRSV-inoculated monocyte-derived macrophages (MDMs). Phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) specific to various regions of porcine TGFβ1 mRNA were synthesized,and those specific to the AUG region efficiently knockdown TGFβ1 mRNA expression and protein translation. Transfection of TGFβAS ODNs in MDMs inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) significantly reduced TGFβ1 mRNA expression and significantly increased mRNA expressions of CD80,CD86,IFNβ,IRGs (i.e. IFN regulatory factor 3 (IRF3),IRF7,myxovirus resistance 1,osteopontin,and stimulator of IFN genes),Toll-like receptor 3,and tumor necrosis factor-alpha. Transfection of TGFβAS ODNs in MDMs inoculated with HP-PRRSV-2 also significantly increased mRNA expressions of IFNα,IFNγ,and 2’-5’-oligoadenylate synthetase 1. The quantity of PRRSV-2 RNA copy numbers was significantly reduced in MDMs transfected with TGFβAS ODNs as compared to untransfected MDMs. Recombinant porcine TGFβ1 (rTGFβ1) and recombinant porcine IFNα (rIFNα) sustained and reduced the yields of PRRSV-2 RNA copy numbers in PRRSV-2 inoculated MDMs,respectively. These findings demonstrate a strategy of PRRSV for innate immune suppression via an induction of TGFβ expression. These findings also suggest TGFβ as a potential parameter that future PRRSV vaccine and vaccine adjuvant candidates should take into consideration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12917-023-03760-8. View Publication

The mitochondrial permeability transition pore (mPTP) is a supramolecular channel that regulates exchange of solutes across cristae membranes,with executive roles in mitochondrial function and cell death. The contribution of the mPTP to normal physiology remains debated,although evidence implicates the mPTP in mitochondrial inner membrane remodeling in differentiating progenitor cells. Here,we demonstrate that strict control over mPTP conductance shapes metabolic machinery as cells transit toward hematopoietic identity. Cells undergoing the endothelial-to-hematopoietic transition (EHT) tightly control chief regulatory elements of the mPTP. During EHT,maturing arterial endothelium restricts mPTP activity just prior to hematopoietic commitment. After transition in cellular identity,mPTP conductance is restored. In utero treatment with NIM811,a molecule that blocks sensitization of the mPTP to opening by Cyclophilin D (CypD),amplifies oxidative phosphorylation (OXPHOS) in hematopoietic precursors and increases hematopoiesis in the embryo. Additionally,differentiating pluripotent stem cells (PSCs) acquire greater organization of mitochondrial cristae and hematopoietic activity following knockdown of the CypD gene,Ppif. Conversely,knockdown of Opa1,a GTPase critical for proper cristae architecture,induces cristae irregularity and impairs hematopoiesis. These data elucidate a mechanism that regulates mitochondrial maturation in hematopoietic precursors and underscore a role for the mPTP in the acquisition of hematopoietic fate. Examination of mitochondria in embryonic blood cell development exposes a mechanism that regulates mitochondrial cristae biogenesis and function during the endothelial-to-hematopoietic transition. View Publication

ObjectiveVitamin D/Vitamin D receptor (VD/VDR) signaling and the Notch pathway are involved in intestinal barrier restoration in colitis; however,their relationship and underlying mechanism are largely unknown. Therefore,this study aimed to investigate the role and mechanism of VD/VDR and the Notch pathways in intestinal barrier protection.MethodsGenetic Vdr knockout (VDR KO) and VD deficient (VDd) mice were established,and colitis was induced by feeding 2.5% dextran sodium sulfate (DSS) water. Mechanistic studies,including real-time PCR,immunofluorescence,Western blotting and dual-luciferase reporter assays,were performed on cultured Caco-2 cells and intestinal organoids.ResultsVD deficiency and VDR genetical KO increased the severity of DSS-induced colitis in mice,which presented a higher disease activity index score,increased intestinal permeability,and more severe intestinal histological damage than controls,accompanied by decreased and disrupted claudin-1 and claudin-3. Moreover,inhibition of Notch pathway by LY411,575 aggravated the severity of DSS-induced colitis and intestinal injury. In Caco-2 cells and intestinal organoids,the expression of Notch-1,N1ICD and Hes1 decreased upon downregulation or KO of VDR but increased upon paricalcitol (PAR,a VDR agonist) treatment. Meanwhile,PAR rescued claudin-1 and claudin-3 impairments that resulted from TNF-α exposure but failed to restore claudin-3 upon Notch inhibition. The dual-luciferase reporter assay further suggested that VD/VDR positively regulated the Notch signaling pathway by modulating Notch-1 transcription.ConclusionVD/VDR positively modulates Notch activation by promoting Notch-1 transcription to maintain intestinal tight junction integrity and barrier function. This highlights the VD/VDR-Notch pathway as a potential new therapeutic target for protecting the intestinal barrier against ulcerative colitis. View Publication

The combination of radiotherapy/chemoradiotherapy and immune checkpoint blockade can result in poor outcomes in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). Here,we show that combining ATR inhibition (ATRi) with radiotherapy (RT) increases the frequency of activated NKG2A+PD-1+ T cells in animal models of HNSCC. Compared with the ATRi/RT treatment regimen alone,the addition of simultaneous NKG2A and PD-L1 blockade to ATRi/RT,in the adjuvant,post-radiotherapy setting induces a robust antitumour response driven by higher infiltration and activation of cytotoxic T cells in the tumour microenvironment. The efficacy of this combination relies on CD40/CD40L costimulation and infiltration of activated,proliferating memory CD8+ and CD4+ T cells with persistent or new T cell receptor (TCR) signalling,respectively. We also observe increased richness in the TCR repertoire and emergence of numerous and large TCR clonotypes that cluster based on antigen specificity in response to NKG2A/PD-L1/ATRi/RT. Collectively,our data point towards potential combination approaches for the treatment of HNSCC. Immune checkpoint blockade can result in good outcomes in patients with metastatic head and neck squamous cell carcinoma (HNSCC),but not those with locally advanced disease. Here,the authors demonstrate that the addition of anti-NKG2A and anti-PD-L1 blockade to ATR inhibition and radiotherapy in the adjuvant setting can induce a robust antitumour immune response in mice. View Publication

Cathepsin C (CatC) is an enzyme which regulates the maturation of neutrophil serine proteases (NSPs) essential for neutrophil activation. Activated neutrophils are key players in the innate immune system,and are also implicated in the etiology of various inflammatory diseases. This study aims to demonstrate a therapeutic potential for CatC inhibitors against disorders in which activated neutrophil-derived neutrophil extracellular traps (NETs) play a significant role. We demonstrate that a CatC inhibitor,MOD06051,dose-dependently suppresses the cellular activity of NSPs,including neutrophil elastase (NE),in vitro. Neutrophils derived from MOD06051-administered rats exhibit significantly lower NE activity and NET-forming ability than controls. Furthermore,MOD06051 dose-dependently ameliorates vasculitis and significantly decreases NETs when administered to a rat model of myeloperoxidase (MPO)-antineutrophil cytoplasmic antibody-associated vasculitis (AAV). These findings suggest that CatC inhibition is a promising strategy to reduce neutrophil activation and improve activated neutrophil-mediated diseases such as MPO-AAV. Neutrophil extracellular traps (NETs) play multifaceted roles in numerous neutrophil-mediated diseases. The authors show that inhibiting cathepsin C ameliorates ANCA-associated vasculitis,a NET-related disorder,by reducing neutrophil serine protease activity and NET formation in a rat model. View Publication

Prenatal exposure to toxins can adversely affect long-term health outcomes of the offspring. Though chemotherapeutics are now standard of care for treating cancer patients during pregnancy,certain compounds are known to cross the placenta and harm placental tissue. The consequences for the fetus are largely unexplored. Here we examined the responses of newborn cord blood mononuclear cells in tissue culture to two chemotherapeutic drugs,cyclophosphamide and epirubicin,when either directly exposed to these drugs,or indirectly after crossing a placenta trophoblast bilayer barrier. Cord blood mononuclear cells exposed to the conditioned media obtained from cyclophosphamide-exposed trophoblast barriers showed a significant 2.4-fold increase of nuclear ROS levels compared to direct exposure to cyclophosphamide. Indirect exposure to epirubicine-exposed trophoblast barriers not only enhanced nuclear ROS levels but also significantly increased the fraction of cord blood cells with double strand breaks,relative to directly exposed cells. Neither apoptosis nor proliferation markers were affected in cord mononuclear blood cells upon direct or indirect exposure to cyclophosphamide or epirubicin. Our data suggests that trophoblast cells exposed to cyclophosphamide or epirubicine may induce an indirect ‘bystander’ effect and can aggravate genotoxicity in the fetal compartment. View Publication

BackgroundParkinson’s disease (PD) is a neurodegenerative condition characterized by the loss of dopaminergic neurons and the accumulation of Lewy-body protein aggregates containing misfolded α-synuclein (α-syn) in a phosphorylated form. The lack of effective models for drug screens has hindered drug development studies for PD. However,the recent development of in vitro brain-like organoids provides a new opportunity for evaluating therapeutic agents to slow the progression of this chronic disease.MethodsIn this study,we used a 3D brain-like organoid model to investigate the potential of repurposing Tilorone,an anti-viral drug,for impeding the propagation of α-synucleinopathy. We assessed the effect of Tilorone on the uptake of fluorescently labeled α-syn preformed fibrils (sPFF) and sPFF-induced apoptosis using confocal microscopy. We also examined Tilorone’s impact on the phosphorylation of endogenous α-syn induced by pathogenic sPFF by immunoblotting midbrain-like organoid extracts. Additionally,quantitative RT-PCR and proteomic profiling of sPFF-treated organoids were conducted to evaluate the global impact of Tilorone treatment on tissue homeostasis in the 3D organoid model.ResultsTilorone inhibits the uptake of sPFF in both mouse primary neurons and human midbrain-like organoids. Tilorone also reduces the phosphorylation of endogenous α-syn induced by pathogenic α-syn fibrils and mitigates α-syn fibril-induced apoptosis in midbrain-like organoids. Proteomic profiling of fibril-treated organoids reveals substantial alterations in lipid homeostasis by α-syn fibrils,which are reversed by Tilorone treatment. Given its safety profile in clinics,Tilorone may be further developed as a therapeutic intervention to alleviate the propagation of synucleinopathy in PD patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-024-05551-7. View Publication