技术资料

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy,but the overall metabolic properties of HSCs remain elusive. Using combined approaches,including single-cell RNA sequencing (RNA-seq),we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed,but not quiescent,HSCs relied readily on glycolysis. Notably,in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant. View Publication

Background Premature ovarian insufficiency (POI) represents the hypergonadotropic hypoestrogenic symptoms that result in the loss of ovarian follicles. 5-30{\%} POI cases are suggested to be involved in autoimmune etiology. MicroRNA-21 (miR-21) plays a vital role in ovarian folliculogenesis via regulating and interacting with multiple target genes. Here,we conduct the target prediction of miR-21,identify the expression and correlation of miR-21 and its putative target Pellino-1 (Peli1),and confirm their relationship with clinical characteristics in autoimmune POI. Methods Bioinformatic analysis was conducted to screen the miR-21 putative target gene. Autoimmune POI mouse models were established by ZP3 immunization. Serum miR-21,Peli1 mRNA of peripheral blood mononuclear cells (PBMCs) and regulatory T cells (Tregs),general status,spleen Tregs ratio,inflammatory factors,ovarian endocrine function,and ovarian structure were evaluated. For autoimmune POI patients,serum miR-21,PBMCs Peli1 mRNA levels,general data,immune parameters,hormone levels,and ultrasound examinations were obtained. The correlations of miR-21 with Peli1 and clinical characteristics in patients were analyzed. Results Peli1 was selected based on four microRNA prediction databases and literature retrieval. In mouse models,serum miR-21 level,PBMCs and Tregs Peli1 mRNA,and spleen Tregs ratio were 0.61 ± 0.09,0.12 ± 0.12,0.27±0.23 and 4.82 ± 0.58,respectively,lower than those in the control group. In patients,miR-21 level (0.60 ± 0.14) and Peli1 mRNA (0.30 ± 0.14) were lower than those in the control group (1.01 ± 0.07 and 1.63 ± 0.54); miR-21 was positively related with Peli1,AMH,E2,the size of the uterus,and ovarian volume and negatively related with FSH,LH,and the number of positive immune parameters (AOAb,EMAb,ACL,ANA,ds-DNA,ACA,IgG,IgA,IgM,IgE,C3,and C4). Conclusions Low expressions of miR-21 and Peli1 were detected in autoimmune POI mice and patients. Positive correlation between miR-21 and Peli1 was observed in autoimmune POI patients,suggesting that miR-21 and Peli1 might be associated with the pathogenesis of autoimmune POI. View Publication

Control of established chronic lymphocytic choriomeningitis virus (LCMV) infection requires the production of neutralizing antibodies,but it remains unknown how the ensemble of antibodies evolves during ongoing infection. Here,we analyze the evolution of antibody responses during acute or chronic LCMV infection,combining quantitative functional assays and time-resolved antibody repertoire sequencing. We establish that antibody responses initially converge in both infection types on a functional and repertoire level,but diverge later during chronic infection,showing increased clonal diversity,the appearance of mouse-specific persistent clones,and distinct phylogenetic signatures. Chronic infection is characterized by a longer-lasting germinal center reaction and a continuous differentiation of plasma cells,resulting in the emergence of higher-affinity plasma cells exhibiting increased antibody secretion rates. Taken together,our findings reveal the emergence of a personalized antibody response in chronic infection and support the concept that maintaining B cell diversity throughout chronic LCMV infection correlates with the development of infection-resolving antibodies. View Publication

Personalized cancer therapy is based on a patient's tumor lineage,histopathology,expression analyses,and/or tumor DNA or RNA analysis. Here,we aim to develop an in vitro functional assay of a patient's living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care,we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care. Kodack et al. report on the development of cancer models from tumor biopsies and technologies toward a functional approach for personalized medicine. They describe the ability to reliably test drug response in patient-derived samples of mixed cell populations. In doing so,they show that patient biopsy cultures may predict patient clinical responses. View Publication

Much of our understanding of chromosome segregation is based on cell culture systems. Here,we examine the importance of the tissue environment for chromosome segregation by comparing chromosome segregation fidelity across several primary cell types in native and nonnative contexts. We discover that epithelial cells have increased chromosome missegregation outside of their native tissues. Using organoid culture systems,we show that tissue architecture,specifically integrin function,is required for accurate chromosome segregation. We find that tissue architecture enhances the correction of merotelic microtubule-kinetochore attachments,and this is especially important for maintaining chromosome stability in the polyploid liver. We propose that disruption of tissue architecture could underlie the widespread chromosome instability across epithelial cancers. Moreover,our findings highlight the extent to which extracellular context can influence intrinsic cellular processes and the limitations of cell culture systems for studying cells that naturally function within a tissue. Tissue architecture and integrin function are critical factors that support chromosome segregation fidelity in epithelial tissues. View Publication

The process of oligodendrogenesis has been relatively well delineated in the rodent brain. However,it remains unknown whether analogous developmental processes are manifested in the human brain. Here we report oligodendrogenesis in forebrain organoids,generated by using OLIG2-GFP knockin human pluripotent stem cell (hPSC) reporter lines. OLIG2/GFP exhibits distinct temporal expression patterns in ventral forebrain organoids (VFOs) versus dorsal forebrain organoids (DFOs). Interestingly,oligodendrogenesis can be induced in both VFOs and DFOs after neuronal maturation. Assembling VFOs and DFOs to generate fused forebrain organoids (FFOs) promotes oligodendroglia maturation. Furthermore,dorsally derived oligodendroglial cells outcompete ventrally derived oligodendroglia and become dominant in FFOs after long-term culture. Thus,our organoid models reveal human oligodendrogenesis with ventral and dorsal origins. These models will serve to study the phenotypic and functional differences between human ventrally and dorsally derived oligodendroglia and to reveal mechanisms of diseases associated with cortical myelin defects. View Publication

Interferon regulatory factor 1 (IRF1) regulates diverse biological functions,including modulation of cellular responses involved in tumorigenesis. Genetic mutations and altered IRF1 function are associated with several cancers. Although the function of IRF1 in the immunobiology of cancer is emerging,IRF1-specific mechanisms regulating tumorigenesis and tissue homeostasis in vivo are not clear. Here,we found that mice lacking IRF1 were hypersusceptible to colorectal tumorigenesis. IRF1 functions in both the myeloid and epithelial compartments to confer protection against AOM/DSS-induced colorectal tumorigenesis. We further found that IRF1 also prevents tumorigenesis in a spontaneous mouse model of colorectal cancer. The attenuated cell death in the colons of Irf1-/- mice was due to defective pyroptosis,apoptosis,and necroptosis (PANoptosis). IRF1 does not regulate inflammation and the inflammasome in the colon. Overall,our study identified IRF1 as an upstream regulator of PANoptosis to induce cell death during colitis-associated tumorigenesis. View Publication

Recent developments in understanding how the functional phenotype of the innate immune system is programmed has led to paradigm-shifting views on immunomodulation. These advances have overturned two long-held dogmas: (1) only adaptive immunity confers immunological memory; and,(2) innate immunity lacks specificity. This work describes the observation that innate immune effector cells appear to be differentially recruited to specific pathological sites when mobilized by distinct inactivated bacterial-based stimuli administered subcutaneously. The studies presented suggest that the immune system,upon detecting the first signs of a potential infection by a specific pathogen,tends to direct its resources to the compartment from which that pathogen is most likely originating. The findings from this work puts forth the novel hypothesis that the immunotherapeutic efficacy of a microbial-based stimulus for innate immune mobilization depends on the correct selection of the microbial species used as the stimulant and its relationship to the organ in which the pathology is present. View Publication

This study reports the use of cell-type-specific in vivo bioluminescence to measure intraocular immune cell population dynamics during the course of inflammation in a mouse model of uveitis. Transgenic lines expressing luciferase in inflammatory cell subsets (myeloid cells,T cells,and B cells) were generated and ocular bioluminescence was measured serially for 35 days following uveitis induction. Ocular leukocyte populations were identified using flow cytometry and compared to the ocular bioluminescence profile. Acute inflammation is neutrophilic (75{\%} of ocular CD45 + cells) which is reflected by a significant increase in ocular bioluminescence in one myeloid reporter line on day 2. By day 7,the ocular T cell population increases to 50{\%} of CD45 + cells,leading to a significant increase in ocular bioluminescence in the T cell reporter line. While initially negligible ({\textless} 1{\%} of CD45 + cells),the ocular B cell population increases to {\textgreater} 4{\%} by day 35. This change is reflected by a significant increase in the ocular bioluminescence of the B cell reporter line starting on day 28. Our data demonstrates that cell-type-specific in vivo bioluminescence accurately detects changes in multiple intraocular immune cell populations over time in experimental uveitis. This assay could also be useful in other inflammatory disease models. View Publication

The inability of Nef to downmodulate the CD3-T cell receptor (TCR) complex distinguishes HIV-1 from other primate lentiviruses and may contribute to its high virulence. However,the role of this Nef function in virus-mediated immune activation and pathogenicity remains speculative. Here,we selectively disrupted this Nef activity in SIVmac239 and analyzed the consequences for the virological,immunological,and clinical outcome of infection in rhesus macaques. The inability to downmodulate CD3-TCR does not impair viral replication during acute infection but is associated with increased immune activation and antiviral gene expression. Subsequent early reversion in three of six animals suggests strong selective pressure for this Nef function and is associated with high viral loads and progression to simian AIDS. In the absence of reversions,however,viral replication and the clinical course of infection are attenuated. Thus,Nef-mediated downmodulation of CD3 dampens the inflammatory response to simian immunodeficiency virus (SIV) infection and seems critical for efficient viral immune evasion. View Publication

Proliferation of dendritic cell (DC)-restricted progenitor cells in bone marrow compartment is tightly regulated at steady state and responds to multiple tissue-specific triggers during disturbed homeostasis such as obesity. DCs in the lung stem from a rapidly dividing DC-restricted progenitor cells and are effective at generating adaptive immune responses in allergic airway inflammation. Precisely,how DC-restricted progenitor expansion and differentiation are influenced by airway inflammation to maintain constant supply of myeloid DCs is poorly understood. Here we show that a high fat diet (HFD) induces oxidative stress and accelerates the expansion of DC- restricted progenitor cells in bone marrow and correlates with persistent induction of p38 mitogen activated protein kinase (MAPK),which is blocked with a selective p38$\alpha$/$\beta$ MAPK inhibitor. Mice fed a HFD and sensitized to inhaled allergen house dust mite (HDM) led to alterations of DC- restricted progenitor cells that were characterized by increased expansion and seeding of lung DCs in airway inflammation. Mechanistically,we establish that the expansion induced by HFD dysregulates the expression of a disintegrin and metallopeptidase domain 17 (Adam17) and is required for p38 MAPK activation in DC-restricted progenitors. These results demonstrates that obesity produces persistent changes in DC precursors and that elevation of Adam17 expression is tightly coupled to p38 MAPK and is a key driver of proliferation. Altogether,these data provide phenotypic and mechanistic insight into dendritic cell supply chain in obesity-associated airway inflammation. View Publication

The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise questions regarding the unclarified heterogeneity of pathological mechanisms promoting disease progression. While biomarkers for the differentiation of Crohn's disease (CD) versus ulcerative colitis (UC) have been suggested,specific markers for a CD subclassification in ileal CD versus colonic CD are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease,we sought to characterize potential biomarkers by focusing on the downstream enzymes and metabolites of kynurenine metabolism. Using immunohistochemical stainings,we analyzed and compared the mucosal tryptophan immune metabolism in bioptic samples from patients with active inflammation due to UC or CD versus healthy controls. Localization-specific quantification of immune cell infiltration,tryptophan-metabolizing enzyme expression and mucosal tryptophan downstream metabolite levels was performed. We found generally increased immune cell infiltrates in the tissue of all patients with IBD. However,in patients with CD,significant differences were found between regulatory T cell and neutrophil granulocyte infiltration in the ileum compared with the colon. Furthermore,we observed decreased kynurenine levels as well as strong kynureninase (KYNU) expression specifically in patients with ileal CD. Correspondingly,significantly elevated levels of the kynurenine metabolite 3-hydroxyanthranilic acid were detected in the ileal CD samples. Highlighting the heterogeneity of the different phenotypes of CD,we identified KYNU as a potential mucosal biomarker allowing the localization-specific differentiation of ileal CD versus colonic CD. View Publication