技术资料

IntroductionAnandamide (AEA) is an endocannabinoid that has recently been recognized as a regulator of various inflammatory diseases as well as cancer. While AEA was thought to predominantly engage cannabinoid (CB) receptors,recent findings suggest that,given its protective anti-inflammatory role in pathological conditions,anandamide may engage not only CB receptors.MethodsIn this study,we studied the role of exogenous AEA in a mouse AirPouch model of acute inflammation by examining immune cell infiltrates by flow cytometry. Human primary immune cells were used to validate findings towards immune cell activation and migration by flow cytometry and bead-based ELISA.ResultsWe found that AEA decreases the acute infiltration of myeloid cells including granulocytes and monocytes into the inflamed area,but unexpectedly increases the number of T cells at the site of inflammation. This was related to AEA signaling through nuclear receptor subfamily 4A (NR4A) transcription factors rather than CB receptors. Exploring regulatory mechanisms in the human system,we found that AEA broadly inhibits the migratory capacity of immune cells,arguing for blocked emigration of T cells from the inflamed tissue. Taking a closer look at the impact of AEA on T cells revealed that AEA profoundly alters the activation and exhaustion status of CD4+ T and CD8+ T cells,thereby strongly inhibiting TH17 responses,while not altering TH1 differentiation.DiscussionThese data suggest that AEA has the potential to block chronic inflammation without influencing crucial anti-viral and anti-microbial immune defense mechanisms,and may therefore be an attractive molecule to interfere with the establishment of chronic inflammation. View Publication

SummaryHere,we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling.For complete details on the use and execution of this protocol,please refer to Foltz et al.1 Graphical abstract Highlights•Protocol for inducing hESCs or iPSCs to form neural crest stem cells (NCSCs)•Steps for differentiating NCSCs into craniofacial cartilage organoids•Instructions for preparing appropriate media and conditions for differentiation•Guidance for assessing changes in cell and organoid morphology during differentiation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here,we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling. View Publication

IntroductionThe role of miRNAs in regulating variable molecular functions has been sought by scientists for its promising utility in regulating the immune response and,hence,in treating various diseases. In hepatocellular carcinoma (HCC) specifically,a reduction in the number and efficiency of circulating and intrahepatic natural killer (NK) cells has been reported. Our project aims to investigate the role of miR-216a-3p in the regulation of NK cell cytotoxicity,especially since it plays a tumor suppressor role in the context of HCC.MethodsTo achieve our aim,we isolated NK cells from the whole blood of 86 patients with HCC and 23 healthy controls. We assessed the expression profile of miR-216a-3p in NK cells of patients and controls. Furthermore,we induced the expression of miR-216a-3p in NK cells isolated from healthy controls,followed by measuring the release of interferon-gamma (IFN-γ),tumor necrosis factor-alpha (TNF-α),perforins (PRF) and granzyme B (GrB) using ELISA as well as NK cells cytolytic activity against Huh7 cells using lactate dehydrogenase (LDH) cytotoxicity assay. After that,we performed an in silico analysis to understand the mechanistic regulation imposed by miR-216a-3p on NK cells to study its impact on one of its potential downstream targets.ResultsOur results have indicated that miR-216a-3p has higher expression in NK cells of patients with HCC,and simulating this elevated expression pattern via forcing miR-216a-3p expression in normal NK cells has negatively impacted the release of TNF- α,IFN- γ,GrB,and PRF. Consequently,a decrease in cell cytolysis was observed. Our in silico analysis revealed that the predicted downstream targets of miR-216a-3p are enriched in the FOXO-signaling pathway. Among those targets is FOXO-1,which has been reported to play a role in NK cell maturation. Thus,we evaluated FOXO-1 expression upon mimicking miR-216a-3p in control NK cells that showed significant downregulation of FOXO-1 on both RNA and protein levels.ConclusionIn conclusion,we report miR-216-3p as a negative regulator of NK cell cytotoxicity. View Publication

The persistence of HIV-1 latency reservoirs in CD4+ T cells is a significant obstacle for curing HIV-1. Shock-and-kill strategies,which aim to reactivate latent HIV-1 followed by cytotoxic clearance,have shown limited success in vivo due to insufficient efficacy of latency reversal agents (LRAs) and off-target effects. Natural killer (NK) cells,with their ability to mediate cytotoxicity independent of antigen specificity,offer a promising avenue for enhancing the shock-and-kill approach. Previously,we observed that pan-caspase inhibitors induce NK cells to secrete an LRA in vitro. Here,we aimed to identify this LRA using a targeted proteomic approach. We identified lymphotoxin-α (LTα) as the key LRA secreted by NK cells following pan-caspase inhibitor treatment. LTα was shown to significantly induce HIV-1 LTR promoter activity,a hallmark of viral reactivation. Neutralization of LTα effectively abolished the observed LRA activity,confirming its central role. Moreover,cytokine-primed but not resting human primary NK cells exhibited LRA activity that could be neutralized with LTα neutralizing antibodies. Finally,pan-caspase inhibitor treatment did not decrease the ability of the cytokine-primed NK cells to kill target cells. These findings demonstrate that cytokine-primed NK cells,through LTα secretion,can effectively reactivate latent HIV-1 following pan-caspase inhibitor treatment,without compromising NK cell cytotoxicity. This highlights a potential enhancement strategy utilizing NK cells for shock-and-kill approaches in HIV-1 cure research. View Publication

Post-transcriptional modifications to RNA,which comprise the epitranscriptome,play important roles in RNA metabolism,gene regulation,and human disease,including viral pathogenesis. Modifications to the RNA viral genome and transcripts of human immunodeficiency virus 1 (HIV-1) have been reported and investigated in the context of virus and host biology. However,the diversity of experimental approaches used has made clear correlations across studies,as well as the significance of the HIV-1 epitranscriptome in biology and disease,difficult to assess. Therefore,we established a reference HIV-1 epitranscriptome. We sequenced the model NL4–3 HIV-1 genome from infected primary CD4+ T cells and the Jurkat cell line using the latest nanopore chemistry,optimized RNA preparation methods,and the most current and readily available base-calling algorithms. A highly reproducible sense and a preliminary antisense HIV-1 epitranscriptome were created,where N6-methyladenosine (m6A),5-methylcytosine (m5C),pseudouridine (psi),inosine,and 2’-O-methyl (Nm) modifications could be identified by rapid multiplexed base-calling. We observed that sequence and neighboring modification contexts induced modification miscalling,which could be corrected with synthetic HIV-1 RNA fragments. We validated m6A modification sites with STM2457,a small molecule inhibitor of methyltransferase-like 3 (METTL3). We find that modifications are quite stable under combination antiretroviral therapy (cART) treatment,in primary CD4+ T cells,and in HIV-1 virions. Sequencing samples from people living with HIV (PLWH) revealed conservation of m6A modifications. However,analysis of spliced transcript variants suggests transcript-dependent modification levels. Our approach and reference data offer a straightforward benchmark that can be adopted to help advance rigor,reproducibility,and uniformity across HIV-1 epitranscriptomics studies. They also provide a roadmap for the creation of reference epitranscriptomes for many other viruses or pathogens. View Publication

Background/Objectives: The SARS-CoV-2’s high mutations and replication rates contribute to its high infectivity and resistance to current vaccinations and treatments. The primary cause of resistance to most current treatments aligns within the coding regions for the spike S protein of SARS-CoV-2 that has mutated. As a potential novel immunotherapy,we generated a novel fusion protein composed of a soluble ACE2 (sACE2) linked to llama-derived anti-CD16 that targets different variants of spike proteins and enhances natural killer cells to target infected cells. Methods: Here,we generated a novel sACE2-AntiCD16VHH fusion protein using a Gly4Ser linker,synthesized and cloned into the pLVX-EF1alpha-IRES-Puro vector,and further expressed in ExpiCHO-S cells and purified using Ni+NTA chromatography. Results: The fusion protein significantly blocked SARS-CoV-2 alpha,beta,delta,gamma,and omicron S-proteins binding and activating angiotensin-converting enzyme receptor-2 (ACE2) on ACE2-expressing RAW-Blue macrophage cells and the secretion of several key inflammatory cytokines,G-CSF,MIP-1A,and MCP-1,implicated in the cytokine release storm (CRS). The sACE2-Anti-CD16VHH fusion protein also bridged NK cells to ACE2-expressing human lung carcinoma A549 cells and significantly activated NK-dependent cytotoxicity. Conclusions: The findings show that a VHH directed against CD16 could be an excellent candidate to be linked to soluble ACE2 to generate a bi-specific molecule (sACE2-AntiCD16VHH) suitable for bridging effector cells and infected target cells to inhibit SARS-CoV-2 variant spike proteins binding to the ACE2 receptor in the RAW-Blue cell line and pro-inflammatory cytokines and to activate natural killer cell cytotoxicity. View Publication

Immune evasion by cancer cells involves reshaping the tumor microenvironment (TME) via communication with non-malignant cells. However,resistance-promoting interactions during treatment remain lesser known. Here we examine the composition,communication,and phenotypes of tumor-associated cells in serial biopsies from stage II and III high-risk estrogen receptor positive (ER+ ) breast cancers of patients receiving endocrine therapy (letrozole) as single agent or in combination with ribociclib,a CDK4/6-targeting cell cycle inhibitor. Single-cell RNA sequencing analyses on longitudinally collected samples show that in tumors overcoming the growth suppressive effects of ribociclib,first cancer cells upregulate cytokines and growth factors that stimulate immune-suppressive myeloid differentiation,resulting in reduced myeloid cell- CD8 + T-cell crosstalk via IL-15/18 signaling. Subsequently,tumors growing during treatment show diminished T-cell activation and recruitment. In vitro,ribociclib does not only inhibit cancer cell growth but also T cell proliferation and activation upon co-culturing. Exogenous IL-15 improves CDK4/6 inhibitor efficacy by augmenting T-cell proliferation and cancer cell killing by T cells. In summary,response to ribociclib in stage II and III high-risk ER +  breast cancer depends on the composition,activation phenotypes and communication network of immune cells. The CDK4/6 inhibitor ribociclib holds promise in cancer therapy but how cell cycle inhibitory drugs affect the anti-tumor immune response remains a question. Here authors show that poor response of early-stage estrogen receptor positive breast cancers to ribociclib is caused by changes in the immune cell composition and cancer-cell-immune-cell communication in the tumors rather than intrinsic cancer cell resistance to cell cycle inhibition. View Publication

The levels of transcription factor Ets1 are high in resting B and T cells,but are downregulated by signaling through antigen receptors and Toll-like receptors (TLRs). Loss of Ets1 in mice leads to excessive immune cell activation and development of an autoimmune syndrome and reduced Ets1 expression has been observed in human PBMCs in the context of autoimmune diseases. In B cells,Ets1 serves to prevent premature activation and differentiation to antibody-secreting cells. Given these important roles for Ets1 in the immune response,stringent control of Ets1 gene expression levels is required for homeostasis. However,the genetic regulatory elements that control expression of the Ets1 gene remain relatively unknown. Here we identify a topologically-associating domain (TAD) in the chromatin of B cells that includes the mouse Ets1 gene locus and describe an interaction hub that extends over 100 kb upstream and into the gene body. Additionally,we compile epigenetic datasets to find several putative regulatory elements within the interaction hub by identifying regions of high DNA accessibility and enrichment of active enhancer histone marks. Using reporter constructs,we determine that DNA sequences within this interaction hub are sufficient to direct reporter gene expression in lymphoid tissues of transgenic mice. Further analysis indicates that the reporter construct drives faithful expression of the reporter gene in mouse B cells,but variegated expression in T cells,suggesting the existence of T cell regulatory elements outside this region. To investigate how the downregulation of Ets1 transcription is associated with alterations in the epigenetic landscape of stimulated B cells,we performed ATAC-seq in resting and BCR-stimulated primary B cells and identified four regions within and upstream of the Ets1 locus that undergo changes in chromatin accessibility that correlate to Ets1 gene expression. Interestingly,functional analysis of several putative Ets1 regulatory elements using luciferase constructs suggested a high level of functional redundancy. Taken together our studies reveal a complex network of regulatory elements and transcription factors that coordinate the B cell-specific expression of Ets1. View Publication

Large-scale cancer genetic/genomic studies demonstrated that papillary renal cell carcinoma (pRCC) is featured with a frequent shallow deletion of the upstream tumor suppressors of the Hippo/YAP signaling pathway,suggesting that this signaling pathway may play a role in pRCC development. Here we develop a transgenic mouse model with a renal epithelial cell-specific hyperactivation of YAP1 and find that hyperactivation of YAP1 can induce dedifferentiation and transformation of renal tubular epithelial cells leading to the development of pRCC. We analyze at the single-cell resolution the cellular landscape alterations during cancer initiation and progression. Our data indicate that the hyperactivated YAP1,via manipulating multiple signaling pathways,induces epithelial cell transformation,MDSC (Myeloid-derived suppressor cells) accumulation,and pRCC development. Interestingly,we find that depletion of MDSC blocks YAP1-induced kidney overgrowth and tumorigenesis. Inhibiting YAP1 activity with MGH-CP1,a recently developed TEAD inhibitor,impedes MDSC accumulation and suppresses tumor development. Our results identify the disrupted Hippo/YAP signaling as a major contributor to pRCC and suggest that targeting the disrupted Hippo pathway represents a plausible strategy to prevent and treat pRCC. Deletion of upstream tumor suppressors of the Hippo/YAP pathway is frequent in papillary renal cell carcinoma (pRCC). Here,the authors employ a transgenic mouse model,single-cell transcriptomics and public genomic datasets to show that targeting hyperactivated YAP1 prevents neoplastic renal epithelial cell immune evasion and impairs the development of pRCC. 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

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

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