技术资料

The SARS-CoV-2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID-19 pathogenesis,especially host factors facilitating virus infection and replication. SARS-CoV-2 was reported to enter cells via binding to ACE2,followed by its priming by TMPRSS2. Here,we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors,39,778 cells) and in cells derived from subsegmental bronchial branches (four donors,17,521 cells) by single nuclei and single cell RNA sequencing,respectively. While TMPRSS2 is strongly expressed in both tissues,in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly,these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. Our data provide a rich resource for future investigations of COVID-19 infection and pathogenesis. View Publication

Why ocular mucosa is paucibacterial is unknown. Many different mechanisms have been suggested but the comprehensive experimental studies are sparse. We found that a deficiency in L-plastin (LCP1),an actin bundling protein,resulted in an ocular commensal overgrowth,characterized with increased presence of conjunctival Streptococcal spp. The commensal overgrowth correlated with susceptibility to P. aeruginosa-induced keratitis. L-plastin knock-out (KO) mice displayed elevated bacterial burden in the P. aeruginosa-infected corneas,altered inflammatory responses,and compromised bactericidal activity. Mice with ablation of LPL under the LysM Cre (LysM. Cre pos LPLfl/fl ) and S100A8 Cre (S100A8.Cre pos LPLfl/fl ) promoters had a similar phenotype to the LPL KOs mice. In contrast,infected CD11c.Cre pos LPLfl/fl mice did not display elevated susceptibility to infection,implicating the myeloid L-plastin-sufficient cells (e.g.,macrophages and neutrophils) in maintaining ocular homeostasis. Mechanistically,the elevated commensal burden and the susceptibility to infection were linked to defects in neutrophil frequencies at steady state and during infection and compromised bactericidal activities upon priming. Macrophage exposure to commensal organisms primed neutrophil responses to P. aeruginosa,augmenting PMN bactericidal capacity in an L-plastin dependent manner. Cumulatively,our data highlight the importance of neutrophils in controlling ocular paucibacteriality,reveal molecular and cellular events involved in the process,and suggest a link between commensal exposure and resistance to infection. View Publication

Historically,it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However,in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research,including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR),which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications,including regenerative medicine,drug sensitivity testing,gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue,and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d,the technique is directly applicable to diagnostic and predictive medicine. Moreover,the epithelial cells can be propagated indefinitely in vitro,yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment. View Publication

Stem cell therapy is widely employed in treating osteoarthritis (OA),and bone marrow-derived mesenchymal stem cells (BMSCs) has gradually become the most attractive new method for treating OA due to the benefit for cartilage tissue repair. However,the apoptosis in the neural stem cell transplantation severely decreases repairing efficacy. Icariin has been reported to exert multiple effects on BMSCs,including its proliferation,osteogenic,and chondrogenic differentiation. However,its effects on the injury induced by oxygen,glucose and serum deprivation (OGD) remains unknown. We prospectively investigated the role of ICA on rabbit BMSCs under conditions of OGD. Firstly,BMSCs were cultured under conditions of OGD,ICA relieved OGD-induced cell damage by promoting cell proliferation and suppressing apoptosis. Secondly,Markers of endoplasmic reticulum stress (ERs),ER stress IRE-1 pathway,and autophagy were both inhibited by ICA via inhibition of phosphor-extracellular regulated protein kinases (p-ERKs),p-P38,p-c-Jun N-terminal kinase (p-JNK) or si-MAPK. Finally,decrease of ERs marker levels enhanced protective effect of ICA against OGD-induced injury by limiting apoptosis and autophagy. Moreover,an autophagy inhibitor (3-methyladenine: 3-MA) contributed to a synergistic effect in conjunction with ICA,in promoting cell proliferation,suggesting that ICA exerts anti-ERs and anti-autophagy effects in OGD-treated BMSCs. Therefore,ICA protected rabbit BMSCs from OGD-induced apoptosis through inhibitory regulation of ERs-mediated autophagy related to the MAPK signaling pathway,which provided insights for a potential therapeutic strategy in OA. View Publication

S100A12 is a calcium-binding protein of the S100 subfamily of myeloid-related proteins that acts as an alarmin to induce a pro-inflammatory innate immune response. It has been linked to several chronic inflammatory diseases,however its role in the common oral immunopathology periodontitis is largely unknown. Previous in vitro monoculture experiments indicate that S100A12 production decreases during monocyte differentiation stages,while the regulation within tissue is poorly defined. This study evaluated S100A12 expression in monocyte subsets,during monocyte-to-macrophage differentiation and following polarization,both in monoculture and in a tissue context,utilizing a three-dimensional co-culture oral tissue model. Further,we explored the involvement of S100A12 in periodontitis by analyzing its expression in peripheral circulation and gingival tissue,as well as in saliva. We found that S100A12 expression was higher in classical than in non-classical monocytes. S100A12 expression and protein secretion declined significantly during monocyte-to-macrophage differentiation,while polarization of monocyte-derived macrophages had no effect on either. Peripheral monocytes from periodontitis patients had higher S100A12 expression than monocytes from controls,a difference particularly observed in the intermediate and non-classical monocyte subsets. Further,monocytes from periodontitis patients displayed an increased secretion of S100A12 compared with monocytes from controls. In oral tissue cultures,monocyte differentiation resulted in increased S100A12 secretion over time,which further increased after inflammatory stimuli. Likewise,S100A12 expression was higher in gingival tissue from periodontitis patients where monocyte-derived cells exhibited higher expression of S100A12 in comparison to non-periodontitis tissue. In line with our findings,patients with severe periodontitis had significantly higher levels of S100A12 in saliva compared to non-periodontitis patients,and the levels correlated to clinical periodontal parameters. Taken together,S100A12 is predominantly secreted by monocytes rather than by monocyte-derived cells. Moreover,S100A12 is increased in inflamed tissue cultures,potentially as a result of enhanced production by monocyte-derived cells. This study implicates the involvement of S100A12 in periodontitis pathogenesis,as evidenced by increased S100A12 expression in inflamed gingival tissue,which may be due to altered circulatory monocytes in periodontitis. View Publication

Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of miRNAs in regulating drug resistance and leukemic stem cell (LSCs) fate,we performed global transcriptome profiling in treatment-na{\{i}}ve chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor; its restored expression impaired survival of drug-resistant cells sensitized them to TKIs in vitro and markedly eliminated long-term repopulating LSCs and infiltrating blast cells conferring a survival advantage in pre-clinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185 and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity sensitizing therapy-resistant cells to TKIs. Thus miR-185 presents as a potential predictive biomarker and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL may provide a valuable strategy for overcoming drug resistance in patients." View Publication

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

Generating robust CD4+ T-helper cell type 1 (Th1) responses is essential for protective vaccine-induced type 1 immunity. Here,we examine whether immunization formulation associated with enhanced vaccine efficacy promotes antigen targeting and cell recruitment into lymph node (LN) niches associated with optimal type 1 responses. Immunization with antigen and Toll-like receptor agonist emulsified in oil leads to an increased differentiation of IFN$\gamma$/TNF-$\alpha$+ polyfunctional Th1 cells compared to an identical immunization in saline. Oil immunization results in a rapid delivery and persistence of antigen in interfollicular regions (IFRs) of the LN,whereas without oil,antigen is distributed in the medullary region. Following oil immunization,CXCL10-producing inflammatory monocytes accumulate in the IFR,which mobilizes antigen-specific CD4+ T cells into this niche. In this microenvironment,CD4+ T cells are advantageously positioned to encounter arriving IL-12-producing inflammatory dendritic cells (DCs). These data suggest that formulations delivering antigen to the LN IFR create an inflammatory niche that can improve vaccine efficacy. 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

Both antitumor and protumor property of mesenchymal stem cells (MSCs) have been demonstrated. We hypothesize that this contradiction is due to the heterogeneity of MSC subsets and that extracellular vesicles (EVs) from distinct MSC subsets can transfer the corresponding antitumor activities. Here we evaluated the antitumor activities of two subsets of adipose-derived mesenchymal stem cells (ADSCs) and ADSC-derived EVs (ADSC-EVs) in immunocompetent syngeneic mouse models of breast cancer. We identified CD90high and CD90low ADSC subsets and demonstrated that CD90high ADSCs could be converted into CD90low ADSCs by stimulation with LPS. CD90low ADSCs and its derived EVs significantly inhibited tumor growth in tumor-bearing mice. Benefit of tumor control were associated with decreased tumor cell proliferation and migration,and enhanced tumor cell apoptosis mediated by ADSC-EVs. Antioncogenic miRNA-16-5p loaded CD90low ADSC-EVs further significantly enhanced antitumor activities. Taken together,this study represents the first attempt to apply our newly identified antitumor ADSCs and its derived EVs in preclinical treatment of breast cancer. This study also provides the evidence that EVs can serve as a novel and effective therapeutics or drug delivery vesicle. This new therapeutic approach could be potentially applicable to breast cancer and many other types of cancer. View Publication

Glioblastoma is the most aggressive and deadly brain cancer. There is growing interest to develop drugs that specifically target to glioblastoma tumor-initiating cells (TICs). However,the cost-effective production of large numbers of high quality glioblastoma TICs for drug discovery with current cell culturing technologies remains very challenging. Here,we report a new method that cultures glioblastoma TICs in microscale alginate hydrogel tubes (or AlgTubes). The AlgTubes allowed long-term culturing ({\~{}}50 days,10 passages) of glioblastoma TICs with high growth rate ({\~{}}700-fold expansion/14 days),high cell viability and high volumetric yield ({\~{}}3.0 × 108 cells/mL) without losing the stem cell properties,all offered large advancements over current culturing methods. This method can be applied for the scalable production of glioblastoma TICs at affordable cost for drug discovery. View Publication

Type III interferon-lambda (IFN-$\lambda$) plays a critical role against infection,particularly in mucosal infection in the respiratory and gastrointestinal tract. Our study and other previous studies have shown that porcine IFN-$\lambda$ more efficiently curtails the infection of porcine epidemic diarrhea virus (PEDV) in the intestine epithelia than type I IFN,whereas IFN-$\lambda$3 exerts a more potent effect than IFN-$\lambda$1. However,the underlying mechanism remains elusive,and in particular,the transcriptional profile induced by IFN-$\lambda$3 has not been reported. Here,to resolve the mechanism responsible for the disparity between IFN-$\lambda$3 and type I IFN in anti-mucosal virus infection,we compared the transcription profiles induced by the two IFNs in porcine intestinal epithelial (IPEC-J2) cells by RNA-Seq. Our results showed that the pretreatment of IPEC-J2 cells with IFN-$\lambda$3 resulted in the differential expression of 983 genes. In contrast,IFN-$\alpha$ only modified the expression of 134 genes,and 110 of these genes were also observed in the response to IFN-$\lambda$3. A transcriptional enrichment analysis indicated that IFN-$\lambda$3 or IFN-$\alpha$ regulates multiple cellular processes and that IFN-$\lambda$3 activates more robust signaling pathways,particularly the antiviral Jak-STAT signaling pathway,than IFN-$\alpha$. Furthermore,we verified the RNA-Seq results through an RT-qPCR analysis of IPEC-J2 cells and porcine enteroids. Moreover,transient expression of the porcine rsad2 and mx2 genes among the top 10 genes induced by IFN-$\lambda$3 significantly inhibited PEDV infection. Collectively,the data showed that IFN-$\lambda$3 induces a unique transcriptional profile that does not completely overlap with that induced by IFN-$\alpha$ and strongly elicits a set of genes responsible for the antiviral activity of IFN-$\lambda$3. These findings provide important knowledge regarding the elicited ISGs of type I and III IFNs in restricting porcine intestinal viral infection. View Publication