技术资料

We introduce machine learning (ML) to perform classification and quantitation of images of nuclei from human blood neutrophils. Here we assessed the use of convolutional neural networks (CNNs) using free,open source software to accurately quantitate neutrophil NETosis,a recently discovered process involved in multiple human diseases. CNNs achieved {\textgreater}94{\%} in performance accuracy in differentiating NETotic from non-NETotic cells and vastly facilitated dose-response analysis and screening of the NETotic response in neutrophils from patients. Using only features learned from nuclear morphology,CNNs can distinguish between NETosis and necrosis and between distinct NETosis signaling pathways,making them a precise tool for NETosis detection. Furthermore,by using CNNs and tools to determine object dispersion,we uncovered differences in NETotic nuclei clustering between major NETosis pathways that is useful in understanding NETosis signaling events. Our study also shows that neutrophils from patients with sickle cell disease were unresponsive to one of two major NETosis pathways. Thus,we demonstrate the design,performance,and implementation of ML tools for rapid quantitative and qualitative cell analysis in basic science. View Publication

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent,submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages,reduces tumor cell proliferation and angiogenesis,and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33,an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11,which is required for the growth of gastric cancers in mice. Accordingly,ablation of the cognate IL-33 receptor St2 limits tumor growth,and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2,Ccl3,and Il6. Conversely,genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore,tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer. View Publication

The objective of the study was to identify the mechanism of action for a radiation mitigator of the gastrointestinal (GI) acute radiation syndrome (ARS),identified in an unbiased high-throughput screen. We used mice irradiated with a lethal dose of radiation and treated with daily injections of the radiation mitigator 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine to study its effects on key pathways involved in intestinal stem cell (ISC) maintenance. RNASeq,quantitative reverse transcriptase-polymerase chain reaction,and immunohistochemistry were performed to identify pathways engaged after drug treatment. Target validation was performed with competition assays,reporter cells,and in silico docking. 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine activates Hedgehog signaling by binding to the transmembrane domain of Smoothened,thereby expanding the ISC pool,increasing the number of regenerating crypts and preventing the GI-ARS. We conclude that Smoothened is a target for radiation mitigation in the small intestine that could be explored for use in radiation accidents as well as to mitigate normal tissue toxicity during and after radiotherapy of the abdomen. View Publication

Inflammatory bowel diseases (IBDs) are chronic and relapsing immune disorders that result,or possibly originate,from epithelial barrier defects. Intestinal organoids are a new reliable tool to investigate epithelial response in models of chronic inflammation. We produced organoids from the ulcerative colitis murine model Winnie to explore if the chronic inflammatory features observed in the parental intestine were preserved by the organoids. Furthermore,we investigated if quercetin administration to in vitro cultured organoids could suppress LPS-induced inflammation in wild-type organoids (WT-organoids) and spontaneous inflammation in ulcerative colitis organoids (UC-organoids). Our data demonstrate that small intestinal organoids obtained from Winnie mice retain the chronic intestinal inflammatory features characteristic of the parental tissue. Quercetin administration was able to suppress inflammation both in UC-organoids and in LPS-treated WT-organoids. Altogether,our data demonstrate that UC-organoids are a reliable experimental system for investigating chronic intestinal inflammation and pharmacological responses. View Publication

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression,yet their role in cell fate transitions remains largely unexplored. Here,we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant,hyper-pluripotent" state which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer heterochromatin and DNA methylation landscapes of undifferentiated cell types. Collectively our data establish a link between P-body homeostasis chromatin organization and stem cell potency." View Publication

mTORC1 regulates cellular growth and is activated by growth factors and by essential amino acids such as Leu. Leu enters cells via the Leu transporter LAT1-4F2hc (LAT1). Here we show that the Na+/K+/2Cl- cotransporter NKCC1 (SLC12A2),a known regulator of cell volume,is present in complex with LAT1. We further show that NKCC1 depletion or deletion enhances LAT1 activity,as well as activation of Akt and Erk,leading to activation of mTORC1 in cells,colonic organoids,and mouse colon. Moreover,NKCC1 depletion reduces intracellular Na+ concentration and cell volume (size) and mass and stimulates cell proliferation. NKCC1,therefore,suppresses mTORC1 by inhibiting its key activating signaling pathways. Importantly,by linking ion transport and cell volume regulation to mTORC1 function,NKCC1 provides a long-sought link connecting cell volume (size) to cell mass regulation. View Publication

BACKGROUND Vascularization is important for the clinical application of tissue engineered products. Both adipose-derived stem cells (ASCs) and surgical prefabrication can be used to induce angiogenesis in scaffolds. Our aim was to compare the angiogenic potential of ASC-seeded scaffolds combined with scaffold prefabrication with that of non-seeded,non-prefabricated scaffolds. METHODS For prefabrication,functional blood vessels were introduced into the scaffold using a flow-through pedicle system. ASCs were isolated from rat fat deposits. Three-dimensional-printed cylindrical poly-$\epsilon$-caprolactone scaffolds were fabricated by fused deposition modelling. Three groups,each containing six rats,were investigated by using non-seeded,ASC-seeded,and osteogenic induced ASC-seeded scaffolds. In each group,one rat was implanted with two scaffolds in the inguinal region. On the right side,a scaffold was implanted subcutaneously around the inferior epigastric vessels (classic prefabrication group). On the left side,the inferior epigastric vessels were placed inside the prefabricated scaffold in the flow-through pedicle system (flow-through prefabrication group). The vessel density and vascular architecture were examined histopathologically and by $\mu$CT imaging,respectively,at 2 months after implantation. RESULTS The mean vessel densities were 10- and 5-fold higher in the ASC-seeded and osteogenic induced ASC-seeded scaffolds with flow-through prefabrication,respectively,than in the non-seeded classic prefabricated group (p {\textless} 0.001). $\mu$CT imaging revealed functional vessels within the scaffold. CONCLUSION ASC-seeded scaffolds with prefabrication showed significantly improved scaffold vasculogenesis and could be useful for application to tissue engineering products in the clinical settings. View Publication

Bone marrow mesenchymal stem/stromal cells (BM-MSCs) have immunoregulatory properties and have been used as immune regulators for the treatment of graft-versus-host disease (GVHD). Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to BM-MSCs for potential clinical applications because of their accessibility and easy preparation. The aim of this in vitro study was to compare MSCs from dental pulp (DP-MSCs),gingival tissue (G-MSCs),and periodontal ligament (PDL-MSCs) in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3+ T cells to determine which MSCs would be the most appropriate for in vivo immunoregulatory applications. BM-MSCs were included as the gold standard. Our results demonstrated,in vitro,that MSCs from DP,G,and PDL showed immunoregulatory properties similar to those from BM,in terms of the cellular proliferation inhibition of both CD4+- and CD8+-activated T-cells. This reduced proliferation in cell co-cultures correlated with the production of interferon-$\gamma$ and tumor necrosis factor alpha (TNF-$\alpha$) and the upregulation of programmed death ligand 1 (PD-L1) in MSCs and cytotoxic T-cell-associated Ag-4 (CTLA-4) in T-cells and increased interleukin-10 and prostaglandin E2 production. Interestingly,we observed differences in the production of cytokines and surface and secreted molecules that may participate in T-cell immunosuppression in co-cultures in the presence of DT-MSCs compared with BM-MSCs. Importantly,MSCs from four sources favored the generation of T-cell subsets displaying the regulatory phenotypes CD4+CD25+Foxp3+ and CD4+CD25+CTLA-4+. Our results in vitro indicate that,in addition to BM-MSCs,MSCs from all of the dental sources analyzed in this study might be candidates for future therapeutic applications. View Publication

BACKGROUND The inhibition of Janus kinases (JAKs) and subsequent signal transducers and activators of transcription (STATs) by tofacitinib represents a new therapeutic strategy in inflammatory bowel diseases (IBD) as clinical trials have led to approval of tofacitinib for ulcerative colitis (UC) and hint at a possible efficacy for Crohn`s disease (CD). However,the impact of tofacitinib on cellular response of monocytes,which are key players in inflammatory responses,has not been investigated so far. We aimed to analyze JAK/STAT-inhibition by tofacitinib in monocytes of IBD patients and healthy controls. METHODS Primary monocytes of IBD patients with active disease and healthy controls (n = 18) were analyzed for cytokine expression and phenotype after granulocyte macrophage colony-stimulating factor (GM-CSF)/interferon (IFN)$\gamma$-stimulation and tofacitinib pretreatment (1-1000 nM) and capacity to induce Foxp3+-regulatory T cells (Tregs) in cocultures. In total,20 UC patients and 21 CD patients were included. Additionally,dose-dependent inhibition of JAK/STAT-phosphorylation was analyzed in controls. RESULTS Pro-inflammatory costimulation with GM-CSF/IFN$\gamma$ resulted in significant tumor necrosis factor (TNF$\alpha$) and interleukin (IL)-6 increase,whereas IL-10 expression decreased in monocytes. Tofacitinib modulated the responses of activated monocytes toward a regulatory phenotype through reduced TNF$\alpha$ and IL-6 secretion and enhanced Treg induction in cocultures. However,in monocytes from active IBD patients,higher tofacitinib dosages were needed for blockade of pro-inflammatory cytokines. Tofacitinib induced stronger regulatory phenotypes in monocytes of UC patients,including more effective inhibition of pro-inflammatory pathways and better restoration of anti-inflammatory mechanisms as compared with CD-derived monocytes. CONCLUSION Tofacitinib dose-dependently reprograms monocytes toward a more regulatory cell type. This beneficial effect possibly results from selective JAK/STAT-blockade by adequate tofacitinib dosage with inhibition of pro-inflammatory responses and permission of a balance-shift toward regulatory pathways. View Publication

INTRODUCTION Bone marrow mesenchymal stem cells (BMSCs) have been extensively investigated from a perspective on cardiac regeneration therapy. The current study aimed to investigate the protective effect conferred by BMSCs in subacute myocardial injury,and to identify an appropriate BMSC reinfusion time. METHODS BMSCs were isolated from human bone marrow blood. Daunorubicin (DNR)-induced subacute myocardial models were subsequently established. The rats with DNR-induced subacute myocardial injury were injected with dexrazoxane (DZR) and/or BMSCs at varying time points,after which cardiac function was evaluated by assessing left ventricular ejection fraction (LVEF) and fraction shortening (FS). The myocardial structural changes were analyzed,after which the levels of CD3 and human leukocyte antigen DR (HLA-DR) were examined to further validate the mechanism by which BMSCs could influence subacute myocardial injury. RESULTS BMSCs combined with DZR treatment enhanced the cardiac function of rats with DNR-induced myocardial injury,as reflected by increased LVEF and FS. DNR-induced myocardial injuries were mitigated via the application of BMSCs combined with treatment of DZR,accompanied by diminished infiltration or vacuolization. Moreover,BMSCs were observed to alleviate infiltration of T lymphocyte and antigen-presenting cells,as evidenced by reduced expression of CD3 and HLA-DR. CONCLUSION Taken together,this study demonstrates that BMSCs could protect against DNR-induced myocardial injury,especially in the first three days of DNR administration. BMSCs combined with DZR exert a better therapeutic effect,but there are individual differences. View Publication

Glycyrrhetinic acid (GA) is one of the main components of the traditional Chinese medicine of licorice,which can coordinate and promote the effects of other medicines in the traditional prescription. We found that GA could promote the proliferation,decrease the apoptotic rate,and attenuate DFMO-elicited growth arrest and delay in restitution after wounding in IEC-6 cells via HuR. GA failed to promote proliferation and to suppress apoptosis after silencing HuR by siRNA in IEC-6 cells. Furthermore,with the model of small intestinal organoids developed from intestinal crypt stem cells,we found that GA could increase HuR and its downstream ki67 levels to promote intestinal organoid development. In the in vivo assay,GA was shown to maintain the integrity of the intestinal epithelium under the circumstance of 48 h-fasting in rats via raising HuR and its downstream genes such as EGF,EGFR,and MEK. These results suggested that via HuR modulation,GA could promote intestinal epithelium homeostasis,and therefore contribute to the absorption of constituents from other medicines co-existing in the traditional prescription with licorice in the small intestine. Our results provide a new perspective for understanding the effect of licorice on enhancing the therapeutic effect of traditional prescriptions according to the traditional Chinese medicine theory. View Publication

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood,initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however,the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome,RNA-seq),Colony forming assay and xenograft studies,we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones,both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells,JMML-PCs are not always restricted to this compartment,highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML,and the identity of JMML-PCs,and provides robust models to monitor the disease and test novel therapeutic approaches. View Publication