技术资料

Dysplasia is considered a key transition state between pre-cancer and cancer in gastric carcinogenesis. However,the cellular or phenotypic heterogeneity and mechanisms of dysplasia progression have not been elucidated. We have established metaplastic and dysplastic organoid lines,derived from Mist1-Kras(G12D) mouse stomach corpus and studied distinct cellular behaviors and characteristics of metaplastic and dysplastic organoids. We also examined functional roles for Kras activation in dysplasia progression using Selumetinib,a MEK inhibitor,which is a downstream mediator of Kras signaling. Here,we report that dysplastic organoids die or show altered cellular behaviors and diminished aggressive behavior in response to MEK inhibition. However,the organoids surviving after MEK inhibition maintain cellular heterogeneity. Two dysplastic stem cell (DSC) populations are also identified in dysplastic cells,which exhibited different clonogenic potentials. Therefore,Kras activation controls cellular dynamics and progression to dysplasia,and DSCs might contribute to cellular heterogeneity in dysplastic cell lineages. View Publication

Tissue-resident memory CD8+ T (Trm) cells mediate potent local innate and adaptive immune responses and play a central role against solid tumors. However,whether Trm cells cross-talk with dendritic cells (DCs) to support anti-tumor immunity remains unclear. Here we show that antigen-specific activation of skin Trm cells leads to maturation and migration to draining lymph nodes of cross-presenting dermal DCs. Tumor rejection mediated by Trm cells triggers the spread of cytotoxic CD8+ T cell responses against tumor-derived neo- and self-antigens via dermal DCs. These responses suppress the growth of intradermal tumors and disseminated melanoma lacking the Trm cell-targeted epitope. Moreover,analysis of RNA sequencing data from human melanoma tumors reveals that enrichment of a Trm cell gene signature associates with DC activation and improved survival. This work unveils the ability of Trm cells to amplify the breath of cytotoxic CD8+ T cell responses through DCs,thereby strengthening anti-tumor immunity. View Publication

Gastrointestinal dysfunction in cystic fibrosis (CF) is a prominent source of pain among patients with CF. Linaclotide,a guanylate cyclase C (GCC) receptor agonist,is a US Food and Drug Administration-approved drug prescribed for chronic constipation but has not been widely used in CF,as the cystic fibrosis transmembrane conductance regulator (CFTR) is the main mechanism of action. However,anecdotal clinical evidence suggests that linaclotide may be effective for treating some gastrointestinal symptoms in CF. The goal of this study was to determine the effectiveness and mechanism of linaclotide in treating CF gastrointestinal disorders using CF mouse models. Intestinal transit,chloride secretion,and intestinal lumen fluidity were assessed in wild-type and CF mouse models in response to linaclotide. CFTR and sodium/hydrogen exchanger 3 (NHE3) response to linaclotide was also evaluated. Linaclotide treatment improved intestinal transit in mice carrying either F508del or null Cftr mutations but did not induce detectable Cl- secretion. Linaclotide increased fluid retention and fluidity of CF intestinal contents,suggesting inhibition of fluid absorption. Targeted inhibition of sodium absorption by the NHE3 inhibitor tenapanor produced improvements in gastrointestinal transit similar to those produced by linaclotide treatment,suggesting that inhibition of fluid absorption by linaclotide contributes to improved gastrointestinal transit in CF. Our results demonstrate that linaclotide improves gastrointestinal transit in CF mouse models by increasing luminal fluidity through inhibiting NHE3-mediated sodium absorption. Further studies are necessary to assess whether linaclotide could improve CF intestinal pathologies in patients. GCC signaling and NHE3 inhibition may be therapeutic targets for CF intestinal manifestations. NEW {\&} NOTEWORTHY Linaclotide's primary mechanism of action in alleviating chronic constipation is through cystic fibrosis transmembrane conductance regulator (CFTR),negating its use in patients with cystic fibrosis (CF). For the first time,our findings suggest that in the absence of CFTR,linaclotide can improve fluidity of the intestinal lumen through the inhibition of sodium/hydrogen exchanger 3. These findings suggest that linaclotide could improve CF intestinal pathologies in patients. View Publication

Secreted factors derived from Lactobacillus are able to dampen pro-inflammatory cytokine responses. Still,the nature of these components and the underlying mechanisms remain elusive. Here,we aimed to identify the components and the mechanism involved in the Lactobacillus-mediated modulation of immune cell activation. PBMC were stimulated in the presence of the cell free supernatants (CFS) of cultured Lactobacillus rhamnosus GG and Lactobacillus reuteri DSM 17938,followed by evaluation of cytokine responses. We show that lactobacilli-CFS effectively dampen induced IFN-$\gamma$ and IL-17A responses from T- and NK cells in a monocyte dependent manner by a soluble factor. A proteomic array analysis highlighted Lactobacillus-induced IL-1 receptor antagonist (ra) as a potential candidate responsible for the IFN-$\gamma$ dampening activity. Indeed,addition of recombinant IL-1ra to stimulated PBMC resulted in reduced IFN-$\gamma$ production. Further characterization of the lactobacilli-CFS revealed the presence of extracellular membrane vesicles with a similar immune regulatory activity to that observed with the lactobacilli-CFS. In conclusion,we have shown that lactobacilli produce extracellular MVs,which are able to dampen pro-inflammatory cytokine responses in a monocyte-dependent manner. View Publication

Sustained,indolent immune injury of the vasculature of a heart transplant limits long-term graft and recipient survival. This injury is mitigated by a poorly characterized,maladaptive repair response. Vascular endothelial cells respond to proangiogenic cues in the embryo by differentiation to specialized phenotypes,associated with expression of apelin. In the adult,the role of developmental proangiogenic cues in repair of the established vasculature is largely unknown. We found that human and minor histocompatibility-mismatched donor mouse heart allografts with alloimmune-mediated vasculopathy upregulated expression of apelin in arteries and myocardial microvessels. In vivo,loss of donor heart expression of apelin facilitated graft immune cell infiltration,blunted vascular repair,and worsened occlusive vasculopathy in mice. In vitro,an apelin receptor agonist analog elicited endothelial nitric oxide synthase activation to promote endothelial monolayer wound repair and reduce immune cell adhesion. Thus,apelin acted as an autocrine growth cue to sustain vascular repair and mitigate the effects of immune injury. Treatment with an apelin receptor agonist after vasculopathy was established markedly reduced progression of arterial occlusion in mice. Together,these initial data identify proangiogenic apelin as a key mediator of coronary vascular repair and a pharmacotherapeutic target for immune-mediated injury of the coronary vasculature. View Publication

Peripheral serotonin (5-hydroxytryptamine: 5-HT) synthesized in the intestine by enterochromaffin cells (ECs),plays an important role in the regulation of peristaltic of the gut,epithelial secretion and promotes the development and maintenance of the enteric neurons. Recent studies showed that the indigenous gut microbiota modulates 5-HT signalling and that ECs use sensory receptors to detect dietary and microbiota-derived signals from the lumen to subsequently transduce the information to the nervous system. We hypothesized that Clostridium ramosum by increasing gut 5-HT availability consequently contributes to high-fat diet-induced obesity. Using germ-free mice and mice monoassociated with C. ramosum,intestinal cell lines and mouse organoids,we demonstrated that bacterial cell components stimulate host 5-HT secretion and program the differentiation of colonic intestinal stem progenitors toward the secretory 5-HT-producing lineage. An elevated 5-HT level regulates the expression of major proteins involved in intestinal fatty acid absorption in vitro,suggesting that the presence of C. ramosum in the gut promotes 5-HT secretion and thereby could facilitates intestinal lipid absorption and the development of obesity. View Publication

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner,thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation,differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10,IRF8,KLF4,MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML. View Publication

Despite the key role that antibodies play in protection,the cellular processes mediating the acquisition of humoral immunity against malaria are not fully understood. Using an infection model of severe malaria,we find that germinal center (GC) B cells upregulate the transcription factor T-bet during infection. Molecular and cellular analyses reveal that T-bet in B cells is required not only for IgG2c switching but also favors commitment of B cells to the dark zone of the GC. T-bet was found to regulate the expression of Rgs13 and CXCR3,both of which contribute to the impaired GC polarization observed in the absence of T-bet,resulting in reduced IghV gene mutations and lower antibody avidity. These results demonstrate that T-bet modulates GC dynamics,thereby promoting the differentiation of B cells with increased affinity for antigen. View Publication

When transmitted through the oral route,Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication,as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However,due to a lack of tractable infection models,we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However,the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here,we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular,we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization,and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with,and support replication of,T. gondii. Using quantitative label-free mass spectrometry,we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism,extracellular exosomes,intermicrovillar adhesion,and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses,indicating that de novo synthesis may support,but is not required for,parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection. View Publication

Apoptosis and autophagy are dynamic processes that determine the fate of cells. Vitamin D receptor (VDR) deficiency in the intestine leads to abnormal Paneth cells and impaired autophagy function. Here,we will elucidate the mechanisms of the intestinal epithelial VDR regulation of autophagy and apoptosis. We used in vivo VDRlox and VDR∆IEC mice and ex vivo organoids generated from small intestine and colon tissues. We found that VDR deficiency induced more apoptotic cells and significantly increased cell death in the small intestine and colon of VDR∆IEC mice. The proapoptotic protein B-cell lymphoma 2 (BCL-2) associated X protein (Bax) was enhanced,whereas autophagy related 16 like 1 (ATG16L1) and Beclin-1 were decreased in the intestines of VDR$\Delta$IEC mice. Apoptosis induced by Bax reduced autophagy by decreasing Beclin-1. Physical interactions between Beclin-1 and Bcl-2 were increased in the VDR-deficient epithelia from mice. The growth of VDR∆IEC organoids was significantly slower with fewer Paneth cells than that of VDR+/+ organoids. The expression levels of Beclin-1 and lysozyme were decreased in VDR∆IEC organoids. Bacterial endotoxin levels were high in the serum from VDR∆IEC mice and made mice susceptible to colitis. In the organoids and colitis IL-10-/- mice,vitamin D3 treatment increased VDR and ATG16L1 protein expression levels,which activated autophagic responses. In summary,intestinal epithelial VDR regulates autophagy and apoptosis through ATG16L1 and Beclin-1. Our studies provide fundamental insights into the tissue-specific function of VDR in modulating the balance between autophagy and apoptosis.-Lu,R.,Zhang,Y.-G.,Xia,Y.,Sun,J. Imbalance of autophagy and apoptosis in intestinal epithelium lacking the vitamin D receptor. View Publication

BACKGROUND MiR-199a-3p (miR-199a) can enhance the chemosensitivity of hepatocellular carcinoma (HCC). Because of the easy degradation of miRNA by direct infusion,effective vehicle-mediated delivery of miR-199a may represent a new strategy for improving HCC chemotherapy. Considering mesenchymal stem cell (MSC)-derived exosomes as promising natural nanovectors for drug and molecule delivery,we aimed to determine whether exosomes from adipose tissue-derived MSCs (AMSCs) could be used to deliver miR-199a and improve HCC chemosensitivity. METHODS MiR-199a-modified AMSCs (AMSC-199a) were constructed by miR-199a lentivirus infection and puromycin selection. MiR-199-modified exosomes (AMSC-Exo-199a) were isolated from the supernatant of AMSC-199a and were assessed by transmission electron microscopy,nanoparticle tracking analysis,and flow cytometry analysis. The expression levels of miR-199a in HCC samples,AMSCs,exosomes,and HCC cells were quantified by real-time PCR. The effects of AMSC-Exo-199a on HCC chemosensitivity were determined by cell proliferation and apoptosis assays and by i.v. injection into orthotopic HCC mouse models with doxorubicin treatment. MTOR,p-4EBP1 and p-70S6K levels in HCC cells and tissues were quantified by Western blot. RESULTS AMSC-Exo-199a had the classic characteristics of exosomes and could effectively mediate miR-199a delivery to HCC cells. Additionally,AMSC-Exo-199a significantly sensitized HCC cells to doxorubicin by targeting mTOR and subsequently inhibiting the mTOR pathway. Moreover,i.v.-injected AMSC-Exo-199a could distribute to tumor tissue and markedly increased the effect of Dox against HCC in vivo. CONCLUSIONS AMSC-Exo-199a can be an effective vehicle for miR-199a delivery,and they effectively sensitized HCC to chemotherapeutic agents by targeting mTOR pathway. AMSC-Exo-199a administration may provide a new strategy for improving HCC chemosensitivity. View Publication

The gut microbiome contributes to inflammatory bowel disease (IBD),in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD,and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here,we treated human colonic biopsies,epithelial colonoids,and epithelial cells with an uncoupler of oxidative phosphorylation,dinitrophenol (DNP),with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6,an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis,induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather,specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note,epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria,thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation. View Publication