参考文献

Myeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD),donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer,resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen-induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival,indicating in vivo biologic effects,no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1$\beta$ (IL-1$\beta$),we expected that IL-1$\beta$ KO donor MDSCs would be superior in subverting GVHD,but such MDSCs proved inferior relative to WT. IL-1$\beta$ release and IL-1 receptor expression was required for optimal MDSC function,and exogenous IL-1$\beta$ added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1$\beta$ could diminish survival in GVHD. However,loss of inflammasome activation and IL-1$\beta$ release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1$\beta$ signaling,improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum,the ATP release site,synergized with WT MDSCs,as did regulatory T-cell infusion,which we showed reduced but did not eliminate MDSC inflammasome activation,as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments. View Publication

BACKGROUND The combination of serum $\beta$2-microglubulin and albumin levels is highly prognostic in multiple myeloma (MM),defined as the International Staging System (ISS). Recurrent genomic abnormalities present in myeloma cells also have a strong prognostic power. This study aimed to assess,in a routine diagnostic setting,whether genomic aberrations can be used to identify sub-groups in ISS staging,as this system does not incorporate intrinsic myeloma cell variability at the molecular level. MATERIALS AND METHODS A prospective population-based study of 123 patients newly diagnosed with MM with ISS staging were included for karyotyping,interphase nuclei fluorescence in situ hybridization (iFISH) and oligo-based array comparative genomic hybridization (oaCGH) analyses. RESULTS Clonal abnormalities were identified in 27{\%} of analyses by karyotyping,in 83{\%} by iFISH,and in 99{\%} by oaCGH analysis. ISS staging combined with oaCGH aberrations identified ISS sub-groups. CONCLUSION oaCGH analysis is a valuable asset in detecting prognostically relevant genomic abnormalities. The combination of oaCGH data with ISS staging might help define new sub-groups in MM. View Publication

Microfold (M) cells residing in the follicle-associated epithelium (FAE) of the gut-associated lymphoid tissue are specialized for antigen uptake to initiate mucosal immune responses. The molecular machinery and biological significance of M cell differentiation,however,remain to be fully elucidated. Here,we demonstrate that Sox8,a member of the SRY-related HMG box transcription factor family,is specifically expressed by M cells in the intestinal epithelium. The expression of Sox8 requires activation of RANKL-RelB signaling. Chromatin immunoprecipitation and luciferase assays revealed that Sox8 directly binds the promoter region of Gp2 to increase Gp2 expression,which is the hallmark of functionally mature M cells. Furthermore,genetic deletion of Sox8 causes a marked decrease in the number of mature M cells,resulting in reduced antigen uptake in Peyer's patches. Consequently,juvenile Sox8-deficient mice showed attenuated germinal center reactions and antigen-specific IgA responses. These findings indicate that Sox8 plays an essential role in the development of M cells to establish mucosal immune responses. View Publication

BACKGROUND {\&} AIMS The nuclear receptor subfamily 0 group B member 2 (NR0B2,also called SHP) is expressed at high levels in the liver and intestine. Postprandial fibroblast growth factor 19 (human FGF19,mouse FGF15) signaling increases the transcriptional activity of SHP. We studied the functions of SHP and FGF19 in the intestines of mice,including their regulation of expression of the cholesterol transporter NPC1L1 )NPC1-like intracellular cholesterol transporter 1) and cholesterol absorption. METHODS We performed histologic and biochemical analyses of intestinal tissues from C57BL/6 and SHP-knockout mice and performed RNA-sequencing analyses to identify genes regulated by SHP. The effects of fasting and refeeding on intestinal expression of NPC1L1 were examined in C57BL/6,SHP-knockout,and FGF15-knockout mice. Mice were given FGF19 daily for 1 week; fractional cholesterol absorption,cholesterol and bile acid (BA) levels,and composition of BAs were measured. Intestinal organoids were generated from C57BL/6 and SHP-knockout mice,and cholesterol uptake was measured. Luciferase reporter assays were performed with HT29 cells. RESULTS We found that the genes that regulate lipid and ion transport in intestine,including NPC1L1,were up-regulated and that cholesterol absorption was increased in SHP-knockout mice compared with C57BL/6 mice. Expression of NPC1L1 was reduced in C57BL/6 mice after refeeding after fasting but not in SHP-knockout or FGF15-knockout mice. SHP-knockout mice had altered BA composition compared with C57BL/6 mice. FGF19 injection reduced expression of NPC1L1,decreased cholesterol absorption,and increased levels of hydrophilic BAs,including tauro-$\alpha$- and -$\beta$-muricholic acids; these changes were not observed in SHP-knockout mice. SREBF2 (sterol regulatory element binding transcription factor 2),which regulates cholesterol,activated transcription of NPC1L1. FGF19 signaling led to phosphorylation of SHP,which inhibited SREBF2 activity. CONCLUSIONS Postprandial FGF19 and SHP inhibit SREBF2,which leads to repression of intestinal NPC1L1 expression and cholesterol absorption. Strategies to increase FGF19 signaling to activate SHP might be developed for treatment of hypercholesterolemia. View Publication

BACKGROUND Human pancreata contain many types of cells,such as endocrine islets,acinar,ductal,fat,and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions. METHODS We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore,whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated. RESULTS IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5{\%} human platelet lysate (hPL). IPTD-MSCs were found to be highly pure,with {\textgreater} 95{\%} positive for CD90,CD105,and CD73,and negative for CD45,CD34,CD14,and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes,chondrocytes,and osteoblasts in vitro,underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-$\alpha$,gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased,compared to control. In contrast,treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly,a combination of TNF-$\alpha$ and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-$\alpha$ contained higher levels of pro-angiogenic (VEGF,IL-6,and IL-8) compared to controls,promoting angiogenesis of human endothelial cells in vitro. In contrast,levels of MCP-1,a pro-inflammatory cytokine,were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-$\alpha$. CONCLUSIONS The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application. View Publication

Adherens junctions (AJs),together with tight junctions (TJs),form an apical junctional complex that regulates intestinal epithelial cell-to-cell adherence and barrier homeostasis. Within the AJ,membrane-bound E-cadherin binds $\beta$-catenin,which functions as an essential intracellular signaling molecule. We have previously identified a novel protein in the region of the apical junction complex,chloride channel protein-2 (ClC-2),that we have used to study TJ regulation. In this study,we investigated the possible effects of ClC-2 on the regulation of AJs in intestinal mucosal epithelial homeostasis and tumorigenicity. Mucosal homeostasis and junctional proteins were examined in wild-type (WT) and ClC-2 knockout (KO) mice as well as associated colonoids. Tumorigenicity and AJ-associated signaling were evaluated in a murine colitis-associated tumor model and in a colorectal cancer cell line (HT-29). Colonic tissues from ClC-2 KO mice had altered ultrastructural morphology of intercellular junctions with reduced colonocyte differentiation,whereas jejunal tissues had minimal changes. Colonic crypts from ClC-2 KO mice had significantly higher numbers of less-differentiated forms of colonoids compared with WT. Furthermore,the absence of ClC-2 resulted in redistribution of AJ proteins and increased $\beta$-catenin activity. Downregulation of ClC-2 in colorectal cells resulted in significant increases in proliferation associated with disruption of AJs. Colitis-associated tumors in ClC-2 KO mice were significantly increased,associated with $\beta$-catenin transcription factor activation. The absence of ClC-2 results in less differentiated colonic crypts and increased tumorigenicity associated with colitis via dysregulation of AJ proteins and activation of $\beta$-catenin-associated signaling. NEW {\&} NOTEWORTHY Disruption of adherens junctions in the absence of chloride channel protein-2 revealed critical functions of these junctional structures,including maintenance of colonic homeostasis and differentiation as well as driving tumorigenicity by regulating $\beta$-catenin signaling. View Publication

Recurrent oncogenic mutations of MyD88 have been identified in a variety of lymphoid malignancies. Gain-of-function mutations of MyD88 constitutively activate downstream NF-$\kappa$B signaling pathways,resulting in increased cellular proliferation and survival. However,whether MyD88 activity can be aberrantly regulated in MyD88-wild-type lymphoid malignancies remains poorly understood. SPOP is an adaptor protein of CUL3-based E3 ubiquitin ligase complex and frequently mutated genes in prostate and endometrial cancers. In this study,we reveal that SPOP binds to and induces the nondegradative ubiquitination of MyD88 by recognizing an atypical SPOP-binding motif in MyD88. This modification blocks Myddosome assembly and downstream NF-$\kappa$B activation. SPOP is mutated in a subset of lymphoid malignancies,including diffuse large B-cell lymphoma (DLBCL). Lymphoid malignancies-associated SPOP mutants exhibited impaired binding to MyD88 and suppression of NF-$\kappa$B activation. The DLBCL-associated,SPOP-binding defective mutants of MyD88 escaped from SPOP-mediated ubiquitination,and their effect on NF-$\kappa$B activation is stronger than that of wild-type MyD88. Moreover,SPOP suppresses DLBCL cell growth in vitro and tumor xenograft in vivo by inhibiting the MyD88/NF-$\kappa$B signaling. Therefore,SPOP acts as a tumor suppressor in DLBCL. Mutations in the SPOP-MyD88 binding interface may disrupt the SPOP-MyD88 regulatory axis and promote aberrant MyD88/NF-$\kappa$B activation and cell growth in DLCBL. View Publication

Mesenchymal stem cells (MSCs) in bone marrow and adipose tissues are expected to be effective tools for regenerative medicine to treat various diseases. To obtain MSCs that possess both high differentiation and tissue regenerative potential,it is necessary to establish an isolation system that does not require long-term culture. It has previously been reported that the cytoskeletal protein vimentin,expressed on the surfaces of multiple cell types,possesses N-acetylglucosamine- (GlcNAc-) binding activity. Therefore,we tried to exploit this interaction to efficiently isolate MSCs from rat bone marrow cells using GlcNAc-bearing polymer-coated dishes. Cells isolated by this method were identified as MSCs because they were CD34-,CD45-,and CD11b/c-negative and CD90-,CD29-,CD44-,CD54-,CD73-,and CD105-positive. Osteoblast,adipocyte,and chondrocyte differentiation was observed in these cells. In total,yields of rat MSCs were threefold to fourfold higher using GlcNAc-bearing polymer-coated dishes than yields using conventional tissue-culture dishes. Interestingly,MSCs isolated with GlcNAc-bearing polymer-coated dishes strongly expressed CD106,whereas those isolated with conventional tissue-culture dishes had low CD106 expression. Moreover,senescence-associated $\beta$-galactosidase activity in MSCs from GlcNAc-bearing polymer-coated dishes was lower than that in MSCs from tissue-culture dishes. These results establish an improved isolation method for high-quality MSCs. View Publication

Therapeutic drug monitoring is routinely performed to maintain optimal tacrolimus concentrations in kidney transplant recipients. Nonetheless,toxicity and rejection still occur within an acceptable concentration-range. To have a better understanding of the relationship between tacrolimus dose,tacrolimus concentration,and its effect on the target cell,we developed functional immune tests for the quantification of the tacrolimus effect. Twelve healthy volunteers received a single dose of tacrolimus,after which intracellular and whole blood tacrolimus concentrations were measured and were related to T cell functionality. A significant correlation was found between tacrolimus concentrations in T cells and whole blood concentrations (r = 0.71,p = 0.009),while no correlation was found between tacrolimus concentrations in peripheral blood mononuclear cells (PBMCs) and whole blood (r = 0.35,p = 0.27). Phytohemagglutinin (PHA) induced the production of IL-2 and IFN$\gamma$,as well as the inhibition of CD71 and CD154 expression on T cells at 1.5 h post-dose,when maximum tacrolimus levels were observed. Moreover,the in vitro tacrolimus effect of the mentioned markers corresponded with the ex vivo effect after dosing. In conclusion,our results showed that intracellular tacrolimus concentrations mimic whole blood concentrations,and that PHA-induced cytokine production (IL-2 and IFN$\gamma$) and activation marker expression (CD71 and CD154) are suitable readout measures to measure the immunosuppressive effect of tacrolimus on the T cell. View Publication

We study the effect of antifreeze glycoproteins (AFGPs) on the survival of organoids under hypothermic conditions. We find that the survival of organoids in cold conditions depends on their developmental stage. Mature organoids die within 24 h when being stored at 4 °C,while cystic organoids can survive up to 48 h. We find that in the presence of AFGPs,the organoid survival is prolonged up to 72 h,irrespective of their developmental stage. Fluorescence microscopy experiments reveal that the AFGPs predominately localize at the cell surface and cover the cell membranes. Our findings support a mechanism in which the positive effect of AFGPs on cell survival during hypothermic storage involves the direct interaction of AFGPs with the cell membrane. Our research highlights organoids as an attractive multicellular model system for studying the action of AFGPs that bridges the gap between single-cell and whole-organ studies. View Publication