技术资料

Spinal cord injury (SCI) is one of the most debilitating injuries,and transplantation of stem cells in a scaffold is a promising strategy for treatment. However,stem cell treatment of SCI has been severely impaired by the increased generation of reactive oxygen species in the lesion microenvironment,which can lead to a high level of stem cell death and dysfunction. Herein,a MnO2 nanoparticle (NP)-dotted hydrogel is prepared through dispersion of MnO2 NPs in a PPFLMLLKGSTR peptide modified hyaluronic acid hydrogel. The peptide-modified hydrogel enables the adhesive growth of mesenchymal stem cells (MSCs) and nerve tissue bridging. The MnO2 NPs alleviate the oxidative environment,thereby effectively improving the viability of MSCs. Transplantation of MSCs in the multifunctional gel generates a significant motor function restoration on a long-span rat spinal cord transection model and induces an in vivo integration as well as neural differentiation of the implanted MSCs,leading to a highly efficient regeneration of central nervous spinal cord tissue. Therefore,the MnO2 NP-dotted hydrogel represents a promising strategy for stem-cell-based therapies of central nervous system diseases through the comprehensive regulation of pathological microenvironment complications. View Publication

Reduced serum testosterone (T),or hypogonadism,affects millions of men and is associated with many pathologies,including infertility,cardiovascular diseases,metabolic syndrome,and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However,TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus,there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs),proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs,the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively,human induced pluripotent stem cells (hiPSCs),which are expandable in culture and have the potential to differentiate into all somatic cell types,have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis,synthesized T rather than cortisol,secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol,and displayed ultrastructural features resembling LCs. By contrast,hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration. View Publication

Bone loss in postmenopausal osteoporosis is induced chiefly by an imbalance of bone-forming osteoblasts and bone-resorbing osteoclasts. Salubrinal is a synthetic compound that inhibits de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2$\alpha$). Phosphorylation of eIF2$\alpha$ alleviates endoplasmic reticulum (ER) stress,which may activate autophagy. We hypothesized that eIF2$\alpha$ signaling regulates bone homeostasis by promoting autophagy in osteoblasts and inhibiting osteoclast development. To test the hypothesis,we employed salubrinal to elevate the phosphorylation of eIF2$\alpha$ in an ovariectomized (OVX) mouse model and cell cultures. In the OVX model,salubrinal prevented abnormal expansion of rough ER and decreased the number of acidic vesiculars. It regulated ER stress-associated signaling molecules such as Bip,p-eIF2$\alpha$,ATF4 and CHOP,and promoted autophagy of osteoblasts via regulation of eIF2$\alpha$,Atg7,LC3,and p62. Salubrinal markedly alleviated OVX-induced symptoms such as reduction of bone mineral density and bone volume fraction. In primary bone-marrow-derived cells,salubrinal increased the differentiation of osteoblasts,and decreased the formation of osteoclasts by inhibiting nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Live cell imaging and RNA interference demonstrated that suppression of osteoclastogenesis is in part mediated by Rac1 GTPase. Collectively,this study demonstrates that ER stress-autophagy axis plays an important role in OVX mice. Bone-forming osteoblasts are restored by maintaining phosphorylation of eIF2$\alpha$,and bone-resorbing osteoclasts are regulated by inhibiting NFATc1 and Rac1 GTPase. View Publication

Glucocorticoids (GCs) play an important role in controlling acute graft-versus-host disease (aGvHD),a frequent complication of allogeneic hematopoietic stem cell transplantation. The anti-inflammatory activity of GCs is mainly ascribed to the modulation of T cells and macrophages,for which reason a genetically induced GC resistance of either of these cell types causes aggravated aGvHD. Since only a few genes are currently known that are differentially regulated under these conditions,we analyzed the expression of 54 candidate genes in the inflamed small intestine of mice suffering from aGvHD when either allogeneic T cells or host myeloid cells were GC resistant using a microfluidic dynamic array platform for high-throughput quantitative PCR. The majority of genes categorized as cytokines (e.g. Il2,Il6),chemokines (e.g. Ccl2,Cxcl1),cell surface receptors (e.g. Fasl,Ctla4) and intracellular molecules (e.g. Dusp1,Arg1) were upregulated in mice transplanted with GC resistant allogeneic T cells. Moreover,the expression of several genes linked to energy metabolism (e.g. Glut1) was altered. Surprisingly,mice harboring GC resistant myeloid cells showed almost no changes in gene expression despite their fatal disease course after aGvHD induction. To identify additional genes in the inflamed small intestine that were affected by a GC resistance of allogeneic T cells,we performed an RNAseq analysis,which uncovered more than 500 differentially expressed transcripts (e.g. Cxcr6,Glut3,Otc,Aoc1,Il1r1,Sphk1) that were enriched for biological processes associated with inflammation and tissue disassembly. The changes in gene expression could be confirmed during full-blown disease but hardly any of them in the preclinical phase using high-throughput quantitative PCR. Further analysis of some of these genes revealed a highly selective expression pattern in T cells,intestinal epithelial cells and macrophages,which correlated with their regulation during disease progression. Collectively,we identified an altered gene expression profile caused by GC resistance of transplanted allogeneic T cells,which could help to define new targets for aGvHD therapy. View Publication

Necrotizing enterocolitis (NEC) is a devastating neonatal disease characterized by acute intestinal injury. Intestinal stem cell (ISC) renewal is required for gut regeneration in response to acute injury. The Wnt/$\beta$-catenin pathway is essential for intestinal renewal and ISC maintenance. We found that ISC expression,Wnt activity and intestinal regeneration were all decreased in both mice with experimental NEC and in infants with acute active NEC. Moreover,intestinal organoids derived from NEC-injured intestine of both mice and humans failed to maintain proliferation and presented more differentiation. Administration of Wnt7b reversed these changes and promoted growth of intestinal organoids. Additionally,administration of exogenous Wnt7b rescued intestinal injury,restored ISC,and reestablished intestinal epithelial homeostasis in mice with NEC. Our findings demonstrate that during NEC,Wnt/$\beta$-catenin signaling is decreased,ISC activity is impaired,and intestinal regeneration is defective. Administration of Wnt resulted in the maintenance of intestinal epithelial homeostasis and avoidance of NEC intestinal injury. View Publication

In addition to antigen-driven signals,T cells need co-stimulatory signals for robust activation. Several receptors,including members of the tumor necrosis factor receptor superfamily (TNFRSF),can deliver co-stimulatory signals to T cells. Thioredoxin interacting protein (TXNIP) is an important inhibitor of glucose uptake and cell proliferation,but it is unknown how TXNIP is regulated in T cells. The aim of this study was to determine expression levels and regulation of TXNIP in human T cells. We found that na{\{i}}ve T cells express high levels of TXNIP and that treatment of blood samples with TNF results in rapid down-regulation of TXNIP in the T cells. TNF-induced TXNIP down-regulation correlated with increased glucose uptake. Furthermore we found that density gradient centrifugation (DGC) induced down-regulation of TXNIP. We demonstrate that DGC induced TNF production that paralleled the TXNIP down-regulation. Treatment of blood with toll-like receptor (TLR) ligands induced TNF production and TXNIP down-regulation suggesting that damage-associated molecular patterns (DAMPs) such as endogenous TLR ligands released during DGC play a role in DGC-induced TXNIP down-regulation. Finally we demonstrate that TNF-induced TXNIP down-regulation is dependent on caspase activity and is caused by caspase-mediated cleavage of TXNIP." View Publication

BACKGROUND Vitamin D deficiency is associated with intestinal barrier dysfunction,which contributes to pathogenesis of acute intestinal injury in children. We aim to investigate the effects of vitamin D on intestinal injury in intestinal epithelial cells and organoids. METHODS Lipopolysaccharide (LPS) was used to induce injury in intestinal epithelial cells (IEC-18) and organoids,and the effect of vitamin D was assessed. Cell viability was measured and inflammation cytokines TNF$\alpha$ and IL-8 were quantified. FITC-dextran 4 kDa (FD4) permeability was measured using Transwell while tight junction markers were assessed by immunofluorescence staining in IEC-18 and intestinal organoids. Data were compared using one-way ANOVA with Bonferroni post-test. RESULTS IEC-18 viability was decreased by LPS treatment,but was prevented by vitamin D. The upregulation of inflammation was inhibited by vitamin D,which also decreased epithelium permeability. Vitamin D restored tight junction ZO-1 and claudin 2. In addition,vitamin D decreased TNF$\alpha$ expression and prevented the disruption of ZO-1 in injured organoids. CONCLUSIONS Vitamin D rescued epithelial barrier function by improving permeability and restoring tight junctions,leading to decrease inflammation. This study confirms the protective effects of vitamin D,which could be used as a treatment strategy for infants at risk of developing intestinal injury. View Publication

Epitope-specific CD4+ T cells can be labeled in complex cell mixtures from secondary lymphoid organs with fluorophore-labeled peptide:major histocompatibility complex class II (p:MHCII) tetramers and then detected by flow cytometry. Magnetic enrichment of tetramer-bound cells before flow cytometry increases the sensitivity of detection to the point where epitope-specific cells can be studied even when very rare at early and late times after the host has been exposed to the epitope. This method is very useful for studying polyclonal epitope-specific CD4+ T cells under physiological conditions. {\textcopyright} 2019 by John Wiley {\&} Sons,Inc. View Publication

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