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Bronchial epithelial cells (BECs) from healthy children inhibit human lung fibroblast (HLF) expression of collagen and fibroblast-to-myofibroblast transition (FMT),whereas asthmatic BECs do so less effectively,suggesting that diminished epithelial-derived regulatory factors contribute to airway remodeling. Preliminary data demonstrated that secretion of the activin A inhibitor follistatin-like 3 (FSTL3) by healthy BECs was greater than that by asthmatic BECs. We sought to determine the relative secretion of FSTL3 and activin A by asthmatic and healthy BECs,and whether FSTL3 inhibits FMT. To quantify the abundance of the total proteome FSTL3 and activin A in supernatants of differentiated BEC cultures from healthy children and children with asthma,we performed mass spectrometry and ELISA. HLFs were cocultured with primary BECs and then HLF expression of collagen I and alpha$-smooth muscle actin (alpha$-SMA) was quantified by qPCR,and FMT was quantified by flow cytometry. Loss-of-function studies were conducted using lentivirus-delivered shRNA. Using mass spectrometry and ELISA results from larger cohorts,we found that FSTL3 concentrations were greater in media conditioned by healthy BECs compared with asthmatic BECs (4,012 vs. 2,553 pg/ml; P = 0.002),and in media conditioned by asthmatic BECs from children with normal lung function relative to those with airflow obstruction (FEV1/FVC ratio {\textless} 0.8; n = 9; 3,026 vs. 1,922 pg/ml; P = 0.04). shRNA depletion of FSTL3 in BECs (n = 8) increased HLF collagen I expression by 92{\%} (P = 0.001) and alpha$-SMA expression by 88{\%} (P = 0.02),and increased FMT by flow cytometry in cocultured HLFs,whereas shRNA depletion of activin A (n = 6) resulted in decreased alpha$-SMA (22{\%}; P = 0.01) expression and decreased FMT. Together,these results indicate that deficient FSTL3 expression by asthmatic BECs impairs epithelial regulation of HLFs and FMT. View Publication

Suprachiasmatic nucleus circadian oscillatory protein (SCOP) (a.k.a. PHLPP1) regulates long-term memory consolidation in the brain. Using a mouse model of controlled cortical impact (CCI) we tested if (1) brain tissue levels of SCOP/PHLPP1 increase after a traumatic brain injury (TBI),and (2) if SCOP/PHLPP1 gene knockout (KO) mice have improved (or worse) neurologic outcomes. Blood chemistry (pH,pCO2,pO2,pSO2,base excess,sodium bicarbonate,and osmolarity) and arterial pressure (MAP) differed in isoflurane anesthetized WT vs. KOs at baseline and up to 1 h post-injury. CCI injury increased cortical/hippocampal SCOP/PHLPP1 levels in WTs 7d and 14d post-injury. Injured KOs had higher brain tissue levels of phosphorylated AKT (pAKT) in cortex (14d post-injury),and higher levels of phosphorylated MEK (pMEK) in hippocampus (7d and 14d post-injury) and in cortex (7d post-injury). Consistent with an important role of SCOP/PHLPP1 on memory function,injured-KOs had near normal performance on the probe trial of the Morris water maze,whereas injured-WTs were impaired. CA1/CA3 hippocampal survival was lower in KOs vs. WTs 24 h post-injury but equivalent by 7d. No difference in 21d cortical lesion volume was detected. SCOP/PHLPP1 overexpression in cultured rat cortical neurons had no effect on 24 h cell death after a mechanical stretch-injury. View Publication

Background and Aims Disturbance of intestinal homeostasis is associated with the development of inflammatory bowel disease (IBD),and TGF-beta$ signaling impairment in mononuclear phagocytes (MPs) causes murine colitis with goblet cell depletion. Here,we examined an organoid-MP co-culture system to study the role of MPs in intestinal epithelial differentiation and homeostasis. Methods Intestinal organoids were co-cultured with lamina propria leukocytes and bone marrow-derived dendritic cells (BMDCs) from CD11c-cre Tgfbr2fl/fl mice. Organoid-MP adhesive interactions were evaluated by microscopy,RT-PCR,and flow cytometry. Murine colitis models (dextran sodium sulphate (DSS),CD11c-cre Tgfbr2fl/fl,T-cell-transfer) were used for histological and immunohistochemical analysis. Anti-E-cadherin antibody treatment or CD11c+-cell-specific CDH1 gene deletion were performed for E-cadherin neutralization or knockout. Colonic biopsies from patients with ulcerative colitis were analyzed by flow cytometry. Results Intestinal organoids co-cultured with CD11c+ lamina propria leukocytes or BMDCs from CD11c-cre Tgfbr2fl/fl mice showed morphological changes and goblet cell depletion with Notch signal activation,analogous to CD11c-cre Tgfbr2fl/fl colitis. E-cadherin was upregulated in CD11c+ MPs,especially CX3CR1+CCR2+ monocytes,of CD11c-cre Tgfbr2fl/fl mice. E-cadherin-mediated BMDC adhesion promoted Notch activation and cystic changes in organoids. Anti-E-cadherin antibody treatment attenuated colitis in CD11c-cre Tgfbr2fl/fl and T-cell-transferred mice. In addition,E-cadherin deletion in CD11c+ cells attenuated colitis in both CD11c-cre Tgfbr2fl/fl and DSS-treated mice. In patients with ulcerative colitis,E-cadherin expressed by intestinal CD11c+ leukocytes was enhanced compared with that in healthy controls. Conclusions E-cadherin-mediated MP-epithelium adhesion is associated with the development of colitis,and blocking these adhesions may have therapeutic potential for IBD. View Publication

BackgroundThere is substantial evidence that signaling through Toll-like receptor 4 (TLR4) contributes to the pathogenesis of necrotizing enterocolitis (NEC). Pregnane X receptor (PXR),a xenobiotic sensor and signaling intermediate for certain host-bacterial metabolites,has been shown to negatively regulate TLR4 signaling. Here we investigated the relationship between PXR and TLR4 in the developing murine intestine and explored the capacity of PXR to modulate inflammatory pathways involved in experimental NEC.MethodsWild-type and PXR-/- mice were studied at various time points of development in an experimental model of NEC. In addition,we studied the ability of the secondary bile acid lithocholic acid (LCA),a known PXR agonist in liver,to activate intestinal PXR and reduce NEC-related intestinal inflammation.ResultsWe found a reciprocal relationship between the developmental expression of PXR and TLR4 in wild-type murine intestine,with PXR acting to reduce TLR4 expression by decreasing TLR4 mRNA stability. In addition,PXR-/- mice exhibited a remarkably heightened severity of disease in experimental NEC. Moreover,LCA attenuated intestinal proinflammatory responses in the early stages of experimental NEC.ConclusionThese findings provide proactive insights into the regulation of TLR4 in the developing intestine. Targeting PXR may be a novel approach for NEC prevention. View Publication

Intraepithelial lymphocytes (IELs) expressing the gamma$delta$ TCR (gamma$delta$ IELs) provide continuous surveillance of the intestinal epithelium. However,the mechanisms regulating the basal motility of these cells within the epithelial compartment have not been well defined. We investigated whether IL-15 contributes to gamma$delta$ IEL localization and migratory behavior in addition to its role in IEL differentiation and survival. Using advanced live cell imaging techniques in mice,we find that compartmentalized overexpression of IL-15 in the lamina propria shifts the distribution of gamma$delta$ T cells from the epithelial compartment to the lamina propria. This mislocalization could be rescued by epithelial IL-15 overexpression,indicating that epithelial IL-15 is essential for gamma$delta$ IEL migration into the epithelium. Furthermore,in vitro analyses demonstrated that exogenous IL-15 stimulates gamma$delta$ IEL migration into cultured epithelial monolayers,and inhibition of IL-2Rbeta$ significantly attenuates the basal motility of these cells. Intravital microscopy showed that impaired IL-2Rbeta$ signaling induced gamma$delta$ IEL idling within the lateral intercellular space,which resulted in increased early pathogen invasion. Similarly,the redistribution of gamma$delta$ T cells to the lamina propria due to local IL-15 overproduction also enhanced bacterial translocation. These findings thus reveal a novel role for IL-15 in mediating gamma$delta$ T cell localization within the intestinal mucosa and regulating gamma$delta$ IEL motility and patrolling behavior as a critical component of host defense. View Publication

Regulation of AMPA receptor (AMPAR) trafficking is a key modulator of excitatory synaptic transmission; however,intracellular vesicular transport of newly synthesized AMPARs has been little studied due to technical limitations. By combining molecular tools with imaging strategies in cultured rat hippocampal neurons,we found that vesicles containing newly synthesized,GluA1-subunit-containing AMPARs are transported antero- and retrogradely at a mean speed of 1.5 mu$m.s-1. Synaptic activity and variations in intracellular calcium levels bidirectionally modulate GluA1 transport. Chemical long-term potentiation (cLTP) initially induces a halt in GluA1 transport,followed by a sustained increase,while acute glutamate uncaging on synaptic spines arrests vesicular movements. GluA1 phosphomimetic mutants preferentially travel to the dendritic tip,probably to replenish extrasynaptic pools,distal to the soma. Our findings indicate that AMPAR intracellular transport is highly regulated during synaptic plasticity and likely controls AMPAR numbers at the plasma membrane. View Publication

Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However,few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays,which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing,endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall,we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation,which may be important in future regenerative medicine applications requiring vascularization. View Publication

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease of unknown etiopathogenesis with limited therapeutic options. IPF is characterized by an abundance of fibroblasts and loss of epithelial progenitors,which cumulates in unrelenting fibrotic lung remodeling and loss of normal oxygenation. IPF has been challenging to model in rodents; nonetheless,mouse models of lung fibrosis provide clues as to the natural progression of lung injury and remodeling,but many have not been useful in predicting efficacy of therapeutics in clinical IPF. We provide a detailed methodologic description of various iterations of humanized mouse models,initiated by the i.v. injection of cells from IPF lung biopsy or explants specimens into severe combined immunodeficiency (SCID)/beige or nonobese diabetic SCID gamma$ mice. Unlike cells from normal lung samples,IPF cells promote persistent,nonresolving lung remodeling in SCID mice. Finally,we provide examples and discuss potential advantages and pitfalls of human-specific targeting approaches in a humanized SCID model of pulmonary fibrosis. View Publication

Short-term outcomes of kidney transplantation have improved dramatically,but chronic rejection and regimen-related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless,the immunomodulatory effects of end-stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study,we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover,these Tregs maintain a CD3(+) CD4(+) CD25(+) CD127(lo) surface phenotype,high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg-specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple,brief Treg production strategy in clinical trials of mismatched kidney transplantation. View Publication

Huntington's disease (HD) is caused by an expanded CAG repeat in the Huntingtin (HTT) gene. The mechanism(s) by which mutant HTT (mHTT) causes disease is unclear. Nucleocytoplasmic transport,the trafficking of macromolecules between the nucleus and cytoplasm,is tightly regulated by nuclear pore complexes (NPCs) made up of nucleoporins (NUPs). Previous studies offered clues that mHTT may disrupt nucleocytoplasmic transport and a mutation of an NUP can cause HD-like pathology. Therefore,we evaluated the NPC and nucleocytoplasmic transport in multiple models of HD,including mouse and fly models,neurons transfected with mHTT,HD iPSC-derived neurons,and human HD brain regions. These studies revealed severe mislocalization and aggregation of NUPs and defective nucleocytoplasmic transport. HD repeat-associated non-ATG (RAN) translation proteins also disrupted nucleocytoplasmic transport. Additionally,overexpression of NUPs and treatment with drugs that prevent aberrant NUP biology also mitigated this transport defect and neurotoxicity,providing future novel therapy targets. View Publication

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML,they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors,we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism,resulting in impaired production of the antioxidant factor glutathione,which was further impaired by ATM or G6PD inactivation. Moreover,FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo,revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML. View Publication

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article,we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro,respectively. Furthermore,our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells,along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover,we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion,our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system. View Publication