参考文献

Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages1,2. In mouse myeloid cells,Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b,which in turn activates pro-caspase-1 to mediate cell death3,but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples,but not in cells from many other lineages,and that these inhibitors inhibit human AML progression in mouse models. Overall,this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML. View Publication

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

Understanding the mechanisms of CD4 memory T cell (Tmem) differentiation in malaria is critical for vaccine development. However,the metabolic regulation of CD4 Tmem differentiation is not clear,particularly in persistent infections. In this study,we investigated the role of fatty acid synthesis (FAS) in Tmem development in Plasmodium chabaudi chronic mouse malaria infection. We show that T cell-specific deletion and early pharmaceutical inhibition of acetyl CoA carboxylase 1,the rate limiting step of FAS,inhibit generation of early memory precursor effector T cells (MPEC). To compare the role of FAS during early differentiation or survival of Tmem in chronic infection,a specific inhibitor of acetyl CoA carboxylase 1,5-(tetradecyloxy)-2-furoic acid,was administered at different times postinfection. Strikingly,the number of Tmem was only reduced when FAS was inhibited during T cell priming and not during the Tmem survival phase. FAS inhibition during priming increased effector T cell (Teff) proliferation and strongly decreased peak parasitemia,which is consistent with improved Teff function. Conversely,MPEC were decreased,in a T cell-intrinsic manner,upon early FAS inhibition in chronic,but not acute,infection. Early cure of infection also increased mitochondrial volume in Tmem compared with Teff,supporting previous reports in acute infection. We demonstrate that the MPEC-specific effect was due to the higher fatty acid content and synthesis in MPEC compared with terminally differentiated Teff. In conclusion,FAS in CD4 T cells regulates the early divergence of Tmem from Teff in chronic infection. 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