技术资料

Uncoupling of oxidative phosphorylation in epithelial mitochondria results in decreased epithelial barrier function as characterized by increased internalization of non-invasive Escherichia coli and their translocation across the epithelium. We hypothesized that the increased burden of intracellular commensal bacteria would activate the enterocyte,with the potential to promote inflammation. Treatment of human colon-derived epithelial cell lines in vitro with dinitrophenol (DNP) and commensal E. coli (strains F18,HB101) provoked increased production of interleukin (IL-8),which was not observed with conditioned medium from the bacteria,lipopolysaccharide or inert beads. The IL-8 response was inhibited by co-treatment with cytochalasin-D (blocks F-actin rearrangement),chloroquine (blocks phagosome acidification) and a MyD88 inhibitor (blocks TLR signaling),consistent with TLR-signaling mediating IL-8 synthesis subsequent to bacterial internalization. Use of the mitochondria-targeted antioxidant,mitoTEMPO,or U0126 to block ERK1/2 MAPK signalling inhibited DNP+E. coli-evoked IL-8 production. Mutations in the NOD2 (the intracellular sensor of bacteria) or ATG16L1 (autophagy protein) genes are susceptibility traits for Crohn's,and epithelia lacking either protein displayed enhanced IL-8 production in comparison to wild-type cells when exposed to DNP + E coli. Thus,metabolic stress perturbs the normal epithelial-bacterial interaction resulting in increased IL-8 production due to uptake of bacteria into the enterocyte: this potentially pro-inflammatory event is enhanced in cells lacking NOD2 or ATG16L1 that favor increased survival of bacteria within the enterocyte. We speculate that by increasing epithelial permeability and IL-8 production,reduced mitochondria function in the enteric epithelium would contribute to the initiation,pathophysiology,and reactivation of inflammatory disease in the gut. View Publication

Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin in response to epithelial forces,and thereby regulate intestinal fluid secretion. However,it is unknown whether EE and EC cells are directly mechanosensitive,and if so,what the molecular mechanism of their mechanosensitivity is. Consequently,the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors,and they are expressed in mouse and human EC cells. Here,we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells,and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current,which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca2+ increase in isolated cells as well as in EE cells within intestinal organoids,and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive,uncovers Piezo2 as their primary mechanotransducer,defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release,and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology. View Publication

Traumatic spinal cord injury results in persistent disability due to disconnection of surviving neural elements. Neural stem cell transplantation has been proposed as a therapeutic option,but optimal cell type and mechanistic aspects remain poorly defined. Here,we describe robust engraftment into lesioned immunodeficient mice of human neuroepithelial stem cells derived from the developing spinal cord and maintained in self-renewing adherent conditions for long periods. Extensive elongation of both graft and host axons occurs. Improved functional recovery after transplantation depends on neural relay function through the grafted neurons,requires the matching of neural identity to the anatomical site of injury,and is accompanied by expression of specific marker proteins. Thus,human neuroepithelial stem cells may provide an anatomically specific relay function for spinal cord injury recovery. View Publication

Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here,we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the $\beta$-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation,there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70{\%} of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting,HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules,UM171 and SR1,stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of $\beta$-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research. View Publication

Proteasome inhibitors bortezomib,carfilzomib and ixazomib (approved by the US Food and Drug Administration [FDA]) induce remissions in patients with multiple myeloma (MM),but most patients eventually become resistant. MM and other hematologic malignancies express ubiquitous constitutive proteasomes and lymphoid tissue-specific immunoproteasomes; immunoproteasome expression is increased in resistant patients. Immunoproteasomes contain 3 distinct pairs of active sites,$\beta$5i,$\beta$1i,and $\beta$2i,which are different from their constitutive $\beta$5c,$\beta$1c,and $\beta$2c counterparts. Bortezomib and carfilzomib block $\beta$5c and $\beta$5i sites. We report here that pharmacologically relevant concentrations of $\beta$5i-specific inhibitor ONX-0914 show cytotoxicity in MM cell lines similar to that of carfilzomib and bortezomib. In addition,increasing immunoproteasome expression by interferon-$\gamma$ increases sensitivity to ONX-0914 but not to carfilzomib. LU-102,an inhibitor of $\beta$2 sites,dramatically sensitizes MM cell lines and primary cells to ONX-0914. ONX-0914 synergizes with all FDA-approved proteasome inhibitors in MM in vitro and in vivo. Thus,immunoproteasome inhibitors,currently in clinical trials for the treatment of autoimmune diseases,should also be considered for the treatment of MM. View Publication

Tolerogenic dendritic cell (tolDC)-based therapies have become a promising approach for the treatment of autoimmune diseases by their potential ability to restore immune tolerance in an antigen-specific manner. However,the broad variety of protocols used to generate tolDC in vitro and their functional and phenotypical heterogeneity are evidencing the need to find robust biomarkers as a key point towards their translation into the clinic,as well as better understanding the mechanisms involved in the induction of immune tolerance. With that aim,in this study we have compared the transcriptomic profile of tolDC induced with either vitamin D3 (vitD3-tolDC),dexamethasone (dexa-tolDC) or rapamycin (rapa-tolDC) through a microarray analysis in 5 healthy donors. The results evidenced that common differentially expressed genes could not be found for the three different tolDC protocols. However,individually,CYP24A1,MUCL1 and MAP7 for vitD3-tolDC; CD163,CCL18,C1QB and C1QC for dexa-tolDC; and CNGA1 and CYP7B1 for rapa-tolDC,constituted good candidate biomarkers for each respective cellular product. In addition,a further gene set enrichment analysis of the data revealed that dexa-tolDC and vitD3-tolDC share several immune regulatory and anti-inflammatory pathways,while rapa-tolDC seem to be playing a totally different role towards tolerance induction through a strong immunosuppression of their cellular processes. View Publication

Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming,but systematic interrogation of transcription factors,and their interactions with each other,has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells,revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation,with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons,which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition. View Publication

Regulatory T (Treg) cells induce an immunosuppressive microenvironment that is a major obstacle for successful tumor immunotherapy. Dissecting the regulatory mechanisms between energy metabolism and functionality in Treg cells will provide insight toward developing novel immunotherapies against cancer. Here we report that human naturally occurring and tumor-associated Treg cells exhibit distinct metabolic profiles with selectivity for glucose metabolism compared with effector T cells. Treg-mediated accelerated glucose consumption induces cellular senescence and suppression of responder T cells through cross-talk. TLR8 signaling selectively inhibits glucose uptake and glycolysis in human Treg cells,resulting in reversal of Treg suppression. Importantly,TLR8 signaling-mediated reprogramming of glucose metabolism and function in human Treg cells can enhance anti-tumor immunity in vivo in a melanoma adoptive transfer T cell therapy model. Our studies identify mechanistic links between innate signaling and metabolic regulation of human Treg suppression,which may be used as a strategy to advance tumor immunotherapy. View Publication

Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However,CTC investigation is hampered by their low number,making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy,and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype,stem-cell like characteristics,angiogenesis potential,an osteomimetic signature and the capacity to escape from the immune system. Moreover,they showed changes in mRNA and protein expression (e.g.,DEFA6,ABCB1 and GAL),whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits,treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time. View Publication

Chronic periodontitis (CP) is a microbial dysbiotic disease linked to increased risk of oral squamous cell carcinomas (OSCCs). To address the underlying mechanisms,mouse and human cell infection models and human biopsy samples were employed. We show that the 'keystone' pathogen Porphyromonas gingivalis,disrupts immune surveillance by generating myeloid-derived dendritic suppressor cells (MDDSCs) from monocytes. MDDSCs inhibit CTLs and induce FOXP3 + Tregs through an anti-apoptotic pathway. This pathway,involving pAKT1,pFOXO1,FOXP3,IDO1 and BIM,is activated in humans with CP and in mice orally infected with Mfa1 expressing P. gingivalis strains. Mechanistically,activation of this pathway,demonstrating FOXP3 as a direct FOXO1-target gene,was demonstrated by ChIP-assay in human CP gingiva. Expression of oncogenic but not tumor suppressor markers is consistent with tumor cell proliferation demonstrated in OSCC-P. gingivalis cocultures. Importantly,FimA + P. gingivalis strain MFI invades OSCCs,inducing inflammatory/angiogenic/oncogenic proteins stimulating OSCCs proliferation through CXCR4. Inhibition of CXCR4 abolished Pg-MFI-induced OSCCs proliferation and reduced expression of oncogenic proteins SDF-1/CXCR4,plus pAKT1-pFOXO1. Conclusively,P. gingivalis,through Mfa1 and FimA fimbriae,promotes immunosuppression and oncogenic cell proliferation,respectively,through a two-hit receptor-ligand process involving DC-SIGN+hi/CXCR4+hi,activating a pAKT+hipFOXO1+hiBIM-lowFOXP3+hi and IDO+hi- driven pathway,likely to impact the prognosis of oral cancers in patients with periodontitis. View Publication

While it is now acknowledged that CD4+ T cells expressing CD25 and Foxp3 (Treg cells) regulate immune responses and,consequently,influence the pathogenesis of infectious diseases,the regulatory response mediated by Treg cells upon infection by Trypanosoma cruzi was still poorly characterized. In order to understand the role of Treg cells during infection by this protozoan parasite,we determined in time and space the magnitude of the regulatory response and the phenotypic,functional and transcriptional features of the Treg cell population in infected mice. Contrary to the accumulation of Treg cells reported in most chronic infections in mice and humans,experimental T. cruzi infection was characterized by sustained numbers but decreased relative frequency of Treg cells. The reduction in Treg cell frequency resulted from a massive accumulation of effector immune cells,and inversely correlated with the magnitude of the effector immune response as well as with emergence of acute immunopathology. In order to understand the causes underlying the marked reduction in Treg cell frequency,we evaluated the dynamics of the Treg cell population and found a low proliferation rate and limited accrual of peripheral Treg cells during infection. We also observed that Treg cells became activated and acquired a phenotypic and transcriptional profile consistent with suppression of type 1 inflammatory responses. To assess the biological relevance of the relative reduction in Treg cells frequency observed during T. cruzi infection,we transferred in vitro differentiated Treg cells at early moments,when the deregulation of the ratio between regulatory and conventional T cells becomes significant. Intravenous injection of Treg cells dampened parasite-specific CD8+ T cell immunity and affected parasite control in blood and tissues. Altogether,our results show that limited Treg cell response during the acute phase of T. cruzi infection enables the emergence of protective anti-parasite CD8+ T cell immunity and critically influences host resistance. View Publication

Common variable immunodeficiency disorders (CVID) represent a group of primary immunodeficiency diseases characterized by hypogammaglobulinemia and impaired specific Ab response,resulting in recurrent infections due to dysfunctional immune response. The specific mechanisms mediating immune deficiency in CVID remain to be determined. Previous studies indicated that immune dysregulation in CVID patients is associated with chronic microbial translocation,systemic immune activation,and altered homeostasis of lymphocytic and myeloid lineages. A detailed phenotypic,functional characterization of plasma markers and immune cell populations was performed in 46 CVID patients and 44 healthy donors. CVID patients displayed significantly elevated plasma levels of a marker of neutrophil activation neutrophil gelatinase-associated lipocalin. Neutrophils from CVID patients exhibited elevated surface levels of CD11b and PD-L1 and decreased levels of CD62L,CD16,and CD80,consistent with a phenotype of activated neutrophils with suppressive properties. Neutrophils from CVID patients actively suppressed T cell activation and release of IFN-$\gamma$ via the production of reactive oxygen species. Furthermore,CVID was associated with an increased frequency of low-density neutrophils (LDNs)/granulocytic myeloid-derived suppressor cells. LDN/granulocytic myeloid-derived suppressor cell frequency in CVID patients correlated with reduced T cell responsiveness. Exogenous stimulation of whole blood with bacterial LPS emulated some but not all of the phenotypic changes observed on neutrophils from CVID patients and induced neutrophil population with LDN phenotype. The presented data demonstrate that neutrophils in the blood of CVID patients acquire an activated phenotype and exert potent T cell suppressive activity. Specific targeting of myeloid cell-derived suppressor activity represents a novel potential therapeutic strategy for CVID. View Publication