技术资料

Both anti-tumoral and pro-tumoral effects of mesenchymal stem cells (MSCs) in preclinical treatment of ovarian cancer have been controversially demonstrated. In this study,we profiled the phenotypes of mouse compact bone-derived MSCs (CB-MSCs) and bone marrow-derived MSCs (BM-MSCs) and found that CB-MSCs expressed lower CD90 compared to BM-MSCs. We examined gene expression of immune regulating cytokines of CB-MSCs in 2D and 3D culture and under stimulation with TLR4 agonist LPS or immune activator VIC-008. Our data showed that when CB-MSCs were cultured in simulated in vivo 3D condition,CD90 expression was further decreased. Moreover,gene expressions of immune activating cytokines IL-12,IL-21,IFNgamma and a pro-inflammatory cytokine CXCL10 in CB-MSCs were increased in 3D culture whereas gene expression of anti-inflammatory cytokines IL-10 and CCL5 were downregulated. Stimulation of CB-MSCs by LPS or VIC-008 presented similar profile of the cytokine gene expressions to that in 3D culture which might benefit the anti-tumor efficacy of CD90low MSCs. The anti-tumor effects of CD90low CB-MSCs alone or in combination with VIC-008 were evaluated in a syngeneic orthotopic mouse model of ovarian cancer. Treatment that combines CB-MSCs and VIC-008 significantly decreased tumor growth and prolonged mouse survival. This was associated with the increase of activated anti-tumoral CD4+ and CD8+ T cells and the decrease of Treg cells in the tumor microenvironment. Taken together,our study demonstrates the synergistic anti-tumoral efficacy by application of CB-MSCs combined with immune activator VIC-008 and provides new insight into CD90low MSCs as a new anti-tumor arsenal. View Publication

Background and Aims Epithelial expression of the insulin receptor in the colon has previously been reported to correlate with extent of colonic inflammation. However,the impact of insulin signalling in the intestinal mucosa is still unknown. Here,we investigated the effects of inactivating the epithelial insulin receptor in the intestinal tract,in an experimental model of inflammation-induced colorectal cancer. Methods The mice were generated by utilizing the intestinal- and epithelial-specific villin promoter and the Cre-Lox technology. All mice included in the cohorts were generated by crossing [vil-Cre-INSR+/-] × [INSRfl/fl] to obtain [vil-Cre-INSR-/-],and their floxed littermates [INSRfl/fl] served as the control group. For the intervention study,phosphate-buffered saline with or without insulin was instilled rectally in anaesthetized wild-type mice with chemically induced colitis. Results We found higher endoscopic colitis scores together with potentiated colonic tumorigenesis in the knockout mice. Furthermore,we showed that topically administered insulin in inflamed colons of wild-type mice reduced inflammation-induced weight loss and improved remission in a dose-dependent manner. Mice receiving rectal insulin enemas exhibited lower colitis endoscopic scores and reduced cyclooxygenase 2 mRNA expression,and developed significantly fewer and smaller tumours compared with the control group receiving phosphate-buffered saline only. Conclusions Rectal insulin therapy could potentially be a novel treatment,targeting the epithelial layer to enhance mucosal healing in ulcerated areas. Our findings open up new possibilities for combination treatments to synergize with the existing anti-inflammatory therapies. View Publication

Human adipose-derived stem cells (ADSCs) can release exosomes; however,their specific functions remain elusive. In this study,we verified that exosomes derived from osteogenically differentiated ADSCs can promote osteogenic differentiation of ADSCs. Furthermore,in order to investigate the importance of exosomal microRNAs (miRNAs) in osteogenic differentiation of ADSCs,we used microarray assays to analyze the expression profiles of exosomal miRNAs derived from undifferentiated as well as osteogenically differentiated ADSCs; 201 miRNAs were upregulated and 33 miRNAs were downregulated between the two types of exosomes. Additionally,bioinformatic analyses,which included gene ontology analyses,pathway analysis,and miRNA-mRNA-network investigations,were performed. The results of these analyses revealed that the differentially expressed exosomal miRNAs participate in multiple biological processes,such as gene expression,synthesis of biomolecules,cell development,differentiation,and signal transduction,among others. Moreover,we found that these differentially expressed exosomal miRNAs connect osteogenic differentiation to processes such as axon guidance,MAPK signaling,and Wnt signaling. To the best of our knowledge,this is the first study to identify and characterize exosomal miRNAs derived from osteogenically differentiated ADSCs. This study confirms that alterations in the expression of exosomal miRNAs can promote osteogenic differentiation of ADSCs,which also provides the foundation for further research on the regulatory functions of exosomal miRNAs in the context of ADSC osteogenesis. View Publication

Subclinical hypothyroidism is associated with cardiovascular diseases,yet the underlying mechanism remains largely unknown. Herein,in a common population (n = 1,103),TSH level was found to be independently correlated with both carotid plaque prevalence and intima-media thickness. Consistently,TSH receptor ablation in ApoE-/- mice attenuated atherogenesis,accompanied by decreased vascular inflammation and macrophage burden in atherosclerotic plaques. These results were also observed in myeloid-specific Tshr-deficient ApoE-/- mice,which indicated macrophages to be a critical target of the proinflammatory and atherogenic effects of TSH. In vitro experiments further revealed that TSH activated MAPKs (ERK1/2,p38alpha,and JNK) and IkappaB/p65 pathways in macrophages and increased inflammatory cytokine production and their recruitment of monocytes. Thus,the present study has elucidated the new mechanisms by which TSH,as an independent risk factor of atherosclerosis,aggravates vascular inflammation and contributes to atherogenesis. View Publication

Dysregulation of the JAK/STAT signaling pathway is associated with Multiple Sclerosis (MS) and its mouse model,Experimental Autoimmune Encephalomyelitis (EAE). Suppressors Of Cytokine Signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe,brain-targeted,atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3DeltaLysM),which is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE,however,their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3DeltaLysM mice show a hyper-activated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically,Socs3-deficient neutrophils exhibit enhanced STAT3 activation,a hyper-activated phenotype in response to G-CSF,and upon G-CSF priming,increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall,our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3DeltaLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress,which may explain the detrimental role of G-CSF in MS patients. View Publication

Rheumatoid Arthritis (RA) causes chronic inflammation of joints. The cytokines TNFalpha and IFNgamma are central players in RA,however their source has not been fully elucidated. Natural Killer (NK) cells are best known for their role in elimination of viral-infected and transformed cells,and they secrete pro-inflammatory cytokines. NK cells are present in the synovial fluids (SFs) of RA patients and are considered to be important in bone destruction. However,the phenotype and function of NK cells in the SFs of patients with erosive deformative RA (DRA) versus non-deformative RA (NDRA) is poorly characterized. Here we characterize the NK cell populations present in the blood and SFs of DRA and NDRA patients. We demonstrate that a distinct population of activated synovial fluid NK (sfNK) cells constitutes a large proportion of immune cells found in the SFs of DRA patients. We discovered that although sfNK cells in both DRA and NDRA patients have similar phenotypes,they function differently. The DRA sfNK secrete more TNFalpha and IFNgamma upon exposure to IL-2 and IL-15. Consequently,we suggest that sfNK cells may be a marker for more severely destructive RA disease. View Publication

To achieve a functional cure for HIV,treatment regimens that eradicate latently HIV-infected cells must be established. For this,many groups have attempted to reactivate latently-infected cells to induce cytopathic effects and/or elicit cytotoxic T lymphocyte (CTL)/NK cell-mediated immune responses to kill these cells. We believe that not only the reactivation of latently-infected cells,but also the induction of strong CTL responses,would be required for this. Here,we used typical immune activators that target pattern recognition receptors (PRRs). For our experimental model,we identified eight SIV-infected cynomolgus monkeys that became natural controllers of viremia. Although plasma viral loads were undetectable,we could measure SIV-DNA by qPCR in peripheral blood mononuclear cells (PBMCs). Using these PBMCs,we screened 10 distinct PRR ligands to measure IFN-alpha and IFN-gamma production. Among these,STING ligands,cGAMP and c-di-AMP,and the TLR7/8 agonist R848 markedly increased cytokine levels. Both R848 and STING ligands could reactivate latently-infected cells in both cynomolgus monkeys and human PBMCs in vitro. Furthermore,c-di-AMP increased the frequency of SIV Gag-specific CD8+ T cells including polyfunctional CD8+ T cells,as compared to that in untreated control or R848-treated cells. Together,STING ligands might be candidates for HIV treatment. View Publication

The subset of regulatory T (Treg) cells,with its specific transcription Foxp3,is a unique cell type for the maintenance of immune homeostasis by controlling effector T (Teff) cell responses. Although it is common that a defect in Treg cells with Treg/Teff disorder causes autoimmune diseases; however,the precise mechanisms are not thoroughly revealed. Here,we report that miR-34a could attenuate human and murine Foxp3 gene expression via targeting their 3' untranslated regions (3' UTR). The human miR-34a,increased in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) patients,displayed a positive correlation with some serum markers of inflammation including rheumatoid factor (RF),anti-streptolysin antibody (ASO),erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) as well as Th17 signature gene RORgammat,but inversely correlated with the mRNA expression levels of FOXP3. In addition,murine miR-34a levels were downregulated in TGF-beta-induced Treg cells but upregulated in Th17 cells induced in vitro compared to activated CD4+ T cells. It has also been demonstrated that elevated miR-34a disrupting Treg/Th17 balance in vivo contributed to the progress of pathogenesis of collagen induced arthritis (CIA) mice. Furthermore,IL-6 and TNF-alpha were responsible for the upregulation of miR-34a and downregulation of Foxp3,which was reverted by the addition of NF-kappaB/p65 inhibitor BAY11-7082,thus indicating that NF-kappaB/p65 inhibited Foxp3 expression in an miR-34a-dependent manner. Finally,IL-6 or TNF-alpha-activated p65 could bind to the miR-34a promotor and enhance its activity,resulting in upregulation of its transcription. Taken together,we show that NF-kappaB activated by inflammatory cytokines,such as IL-6 and TNF-alpha,ameliorates Foxp3 levels via regulating miR-34a expression,which provides a new mechanistic and therapeutic insight into the ongoing of autoimmune diseases. View Publication

SCOPE Necrotizing enterocolitis (NEC) is a leading cause of morbidity and death in preterm infants,occurring more often in formula-fed than breastfed infants. Studies in both rats and humans show that human milk oligosaccharides (HMOs) lower the incidence of NEC,but the mechanism underlying such protection is currently unclear. METHODS AND RESULTS By extracting HMOs from pooled human breastmilk,the impact of HMOs on the intestinal mucin levels in a murine model of NEC are investigated. To confirm the results,the findings are validated by exposing human intestinal epithelial cells and intestinal organoids to HMOs and evaluated for mucin expression. HMO-gavage to pups increases Muc2 levels and decreases intestinal permeability to macromolecular dextran. HMO-treated cells have increased Muc2 expression,decreased bacterial attachment and dextran permeability during challenge by enteric pathogens. To identify the mediators involved in HMO induction of mucins,it is demonstrated that HMOs directly induce the expression of chaperone proteins including protein disulfide isomerase (PDI). Suppression of PDI activity removes the protective effects of HMOs on barrier function in vitro as well as NEC protection in vivo. CONCLUSIONS Taken together,the results provide insights to the possible mechanisms by which HMOs protect the neonatal intestine through upregulation of mucins. View Publication

Purpose: We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells.Experimental Design: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs,monocytes,and endothelial cells,which express CD105,was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft.Results: ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes,and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs,monocytes,and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added,which depleted human MSCs and murine endothelial cells and macrophages from the TME.Conclusions: Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME,but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells. View Publication

Immunological tolerance removes or inactivates self-reactive B cells,including those that also recognize cross-reactive foreign antigens. Whereas a few microbial pathogens exploit these holes" in the B cell repertoire by mimicking host antigens to evade immune surveillance the extent to which tolerance reduces the B cell repertoire to foreign antigens is unknown. Here we use single-cell cultures to determine the repertoires of human B cell antigen receptors (BCRs) before (transitional B cells) and after (mature B cells) the second B cell tolerance checkpoint in both healthy donors and in patients with systemic lupus erythematosus (SLE) . In healthy donors the majority ({\~{}}70{\%}) of transitional B cells that recognize foreign antigens also bind human self-antigens (foreign+self) and peripheral tolerance halves the frequency of foreign+self-reactive mature B cells. In contrast in SLE patients who are defective in the second tolerance checkpoint frequencies of foreign+self-reactive B cells remain unchanged during maturation of transitional to mature B cells. Patterns of foreign+self-reactivity among mature B cells from healthy donors differ from those of SLE patients. We propose that immune tolerance significantly reduces the scope of the BCR repertoire to microbial pathogens and that cross-reactivity between foreign and self epitopes may be more common than previously appreciated." View Publication

Cancer immunotherapy restores or enhances the effector function of CD8+ T cells in the tumour microenvironment1,2. CD8+ T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin-granzyme and Fas-Fas ligand pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide5,6. Although it has been investigated in vitro7,8,there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios9,10. It is unclear whether,and how,ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumour cells,and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically,interferon gamma (IFNgamma) released from CD8+ T cells downregulates the expression of SLC3A2 and SLC7A11,two subunits of the glutamate-cystine antiporter system xc-,impairs the uptake of cystine by tumour cells,and as a consequence,promotes tumour cell lipid peroxidation and ferroptosis. In mouse models,depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system xc- was negatively associated,in cancer patients,with CD8+ T cell signature,IFNgamma expression,and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNgamma and CD8. Thus,T cell-promoted tumour ferroptosis is an anti-tumour mechanism,and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach. View Publication