Qualitative differences in T‐cell activation by dendritic cell‐derived extracellular vesicle subtypes

Exosomes, nano‐sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC‐peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4⁺ T‐cell activation in vitro. When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN‐γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN‐γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles.     

Wild-type Leishmania donovani promastigotes block maturation, increase integrin expression and inhibit detachment of human monocyte-derived dendritic cells – the influence of phosphoglycans

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.     

Effect of age on surface molecules and cytokine expression in human dendritic cells

Dendritic cells (DCs) are central in regulating both innate and acquired immunity, but their possible age-related functional modifications are still unclear. Here we have analyzed the effect of age on LPS-treated monocyte-derived DCs (MDDCs). A negative correlation between age and cell expression of ICAM-1, CD25 and IL-10 was observed in a group of healthy donors. This has been confirmed by a significantly reduced expression of the same molecules in cells of subgrouped elderly versus younger individuals. On the contrary, a positive correlation between age and cell expression of IL-6 and IL-18 has been reported in all the subjects and further supported by a significant increase of the two pro-inflammatory cytokines in cells of elderly versus young subjects.Our data indicate that aging can impair the expression of ICAM-1 and CD25 and skew the production of cytokines, including IL-18, which concur to make a pro-inflammatory milieu. Altogether, the present results point to additional molecules whose role might be relevant in immunosenescence of human DCs, confirming that these cells undergo functional changes during aging.     

BCG vaccine mediated reduction in the MHC-II expression of macrophages and dendritic cells is reversed by activation of Toll-like receptors 7 and 9

Tuberculosis is a major cause of death in mankind and BCG vaccine protects against childhood but not adult tuberculosis. BCG avoids lysosomal fusion in macrophages decreasing peptides required for activating CD4 T cells and Th1 immunity while suppressing the expression of MHC-II by antigen presenting cells (APCs). An in vitro model of antigen presentation showed that ligands for TLR-9, 7, 4 and 1/2 increased the ability of APCs to present antigen-85B of BCG to CD4 T cells, which correlated with an increase in MHC-II expression. TLR-activation led to a down-regulation of MARCH1 ubiquitin ligase which prevents the degradation of MHC-II and decreased IL-10 also contributed to an increase in MHC-II. TLR-activation induced up-regulation of MHC-II was inhibited by the blockade of IRAK, NF-kB, and MAPKs. TLR-7 and TLR-9 ligands had the most effective adjuvant like effect on MHC-II of APCs which allowed BCG vaccine mediated activation of CD4 T cells.     

Functionality of insect‐cell‐derived colorectal cancer vaccine candidate protein EpCAM‐Fc in human dendritic cells

The baculovirus–insect cell expression system is widely used to produce recombinant proteins for various biomedical applications. Our previous study demonstrated that EpCAM, a colorectal cancer vaccine candidate protein, can be expressed in the baculovirus–insect cell expression system. However, its functionality (the ability to elicit an immune response), which is important for its possible use as a colorectal cancer vaccine for immunotherapy, still needed to be confirmed. In this study, we examined the ability of recombinant EpCAM to induce maturation of immature dendritic cells (DCs) derived from CD34⁺ cells isolated from human umbilical cord blood. We demonstrated that EpCAM induces the expression of four DC maturation markers: CD80, CD83, CD86 and MHC II. These results suggest that EpCAM produced in the baculovirus–insect cell expression system is functional in terms of its ability to trigger maturation of human DCs.     

The avidity of tumor‐specific T cells amplified by a plasmacytoid dendritic cell‐based assay can predict the clinical evolution of melanoma patients

The advent of immune checkpoint blockers and targeted therapies has changed the outcome of melanoma. However, many patients experience relapses, emphasizing the need for predictive and prognostic biomarkers. We developed a strategy based on plasmacytoid dendritic cells (pDCs) loaded with melanoma tumor antigens that allows eliciting highly efficient antitumor T‐cell responses. We used it to investigate antitumor T‐cell functionality in peripheral blood mononuclear cells and tumor‐infiltrating lymphocytes from melanoma patients. The pDCs elicited tumor‐specific T cells in different proportions and displaying diverse functional features, dependent upon the stage of the disease, but independent of the histological parameters at diagnosis. Strikingly, the avidity of the MelA‐specific T cells triggered by the pDCs was found to predict patient relapse time and overall survival. Our findings highlighted unexplored aspects of antitumor T‐cell responsiveness in melanoma, and revealed for the first time the structural avidity of tumor‐specific T cells as a crucial feature for predicting clinical evolution.     

Downregulation of IL-12 and a novel negative feedback system mediated by CD25+CD4+ T cells

CD25(+)CD4(+) regulatory T cells suppress immune responses and are believed to play roles in preventing autoimmune diseases. However, the mechanism(s) underlying the suppression and the regulation of their homeostasis remain to be elucidated. Here we show that these regulatory T cells downregulated CD25(-)CD4(+) T-cell-mediated production of IL-12 from antigen-presenting cells, which can act as a growth factor for CD25(-)CD4(+) T cells. We further found that CD25(+)CD4(+) T cells, despite their well-documented "anergic" nature, proliferate significantly in vitro only when CD25(-)CD4(+) T cells are present. Notably, this proliferation was strongly dependent on IL-2 and relatively independent of IL-12. Thus, CD25(+)CD4(+) T cells suppress CD25(-)CD4(+) T-cell responses, at least in part, by inhibiting IL-12 production while they themselves can undergo proliferation with the mediation of CD25(-)CD4(+) T cells in vitro. These results offer a novel negative feedback system involving a tripartite interaction among CD25(+)CD4(+) and CD25(-)CD4(+) T cells, and APCs that may contribute to the termination of immune responses.     

Regulation of tumor necrosis factor-α expression by CD40 ligation in BV-2 microglial cells

Ligation of CD40 has been shown to induce/stimulate the expression of tumor necrosis factor-alpha (TNF-alpha) in microglial cells. This study delineates the mechanism by which CD40 ligation regulates the expression of TNF-alpha in BV-2 microglial cells. There was very little induction of TNF-alpha by ligation of CD40 alone by either cross-linking antibodies against CD40 or recombinant CD40 ligand (CD154). The absence of any increase in TNF-alpha production by CD40 ligation alone even in CD40-overexpressed BV-2 microglial cells suggest that signal transduced by the ligation of CD40 alone is not sufficient for strong induction of TNF-alpha. However, CD40 ligation markedly induced the production of TNF-alpha as well as the expression of TNF-alpha mRNA in interferon-gamma (IFN-gamma)-stimulated BV-2 glial cells. Ligation of CD40 in CD40-overexpressed cells markedly enhanced the expression of TNF-alpha in the presence of IFN-gamma. To understand the mechanism of CD40 ligation-mediated induction/stimulation of TNF-alpha, we investigated the role of nuclear factor-kappaB (NF-kappaB) and C/EBPbeta. IFN-gamma alone was able to induce the activation of NF-kappaB as well as C/EBPbeta. However, CD40 ligation alone in the presence or absence of CD40 overexpression induced the activation of only NF-kappaB and not that of C/EBPbeta, suggesting that the activation of NF-kappaB alone by CD40 ligation is not sufficient to induce the expression of TNF-alpha and that the activation of C/EBPbeta is also necessary for strong induction of TNF-alpha. Consistently, a dominant-negative mutant of p65 (Delta(p65)) and that of C/EBPbeta (DeltaC/EBPbeta) inhibited the expression of TNF-alpha in BV-2 microglial cells stimulated with the combination of IFN-gamma and CD40 ligand. Taken together, these studies suggest that activation of both NF-kappaB and C/EBPbeta is important for strong induction of TNF-alpha and that CD40 ligation regulates the expression of TNF-alpha by modulating the activation of only NF-kappaB but not that of C/EBPbeta.     

HSCs play a distinct role in different phases of oval cell‐mediated liver regeneration

Hepatic stem cell niche plays an important role in hepatic oval cell‐mediated liver regeneration. As a component of hepatic stem cell niche, the role of hepatic stellate cells (HSCs) in oval cell proliferation needs further studies. In the present study, we isolated HSCs from rats at indicated time point after partial hepatectomy (PH) in 2‐acetylaminofluorene/PH oval cell proliferation model. Conditional medium (CM) from HSCs were collected to detect their effects on proliferation and the mitogen‐activated protein kinase pathway activation of two oval cell lines. We found that CM collected from HSCs at early phase of liver regeneration (4 and 9 days group) contained high levels of hepatocyte growth factor (HGF) and stimulated oval cell proliferation via extracellular signal‐regulated kinase and p38 pathway. CM collected from HSCs at terminal phase of liver regeneration (12 and 15 days group) contained high levels of transforming growth factor (TGF)‐β1, which suppressed DNA synthesis of oval cells. The shift between these two distinct effects depended on the balance between HGF and TGF‐β1 secreted by HSCs. Our study demonstrated that HSCs acted as a positive regulator at the early phase and a negative regulator at the terminal phase of the oval cell‐mediated liver regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

Declining expression of a single epithelial cell‐autonomous gene accelerates age‐related thymic involution

Age‐related thymic involution may be triggered by gene expression changes in lymphohematopoietic and/or nonhematopoietic thymic epithelial cells (TECs). The role of epithelial cell‐autonomous gene FoxN1 may be involved in the process, but it is still a puzzle because of the shortage of evidence from gradual loss‐of‐function and exogenous gain‐of‐function studies. Using our recently generated loxP‐floxed‐FoxN1(fx) mouse carrying the ubiquitous CreER^T (uCreER^T) transgene with a low dose of spontaneous activation, which causes gradual FoxN1 deletion with age, we found that the uCreER^T‐fx/fx mice showed an accelerated age‐related thymic involution owing to progressive loss of FoxN1⁺ TECs. The thymic aging phenotypes were clearly observable as early as at 3–6 months of age, resembling the naturally aged (18–22‐month‐old) murine thymus. By intrathymically supplying aged wild‐type mice with exogenous FoxN1‐cDNA, thymic involution and defective peripheral CD4⁺ T‐cell function could be partially rescued. The results support the notion that decline of a single epithelial cell‐autonomous gene FoxN1 levels with age causes primary deterioration in TECs followed by impairment of the total postnatal thymic microenvironment, and potentially triggers age‐related thymic involution in mice.     

Localization by scanning immunoelectron microscopy of triosephosphate isomerase,the molecules responsible for contact‐mediated killing of Cryptococcus,on the surface of Staphylococcus

In our previous studies, TPI were found to be the molecules responsible for contact‐killing of C. neoformans by S. aureus cells. Since TPI is a glycolytic protein that functions in the cytoplasm, evidence that TPI is present on the surface of S. aureus was required. In the present study, the presence of TPI on the cell surface of S. aureus was demonstrated by agglutination test and scanning immunoelectron microscopy. Furthermore, TPI was found to be present at a lower density than protein A/G molecules on the surface of S. aureus.     

Apigenin,a potent suppressor of dendritic cell maturation and migration,protects against collagen‐induced arthritis

This study aimed to investigate whether apigenin (API) suppresses arthritis development through the modulation of dendritic cell functions. Bone marrow‐derived dendritic cells (BMDCs) were stimulated in vitro with lipopolysaccharide (LPS) and treated with API for 24 hrs; DC functions, including phenotype expressions, cytokine secretion, phagocytosis and chemotaxis, were then investigated. The effects of API on collagen‐induced arthritis (CIA) were examined in vivo, and purified DCs from the lymph nodes (LNs) of API‐treated CIA mice were analysed for phenotypes and subsets. In in vitro, API efficiently restrained the phenotypic and functional maturation of LPS‐stimulated BMDCs while maintaining phagocytotic capabilities. Moreover, API inhibited the chemotactic responses of LPS‐stimulated BMDCs, which may be related to the depressive effect on chemokine receptor 4 (CXCR4). In in vivo, API treatment delayed the onset and reduced the severity of arthritis in CIA mice, and diminished secretion of pro‐inflammatory cytokines in the serum and supernatants from the LN cells of the CIA mice. Similar to the in vitro findings, the API‐treated mice exhibited reduced expression of co‐stimulatory molecules and major histocompatibility complex II on DCs. Furthermore, API treatment strongly down‐regulated the number of Langerhans cells, but not plasmacytoid DCs (pDCs) in LNs, which may be related to the depressive effect of API on the expression of CXCR4 on DCs of peripheral blood. These data provide new insight into the mechanism of action of API on arthritis and indicate that the inhibition of maturation and migration of DCs by API may contribute to its immunosuppressive effects.     

Combination of CD34‐positive cell subsets with infarcted myocardium‐like matrix stiffness: a potential solution to cell‐based cardiac repair

Detection of the optimal cell transplantation strategy for myocardial infarction (MI) has attracted a great deal of attention. Commitment of engrafted cells to angiogenesis within damaged myocardium is regarded as one of the major targets in cell‐based cardiac repair. Bone marrow–derived CD34‐positive cells, a well‐characterized population of stem cells, might represent highly functional endothelial progenitor cells and result in the formation of new blood vessels. Recently, physical microenvironment (extracellular matrix stiffness) around the engrafted cells was found to exert an essential impact on their fate. Stem cells are able to feel and respond to the tissue‐like matrix stiffness to commit to a relevant lineage. Notably, the infarct area after MI experiences a time‐dependent stiffness change from flexible to rigid. Our previous observations demonstrated myocardial stiffness‐dependent differentiation of the unselected bone marrow–derived mononuclear cells (BMMNCs) along endothelial lineage cells. Myocardial stiffness (~42 kPa) within the optimal time domain of cell engraftment (at week 1 to 2) after MI provided a more favourable physical microenvironment for cell specification and cell‐based cardiac repair. However, the difference in tissue stiffness‐dependent cell differentiation between the specific cell subsets expressing and no expressing CD34 phenotype remains uncertain. We presumed that CD34‐positive cell subsets facilitated angiogenesis and subsequently resulted in cardiac repair under induction of infarcted myocardium‐like matrix stiffness compared with CD34‐negative cells. If the hypothesis were true, it would contribute greatly to detect the optimal cell subsets for cell therapy and to establish an optimized therapy strategy for cell‐based cardiac repair.     

Changes in expression of cell‐cycle‐related genes in PC‐3 prostate cancer cells caused by ovine uterine serpin

The hormonal‐regulated serpin, ovine uterine serpin (OvUS), also called uterine milk protein (UTMP), inhibits proliferation of lymphocytes and prostate cancer (PC‐3) cells by blocking cell‐cycle progression. The present aim was to identify cell‐cycle‐related genes regulated by OvUS in PC‐3 cells using the quantitative human cell‐cycle RT² Profiler? PCR array. Cells were cultured ±200 µg/ml recombinant OvUS (rOvUS) for 12 and 24 h. At 12 h, rOvUS increased expression of three genes related to cell‐cycle checkpoints and arrest (CDKN1A, CDKN2B, and CCNG2). Also, 14 genes were down‐regulated including genes involved in progression through S (MCM3, MCM5, PCNA), M (CDC2, CKS2, CCNH, BIRC5, MAD2L1, MAD2L2), G₁ (CDK4, CUL1, CDKN3) and DNA damage checkpoint and repair genes RAD1 and RBPP8. At 24 h, rOvUS decreased expression of 16 genes related to regulation and progression through M (BIRC5, CCNB1, CKS2, CDK5RAP1, CDC20, E2F4, MAD2L2) and G₁ (CDK4, CDKN3, TFDP2), DNA damage checkpoints and repair (RAD17, BRCA1, BCCIP, KPNA2, RAD1). Also, rOvUS down‐regulated the cell proliferation marker gene MKI67, which is absent in cells at G₀. Results showed that OvUS blocks cell‐cycle progression through upregulation of cell‐cycle checkpoint and arrest genes and down‐regulation of genes involved in cell‐cycle progression. J. Cell. Biochem. 107: 1182–1188, 2009. © 2009 Wiley‐Liss, Inc.     

MiR‐16, as a potential NF‐κB‐related miRNA,exerts anti‐inflammatory effects on LPS‐induced myocarditis via mediating CD40 expression: A preliminary study

The purpose of this study was to investigate the biological effect of miR‐16 on myocarditis and the underlying molecular mechanism. H9c2 cells were treated with 10 µg/mL lipopolysaccharide (LPS) for 12 hours to form a myocarditis injury model. We observed that LPS treatment distinctly decreased the level of miR‐16 in H9c2 cells. Upregulation of miR‐16 increased cell proliferation and reduced cell apoptosis. Then, CD40 was predicted and verified as a target gene of miR‐16 by TargetScan and luciferase reporter assay, respectively. Furthermore, the messenger RNA and protein expression of CD40 are negatively regulated by miR‐16. The relative expression of inflammatory factors was dramatically decreased by the miR‐16 mimic. Cells cotransfected with miR‐16 mimic and si‐CD40 could significantly abolish the injury of cardiomyocytes caused by myocarditis. Our study illustrated that the upregulation of miR‐16 has a protective effect on LPS‐damaged H9c2 cells, which may be achieved by regulating CD40 and the nuclear factor kappa B pathway.     

Use of Tregs as a cell‐based therapy via CD39 for benign prostate hyperplasia with inflammation

Benign prostatic hyperplasia (BPH) occurs most commonly among older men, often accompanied by chronic tissue inflammation. Although its aetiology remains unclear, autoimmune dysregulation may contribute to BPH. Regulatory T cells (Tregs) prevent autoimmune responses and maintain immune homeostasis. In this study, we aimed to investigate Tregs frequency, phenotype, and function in BPH patients and to evaluate adoptive transfer Tregs for immunotherapy in mice with BPH via CD39. Prostate specimens and peripheral blood from BPH patients were used to investigate Treg subsets, phenotype and Treg‐associated cytokine production. Sorted CD39^+/? Tregs from healthy mice were adoptively transferred into mice before or after testosterone propionate administration. The Tregs percentage in peripheral blood from BPH patients was attenuated, exhibiting low Foxp3 and CD39 expression with low levels of serum IL‐10, IL‐35 and TGF‐β. Immunohistochemistry revealed Foxp3+ cells were significantly diminished in BPH prostate with severe inflammatory. Although the Tregs subset was comprised of more effector/memory Tregs, CD39 was still down‐regulated on effector/memory Tregs in BPH patients. Before or after testosterone propionate administration, no alterations of BPH symptoms were observed due to CD39‐ Tregs in mice, however, CD39⁺Tregs existed more potency than Tregs to regulate prostatic hyperplasia and inhibit inflammation by decreasing IL‐1β and PSA secretion, and increasing IL‐10 and TGF‐β secretion. Furthermore, adoptive transfer with functional Tregs not only improved prostate hyperplasia but also regulated muscle cell proliferation in bladder. Adoptive transfer with Tregs may provide a novel method for the prevention and treatment of BPH clinically.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.