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Find video protocols related to scientific articles indexed in Pubmed.
Intracellular NAMPT-NAD+-SIRT1 cascade improves post-ischaemic vascular repair by modulating Notch signalling in endothelial progenitors.
Cardiovasc. Res.
PUBLISHED: 10-25-2014
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Intracellular nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD(+)) biosynthesis. This study investigated the role of NAMPT-mediated NAD(+) signalling in post-ischaemic vascular repair.
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Aberrant CD200/CD200R1 expression and its potential role in Th17 cell differentiation, chemotaxis and osteoclastogenesis in rheumatoid arthritis.
Rheumatology (Oxford)
PUBLISHED: 09-28-2014
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CD200/CD200R1 signalling has an immunoregulatory effect on the activation threshold of the inflammatory immune response and maintains immune homeostasis. In this study we evaluated the status of CD200/CD200R1 interaction in patients with RA.
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RasGRP3 limits Toll-like receptor-triggered inflammatory response in macrophages by activating Rap1 small GTPase.
Nat Commun
PUBLISHED: 08-14-2014
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Host immune cells can detect and destruct invading pathogens via pattern-recognition receptors. Small Rap GTPases act as conserved molecular switches coupling extracellular signals to various cellular responses, but their roles as regulators in Toll-like receptor (TLR) signalling have not been fully elucidated. Here we report that Ras guanine nucleotide-releasing protein 3 (RasGRP3), a guanine nucleotide-exchange factor activating Ras and Rap1, limits production of proinflammatory cytokines (especially IL-6) in macrophages by activating Rap1 on activation by low levels of TLR agonists. We demonstrate that RasGRP3, a dominant member of RasGRPs in macrophages, impairs TLR3/4/9-induced IL-6 production and relieves dextrane sulphate sodium-induced colitis and collagen-induced arthritis. In RasGRP3-deficient RAW264.7 cells obtained by CRISPR-Cas9 genome editing, TLR3/4/9-induced activation of Rap1 was inhibited while ERK1/2 activation was enhanced. Our study suggests that RasGRP3 limits inflammatory response by activating Rap1 on low-intensity pathogen infection, setting a threshold for preventing excessive inflammatory response.
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IFN-? primes macrophage activation by increasing phosphatase and tensin homolog via downregulation of miR-3473b.
J. Immunol.
PUBLISHED: 08-04-2014
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The classical activation of macrophages, one of major innate effector cells, requires IFN-? pretreatment (priming) and subsequent TLR stimuli (triggering). The priming effect of IFN-? can promote macrophages to secrete higher level of proinflammatory cytokines but lower level of the anti-inflammatory cytokines, enhancing microbicidal and tumoricidal activity of macrophages. However, the underlying molecular mechanisms for IFN-?-priming effect on macrophage activation remain to be fully understood. microRNAs (miRNAs) are now emerging as important regulators in immune response, including signaling transduction in immune cell function. In this study, we explored the effect of IFN-? on miRNA expression profiling in macrophages and tried to identify the definite miRNA involved in the priming effect of IFN-?. We discovered that miR-3473b, which was significantly downregulated after IFN-? priming, could attenuate the priming effect of IFN-?. miR-3473b promoted Akt/glycogen synthase kinase 3 signaling and IL-10 production through directly targeting phosphatase and tensin homolog (PTEN) to suppress activation of macrophages and inflammatory response. Our data indicate that IFN-? beefs up macrophage innate response and cytotoxicity by downregulating miR-3473b to release PTEN from suppression, and then the increase of PTEN contributes to the full activation of IFN-?-primed macrophages. Our results provide mechanistic insight to priming effect of IFN-? on macrophage classical activation by identifying an IFN-?/miR-3473b/PTEN regulatory loop in the regulation of macrophage function.
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The Roles of Lysosomes in Inflammation and Autoimmune Diseases.
Int. Rev. Immunol.
PUBLISHED: 07-31-2014
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Lysosomes perform a range of functions, some of which, such as degradation, are common to all cell types. Others, such as secretion or lysosomal exocytosis, are more specialised and tend to involve fusion of this organelle with the cell surface to release its contents. This review describes lysosomal regulation of the inflammatory glucocorticoid signaling pathways, and summarizes the roles of lysosomes in negatively or positively modulating the production of inflammatory cytokines. We also review the characteristic changes in lysosomal hydrolases and membrane proteins in common autoimmune diseases. Finally, future directions in lysosome research are proposed, with it being suggested that the role of lysosomes will continue to be of growing interest in immunity research.
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Innate signaling in the inflammatory immune disorders.
Cytokine Growth Factor Rev.
PUBLISHED: 06-04-2014
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Pattern recognition receptors (PRRs) of innate immune cells recognize the conserved molecular signatures on pathogens, termed pathogen-associated molecular patterns. PRRs also recognize endogenous damage-associated molecular patterns. Following pathogen infection or tissue damage, the stimulation of PRRs activates distinct but shared signaling pathways that lead to effector mechanisms in innate host defense. PRR signaling is strictly and finely tuned to ensure the appropriate duration and strength to prevent damaging inflammation to the host. Here we attempt to provide a brief background on the agonists and signal transduction pathways of PRRs and summarize the mechanisms underlying the control of PRR signaling, with a particular focus on the recent progress of the involvement of PRR signaling in the inflammatory immune disorders.
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The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation.
Science
PUBLISHED: 04-19-2014
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Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes; however, few have been identified that regulate immune cell differentiation and function. Here, we identified lnc-DC, which was exclusively expressed in human conventional dendritic cells (DCs). Knockdown of lnc-DC impaired DC differentiation from human monocytes in vitro and from mouse bone marrow cells in vivo and reduced capacity of DCs to stimulate T cell activation. lnc-DC mediated these effects by activating the transcription factor STAT3 (signal transducer and activator of transcription 3). lnc-DC bound directly to STAT3 in the cytoplasm, which promoted STAT3 phosphorylation on tyrosine-705 by preventing STAT3 binding to and dephosphorylation by SHP1. Our work identifies a lncRNA that regulates DC differentiation and also broadens the known mechanisms of lncRNA action.
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The immune potential and immunopathology of cytokine-producing B cell subsets: A comprehensive review.
J. Autoimmun.
PUBLISHED: 04-09-2014
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B lymphocytes are generally recognized for their potential to mediate humoral immunity by producing different antibody isotypes and being involved in opsonization and complement fixation. Nevertheless, the non-classical, antibody-independent immune potential of B cell subsets has attracted much attention especially in the past decade. These B cells can release a broad variety of cytokines (such as IL-2, IL-4, IL-6, IL-10, IL-17, IFN-?, IFN-?, TNF-?, TGF-?, LT), and can be classified into distinct subsets depending on the particular cytokine profile, thus emerging the concept of cytokine-producing B cell subsets. Although there is still controversy surrounding the key cell surface markers, intracellular factors and cellular origins of cytokine-producing B cell subsets, accumulating evidence indicates that these B cells are endowed with great potential to regulate both innate and adaptive arms of immune system though releasing cytokines. On the one hand, they promote immune responses through mounting Th1/Th2/Th17 and neutrophil response, inducing DC maturation and formation of lymphoid structures, increasing NK cell and macrophage activation, enhancing development of themselves and sustaining antibody production. On the other hand, they can negatively regulate immune responses by suppressing Th cell responses, inhibiting Tr1 cell and Foxp3(+) Treg differentiation, impairing APC function and pro-inflammatory cytokine release by monocytes, and inducing CD8(+) T cell anergy and CD4(+) T cell apoptosis. Therefore, cytokine-producing B cell subsets have multifunctional functions in health and diseases, playing pathologic as well as protective roles in autoimmunity, infection, allergy, and even malignancy. In this review, we revisit the history of discovering cytokine-producing B cells, describe the identification of cytokine-producing B cell subsets, introduce the origins of cytokine-producing B cell subsets as well as molecular and cellular mechanisms for their differentiation, and summarize the recent progress made toward understanding the unexpectedly complex and potentially opposing roles of cytokine-producing B cells in immunological disorders.
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Blockade of Fas signaling in breast cancer cells suppresses tumor growth and metastasis via disruption of Fas signaling-initiated cancer-related inflammation.
J. Biol. Chem.
PUBLISHED: 03-13-2014
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Mechanisms for cancer-related inflammation remain to be fully elucidated. Non-apoptotic functions of Fas signaling have been proposed to play an important role in promoting tumor progression. It has yet to be determined if targeting Fas signaling can control tumor progression through suppression of cancer-related inflammation. In the current study we found that breast cancer cells with constitutive Fas expression were resistant to apoptosis induction by agonistic anti-Fas antibody (Jo2) ligation or Fas ligand cross-linking. Higher expression of Fas in human breast cancer tissue has been significantly correlated with poorer prognosis in breast cancer patients. To determine whether blockade of Fas signaling in breast cancer could suppress tumor progression, we prepared an orthotopic xenograft mouse model with mammary cancer cells 4T1 and found that blockade of Fas signaling in 4T1 cancer cells markedly reduced tumor growth, inhibited tumor metastasis in vivo, and prolonged survival of tumor-bearing mice. Mechanistically, blockade of Fas signaling in cancer cells significantly decreased systemic or local recruitment of myeloid derived suppressor cells (MDSCs) in vivo. Furthermore, blockade of Fas signaling markedly reduced IL-6, prostaglandin E2 production from breast cancer cells by impairing p-p38, and activity of the NF?B pathway. In addition, administration of a COX-2 inhibitor and anti-IL-6 antibody significantly reduced MDSC accumulation in vivo. Therefore, blockade of Fas signaling can suppress breast cancer progression by inhibiting proinflammatory cytokine production and MDSC accumulation, indicating that Fas signaling-initiated cancer-related inflammation in breast cancer cells may be a potential target for treatment of breast cancer.
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Small GTPase RBJ mediates nuclear entrapment of MEK1/MEK2 in tumor progression.
Cancer Cell
PUBLISHED: 03-11-2014
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Ras-related small GTPases play important roles in cancer. However, the roles of RBJ, a representative of the sixth subfamily of Ras-related small GTPases, in tumorigenesis and tumor progression remain unknown. Here, we report that RBJ is dysregulated in human gastrointestinal cancers and can promote carcinogenesis and tumor progression via nuclear entrapment of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)1/MEK2 and activation of ERK1/ERK2. Nucleus-localized RBJ interacts with MEK/ERK and prolongs the duration of MEK/ERK activation. Rbj deficiency abrogates nuclear accumulation of MEK1/MEK2, attenuates ERK1/ERK2 activation, and impairs AOM/DSS-induced colonic carcinogenesis. Moreover, Rbj knockdown inhibits growth of established tumors. Our data suggest that RBJ may be an oncogenic Ras-related small GTPase mediating nuclear accumulation of active MEK1/MEK2 in tumor progression.
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Th17 cells play a critical role in the development of experimental Sjogren's syndrome.
Ann. Rheum. Dis.
PUBLISHED: 02-28-2014
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Although Th17 cells have been increasingly recognised as an important effector in various autoimmune diseases, their function in the pathogenesis of Sjögren's syndrome (SS) remains largely uncharacterised. This study aims to determine the role of Th17 cells in the development of experimental SS (ESS).
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Rhbdd3 controls autoimmunity by suppressing the production of IL-6 by dendritic cells via K27-linked ubiquitination of the regulator NEMO.
Nat. Immunol.
PUBLISHED: 02-24-2014
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Excessive activation of dendritic cells (DCs) leads to the development of autoimmune and inflammatory diseases, which has prompted a search for regulators of DC activation. Here we report that Rhbdd3, a member of the rhomboid family of proteases, suppressed the activation of DCs and production of interleukin 6 (IL-6) triggered by Toll-like receptors (TLRs). Rhbdd3-deficient mice spontaneously developed autoimmune diseases characterized by an increased abundance of the TH17 subset of helper T cells and decreased number of regulatory T cells due to the increase in IL-6 from DCs. Rhbdd3 directly bound to Lys27 (K27)-linked polyubiquitin chains on Lys302 of the modulator NEMO (IKK?) via the ubiquitin-binding-association (UBA) domain in endosomes. Rhbdd3 further recruited the deubiquitinase A20 via K27-linked polyubiquitin chains on Lys268 to inhibit K63-linked polyubiquitination of NEMO and thus suppressed activation of the transcription factor NF-?B in DCs. Our data identify Rhbdd3 as a critical regulator of DC activation and indicate K27-linked polyubiquitination is a potent ubiquitin-linked pattern involved in the control of autoimmunity.
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Activated cytotoxic lymphocytes promote tumor progression by increasing the ability of 3LL tumor cells to mediate MDSC chemoattraction via Fas signaling.
Cell. Mol. Immunol.
PUBLISHED: 02-20-2014
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The Fas/FasL system transmits intracellular apoptotic signaling, inducing cell apoptosis. However, Fas signaling also exerts non-apoptotic functions in addition to inducing tumor cell apoptosis. For example, Fas signaling induces lung cancer tumor cells to produce prostaglandin E2 (PGE2) and recruit myeloid-derived suppressor cells (MDSCs). Activated cytotoxic T lymphocytes (CTLs) induce and express high levels of FasL, but the effects of Fas activation initiated by FasL in CTLs on apoptosis-resistant tumor cells remain largely unclear. We purified activated CD8(+) T cells from OT-1 mice, evaluated the regulatory effects of Fas activation on tumor cell escape and investigated the relevant mechanisms. We found that CTLs induced tumor cells to secrete PGE2 and increase tumor cell-mediated chemoattraction of MDSCs via Fas signaling, which was favorable to tumor growth. Our results indicate that CTLs may participate in the tumor immune evasion process. To the best of our knowledge, this is a novel mechanism by which CTLs play a role in tumor escape. Our findings implicate a strategy to enhance the antitumor immune response via reduction of negative immune responses to tumors promoted by CTLs through Fas signaling.Cellular & Molecular Immunology advance online publication, 28 April 2014; doi:10.1038/cmi.2014.21.
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Death domain-associated protein 6 (Daxx) selectively represses IL-6 transcription through histone deacetylase 1 (HDAC1)-mediated histone deacetylation in macrophages.
J. Biol. Chem.
PUBLISHED: 02-18-2014
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As a multifunctional nuclear protein, death domain-associated protein 6 (Daxx) regulates a wide range of biological processes, including cell apoptosis and gene transcription. However, the function of Daxx in innate immunity remains unclear. In our study, we show that Daxx is highly expressed in macrophages and localized in nucleus of macrophages. The expression of Daxx is significantly up-regulated by stimulation with TLR ligands LPS and poly(I:C). Silence of Daxx selectively represses IL-6 expression at transcription level in LPS-activated macrophages. Upon stimulation of LPS, Daxx specifically binds to the promoter of IL-6 and inhibits histone acetylation at IL-6 promoter region. Further mechanism analyses show that histone deacetylase 1 (HDAC1) interacts with Daxx and binds to the promoter of IL-6. Daxx silencing decreases the association of HDAC1 to IL-6 promoter. Therefore, our data reveal that Daxx selectively represses IL-6 transcription through HDAC1-mediated histone deacetylation in LPS-induced macrophages, acting as a negative regulator of IL-6 during innate immunity and potentially preventing inflammatory response because of overproduction of IL-6.
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Phosphatase holoenzyme PP1/GADD34 negatively regulates TLR response by inhibiting TAK1 serine 412 phosphorylation.
J. Immunol.
PUBLISHED: 02-17-2014
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The molecular mechanisms that fine tune TLRs responses need to be fully elucidated. Protein phosphatase-1 (PP1) has been shown to be important in cell death and differentiation. However, the roles of PP1 in TLR-triggered immune response remain unclear. In this study, we demonstrate that PP1 inhibits the activation of the MAPK and NF-?B pathway and the production of TNF-?, IL-6 in macrophages triggered by TLR3, TLR4, and TLR9 in a phosphatase-dependent manner. Conversely, PP1 knockdown increases TLRs-triggered signaling and proinflammatory cytokine production. Tautomycetin, a specific inhibitor of PP1, aggravates LPS-induced endotoxin shock in mice. We further demonstrate that PP1 negatively regulates TLR-triggered signaling by targeting TGF-?-activated kinase 1 (TAK1) serine 412 (Ser412) phosphorylation, which is required for activation of TAK1-mediated IL-1R and TLR signaling. Mutation of TAK1 Serine 412 to alanine (S412A) significantly inhibits TLR/IL-1R-triggered NF-?B and MAPK activation and induction of proinflammatory cytokines in macrophage and murine embryonic fibroblast cells. DNA damage-inducible protein 34 (GADD34) specifies PP1 to dephosphorylate TAK1 at Ser412. GADD34 depletion abolished the interaction between TAK1 and PP1, and it relieved PP1 overexpression-induced inhibition of TLRs signaling and proinflammatory cytokine production. In addition, knockdown of GADD34 significantly promotes TLR-induced TAK1 Ser412 phosphorylation, downstream NF-?B and MAPK activation, and proinflammatory cytokine production. Therefore, PP1, as a physiologic inhibitor, together with its regulatory subunit GADD34, tightly controls TLR-induced TAK1 Ser412 phosphorylation, preventing excessive activation of TLRs and protecting the host from overwhelmed inflammatory immune responses.
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Human hepatocellular carcinoma-infiltrating CD4?CD69?Foxp3? regulatory T cell suppresses T cell response via membrane-bound TGF-?1.
J. Mol. Med.
PUBLISHED: 01-29-2014
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Tumors can recruit, induce, and expand regulatory T cells (Tregs) to suppress antitumor immune responses for survival and progression. The complicated tumor-related Treg subsets and their functional mechanisms are not fully addressed yet. We have previously identified a novel CD4?CD69?Foxp3? Treg subset in tumor-bearing mice, which suppresses CD4 T cell response via membrane-bound transforming growth factor beta 1 (mTGF-?1) and then promotes tumor progression. In hepatocellular carcinoma patients, here, we identified tumor-infiltrating human CD4?CD69? Tregs which represent ~67.2 % of tumor-infiltrating CD4 T cells that is significantly higher than conventional CD4?CD25?Foxp3? Tregs. They expressed mTGF-?1, PD-1, and CTLA-4, but not CD25 or Foxp3, and only produced a little interleukin (IL)-10 and TGF-?1. More importantly, they significantly suppressed CD4 T cell response via mTGF-?1 in vitro. Furthermore, the percentage of these CD4?CD69? Tregs in tumor tissue was significantly correlated with tumor progression, which is more pronounced at the late stage of cancer patients. Thus, we have identified a tumor-induced new population of human CD4?CD69? Tregs in cancer patients with phenotype of CD25?Foxp3?mTGF-?1?CTLA-4?PD-1?, and these Tregs can suppress antitumor immune response via mTGF-?1. Our results not only enrich the family of Treg subsets, providing new mechanistic insight to tumor-induced immune suppression in human, but also suggest a potential target for cancer immunotherapy.
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Induced pluripotent stem cell (iPSCs) and their application in immunotherapy.
Cell. Mol. Immunol.
PUBLISHED: 11-20-2013
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The ever-improving technology to generate induced pluripotent stem cells (iPSCs) has increased their potential use as novel candidates for disease modeling, drug screening, regenerative medicine and cell therapy. Indeed, iPSCs offer extensive capacity for self-renewal without the ethical concerns faced by embryonic stem cells (ESCs). With respect to potential applications in the immune system, many studies provide evidence to support that there are exclusive advantages to using iPSCs over other systems. Both hematopoietic stem cells and several types of mature immune cells have successfully been reprogrammed to iPSCs and vice versa, paving a path toward our ability to effectively model patient-specific diseases and provide potentially alternative cell sources for transfusion medicine. Despite these potential advances, some limitations regarding the use of iPSCs in the clinic still remain, including the immunogenicity of iPSCs and their derivatives, which is currently under debate in the field. In this review, we mainly focus on discussing the recent progress being made in the latest differentiation methods and clinical implications of iPSCs with respect to the immune system. Additionally, current issues regarding the clinical application of iPSCs are addressed, especially the controversy surrounding immunogenicity, along with various other perspectives.Cellular & Molecular Immunology advance online publication, 16 December 2013; doi:10.1038/cmi.2013.62.
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TLR4 is essential for dendritic cell activation and anti-tumor T-cell response enhancement by DAMPs released from chemically stressed cancer cells.
Cell. Mol. Immunol.
PUBLISHED: 10-26-2013
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The combination of immunotherapy and chemotherapy is regarded as a promising approach for the treatment of certain types of cancer. However, the underlying mechanisms need to be fully investigated to guide the design of more efficient protocols for cancer chemoimmunotherapy. It is well known that danger-associated molecular patterns (DAMPs) can activate immune cells, including dendritic cells (DCs), via Toll-like receptors (TLRs); however, the role of DAMPs released from chemical drug-treated tumor cells in the activation of the immune response needs to be further elucidated. Here, we found that colorectal cancer (CRC) cells treated with oxaliplatin (OXA) and/or 5-fluorouracil (5-Fu) released high levels of high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70). After OXA/5-Fu therapy, the sera of CRC patients also exhibited increased levels of HMGB1 and HSP70, both of which are well-known DAMPs. The supernatants of dying CRC cells treated with OXA/5-Fu promoted mouse and human DC maturation, with upregulation of HLA-DR, CD80 and CD86 expression and enhancement of IL-1?, TNF-?, MIP-1?, MIP-1?, RANTES and IP-10 production. Vaccines composed of DCs pulsed with the supernatants of chemically stressed CRC cells induced a more significant IFN-?-producing Th1 response both in vitro and in vivo. However, the supernatants of chemically stressed CRC cells failed to induce phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating an essential role of TLR4 in DAMP-induced DC maturation and activation. Furthermore, pulsing with the supernatants of chemically stressed CRC cells did not efficiently induce an IFN-?-producing Th1 response in TLR4-deficient DCs. Collectively, these results demonstrate that DAMPs released from chemically stressed cancer cells can activate DCs via TLR4 and enhance the induction of an anti-tumor T-cell immune response, delineating a clinically relevant immuno-adjuvant pathway triggered by DAMPs.Cellular & Molecular Immunology advance online publication, 23 December 2013; doi:10.1038/cmi.2013.59.
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Type I IFN inhibits innate IL-10 production in macrophages through histone deacetylase 11 by downregulating microRNA-145.
J. Immunol.
PUBLISHED: 08-26-2013
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Innate immune responses must be tightly regulated to avoid overactivation and subsequent inflammatory damage to host tissue while eliminating invading pathogens. IL-10 is a crucial suppressor of inflammatory responses and its expression is under precise regulation involving complex regulatory networks and multiple feedback loops. MicroRNAs are now emerging as critical regulators in immune response. Our previous work showed that miR-143/145 cluster was markedly downregulated in macrophages upon vesicular stomatitis virus infection. However, the particular role of miR-143/145 cluster in the regulation of innate immune response remains unknown. In this study, we found that miR-143/145 cluster expression was also downregulated dramatically by TLR signals in macrophages, which was dependent on the subsequent type I IFN (IFN-I) production and downstream IFN-I receptor-JAK1-STAT1 signal cascade. Further studies demonstrated that miR-145, but not miR-143, promoted IL-10 expression in TLR4-triggered macrophages through directly targeting the epigenetic Il10 gene silencer histone deacetylase 11. Therefore, we demonstrate that miR-145, downregulated by IFN-I, targets histone deacetylase 11 to promote innate IL-10 expression in macrophages. Our findings suggest a new IFN-I-mediated negative feedback loop in the fine-tuning of innate IL-10 production that creates precise coordination of innate immune responses.
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Identification of IFN-?-producing innate B cells.
Cell Res.
PUBLISHED: 08-21-2013
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Although B cells play important roles in the humoral immune response and the regulation of adaptive immunity, B cell subpopulations with unique phenotypes, particularly those with non-classical immune functions, should be further investigated. By challenging mice with Listeria monocytogenes, Escherichia coli, vesicular stomatitis virus and Toll-like receptor ligands, we identified an inducible CD11a(hi)Fc?RIII(hi) B cell subpopulation that is significantly expanded and produces high levels of IFN-? during the early stage of the immune response. This subpopulation of B cells can promote macrophage activation via generating IFN-?, thereby facilitating the innate immune response against intracellular bacterial infection. As this new subpopulation is of B cell origin and exhibits the phenotypic characteristics of B cells, we designated these cells as IFN-?-producing innate B cells. Dendritic cells were essential for the inducible generation of these innate B cells from the follicular B cells via CD40L-CD40 ligation. Increased Brutons tyrosine kinase activation was found to be responsible for the increased activation of non-canonical NF-?B pathway in these innate B cells after CD40 ligation, with the consequent induction of additional IFN-? production. The identification of this new population of innate B cells may contribute to a better understanding of B cell functions in anti-infection immune responses and immune regulation.Cell Research advance online publication 3 December 2013; doi:10.1038/cr.2013.155.
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Fas signal promotes the immunosuppressive function of regulatory dendritic cells via the ERK/?-catenin pathway.
J. Biol. Chem.
PUBLISHED: 08-13-2013
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Dendritic cells (DCs) play important roles in the initiation of immune response and also in the maintenance of immune tolerance. Now, many kinds of regulatory DCs with different phenotypes have been identified to suppress immune response and contribute to the control of autoimmune diseases. However, the mechanisms by which regulatory DCs can be regulated to exert the immunosuppressive function in the immune microenvironment remain to be fully investigated. In addition, how T cells, once activated, can feedback affect the function of regulatory DCs during immune response needs to be further identified. We previously identified a unique subset of CD11b(hi)Ia(low) regulatory DCs, differentiated from mature DCs or hematopoietic stem cells under a stromal microenvironment in spleen and liver, which can negatively regulate immune response in a feedback way. Here, we show that CD11b(hi)Ia(low) regulatory DCs expressed high level of Fas, and endothelial stromal cell-derived TGF-? could induce high expression of Fas on regulatory DCs via ERK activation. Fas ligation could promote regulatory DCs to inhibit CD4(+) T cell proliferation more significantly. Furthermore, Fas ligation preferentially induced regulatory DCs to produce IL-10 and IP-10 via ERK-mediated inactivation of GSK-3 and subsequent up-regulation of ?-catenin. Interestingly, activated T cells could promote regulatory DCs to secrete more IL-10 and IP-10 partially through FasL. Therefore, our results demonstrate that Fas signal, at least from the activated T cells, can promote the immunosuppressive function of Fas-expressing regulatory DCs, providing a new manner for the regulatory DCs to regulate adaptive immunity.
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Zinc finger protein 64 promotes Toll-like receptor-triggered proinflammatory and type I interferon production in macrophages by enhancing p65 subunit activation.
J. Biol. Chem.
PUBLISHED: 07-15-2013
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The molecular mechanisms that fine-tune the Toll-like receptor (TLR)-triggered innate immune response need further investigation. As an important transcription factor, zinc finger proteins (ZFPs) play important roles in many cell functions, including development, differentiation, tumorigenesis, and functions of the immune system. However, the role of ZFP members in the innate immune responses remains unclear. Here we showed that the expression of C2H2-type ZFP, ZFP64, was significantly up-regulated in macrophages upon stimulation with TLR ligands, including LPS, CpG oligodeoxynucleotides, or poly(I:C). ZFP64 overexpression promoted TLR-triggered TNF-?, IL-6, and IFN-? production in macrophages. Coincidently, knockdown of ZFP64 expression significantly inhibited the production of the above cytokines. However, activation of MAPK and IRF3 was not responsible for the ZFP64-mediated promotion of cytokine production. Interestingly, ZFP64 significantly up-regulated TLR-induced NF-?B activation. ZFP64 could bind to the promoter of the TNF-?, IL-6, and IFN-? genes in macrophages only after TLR ligation. Furthermore, ZFP64 associated with the NF-?B p65 subunit upon LPS stimulation, and TLR-ligated macrophages showed a lower level of p65 recruitment to the TNF-?, IL-6, and IFN-? gene promoter in the absence of ZFP64. The data identify ZFP64 as a downstream positive regulator of TLR-initiated innate immune responses by associating with the NF-?B p65 subunit, enhancing p65 recruitment to the target gene promoters and increasing p65 activation and, thus, leading to the promotion of TLR-triggered proinflammatory cytokine and type I interferon production. Our findings add mechanistic insight into the efficient activation of the TLR innate response against invading pathogens.
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Zinc finger protein ZBTB20 promotes Toll-like receptor-triggered innate immune responses by repressing I?B? gene transcription.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-17-2013
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Toll-like receptor (TLR) signaling is critical in innate response against invading pathogens. However, the molecular mechanisms for full activation of TLR-triggered innate immunity need to be fully elucidated. The broad complex tramtrack bric-a-brac/poxvirus and zinc finger (BTB/POZ) family is a class of transcription factors involved in many biological processes. However, few BTB/POZ proteins were reported to function in innate immune response. Zinc finger and BTB domain-containing 20 (ZBTB20), a member of BTB/POZ family, functions in neurogenesis and represses ?-fetoprotein gene transcription in liver. However, the immunological functions of ZBTB20 remain unknown. Here, we found that myeloid cell-specific ZBTB20 KO mice were resistant to endotoxin shock and Escherichia coli-caused sepsis. ZBTB20 deficiency attenuated TLR-triggered production of proinflammatory cytokines and type I IFN in macrophages, which attributed to higher abundance of I?B? protein and impaired activity of NF-?B. Furthermore, ChIP and next generation high-throughput DNA sequencing assay showed that ZBTB20 specifically bound to I?B? gene promoter (+1 to +60 region) after TLR activation. ZBTB20 could inhibit I?B? gene transcription, govern I?B? protein expression, and then promote NF-?B activation. Therefore, transcriptional repressor ZBTB20 is needed to promote full activation of TLR signaling and TLR-triggered innate immune response by selectively suppressing the suppressor I?B? gene transcription.
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LRRFIP2 negatively regulates NLRP3 inflammasome activation in macrophages by promoting Flightless-I-mediated caspase-1 inhibition.
Nat Commun
PUBLISHED: 05-30-2013
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The NLRP3 inflammasome is the most characterized inflammasome activated by cellular infection or stress, which is responsible for the maturation of proinflammatory cytokines IL-1? and IL-18. The precise molecular mechanism for negative regulation of NLRP3 inflammasome activation needs to be further defined. Here we identify leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) as an NLRP3-associated protein and an inhibitor for NLRP3 inflammasome activation. LRRFIP2 binds to NLRP3 via its N terminus upon NLRP3 inflammasome activation, and also interacts with Flightless-I, a pseudosubstrate of caspase-1, via its Coil motif. Knockdown of Flightless-I significantly promotes NLRP3 inflammasome activation. LRRFIP2 enhances the interaction between Flightless-I and caspase-1, facilitating the inhibitory effect of Flightless-I on caspase-1 activation. Furthermore, silencing of Flightless-I abrogates the inhibitory effect of LRRFIP2 on NLRP3 inflammasome. These data demonstrate that LRRFIP2 inhibits NLRP3 inflammasome activation by recruiting the caspase-1 inhibitor Flightless-I, thus outlining a new mechanism for negative regulation of NLRP3 inflammasome.
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miRNomes of haematopoietic stem cells and dendritic cells identify miR-30b as a regulator of Notch1.
Nat Commun
PUBLISHED: 05-17-2013
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Dendritic cells (DCs) are critical to initiate the immune response and maintain tolerance, depending on different status and subsets. The expression profiles of microRNAs (miRNAs) in various DC subsets and haematopoietic stem cells (HSCs), which generate DCs, remain to be fully identified. Here we examine miRNomes of mouse bone marrow HSCs, immature DCs, mature DCs and IL-10/NO-producing regulatory DCs by deep sequencing. We identify numerous stage-specific miRNAs and histone modification in HSCs and DCs at different differentiation stages. miR-30b, significantly upregulated via a TGF-beta/Smad3-mediated epigenetic pathway in regulatory DCs, can target Notch1 to promote IL-10 and NO production, suggesting that miR-30b is a negative regulator of immune response. We also identify miRNomes of in vivo counterparts of mature DCs and regulatory DCs and systematically compare them with DCs cultured in vitro. These results provide a resource for studying roles of miRNAs in stem cell biology, development and functional regulation of DC subsets.
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CD11c(high)CD8+ regulatory T cell feedback inhibits CD4 T cell immune response via Fas ligand-Fas pathway.
J. Immunol.
PUBLISHED: 05-15-2013
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Regulatory T cells can restrict the uncontrolled immune response and inflammation, avoiding pathologic immune injury to the host and thus playing important roles in the maintenance of immune homeostasis. Until recently, many subsets of CD4 and CD8 regulatory T cells have been reported. In this study, we identified CD11c(high)CD8(+) T cells as a new subset of CD8(+) regulatory T cells. During Listeria monocytogenes and Staphylococcus aureus infection, two subsets of CD8 T cells were classified according to the expression level of CD11c, including CD11c(low)CD8(+) and CD11c(high)CD8(+) T cells. CD11c(low)CD8(+) T cells, existing during the whole period of infection, act as conventional activated T cells to kill target cells in a perforin-dependent manner. Interestingly, CD11c(high)CD8(+) T cells appeared only at a late stage of infection, expressed relatively high CD122 and low CD69, did not secrete IFN-?, IL-10, TGF-?, and exhibited much more potent cytotoxicity against target cells via Fas ligand-Fas pathway in an Ag-independent manner. Ligation of CD11c was important in the cytotoxicity of CD11c(high)CD8(+) T cells. Furthermore, CD11c(high)CD8(+) T cells could directly kill the activated CD4 T cells both in vitro and in vivo, whereas CD11c(low)CD8(+) T cells could not. Thus, we identified an infection-induced new subset of CD11c(high)CD8(+) regulatory T cells, which might contribute to protect host from pathological immune injure. Our results indicate that CD11c(+)CD8(+) T cells are constitute a heterogeneous population that can be divided further into regulatory CD11c(high)CD8(+) T cell subset and effector CD11c(low)CD8(+) T cell subset, thus adding insight to the role of CD8 T cells in immune response and regulation.
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Hepatic RIG-I Predicts Survival and Interferon-? Therapeutic Response in Hepatocellular Carcinoma.
Cancer Cell
PUBLISHED: 05-13-2013
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In hepatocellular carcinoma (HCC), biomarkers for prediction of prognosis and response to immunotherapy such as interferon-? (IFN-?) would be very useful in the clinic. We found that expression of retinoic acid-inducible gene-I (RIG-I), an IFN-stimulated gene, was significantly downregulated in human HCC tissues. Patients with low RIG-I expression had shorter survival and poorer response to IFN-? therapy, suggesting that RIG-I is a useful prognosis and IFN-? response predictor for HCC patients. Mechanistically, RIG-I enhances IFN-? response by amplifying IFN-? effector signaling via strengthening STAT1 activation. Furthermore, we found that RIG-I deficiency promotes HCC carcinogenesis and that hepatic RIG-I expression is lower in men than in women. RIG-I may therefore be a tumor suppressor in HCC and contribute to HCC gender disparity.
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Exosomes with membrane-associated TGF-?1 from gene-modified dendritic cells inhibit murine EAE independently of MHC restriction.
Eur. J. Immunol.
PUBLISHED: 05-09-2013
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We have previously demonstrated that exosomes from dendritic cells (DCs) secreting TGF-?1 (sTGF-?1-EXOs) delay the development of murine inflammatory bowel disease (IBD). In this study, we isolated exosomes from DCs expressing membrane-associated TGF-?1 (mTGF-?1-EXOs) and found mTGF-?1-EXOs had more potent immunosuppressive activity than sTGF-?1-EXOs in vitro. Treatment of mice with mTGF-?1-EXOs inhibited the development and progression of myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE even after disease onset. Treatment of mice with mTGF-?1-EXOs also impaired Ag-specific Th1 and IL-17 responses, but promoted IL-10 responses ex vivo. Treatment with mTGF-?1-EXOs decreased the frequency of Th17 cells in EAE mice, which might be associated with the down-regulation of the p38, ERK, Stat3, and NF-?B activation and IL-6 expression in DCs. Treatment with mTGF-?1-EXOs maintained the regulatory capacity of Treg cells, and adoptive transfer of CD4(+)Foxp3(+)Treg cells from mTGF-?1-EXO-treated EAE mice dramatically prevented the development of EAE in the recipients. Moreover, treatment with mTGF-?1-EXOs from C57BL/6 mice effectively prevented and inhibited proteolipid protein (PLP) peptide-induced EAE in BALB/c mice. These results indicate that mTGF-?1-EXOs possess powerful immunosuppressive ability and can effectively inhibit the development and progression of EAE in different strains of mice.
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Rhomboid domain-containing protein 3 is a negative regulator of TLR3-triggered natural killer cell activation.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-22-2013
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Rhomboid domain-containing protein 3 (Rhbdd3), which belongs to a family of proteins with rhomboid domain, is widely expressed in immune cells; however, the roles of the Rhbdd members, including Rhbdd3, in immunity remain unknown. Natural killer (NK) cells are critical for host immune defense and also can mediate inflammatory diseases such as hepatitis. Although much is known about how NK cells are activated, the detailed mechanisms for negative regulation of NK cell activation remain to be fully understood. Using Rhbdd3-deficient mice, we reveal that Rhbdd3, selectively up-regulated in NK cells upon Toll-like receptor 3 (TLR3) stimulation, negatively regulates TLR3-mediated NK cell activation in a feedback manner. Rhbdd3 inhibits TLR3-triggered IFN-? and granzyme B expression of NK cells in cell-cell contact dependence of accessory cells such as dendritic cells and Kupffer cells. Rhbdd3 interacts with DNAX activation protein of 12 kDa and promotes its degradation, inhibiting MAPK activation in TLR3-triggered NK cells. Furthermore, Rhbdd3 plays a critical role in attenuating TLR3-triggered acute inflammation by controlling NK cell activation and accumulation in liver and disrupting NK cell-Kupffer cell interaction. Therefore, Rhbdd3 is a feedback inhibitor of TLR3-triggered NK cell activation. Our study outlines a mechanism for the negative regulation of NK cell activation and also provides clues for the function of the rhomboid proteins in immunity.
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Immune responsive gene 1 (IRG1) promotes endotoxin tolerance by increasing A20 expression in macrophages through reactive oxygen species.
J. Biol. Chem.
PUBLISHED: 04-22-2013
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Sepsis-associated immunosuppression (SAIS) is regarded as one of main causes for the death of septic patients at the late stage because of the decreased innate immunity with a more opportunistic infection. LPS-tolerized macrophages, which are re-challenged by LPS after prior exposure to LPS, are regarded as the common model of hypo-responsiveness for SAIS. However, the molecular mechanisms of endotoxin tolerance and SAIS remain to be fully elucidated. In addition, negative regulation of the Toll-like receptor (TLR)-triggered innate inflammatory response needs further investigation. Here we show that expression of immune responsive gene 1 (IRG1) was highly up-regulated in the peripheral blood mononuclear cells of septic patients and in LPS-tolerized mouse macrophages. IRG1 significantly suppressed TLR-triggered production of proinflammatory cytokines TNF-?, IL-6, and IFN-? in LPS-tolerized macrophages, with the elevated expression of reactive oxygen species (ROS) and A20. Moreover, ROS enhanced A20 expression by increasing the H3K4me3 modification of histone on the A20 promoter domain, and supplement of the ROS abrogated the IRG1 knockdown function in breaking endotoxin tolerance by increasing A20 expression. Our results demonstrate that inducible IRG1 promotes endotoxin tolerance by increasing A20 expression through ROS, indicating a new molecular mechanism regulating hypoinflammation of sepsis and endotoxin tolerance.
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Human CD14(+) CTLA-4(+) regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma.
Hepatology
PUBLISHED: 04-01-2013
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Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. HCC-induced immunosuppression often leads to ineffectiveness of immuno-promoting therapies. Currently, suppressing the suppressors has become the potential strategy for cancer immunotherapy. So, figuring out the immunosuppressive mechanisms induced and employed by HCC will be helpful to the design and application of HCC immunotherapy. Here, we identified one new subset of human CD14(+) CTLA-4(+) regulatory dendritic cells (CD14(+) DCs) in HCC patients, representing ?13% of peripheral blood mononuclear cells. CD14(+) DCs significantly suppress T-cell response in vitro through interleukin (IL)-10 and indoleamine-2,3-dioxygenase (IDO). Unexpectedly, CD14(+) DCs expressed high levels of cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death-1, and CTLA-4 was found to be essential to IL-10 and IDO production. So, we identified a novel human tumor-induced regulatory DC subset, which suppresses antitumor immune response through CTLA-4-dependent IL-10 and IDO production, thus indicating the important role of nonregulatory T-cell-derived CTLA-4 in tumor-immune escape or immunosuppression. Conclusions: These data outline one mechanism for HCC to induce systemic immunosuppression by expanding CD14(+) DCs, which may contribute to HCC progression. This adds new insight to the mechanism for HCC-induced immunosuppression and may also provide a previously unrecognized target of immunotherapy for HCC. (Hepatology 2013;).
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Apoptotic cell administration enhances pancreatic islet engraftment by induction of regulatory T cells and tolerogenic dendritic cells.
Cell. Mol. Immunol.
PUBLISHED: 03-16-2013
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Apoptotic cell transfer has been found to be able to facilitate engraftment of allograft. However, the underlying mechanisms remain to be fully understood. Here we demonstrate that intravenous administration of donor apoptotic splenocytes can promote pancreatic islet engraftment by inducing generation of tolerogenic dendritic cells (Tol-DCs) and expansion of CD4(+)Foxp3(+) regulatory T cells (Tregs). In vivo clearance of either dendritic cells (DCs) or Tregs prevented the induction of immune tolerance by apoptotic cell administration. Transient elimination of Tregs using anti-CD25, monoclonal antibody (mAb) abrogated the generation of Tol-DCs after administration of apoptotic splenocytes. Reciprocally, depletion of DCs within CD11c-DTR mice using diphtheria toxin (DT) prevented the generation of Tregs in the recipients with administration of apoptotic splenocytes. Induction of Tregs by Tol-DCs required direct cell contact between the two cell types, and programmed death 1 ligand (PD-L1) played important role in the Tregs expansion. Apoptotic cell administration failed to induce Tol-DCs in IL-10-deficient and Smad3-deficient mice, suggesting that IL-10 and transforming growth factor-? (TGF-?) are needed to maintain DCs in the tolerogenic state. Therefore, we demonstrate that Tol-DCs promote the expansion of Tregs via PD-L1 on their surface and reciprocally Tregs facilitate Tol-DCs to maintain transplantation tolerance induced by apoptotic cells via secreting IL-10 and TGF-?.
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Histone methyltransferase Ash1l suppresses interleukin-6 production and inflammatory autoimmune diseases by inducing the ubiquitin-editing enzyme A20.
Immunity
PUBLISHED: 02-27-2013
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Histone modifications play important roles in multiple physiological processes by regulating gene expression. However, the roles of histone modifications in immunity remain poorly understood. Here we report that Ash1l, a H3K4 methyltransferase, suppressed interleukin-6 (IL-6), and tumor necrosis factor (TNF) production in Toll-like receptor (TLR)-triggered macrophages, protecting mice from sepsis. Ash1l-silenced mice were more susceptible to autoimmune disease as a result of enhanced IL-6 production. Ash1l enhanced A20 expression through induction of H3K4 modification at the Tnfaip3 promoter via H3K4 methyltransferase activity of Ash1l SET (Su[var]3-9, E[z] and trithorax) domain. Ash1l suppressed NF-?B, mitogen-activated protein kinase (MAPK) pathways, and subsequent IL-6 production via facilitating A20-mediated NF-?B signal modulator NEMO and transducer TRAF6 deubiquitination. Therefore, Ash1l-mediated H3K4 methylation at the Tnfaip3 promoter is required for controlling innate IL-6 production and suppressing inflammatory autoimmune diseases, providing mechanistic insight into epigenetic modulation of immune responses and inflammation.
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Enteropathogenic Escherichia coli Tir recruits cellular SHP-2 through ITIM motifs to suppress host immune response.
Cell. Signal.
PUBLISHED: 01-28-2013
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Immune responses to pathogens are regulated by immune receptors containing either an immunoreceptor tyrosine-based activation motif (ITAM) or an immunoreceptor tyrosine-based inhibitory motif (ITIM). The important diarrheal pathogen enteropathogenic Escherichia coli (EPEC) require delivery and insertion of the bacterial translocated intimin receptor (Tir) into the host plasma membrane for pedestal formation. The C-terminal region of Tir, encompassing Y483 and Y511, shares sequence similarity with cellular ITIMs. Here, we show that EPEC Tir suppresses the production of inflammatory cytokines by recruitment of SHP-2 and subsequent deubiquitination of TRAF6 in an ITIM dependent manner. Our findings revealed a novel mechanism by which the EPEC utilize its ITIM motifs to suppress and evade the host innate immune response, which could lead to the development of novel therapeutics to prevent bacterial infection.
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MicroRNA-92a negatively regulates Toll-like receptor (TLR)-triggered inflammatory response in macrophages by targeting MKK4 kinase.
J. Biol. Chem.
PUBLISHED: 01-25-2013
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Toll-like receptors (TLRs) play a critical role in the initiation of immune responses against invading pathogens. MicroRNAs have been shown to be important regulators of TLR signaling. In this study, we have found that the stimulation of multiple TLRs rapidly reduced the levels of microRNA-92a (miRNA-92a) and some other members of the miRNA-92a family in macrophages. miR-92a mimics significantly decreased, whereas miR-92a knockdown increased, the activation of the JNK/c-Jun pathway and the production of inflammatory cytokines in macrophages when stimulated with ligands for TLR4. Furthermore, mitogen-activated protein kinase kinase 4 (MKK4), a kinase that activates JNK/stress-activated protein kinase, was found to be directly targeted by miR-92a. Similar to the effects of the miR-92a mimics, knockdown of MKK4 inhibited the activation of JNK/c-Jun signaling and the production of TNF-? and IL-6. In conclusion, we have demonstrated that TLR-mediated miR-92a reduction feedback enhances TLR-triggered production of inflammatory cytokines in macrophages, thus outlining new mechanisms for fine-tuning the TLR-triggered inflammatory response.
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Induction of Siglec-G by RNA viruses inhibits the innate immune response by promoting RIG-I degradation.
Cell
PUBLISHED: 01-07-2013
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RIG-I is a critical RNA virus sensor that serves to initiate antiviral innate immunity. However, posttranslational regulation of RIG-I signaling remains to be fully understood. We report here that RNA viruses, but not DNA viruses or bacteria, specifically upregulate lectin family member Siglecg expression in macrophages by RIG-I- or NF-?B-dependent mechanisms. Siglec-G-induced recruitment of SHP2 and the E3 ubiquitin ligase c-Cbl to RIG-I leads to RIG-I degradation via K48-linked ubiquitination at Lys813 by c-Cbl. By increasing type I interferon production, targeted inactivation of Siglecg protects mice against lethal RNA virus infection. Taken together, our data reveal a negative feedback loop of RIG-I signaling and identify a Siglec-G-mediated immune evasion pathway exploited by RNA viruses with implication in antiviral applications. These findings also provide insights into the functions and crosstalk of Siglec-G, a known adaptive response regulator, in innate immunity.
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Protein tyrosine phosphatase with proline-glutamine-serine-threonine-rich motifs negatively regulates TLR-triggered innate responses by selectively inhibiting I?B kinase ?/NF-?B activation.
J. Immunol.
PUBLISHED: 01-07-2013
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TLRs are essential for sensing the invading pathogens and initiating protective immune responses. However, aberrant activation of TLR-triggered inflammatory innate responses leads to the inflammatory disorders and autoimmune diseases. The molecular mechanisms that fine-tune TLR responses remain to be fully elucidated. Protein tyrosine phosphatase with proline-glutamine-serine-threonine-rich motifs (PTP-PEST) has been shown to be important in cell adhesion, migration, and also T cell and B cell activation. However, the roles of PTP-PEST in TLR-triggered immune response remain unclear. In this study, we report that PTP-PEST expression was upregulated in macrophages by TLR ligands. PTP-PEST inhibited TNF-?, IL-6, and IFN-? production in macrophages triggered by TLR3, TLR4, and TLR9. Overexpression of catalytically inactive mutants of PTP-PEST abolished the inhibitory effects, indicating that PTP-PEST inhibits TLR response in a phosphatase-dependent manner. Accordingly, PTP-PEST knockdown increased TLR3, -4, and -9-triggered proinflammatory cytokine and type I IFN production. PTP-PEST selectively inhibited TLR-induced NF-?B activation, whereas it had no substantial effect on MAPK and IFN regulatory factor 3 activation. Moreover, PTP-PEST directly interacted with I?B kinase ? (IKK?) then inhibited IKK? phosphorylation at Ser(177/181) and Tyr(188/199), and subsequently suppressed IKK? activation and kinase activity as well as downstream NF-?B activation, resulting in suppression of the TLR-triggered innate immune response. Thus, PTP-PEST functions as a feedback-negative regulator of TLR-triggered innate immune responses by selectively impairing IKK?/NF-?B activation.
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Notch signal suppresses Toll-like receptor-triggered inflammatory responses in macrophages by inhibiting extracellular signal-regulated kinase 1/2-mediated nuclear factor ?B activation.
J. Biol. Chem.
PUBLISHED: 12-28-2011
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Multiple signaling pathways are involved in the tight regulation of Toll-like receptor (TLR) signaling, which is important for the tailoring of inflammatory response to pathogens in macrophages. It is widely accepted that TLR signaling can activate Notch pathway; however, whether full activation of Notch signaling can feedback modulate TLR signaling pathway so as to control inflammation response remains unclear. Here, we demonstrated that stimulation with TLR ligands up-regulated Notch1 and Notch2 expression in macrophages. The expression of Notch target genes including Hes1 and Hes5 was also induced in macrophages by LPS, suggesting that TLR4 signaling enhances the activation of Notch pathway. Importantly, overexpression of constituted active form of Notch1 (NICD1) and Notch2 (NICD2) suppressed production of TLR4-triggered proinflammatory cytokines such as TNF-? and IL-6 but promoted production of antiinflammatory cytokine IL-10, which is dependent on the PEST domain of NICD. In addition, NICD1 and NICD2 suppressed TLR-triggered ERK phosphorylation, which is indispensable for Notch-mediated inhibition of TLR4-triggered proinflammatory cytokine production. Furthermore, activation of Notch signaling inhibited NF-?B transcription activity by MyD88/TRAF6 and TRIF pathways, which was dependent on ERK activity. Therefore, our results showed that Notch signaling negatively regulates TLR-triggered inflammation responses, revealing a new mechanism for negative regulation of TLR signaling via Notch pathway.
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Splenic stroma-educated regulatory dendritic cells induce apoptosis of activated CD4 T cells via Fas ligand-enhanced IFN-? and nitric oxide.
J. Immunol.
PUBLISHED: 12-28-2011
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Stromal microenvironments of bone marrow, lymph nodes, and spleen have been shown to be able to regulate immune cell differentiation and function. Our previous studies demonstrate that splenic stroma could drive mature dendritic cells (DC) to further proliferate and differentiate into regulatory DC subset that could inhibit T cell response via NO. However, how splenic stroma-educated regulatory DC release NO and whether other molecules are involved in the suppression of T cell response remain unclear. In this study, we show that splenic stroma educates regulatory DC to express high level of Fas ligand (FasL) by TGF-? via ERK activation. The findings, that inhibition of CD4 T cell proliferation by regulatory DC required cell-to-cell contact and FasL deficiency impaired inhibitory effect of regulatory DC, indicate that regulatory DC inhibit CD4 T cell proliferation via FasL. Then, regulatory DC have been found to be able to induce apoptosis of activated CD4 T cells via FasL in caspase 8- and caspase 3-dependent manner. Interestingly, FasL on regulatory DC enhanced IFN-? production from activated CD4 T cells, and in turn T cell-derived IFN-? induced NO production from regulatory DC, working jointly to induce apoptosis of activated CD4 T cells. Blockade of IFN-? and NO could reduce the apoptosis induction. Therefore, our results demonstrated that splenic stroma-educated regulatory DC induced T cell apoptosis via FasL-enhanced T cell IFN-? and DC NO production, thus outlining a new way for negative regulation of T cell responses and maintenance of immune homeostasis by regulatory DC and splenic stromal microenvironment.
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Revisiting the protective and pathogenic roles of neutrophils: Ly-6G is key!
Eur. J. Immunol.
PUBLISHED: 09-29-2011
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The physiological and pathophysiological roles of neutrophils in immune homeostasis and disease have been investigated extensively by way of anti-Gr-1 mAb-mediated depletion experiments; however, the ability of the anti-Gr-1 mAb to specifically deplete neutrophils has long been questioned and it is now known that this mAb, which binds Ly6C and Ly6G, is also able to deplete monocytes and subsets of CD8(+) T cells. This, therefore, casts doubt on the previous conclusions regarding the role of neutrophils drawn from studies using this mAb. Another mAb, which targets Ly6G only, has recently been shown to deplete neutrophils specifically and a study by Carr et al. (Eur. J. Immunol. 2011. 41: 2666-2676) in this issue of the European Journal of Immunology utilizes this Ly-6G mAb to reveal the precise role of neutrophils during Listeria monocytogenes (LM) infection. Carr et al. find that monocytes/macrophages, rather than neutrophils, dominate the initial control of LM growth in the spleen, whereas neutrophils in the liver are key for host resistance to LM infection. These data suggest that the previously reported protective or pathogenic roles of neutrophils in disease models need to be reconsidered through anti-Ly6G mAb-mediated depletion experiments.
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CMRF-35-like molecule 3 preferentially promotes TLR9-triggered proinflammatory cytokine production in macrophages by enhancing TNF receptor-associated factor 6 ubiquitination.
J. Immunol.
PUBLISHED: 09-21-2011
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TLRs are critical innate immune sensors in the induction of proinflammatory cytokines to eliminate invading pathogens. However, the mechanisms for the full activation of TLR-triggered innate immune response need to be fully understood. The murine CMRF-35-like molecule (CLM)-3 is a representative of CLM family belonging to the Ig superfamily. Considering that CLM-3 is selectively expressed in macrophages and the roles of CLM members in innate immune response remain unclear, in this study we investigated the role of CLM-3 in the regulation of TLR-triggered innate response. We found that CLM-3 was an endosome/lysosome-localized molecule, and was downregulated in macrophages by stimulation with TLR9 ligand, but not TLR4 and TLR3 ligands. Interestingly, CLM-3 selectively promoted production of TNF-? and IL-6 in macrophages triggered by TLR9, but not TLR4 or TLR3. CLM-3 enhanced activation of MAPKs and NF-?B pathways in TLR9-triggered macrophages. Furthermore, CLM-3-transgenic mice were generated, and CLM-3 expression was confirmed by mAb against CLM-3 that we prepared. Accordingly, the macrophages derived from CLM-3-transgenic mice were more sensitive to TLR9 ligand stimulation, with more pronounced production of TNF-?, IL-6, and increased activation of MAPKs and NF-?B pathways. Moreover, ubiquitination of TNFR-associated factor 6, a crucial signaling transducer of TLR-triggered MAPKs and NF-?B activation, was found to be significantly promoted by CLM-3 in macrophages. Collectively, the endosome/lysosome-localized CLM-3 can promote full activation of TLR9-triggered innate responses by enhancing TNFR-associated factor 6 ubiquitination and subsequently activating MAPKs and NF-?B.
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E3 ubiquitin ligase CHIP facilitates Toll-like receptor signaling by recruiting and polyubiquitinating Src and atypical PKC{zeta}.
J. Exp. Med.
PUBLISHED: 09-12-2011
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The carboxyl terminus of constitutive heat shock cognate 70 (HSC70)-interacting protein (CHIP, also known as Stub1) is a U box-containing E3 ubiquitin ligase that is important for protein quality control. The role of CHIP in innate immunity is not known. Here, we report that CHIP knockdown inhibits Toll-like receptor (TLR) 4- and TLR9-driven signaling, but not TLR3-driven signaling; proinflammatory cytokine and type 1 interferon (IFN) production; and maturation of antigen-presenting cells, including macrophages and dendritic cells. We demonstrate that CHIP can recruit the tyrosine kinase Src and atypical protein kinase C ? (PKC?) to the TLR complex, thereby leading to activation of IL-1 receptor-associated kinase 1, TANK-binding kinase 1, and IFN regulatory factors 3 and 7. CHIP acts as an E3 ligase for Src and PKC? during TLR signaling. CHIP-mediated enhancement of TLR signaling is inhibited by IFNAR deficiency or expression of ubiquitination resistant mutant forms of Src or PKC?. These findings suggest that CHIP facilitates the formation of a TLR signaling complex by recruiting, ubiquitinating, and activating Src and PKC?.
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MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma.
J. Biol. Chem.
PUBLISHED: 08-30-2011
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In our in-depth analysis carried out by the Illumina Solexa massive parallel signature sequencing, microRNA-99a (miR-99a) was found to be the sixth abundant microRNA in the miRNome of normal human liver but was markedly down-regulated in hepatocellular carcinoma (HCC). Compelling evidence has suggested the important roles of microRNAs in HCC development. However, the biological function of miR-99a deregulation in HCC remains unknown. In this study, we found that miR-99a was remarkably decreased in HCC tissues and cell lines. Importantly, lower miR-99a expression in HCC tissues significantly correlated with shorter survival of HCC patients, and miR-99a was identified to be an independent predictor for the prognosis of HCC patients. Furthermore, restoration of miR-99a dramatically suppressed HCC cell growth in vitro by inducing the G(1) phase cell cycle arrest. Intratumoral injection of cholesterol-conjugated miR-99a mimics significantly inhibited tumor growth and reduced the ?-fetoprotein level in HCC-bearing nude mice. Insulin-like growth factor 1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) were further characterized as the direct targets of miR-99a. Furthermore, protein levels of IGF-1R and mTOR were found to be inversely correlated with miR-99a expression in HCC tissues. miR-99a mimics inhibited IGF-1R and mTOR pathways and subsequently suppressed expression of cell cycle-related proteins, including cyclin D1 in HCC cells. Conclusively, miR-99a expression was frequently down-regulated in HCC tissues and correlates with the prognosis of HCC patients, thus proposing miR-99a as a prospective prognosis predictor of HCC. miR-99a suppresses HCC growth by inducing cell cycle arrest, suggesting miR-99a as potential tumor suppressor for HCC therapeutics.
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Efficient induction of a Her2-specific anti-tumor response by dendritic cells pulsed with a Hsp70L1-Her2(341-456) fusion protein.
Cell. Mol. Immunol.
PUBLISHED: 07-25-2011
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Heat shock proteins (HSPs) have been shown to interact with antigen-presenting cells (APCs), especially dendritic cells (DCs). HSPs act as potent adjuvants, inducing a Th1 response, as well as antigen-specific CD8(+) cytotoxic T lymphocytes (CTL) via cross-presentation. Our previous work has demonstrated that Hsp70-like protein 1 (Hsp70L1), a new member of the Hsp70 subfamily, can act as a powerful Th1 adjuvant in a DC-based vaccine. Here we report the efficient induction of tumor antigen-specific T cell immune response by DCs pulsed with recombinant fusion protein of Hsp70L1 and Her2(341-456), the latter of which is a fragment of Her2/neu (Her2) containing E75 (a HLA-A2 restricted CTL epitope). The fusion protein Hsp70L1-Her2(341-456) promotes the maturation of DCs and activates them to produce cytokines, such as IL-12 and TNF-?, and chemokines, such as MIP-1?, MIP-1? and RANTES. Taken together, these results indicate that the adjuvant activity of Hsp70L1 is maintained in the fusion protein. Her2-specific HLA-A2.1-restricted CD8(+) CTLs can be generated efficiently either from the Peripheral blood lymphocytes (PBL) of healthy donors or from the splenocytes of immunized HLA-A2.1/K(b) transgenic mice by in vitro stimulation or immunization with DCs pulsed with the Hsp70L1-Her2(341-456) fusion protein. This results in more potent target cell killing in an antigen-specific and HLA-A2.1-restricted manner. Adoptive transfer of splenocytes from transgenic mice immunized with Hsp70L1-Her2(341-456)-pulsed DCs can markedly inhibit tumor growth and prolong the survival of nude mice with Her2(+)/HLA-A2.1(+) human carcinomas. These results suggest that Hsp70L1-Her2(341-456)-pulsed DCs could be a new therapeutic vaccine for patients with Her2(+) cancer.
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Toll-like receptor 4 (TLR4) is essential for Hsp70-like protein 1 (HSP70L1) to activate dendritic cells and induce Th1 response.
J. Biol. Chem.
PUBLISHED: 07-05-2011
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Toll-like receptors (TLRs) play important roles in initiation of innate and adaptive immune responses. Emerging evidence suggests that TLR agonists can serve as potential adjuvant for vaccination. Heat shock proteins (HSPs), functionally serving as TLR4 agonists, have been proposed to act as Th1 adjuvant. We have identified a novel Hsp70 family member, termed Hsp70-like protein 1 (Hsp70L1), shown that Hsp70L1 is a potent T helper cell (Th1) polarizing adjuvant that contributes to antitumor immune responses. However, the underlying mechanism for how Hsp70L1 exerts its Th1 adjuvant activity remains to be elucidated. In this study, we found that Hsp70L1 binds directly to TLR4 on the surface of DCs, activates MAPK and NF-?B pathways, up-regulates I-a(b), CD40, CD80, and CD86 expression and promotes production of TNF-?, IL-1?, and IL-12p70. Hsp70L1 failed to induce such phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating a role for TLR4 in mediating Hsp70L1-induced DC activation. Furthermore, more efficient induction of carcinoembryonic antigen (CEA)-specific Th1 immune response was observed in mice immunized by wild-type DCs pulsed with Hsp70L1-CEA(576-669) fusion protein as compared with TLR4-deficient DCs pulsed with same fusion protein. In addition, TLR4 antagonist impaired induction of CEA-specific human Th1 immune response in a co-culture system of peripheral blood lymphocytes (PBLs) from HLA-A2.1(+) healthy donors and autologous DCs pulsed with Hsp70L1-CEA(576-669) in vitro. Taken together, these results demonstrate that TLR4 is a key receptor mediating the interaction of Hsp70L1 with DCs and subsequently enhancing the induction of Th1 immune response by Hsp70L1/antigen fusion protein.
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The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-?.
Nat. Immunol.
PUBLISHED: 06-20-2011
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Interferon-? (IFN-?) has a critical role in immune responses to intracellular bacterial infection. MicroRNAs (miRNAs) are important in the regulation of innate and adaptive immunity. However, whether miRNAs can directly target IFN-? and regulate IFN-? production post-transcriptionally remains unknown. Here we show that infection of mice with Listeria monocytogenes or Mycobacterium bovis bacillus Calmette-Guérin (BCG) downregulated miR-29 expression in IFN-?-producing natural killer cells, CD4(+) T cells and CD8(+) T cells. Moreover, miR-29 suppressed IFN-? production by directly targeting IFN-? mRNA. We developed mice with transgenic expression of a sponge target to compete with endogenous miR-29 targets (GS29 mice). We found higher serum concentrations of IFN-? and lower L. monocytogenes burdens in L. monocytogenes-infected GS29 mice than in their littermates. GS29 mice had enhanced T helper type 1 (T(H)1) responses and greater resistance to infection with BCG or Mycobacterium tuberculosis. Therefore, miR-29 suppresses immune responses to intracellular pathogens by targeting IFN-?.
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Defining the critical hurdles in cancer immunotherapy.
Bernard A Fox, Dolores J Schendel, Lisa H Butterfield, Steinar Aamdal, James P Allison, Paolo Antonio Ascierto, Michael B Atkins, Jiřina Bartůňková, Lothar Bergmann, Neil Berinstein, Cristina C Bonorino, Ernest Borden, Jonathan L Bramson, Cedrik M Britten, Xuetao Cao, William E Carson, Alfred E Chang, Dainius Characiejus, A Raja Choudhury, George Coukos, Tanja de Gruijl, Robert O Dillman, Harry Dolstra, Glenn Dranoff, Lindy G Durrant, James H Finke, Jerome Galon, Jared A Gollob, Cécile Gouttefangeas, Fabio Grizzi, Michele Guida, Leif Håkansson, Kristen Hege, Ronald B Herberman, F Stephen Hodi, Axel Hoos, Christoph Huber, Patrick Hwu, Kohzoh Imai, Elizabeth M Jaffee, Sylvia Janetzki, Carl H June, Pawel Kalinski, Howard L Kaufman, Koji Kawakami, Yutaka Kawakami, Ulrich Keilholtz, Samir N Khleif, Rolf Kiessling, Beatrix Kotlan, Guido Kroemer, Rejean Lapointe, Hyam I Levitsky, Michael T Lotze, Cristina Maccalli, Michele Maio, Jens-Peter Marschner, Michael J Mastrangelo, Giuseppe Masucci, Ignacio Melero, Cornelius Melief, William J Murphy, Brad Nelson, Andrea Nicolini, Michael I Nishimura, Kunle Odunsi, Pamela S Ohashi, Jill O'Donnell-Tormey, Lloyd J Old, Christian Ottensmeier, Michael Papamichail, Giorgio Parmiani, Graham Pawelec, Enrico Proietti, Shukui Qin, Robert Rees, Antoni Ribas, Ruggero Ridolfi, Gerd Ritter, Licia Rivoltini, Pedro J Romero, Mohamed L Salem, Rik J Scheper, Barbara Seliger, Padmanee Sharma, Hiroshi Shiku, Harpreet Singh-Jasuja, Wenru Song, Per thor Straten, Hideaki Tahara, Zhigang Tian, Sjoerd H van der Burg, Paul von Hoegen, Ena Wang, Marij Jp Welters, Hauke Winter, Tara Withington, Jedd D Wolchok, Weihua Xiao, Laurence Zitvogel, Heinz Zwierzina, Francesco M Marincola, Thomas F Gajewski, Jon M Wigginton, Mary L Disis.
J Transl Med
PUBLISHED: 05-02-2011
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Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.
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MicroRNA-98 negatively regulates IL-10 production and endotoxin tolerance in macrophages after LPS stimulation.
FEBS Lett.
PUBLISHED: 04-01-2011
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Interleukin 10 (IL-10) is a potent anti-inflammatory cytokine that is crucial for dampening the inflammatory response after pathogen invasion, and was found to be produced by macrophages after exposure to lipopolysaccharide (LPS). It remains unclear whether microRNA-mediated regulatory mechanism is involved in LPS-induced IL-10 production. Here we reported that miR-98 expression in macrophages significantly decreased following LPS stimulation. We also found that miR-98 targets the 3untranslated region of IL-10 transcript. Overexpression of miR-98 inhibited TLR4-triggered IL-10 production and promoted COX-2 expression. We further demonstrated that miR-98 significantly mitigated the induction of endotoxin tolerance, suggesting that miR-98-mediated posttranscriptional control could potentially be involved in fine tuning the critical level of IL-10 production in endotoxin tolerance.
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Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk.
Nat. Immunol.
PUBLISHED: 02-28-2011
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The molecular mechanisms involved in the full activation of innate immunity achieved through Toll-like receptors (TLRs) remain to be fully elucidated. In addition to their classical antigen-presenting function, major histocompatibility complex (MHC) class II molecules might mediate reverse signaling. Here we report that deficiency in MHC class II attenuated the TLR-triggered production of proinflammatory cytokines and type I interferon in macrophages and dendritic cells, which protected mice from endotoxin shock. Intracellular MHC class II molecules interacted with the tyrosine kinase Btk via the costimulatory molecule CD40 and maintained Btk activation, but cell surface MHC class II molecules did not. Then, Btk interacted with the adaptor molecules MyD88 and TRIF and thereby promoted TLR signaling. Therefore, intracellular MHC class II molecules can act as adaptors, promoting full activation of TLR-triggered innate immune responses.
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Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma.
Cancer Cell
PUBLISHED: 02-15-2011
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The full scale of human miRNome in specific cell or tissue, especially in cancers, remains to be determined. An in-depth analysis of miRNomes in human normal liver, hepatitis liver, and hepatocellular carcinoma (HCC) was carried out in this study. We found nine miRNAs accounted for ?88.2% of the miRNome in human liver. The third most highly expressed miR-199a/b-3p is consistently decreased in HCC, and its decrement significantly correlates with poor survival of HCC patients. Moreover, miR-199a/b-3p can target tumor-promoting PAK4 to suppress HCC growth through inhibiting PAK4/Raf/MEK/ERK pathway both in vitro and in vivo. Our study provides miRNomes of human liver and HCC and contributes to better understanding of the important deregulated miRNAs in HCC and liver diseases.
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IFN-?-producing PDCA-1+ Siglec-H- B cells mediate innate immune defense by activating NK cells.
Eur. J. Immunol.
PUBLISHED: 02-01-2011
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B cells have multiple functions in adaptive immunity, including antibody production, antigen presentation and regulation of T-cell responses. Recent evidences indicate that B cells have more subsets than previously thought and may have non-classical functions, such as involvement in innate immunity and immune regulation; however, how B cells respond to microbial infection and elicit innate defense remain unclear. In this study, we identified a new subset of PDCA-1(+) Siglec-H(-) CD19(+) B cells in mice during the early period of bacterial infection with Listeria monocytogenes. PDCA-1(+) Siglec-H(-) CD19(+) B cells secreted large amounts of IFN-? and thus facilitated IFN-? production and cytotoxicity function of natural killer (NK) cells via IFN-?. B-cell-deficient Btk(-/-) mice were incapable of producing PDCA-1(+) CD19(+) B cells, and were more sensitive to L. monocytogenes infection. Adoptive transfer of PDCA-1(+) CD19(+) B cells to Btk(-/-) mice normalized their resistance to L. monocytogenes infection. Furthermore, we found that macrophages were essential for the inducible generation of PDCA-1(+) Siglec-H(-) CD19(+) B cells via CD40-CD40L ligation. Therefore, we have identified a new subset of PDCA-1(+) Siglec-H(-) CD19(+) B cells, which enhances innate immune responses against bacterial infection by activating NK cells via secretion of IFN-?.
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Chemokine-containing exosomes are released from heat-stressed tumor cells via lipid raft-dependent pathway and act as efficient tumor vaccine.
J. Immunol.
PUBLISHED: 01-17-2011
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Exosomes derived from dendritic cells or tumor cells are a population of nanometer-sized membrane vesicles that can induce specific antitumor immunity. During investigation of the effects of hyperthermia on antitumor immune response, we found that exosomes derived from heat-stressed tumor cells (HS-TEX) could chemoattract and activate dendritic cells (DC) and T cells more potently than that by conventional tumor-derived exosomes. We show that HS-TEX contain chemokines, such as CCL2, CCL3, CCL4, CCL5, and CCL20, and the chemokine-containing HS-TEX are functionally competent in chemoattracting CD11c(+) DC and CD4(+)/CD8(+) T cells both in vitro and in vivo. Moreover, the production of chemokine-containing HS-TEX could be inhibited by ATP inhibitor, calcium chelator, and cholesterol scavenger, indicating that the mobilization of chemokines into exosomes was ATP- and calcium-dependent and via a lipid raft-dependent pathway. We consistently found that the intracellular chemokines could be enriched in lipid rafts after heat stress. Accordingly, intratumoral injection of HS-TEX could induce specific antitumor immune response more efficiently than that by tumor-derived exosomes, thus inhibiting tumor growth and prolonging survival of tumor-bearing mice more significantly. Therefore, our results demonstrate that exosomes derived from HS-TEX represent a kind of efficient tumor vaccine and can chemoattract and activate DC and T cells, inducing more potent antitumor immune response. Release of chemokines through exosomes via lipid raft-dependent pathway may be a new method of chemokine exocytosis.
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Immune complex enhances tolerogenecity of immature dendritic cells via Fc?RIIb and promotes Fc?RIIb-overexpressing dendritic cells to attenuate lupus.
Eur. J. Immunol.
PUBLISHED: 01-05-2011
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A balance of inhibitory and activating signals determines the function of dendritic cells (DCs) in the immune response, which may be regulatory or stimulatory. Defects of inhibitory receptor Fc?RIIb are involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE), in which high levels of circulating immune complexes (IC) exist. Our previous study showed that IC/Ig can suppress TLR4-triggered inflammatory responses in macrophages via Fc?RIIb. This led us to question whether IC/Ig can polarize Fc?RIIb-overexpressing DCs (DC-Fc?RIIb) to be tolerogenic, thus attenuating lupus progression once infused in vivo. First, we found that IC/Ig markedly inhibited LPS- or CpG-induced DC maturation, enhanced tolerogenicity of DCs via Fc?RIIb, and induced massive prostaglandin E2 (PGE2) secretion from DCs, both contributing to T-cell hyporesponsiveness. Endogenous Ig and lupus-derived IC also exhibited the same effect. DC-Fc?RIIb, transfected with recombinant adenovirus encoding Fc?RIIb, displayed enhanced tolerogenic function and produced more PGE2 in the presence of IC, thus further inhibiting T-cell responses. Importantly, in vivo infusion with DC-Fc?RIIb significantly reduced kidney damage and prolonged the survival of lupus-prone MRL/lpr mice either before or after the onset of clinic lupus. Therefore, administration of DC-Fc?RIIb may be a new approach to attenuate lupus progression.
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MicroRNA-148/152 impair innate response and antigen presentation of TLR-triggered dendritic cells by targeting CaMKII?.
J. Immunol.
PUBLISHED: 11-10-2010
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MicroRNAs (miRNAs) are involved in the regulation of immunity, including the lymphocyte development and differentiation, and inflammatory cytokine production. Dendritic cells (DCs) play important roles in linking innate and adaptive immune responses. However, few miRNAs have been found to regulate the innate response and APC function of DCs to date. Calcium/calmodulin-dependent protein kinase II (CaMKII), a major downstream effector of calcium (Ca(2+)), has been shown to be an important regulator of the maturation and function of DCs. Our previous study showed that CaMKII? could promote TLR-triggered production of proinflammatory cytokines and type I IFN. Inspired by the observations that dicer mutant Drosophila display defect in endogenous miRNA generation and higher CaMKII expression, we wondered whether miRNAs can regulate the innate response and APC function of DCs by targeting CaMKII?. By predicting with software and confirming with functional experiments, we demonstrate that three members of the miRNA (miR)-148 family, miR-148a, miR-148b, and miR-152, are negative regulators of the innate response and Ag-presenting capacity of DCs. miR-148/152 expression was upregulated, whereas CaMKII? expression was downregulated in DCs on maturation and activation induced by TLR3, TLR4, and TLR9 agonists. We showed that miR-148/152 in turn inhibited the production of cytokines including IL-12, IL-6, TNF-?, and IFN-? upregulation of MHC class II expression and DC-initiated Ag-specific T cell proliferation by targeting CaMKII?. Therefore, miRNA-148/152 can act as fine-tuner in regulating the innate response and Ag-presenting capacity of DCs, which may contribute to the immune homeostasis and immune regulation.
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Regulatory dendritic cells program generation of interleukin-4-producing alternative memory CD4 T cells with suppressive activity.
Blood
PUBLISHED: 11-04-2010
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The heterogeneity and mechanisms for the generation of CD4 memory T (CD4 Tm) cells remain elusive. Distinct subsets of dendritic cells (DCs) have been found to regulate a distinct T-helper (Th)-cell subset differentiation by influencing cytokine cues around CD4 T cells; however, whether and how the regulatory DC subset can regulate Tm-cell differentiation remains unknown. Further, there is no ideal in vitro experimental system with which to mimic the 3 phases of the CD4 T-cell immune response (expansion, contraction, memory generation) and/or to culture CD4 Tm cells for more than a month. By analyzing CD4 T cells programmed by long-term coculture with regulatory DCs, we identified a population of long-lived CD4 T cells with a CD44(hi)CD62L(-)CCR7(-) effector memory phenotype and rapid, preferential secretion of the Th2 cytokines interleukin-4 (IL-4), IL-5, IL-10, and IL-13 after antigenic stimulation. These regulatory DC-programmed Tm cells suppress CD4 T-cell activation and proliferation in vitro via IL-10 and inhibit the delayed-type hypersensitivity response once infused in vivo. We also identify their natural counterpart, which is up-regulated by regulatory DC transfusion and negatively regulates the recall response in vivo. Different from interferon-?-producing conventional Tm cells, these IL-4-producing CD4 Tm cells act as alternative Tm cells with a regulatory function, suggesting a new way of negative immune regulation by memory T cells.
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IL-17A-producing gammadeltaT cells promote CTL responses against Listeria monocytogenes infection by enhancing dendritic cell cross-presentation.
J. Immunol.
PUBLISHED: 10-18-2010
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Interleukin-17A-producing T cells, especially Th17, have been shown to be involved in inflammatory autoimmune diseases and host defense against extracellular infections. However, whether and how IL-17A or IL-17A-producing cells can help protection against intracellular bacteria remains controversial, especially how it regulates the adaptive immunity besides recruitment of neutrophils in the innate immune system. By infecting IL-17A-deficient mice with Listeria monocytogenes, we show in this study that IL-17A is required for the generation of Ag-specific CD8(+) CTL response against primary infection, but not for the generation of memory CD8(+) T cells against secondary challenge. Interestingly, we identify ??T cells, but not conventional CD4(+) Th17 cells, as the main cells for innate IL-17A production during L. monocytogenes infection. Furthermore, ??T cells are found to promote Ag-specific CD8(+) T cell proliferation by enhancing cross-presentation of dendritic cells through IL-17A. Adoptive transfer of Il17a(+/+) ??T cells, but not Il17a(-/-) ??T cells or Il17a(+/+) CD4(+) T cells, were sufficient to recover dendritic cells cross-presentation and defective CD8(+) T cell response in Il17a(-/-) mice. Our findings indicate an important role of infection-inducible IL-17A-producing ??T cells and their derived IL-17A against intracellular bacterial infection, providing a mechanism of IL-17A for regulation of innate and adaptive immunity.
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Inducible microRNA-155 feedback promotes type I IFN signaling in antiviral innate immunity by targeting suppressor of cytokine signaling 1.
J. Immunol.
PUBLISHED: 10-11-2010
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Effective recognition of viral infection and subsequent triggering of antiviral innate immune responses are essential for the host antiviral defense, which is tightly regulated by multiple regulators, including microRNAs. Our previous study showed that a panel of microRNAs, including miR-155, was markedly upregulated in macrophages upon vesicular stomatitis virus infection; however, the biological function of miR-155 during viral infection remains unknown. In this paper, we show that RNA virus infection induces miR-155 expression in macrophages via TLR/MyD88-independent but retinoic acid-inducible gene I/JNK/NF-?B-dependent pathway. And the inducible miR-155 feedback promotes type I IFN signaling, thus suppressing viral replication. Furthermore, suppressor of cytokine signaling 1 (SOCS1), a canonical negative regulator of type I IFN signaling, is targeted by miR-155 in macrophages, and SOCS1 knockdown mediates the enhancing effect of miR-155 on type I IFN-mediated antiviral response. Therefore, we demonstrate that inducible miR-155 feedback positively regulates host antiviral innate immune response by promoting type I IFN signaling via targeting SOCS1.
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Ras-related protein Rab10 facilitates TLR4 signaling by promoting replenishment of TLR4 onto the plasma membrane.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-19-2010
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The Toll-like receptor (TLR)4 receptor complex, TLR4/MD-2, plays an important role in the inflammatory response against lipopolysaccharide, a ubiquitous membrane component in Gram-negative bacteria. Ligand recognition by TLR4 initiates multiple intracellular signaling pathways, leading to production of proinflammatory mediators and type I IFN. Ligand interaction also leads to internalization of the surface receptor complex into lysosomes, leading to the degradation of TLR4 and the termination of LPS response. However, surface level of TLR4 receptor complex is maintained via continuous replenishment of TLR4 from intracellular compartments like Golgi and endosomes. Here we show that continuous replenishment of TLR4 from Golgi to plasma membrane is regulated by the small GTPase Rab10, which is essential for optimal macrophage activation following LPS stimulation. Expression of Rab10 is inducible by LPS. Blockade of Rab10 function leads to decreased membrane TLR4 expression and diminished production of inflammatory cytokines and interferons upon LPS stimulation. These findings suggest that Rab10 expression provides a mechanism to refine TLR4 signaling by regulating the trafficking rate of TLR4 onto the plasma membrane. In addition, we show that altered Rab10 expression in macrophages influences disease severity in an in vivo model of LPS-induced acute lung injury, suggesting Rab10 as a possible therapeutic target for human acute respiratory distress syndrome (ARDS).
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Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b.
Nat. Immunol.
PUBLISHED: 06-16-2010
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Integrins are critical for the migration and function of leukocytes in inflammation. However, the interaction between integrin alpha(M) (CD11b), which has high expression in monocytes and macrophages, and Toll-like receptor (TLR)-triggered innate immunity remains unclear. Here we report that CD11b deficiency enhanced TLR-mediated responses in macrophages, rendering mice more susceptible to endotoxin shock and Escherichia coli-caused sepsis. CD11b was activated by TLR-triggered phosphatidylinositol 3-OH kinase (PI(3)K) and the effector RapL and fed back to inhibit TLR signaling by activating the tyrosine kinases Src and Syk. Syk interacted with and induced tyrosine phosphorylation of MyD88 and TRIF, which led to degradation of these adaptor molecules by the E3 ubiquitin ligase Cbl-b. Thus, TLR-triggered, active CD11b integrin engages in crosstalk with the MyD88 and TRIF pathways and subsequently inhibits TLR signaling in innate immune responses.
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Stress for maintaining memory: HSP70 as a mobile messenger for innate and adaptive immunity.
Eur. J. Immunol.
PUBLISHED: 05-15-2010
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HSP are abundant and conserved proteins present in all cells. Upon temperature shock or other stress stimuli, HSP are synthesized intracellularly, which may protect cells from protein denaturation or from death. Although HSP are synthesized intracellularly, HSP can also be mobilized to the plasma membrane or even be released under stress conditions. Elucidating the roles of cell surface and extracellular HSP in immune regulation has attracted much attention in recent years. Extracellularly, HSP can serve a cytokine function to initiate both innate and adaptive immunity through activation of APC. HSP serves also a chaperone function and facilitates presentation of antigen peptide to T cells. Similarly, cell surface HSP may activate APC and promote antigen presentation through cell-cell contact. A study in this issue of the European Journal of Immunology demonstrates that cell surface HSP70 on DC induced by stress can upregulate membrane-associated IL-15, which in turn promotes the proliferation of CD4(+)CD45RA memory T cells. Moreover, a DC-CD4(+) T-cell interacting circuit formed by CD40L on T cells and CD40 on DC is proposed to play a role in the maintenance of memory homeostasis. This study has widened our view of HSP in adaptive immunity as well as their classical functions such as APC activator and antigen carrier.
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Human phosphatidylethanolamine-binding protein 4 promotes transactivation of estrogen receptor alpha (ERalpha) in human cancer cells by inhibiting proteasome-dependent ERalpha degradation via association with Src.
J. Biol. Chem.
PUBLISHED: 05-11-2010
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We identified human phosphatidylethanolamine-binding protein 4 (hPEBP4) as a human-derived novel member of the phosphatidylethanolamine-binding protein family, which is involved in apoptosis resistance of tumor cells. Because of its preferential expression in estrogen-related cancers, we wondered whether hPEBP4 plays a role in estrogen-induced cancer cell growth. Here, we demonstrated that hPEBP4 inhibited the 17beta-estradiol (E(2))-induced, proteasome-dependent estrogen receptor alpha (ERalpha) degradation to increase the protein level of ERalpha. Silencing of hPEBP4 inhibited the recruitment of ERalpha to the promoter of the ERalpha target gene pS2 in MCF-7 breast cancer cells after E(2) treatment. E(2)-induced, ERalpha-mediated transcription via the estrogen-response element, as well as the cellular proliferation, was significantly suppressed in hPEBP4-silenced MCF-7 cells. We found that Src, whose association with ERalpha facilitates the ERalpha binding to components of proteolytic machinery, could associate with hPEBP4 and that overexpression of hPEBP4 prevented the E(2)-induced interaction between ERalpha and Src. ERalpha overexpression, proteasome inhibitor, or Src inhibitor could reverse the suppression of ERalpha-mediated transactivation by hPEBP4 silencing. The inhibition of the proteasome degradation and the promotion of transactivation of ERalpha by hPEBP4 via the Src pathway were further confirmed in HeLa cells. Finally, we found that the promoting effects of hPEBP4 on ERalpha-mediated transactivation and estrogen-induced proliferation of cancer cells did not depend on its regulation of Akt and ERK activity. Our data suggest that hPEBP4 inhibits proteasome-dependent ERalpha degradation through the Src pathway, thus enhancing ERalpha-mediated transactivation and promoting the proliferation of cancer cells in response to estrogen.
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Apoptotic cells attenuate fulminant hepatitis by priming Kupffer cells to produce interleukin-10 through membrane-bound TGF-?.
Hepatology
PUBLISHED: 05-04-2010
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The liver, a unique tolerogenic organ, is regarded as the site to trap and destroy aging erythrocytes and activated T cells. However, to date, the mechanisms for why the liver is tolerogenic and whether liver Kupffer cells (KC) are critical phagocytes for apoptotic cells (AC) contributing to the liver immunosuppression remain unclear. Here we report that KC is the main phagocyte for AC in the liver. Contact of AC inhibits proinflammatory cytokine but enhances anti-inflammatory cytokine production of KC in response to lipopolysaccharide (LPS) stimulation. Membrane-bound transforming growth factor (TGF)-? on AC is responsible for the increased production of interleukin (IL)-10 in KC through extracellular signal-regulated kinase (ERK) activation via the Smad3 pathway. Importantly, KC-derived IL-10 is critical for AC infusion-mediated protection of endotoxin-induced fulminant hepatitis through suppression of tumor necrosis factor (TNF)-? and nitric oxide (NO) production from KC and consequently attenuation of KC-mediated cytolysis of hepatocytes.
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Scaffolding adaptor protein Gab1 is required for TLR3/4- and RIG-I-mediated production of proinflammatory cytokines and type I IFN in macrophages.
J. Immunol.
PUBLISHED: 04-30-2010
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RIG-I-like helicases and TLRs are critical sensors in the induction of type I IFN and proinflammatory cytokines to initiate innate immunity against invading pathogens. However, the mechanisms for the full activation of TLR and RIG-I-triggered innate response remain to be fully investigated. Grb2-associated binder 1 (Gab1), a member of scaffolding/adaptor proteins, can mediate signal transduction from many receptors, however, whether and how Gab1 is required for TLR and RIG-I-triggered innate responses remain unknown. In this study, we demonstrated that Gab1 significantly enhances TLR4-, TLR3-, and RIG-I-triggered IL-6, IL-1beta, and IFN-alpha/beta production in macrophages. Gab1 knockdown in primary macrophages or Gab1 deficiency in mouse embryonic fibroblasts significantly suppresses TLR3/4- and RIG-I-triggered production of IL-6, IL-1beta, and IFN-alpha/beta. Consistently, Gab1 deficiency impairs vesicular stomatitis virus (VSV) infection-induced IFN-alpha/beta production. In addition to promoting both MyD88- and TLR/IL-1 receptor domain-containing adaptor protein inducing IFN-beta-dependent MAPKs and NF-kappaB activation, Gab1 enhances PI3K/Akt activation by directly binding p85 in TLR signaling and VSV infection. Accordingly, Gab1 inhibits VSV replication and VSV infection-induced cell damage by inducing type I IFNs and IFN-inducible gene expression via PI3K/Akt pathway. Therefore, Gab1 is needed for full activation of TLR3/4- and RIG-I-triggered innate responses by promoting activation of PI3K/Akt, MAPKs, and NF-kappaB pathways.
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Adenovirus-mediated LIGHT gene modification in murine B-cell lymphoma elicits a potent antitumor effect.
Cell. Mol. Immunol.
PUBLISHED: 04-26-2010
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Here, we investigated the antitumor effect of adenovirus-mediated gene transfer of LIGHT, the tumor-necrosis factor (TNF) superfamily member also known as TNFSF14, in the murine A20 B-cell lymphoma. LIGHT gene modification resulted in upregulated expression of Fas and the accessory molecule--intercellular adhesion molecule-1 (ICAM-1) on A20 cells and led to enhanced A20 cell apoptosis. LIGHT-modified A20 cells effectively stimulated the proliferation of T lymphocytes and interferon (IFN)-gamma production in vitro. Immunization of BALB/c mice with a LIGHT-modified A20 cell vaccine efficiently elicited protective immunity against challenge with the parental tumor cell line. Adenovirus-mediated gene transfer of LIGHT by intratumoral injection exerted a very potent antitumor effect against pre-existing A20 cell lymphoma in BALB/c mice. This adenovirus-mediated LIGHT therapy induced substantial splenic natural killer (NK) and cytotoxic T lymphocyte (CTL) activity, enhanced tumor infiltration by inflammatory cells and increased chemokine expression of CC chemokine ligand 21 (CCL21), IFN-inducible protein-10 (IP-10) and monokine induced by IFN-gamma (Mig) from tumor tissues. Thus, adenovirus-mediated LIGHT therapy might have potential utility for the prevention and treatment of B-cell lymphoma.
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MicroRNA-466l upregulates IL-10 expression in TLR-triggered macrophages by antagonizing RNA-binding protein tristetraprolin-mediated IL-10 mRNA degradation.
J. Immunol.
PUBLISHED: 04-21-2010
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MicroRNAs (miRNAs) are generally recognized as regulating gene expression posttranscriptionally by inhibiting translation or inducing target mRNA degradation. New mechanisms for miRNAs to regulate gene expression also still attract much attention. More and more novel miRNAs are discovered by the advanced sequencing technology, but yet their biological functions are largely unknown. Up to now, the function of miR-466l, a miRNA discovered in mouse embryonic stem cells, remains unclear. In this study, we report that miR-466l can upregulate both mRNA and protein expression of IL-10 in TLR-triggered macrophages. Furthermore, we show that miR-466l can competitively bind to the IL-10 3 untranslated region AU-rich elements, which is a typical binding site for RNA-binding protein (RBP). Tristetraprolin is a well-known RBP, and mediates rapid degradation of IL-10 mRNA. miRNA always mediates target mRNA degradation or translation repression modestly; thus, the net effect of miR-466ls binding to IL-10 AU-rich elements is to prevent IL-10 mRNA degradation mediated by tristetraprolin, resulting in extended t(1/2) of IL-10 mRNA and elevated IL-10 expression. Thus, competitive binding with RBP to the same target mRNA and subsequent stabilization of target mRNA is an alternative mechanism for gene regulation by miRNAs. Also, a mechanism for regulation of IL-10 by miRNAs is outlined.
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Interleukin-17 and its expanding biological functions.
Cell. Mol. Immunol.
PUBLISHED: 04-12-2010
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Interleukin-17 (IL-17) and IL-17-producing cells have been shown to play important roles in inflammation and the immune response. IL-17 is believed to be mainly produced by T helper 17 (Th17) cells, a unique helper T-cell subset different from Th1 and Th2 cells. Other subsets of T cells such as gammadeltaT and natural killer T (NKT) cells have also been found to produce IL-17 in response to innate stimuli. IL-17 acts as a proinflammatory cytokine that can induce the release of certain chemokines, cytokines, matrix metalloproteinases (MMPs) and antimicrobial peptides from mesenchymal and myeloid cells. This leads to the expansion and accumulation of neutrophils in the innate immune system and links innate and adaptive immunity in vivo. Furthermore, increasing evidence indicates that IL-17 and IL-17-producing cells are involved in the pathogenesis of various diseases such as allergies, autoimmune diseases, allograft transplantation and even malignancy. They may also play protective roles in host defense against infectious diseases and promote induction of cytotoxic T lymphocyte (CTL) responses against cancer. Targeting of the IL-17 axis is under investigation for the treatment of inflammatory disorders.
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The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I interferon via a beta-catenin-dependent pathway.
Nat. Immunol.
PUBLISHED: 04-09-2010
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Intracellular nucleic acid sensors detect microbial RNA and DNA and trigger the production of type I interferon. However, the cytosolic nucleic acid-sensing system remains to be fully identified. Here we show that the cytosolic nucleic acid-binding protein LRRFIP1 contributed to the production of interferon-beta (IFN-beta) induced by vesicular stomatitis virus (VSV) and Listeria monocytogenes in macrophages. LRRFIP1 bound exogenous nucleic acids and increased the expression of IFN-beta induced by both double-stranded RNA and double-stranded DNA. LRRFIP1 interacted with beta-catenin and promoted the activation of beta-catenin, which increased IFN-beta expression by binding to the C-terminal domain of the transcription factor IRF3 and recruiting the acetyltransferase p300 to the IFN-beta enhanceosome via IRF3. Therefore, LRRFIP1 and its downstream partner beta-catenin constitute another coactivator pathway for IRF3-mediated production of type I interferon.
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Small Rab GTPase Rab7b promotes megakaryocytic differentiation by enhancing IL-6 production and STAT3-GATA-1 association.
J. Mol. Med.
PUBLISHED: 04-01-2010
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Induction of the differentiation of human leukemia cells is a useful strategy in treatment of human leukemia. However, the molecular mechanisms involved in leukemia cell differentiation have not been fully elucidated. Interleukin 6 (IL-6) is a pleiotropic cytokine acting on a variety of cell types, and plays important roles in hematopoiesis. GATA binding protein 1 (GATA-1) is an important transcription factor involved in either megakaryocytic or erythrocytic differentiation. Herein we report that Rab7b, a late endosome/lysosome-localized myeloid small GTPase, promotes phorbol-12-myristate-13-acetate (PMA)-induced megakaryocytic differentiation by increasing nuclear factor ?B (NF-?B)-dependent IL-6 production and subsequently enhancing the association of activated signal transducer and activator of transcription 3 (STAT3) with GATA-1. By using PMA-induced megakaryocytic differentiation of leukemia cells as a model, we investigated the roles of Rab7b in megakaryocytic differentiation. We find that Rab7b can potentiate PMA-induced upregulation of megakaryocytic markers, production of IL-6, and activation of NF-?B. Inhibitor of NF-?B and neutralizing antibodies for IL-6 or the IL-6 signaling receptor gp130 can block the effects of Rab7b in megakaryocytic differentiation. In Rab7b-silenced cells, PMA-induced activation of NF-?B, IL-6 production, and megakaryocytic differentiation are impaired. Furthermore, we demonstrate that IL-6-induced activation of STAT3 and the subsequent association of STAT3 with GATA-1 may contribute to PMA-induced and Rab7b-mediated transcriptional upregulation of megakaryocytic differentiation markers. Therefore, our data suggest that Rab7b may play important roles in megakaryopoiesis by activating NF-?B and promoting IL-6 production. Our study also indicates that the IL-6-induced association of STAT3 with GATA-1 may regulate megakaryocytic differentiation.
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Novel function of B cell-activating factor in the induction of IL-10-producing regulatory B cells.
J. Immunol.
PUBLISHED: 03-05-2010
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Although B cells have been shown to possess a regulatory function, microenvironmental factors or cytokines involved in the induction of regulatory B cells remain largely uncharacterized. B cell-activating factor (BAFF), a member of TNF family cytokines, is a key regulator for B cell maturation and function. In this study, we detected significantly increased numbers of IL-10-producing B cells in BAFF-treated B cell cultures, an effect specifically abrogated by neutralization of BAFF with TACI-Fc. BAFF-induced IL-10-producing B cells showed a distinct CD1d(hi)CD5(+) phenotype, which were mainly derived from marginal zone B cells. Moreover, BAFF activated transcription factor AP-1 for binding to IL-10 promoter. Notably, BAFF treatment in vivo increased the number of IL-10-producing B cells in marginal zone regions. Furthermore, BAFF-induced IL-10-producing B cells possess a regulatory function both in vitro and in vivo. Taken together, our findings identify a novel function of BAFF in the induction of IL-10-producing regulatory B cells.
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