Natural type I interferon-producing cells as a link between innate and adaptive immunity

Type I interferons (IFNs) are promptly produced upon invasion of pathogens, and activate a broad range of effector cells in the innate and adaptive immune system. Lin⁻CD4⁺CD11c⁻ plasmacytoid dendritic cell precursors (plasmacytoid pre-DCs) produce enormous amounts of type I IFNs in response to viruses and CpG DNA, thus corresponding to the previously described but not fully defined natural type I IFN-producing cells (IPCs). Plasmacytoid pre-DCs strongly express toll-like receptor (TLR) 7 and TLR9, in contrast to monocytes, which mainly express TLR1, 2, 4, 5, and 8, suggesting that these two DC precursors recognize different microbial molecules and that they may have developed through different evolutionary trails. Three different stimuli, CpG DNA plus CD40 ligand, interleukin-3 (IL-3), and herpes simplex virus, stimulate plasmacytoid pre-DCs to differentiate into DCs that induce distinct types of T helper cells, i.e., Th1, Th2, and IFN-γ- and IL-10-producing T cells, respectively. The remarkable versatility of plasmacytoid pre-DCs distinguishes them from other cell types in the immune system that have only limited functions, and suggests that these cells may play a key role in integrating the innate and adaptive aspects of various immune responses.     

The emerging roles of the STING adaptor protein in immunity and diseases

DNA that gains access to the cytoplasm generally serves as a danger signal for the hosts. An emerging paradigm for responding to cytosolic DNAs centres on the endoplasmic reticulum‐resident protein stimulator of interferon genes (STING, also known as MITA, ERIS or MPYS), the hub adaptor of the recently identified DNA sensors. Dynamic regulations of STING action are critical for shaping innate immune responses against microbial infections, as well as for preventing autoimmune diseases. STING is also indispensable for the detection of immunogenic tumours. A deeper understanding of STING modulations could be instrumental for developing novel immunotherapeutic strategies against infectious, autoimmune and cancerous diseases. In this review, we summarize the latest advances on the role of STING in the DNA‐triggered immune reactions, and underscore the critical issues that remain to be resolved in future studies.     

Cytosolic d-type CpG-oligonucleotides induce a type I interferon response by activating the cGAS-STING signaling pathway

Cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) has been shown to be critically involved in the detection of cytosolic, self- and non-self-DNA, initiating a type I IFN response through the adaptor protein Stimulator of Interferon Genes (STING) and interferon regulatory factor 3 (IRF3). Current studies propose that canonical binding of dsDNA by cGAS depends on DNA length, but not on base sequence. In contrast, activation of TLR9 is sequence dependent. It requires unmethylated CpG dinucleotides in microbial DNA, which is mimicked by synthetic oligodeoxynucleotides (ODN). Here, we provide evidence that d -type ODN (D-ODN), but not K-type ODN (K-ODN), bind to human cGAS and activate downstream signaling. Transfection of D-ODN into a TLR9-deficient, human monocytic cell line (THP-1) induced phosphorylation of IRF3 and secretion of IFN. This response was absent in cells with CRISPR/Cas9-mediated cGAS- or STING-deficiency. Utilizing a protein pulldown approach, we further demonstrate direct binding of D-ODN to cGAS. Induction of a type I IFN response by D-ODN was confirmed in human primary monocytes and monocyte-derived macrophages. These results are relevant to our understanding of self–nonself-discrimination by cGAS and to the pharmacologic effects of ODN, which currently are investigated in clinical studies.     

RNA recognition and signal transduction by RIG-I-like receptors

Summary:  Viral infection is detected by cellular sensor molecules as foreign nucleic acids and initiates innate antiviral responses, including the activation of proinflammatory cytokines and type I interferon (IFN). Recent identification of cytoplasmic viral sensors, such as retinoic acid-inducible gene-I-like receptors (RLRs), highlights their significance in the induction of antiviral innate immunity. Moreover, it is intriguing to understand how they can discriminate endogenous RNA from foreign viral RNA and initiate signaling cascades leading to the induction of type I IFNs. This review focuses on the current understanding of the molecular machinery underlying RNA recognition and subsequent signal transduction by RLRs.     

Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7R-Dependent STING Activation by Tumor-Derived cGAMP

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乳腺癌组织中STING蛋白的表达及其临床意义

目的探讨干扰素基因刺激因子(stimulator of interferon,STING)在乳腺癌组织中的表达及其临床意义。方法采用免疫组化SP法检测42例乳腺癌及其癌旁正常组织中STING蛋白的表达,分析STING蛋白表达与乳腺癌临床病理特征的关系。结果STING蛋白在乳腺癌组织的阳性率(23.8%,10/42)低于癌旁正常组织(52.4%,22/42)(χ^2=7.269,P<0.05),其表达与患者的HER-2阳性率、Lumina分型相关(χ^2=8.839,P<0.05)。STING蛋白低表达是乳腺癌患者术后复发的危险因素之一(OR=7.472,95%CI:1.279~43.637,P=0.026)。结论乳腺癌组织中STING蛋白表达降低,与乳腺癌预后相关,可能参与乳腺癌的发生、发展,有望成为乳腺癌治疗的潜在靶点。     

RIG-I-like receptors: cytoplasmic sensors for non-self RNA

Viral infection results in the generation of non-self RNA species in the cells, which is recognized by retinoic acid inducible gene-I-like receptors (RLRs), and initiates innate antiviral responses, including the production of proinflammatory cytokines and type I interferon. In this review, we summarize reports on virus-specificity of RLRs, structures of non-self RNA patterns, structural biology of RLRs, and the signaling adapter molecules involved in antiviral innate immunity.     

Antiviral effect of ovotransferrin in mouse peritoneal macrophages by up-regulating type I interferon expression

Ovotransferrin (OVT) is one of the major proteins in avian albumen that exerts diverse biological activities. This study is aimed to investigate the antiviral effect and mechanism of OVT from the avian egg. Effects of OVT on vesicular stomatitis virus (VSV)-infected mouse peritoneal macrophage were detected by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and western blot. In OVT-pretreated mouse peritoneal macrophages, results showed that replication of VSV was significantly suppressed and production of type I interferon (IFN) was increased. OVT down-regulated ring finger protein 125 (RNF125) production and up-regulated retinoic-acid-inducible gene I (RIG-I) expression by the nuclear factor-κB signalling pathway, resulting in the enhancement of the production of type I IFN. It demonstrated that the antiviral effect of OVT on innate immunity is enhanced by promoting type I IFN expression, indicating its potential as a functional food ingredient.     

TRIM10 binds to IFN-α/β receptor 1 to negatively regulate type I IFN signal transduction

The type I interferon (IFN-I) system is important for antiviral and anticancer immunity. Prolonged activation of IFN/JAK/STAT signaling is closely associated with autoimmune diseases. TRIM10 dysfunction may be associated closely with certain autoimmune disorders. Here, we observed that the serum TRIM10 protein level is lower in patients with systemic lupus erythematosus than in healthy control subjects. We speculated the possible involvement of TRIM10-induced modulation of the IFN/JAK/STAT signaling pathway in systemic lupus erythematosus. In line with our hypothesis, TRIM10 inhibited the activation of JAK/STAT signaling pathway triggered by various stimuli. TRIM10 restricted the IFN-I/JAK/STAT signaling pathway, which was independent of its E3 ligase activity. Mechanistically, TRIM10 interacted with the intracellular domain of IFNAR1 and blocked the association of IFNAR1 with TYK2. These data suggest the possible TRIM10 suppresses IFN/JAK/STAT signaling pathway through blocking the interaction between IFNAR1 and TYK2. Targeting TRIM10 is a potential strategy for treating autoimmune diseases.     

Phosphatidylinositol-3-kinase and Akt are required for RIG-I-mediated anti-viral signalling through cross-talk with IPS-1

Retinoic acid-inducible gene I (RIG-I) is a cytosolic pattern-recognition receptor that recognizes viruses and triggers anti-viral immune responses. Activation of intracellular RIG-I signalling is mediated through interferon-β (IFN-β) promoter stimulator-1 (IPS-1), an adaptor of RIG-I, which induces IFN regulatory factor (IRF) 3 activation and type I IFN expression. The phosphatidylinositol-3-kinase (PI3K) and Akt pathway is activated in host immune cells upon viral infection. However, the mechanism as to how they work in RIG-I signalling has not been fully elucidated. Therefore, we investigated the role of PI3K and Akt in the regulation of RIG-I-mediated IRF3 activation and type I IFN expression in macrophages. Our results show that Sendai virus infection, which is recognized by RIG-I, led to IRF3 activation and IFN-β expression and these responses were attenuated by the PI3K inhibitor (LY294002) and an Akt dominant-negative mutant in the macrophage cell line(RAW264.7). IRF3 phosphorylation and dimerization as well as IFN-β expression induced by a synthetic RIG-I agonist, short poly(I:C), were suppressed by LY294002 or siRNA-Akt in bone marrow-derived macrophages. Suppression of PI3K and Akt using a dominant-negative mutant and siRNA knockdown resulted in attenuation of IRF3 activation and IFN-β expression induced by RIG-I itself or its adaptor, IPS-1. Association of Akt with IPS-1 increased with short poly(I:C) stimulation and required the pleckstrin homology domain of Akt and caspase-recruitment domain in IPS-1. Collectively, our results show that PI3K and Akt are required downstream of IPS-1 for RIG-I-mediated anti-viral immune responses. The results describe a novel, interactive relationship between RIG-I downstream signalling molecules resulting in efficient anti-viral immunity.     

Immunobiology and immunotherapy of HCC: spotlight on innate and innate-like immune cells

Immune-based therapies such as immune checkpoint inhibitors have revolutionized the systemic treatment of various cancer types. The therapeutic application of monoclonal antibodies targeting inhibitory pathways such as programmed cell death-1(PD-1)/programmed cell death ligand 1 (PD-L1) and CTLA-4 to cells of the adaptive immune system has recently been shown to generate meaningful improvement in the clinical outcome of hepatocellular carcinoma (HCC). Nevertheless, current immunotherapeutic approaches induce durable responses in only a subset of HCC patients. Since immunologic mechanisms such as chronic inflammation due to chronic viral hepatitis or alcoholic and nonalcoholic fatty liver disease play a crucial role in the initiation, development, and progression of HCC, it is important to understand the underlying mechanisms shaping the unique tumor microenvironment of liver cancer. The liver is an immunologic organ with large populations of innate and innate-like immune cells and is exposed to bacterial, viral, and fungal antigens through the gut–liver axis. Here, we summarize and highlight the role of these cells in liver cancer and propose strategies to therapeutically target them. We also discuss current immunotherapeutic strategies in HCC and outline recent advances in our understanding of how the therapeutic potential of these agents might be enhanced.     

The cytoplasmic 'linker region' in Toll-like receptor 3 controls receptor localization and signaling

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA and transmits signals to activate NF-kappaB and the interferon (IFN)-beta promoter via the newly identified adaptor, TICAM-1. The extracellular LRR domain of TLR3 is engaged in the ligand recognition, while the intracellular TIR domain is crucial for the adaptor binding and signal transduction upon ligand stimulation. Here, we analyzed TLR3 localization in human monocyte-derived immature dendritic cells (iDCs) and stable transfectants expressing human TLR3 by immunofluorescence staining and confocal microscopy. TLR3 was predominantly localized in specific but as yet unidentified intracellular vesicles where TLR3 signaling was initiated. Expression analysis of TLR3-tail-truncated mutants revealed that the cytoplasmic 'linker' region (residues 730-755) determines the intracellular localization of TLR3. Site-directed mutagenesis of the linker region allowed us to identify the relevant determinants as Arg(740) and Val(741) residues for intracellular expression of TLR3. Furthermore, alanine scanning of the linker region demonstrated that the Phe(732), Leu(742) and Gly(743) in the TLR3 cytoplasmic linker region are essential for ligand-induced NF-kappaB and IFN-beta promoter activation. Thus, the cytoplasmic linker region of TLR3 regulates receptor retention inside the organelle and signaling, which may be closely linked to TLR3 function in DCs.     

Type I interferon activates MHC class I-dressed CD11b+ conventional dendritic cells to promote protective anti-tumor CD8+ T cell immunity

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The innate immune architecture of lung tumors and its implication in disease progression

Lung malignancies are the leading cause of cancer-related mortality. By virtue of its unique physiological function, the lung microenvironment is highly dynamic and constantly subjected to mechanical, chemical and pathogenic stimuli. Thus, the airways rely on highly organized innate defense mechanisms to rapidly protect against pathogens and maintain pulmonary homeostasis. However, in the context of lung malignancy, these defenses often provide collateral inflammatory insults that can foster tumor progression. This review summarizes the interactions between cancer cells, recruited immune cells and tissue-resident cell subpopulations, such as airway epithelial cells and alveolar macrophages, during homeostasis and disease. Furthermore, we examine the role of the lung immune landscape in response to current therapeutic interventions for cancer. Given the prevalence of lung malignancies, we propose that consideration of lung physiology as a whole is necessary to understand and treat these lethal diseases. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Controlled infection with a therapeutic virus defines the activation kinetics of human natural killer cells in vivo

Human natural killer (NK) cells play an important role in anti‐viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon‐inducible molecules CD69 and tetherin peaked 24–48 h post‐infection, coincident with a peak of interferon‐induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post‐infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus‐based therapy. Furthermore, our results suggest the existence of a post‐infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection.     

A paradigm shift in therapeutic vaccination of cancer patients: the need to apply therapeutic vaccination strategies in the preventive setting

Summary: An extraordinary variety of potential therapeutic vaccine strategies directed against a wide variety of tumor antigens has been explored in clinical trials. To date, none of these cancer immunotherapies have been approved by the Food and Drug Administration for use in humans. A significant problem is that the vast majority of such clinical trials are carried out in patients with advanced or metastatic cancer. The immune systems of these patients are considerably compromised as a result of tumor- and treatment-mediated immunosuppression. Even in cases where patients are immunized in the adjuvant setting, where there is minimal residual disease, vaccines directed against tumor-associated antigens have failed to mediate eradication of tumors in the overwhelming majority of cases. Recently, we and others have experimented with administering therapeutic cancer vaccines in the preventive setting. This is achieved by vaccinating at the earliest possible stage of carcinogenesis. These studies have demonstrated that early vaccination is extremely effective in eliciting an anti-tumor immune response that leads to unprecedented improvements in the survival of mice that spontaneously develop cancer. Certain human cancers, notably prostate adenocarcinoma and cervical cancer, can currently be detected at very early stages of carcinogenesis. Therapeutic vaccines are available for these diseases, opening up the possibility of administering vaccinations early to patients diagnosed with pre-malignant lesions to halt disease progression. In addition, new technologies have become available in the past decade that will soon yield very sensitive and specific diagnostic tests for a plethora of other cancers. Earlier detection of these cancers, combined with existing vaccines directed against them, will soon make them targets for therapeutic vaccination in the preventive setting. The ability to immunize patients at the very earliest stages of carcinogenesis, when they have fully competent immune systems, has the potential to cause a paradigm shift in how therapeutic cancer vaccines are tested and used clinically.     

The Unfolded Protein Response Mediator PERK Governs Myeloid Cell-Driven Immunosuppression in Tumors through Inhibition of STING Signaling

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NK cells – Versatile tools for viral defense and cancer treatment

About 150 NK cell researchers met at the German Cancer Research Center (DKFZ) in Heidelberg, Germany from 26–28 September 2012 for the Natural Killer Cell Symposium which was organized by the NK cell study group of the German Society for Immunology (DGfI) and sponsored by the European Journal of Immunology (EJI), the European Federation of Immunological Societies (EFIS) and the DGfI. The meeting was a forum for the discussion of the function and regulation of these fascinating innate immune cells and the opportunities for the transfer of this knowledge to cancer immunotherapy.