Retinoic acid-inducible gene-I is induced by interferon-gamma and regulates the expression of interferon-gamma stimulated gene 15 in MCF-7 cells.

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family proteins, which have diverse roles in regulation of gene expression and cellular functions. We found RIG-I mRNA and protein were expressed in MCF-7 human breast cancer cells stimulated with interferon-gamma (IFN-gamma). This effect of IFN-gamma was observed in concentration- and time-dependent manners, and IFN-gamma also induced promoter activity of RIG-I. Transfection of GFP-RIG-I cDNA into MCF-7 cells resulted in the expression of RIG-I protein in cytoplasm. Overexpression of RIG-I induced the upregulation of IFN-gamma stimulated gene 15, which has the potential to amplify the immunomodulatory effects. We conclude that IFN-gamma induces the expression of RIG-I, which may play a role in the immunological effects of IFN-gamma.     

Retinoic acid-inducible gene-I is induced by interferon-gamma and regulates CXCL11 expression in HeLa cells

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family proteins, which have diverse roles in the regulation of gene expression and cellular functions. RIG-I is one of the factors regulated by interferon (IFN)-gamma and regarded as an intracellular signaling molecule for IFN-gamma. IFN-gamma is a major cytokine and also suggested to be involved in embryonal implantation and pregnancy. It is demonstrated that IFN-gamma stimulates endometrial epithelial cells to produce CXCL11, which is implicated in implantation. The aim of the present study was to investigate the effect of IFN-gamma on RIG-I expression in HeLa cells, a cell line derived from human uterine carcinoma. We found that RIG-I mRNA and protein were expressed in HeLa cells stimulated with IFN-gamma. The effect of IFN-gamma was observed in concentration- and time-dependent manners. The RNA interference against RIG-I resulted in the suppression of the IFN-gamma-induced CXCL11 expression. Immunohistochemical studies revealed the RIG-I expression in the normal human endometrium, suggesting a possible role of RIG-I in human reproductive organs.     

Listeria monocytogenes induces the expression of retinoic acid-inducible gene-I

Retinoic acid-inducible gene-I (RIG-I) is considered to play a role in innate immunity against virus infections. We showed by immunohistochemical study that RIG-I expression is upregulated in vivo in hepatic Kupffer cells and in splenic reticular cells of mice infected with Listeria monocytogenes. Both heat-killed L. monocytogenes and live L. monocytogenes induced the expression of RIG-I in cultured RAW264.7 murine macrophage-like cells in vitro. RIG-I may also be involved in innate immunity against Listeria infection.     

RIG-I敲除293T细胞系的建立及其对B型流感病毒复制的影响

CRISPR/Cas9基因编辑技术是通过人工设计的单向导RNA(Single-guide RNA,sgRNA)指导Cas9蛋白对目的基因靶位点进行特异性的识别、结合和切割后,通过细胞的非同源末端连接或同源末端重组修复机制来完成对基因组的敲除与敲入的编辑技术。RIG-I是机体的一种模式识别受体,能够识别胞质中的含5′-三磷酸基团的RNA,并通过与下游信号分子MAVS相互作用,激活IRF3/7和NF-κB,从而启动I型干扰素和炎性因子的表达。已有研究表明,B型流感病毒(IBV)在感染早期能够上调RIG-I的表达水平。为了探索RIG-I是否为B型流感病毒激活抗病毒天然免疫信号通路的主要受体及其对IBV复制的影响,本研究利用CRISPR-Cas9技术对293T细胞中的RIG-I基因进行了敲除,经嘌呤霉素压力筛选到了一株稳定敲除RIG-I基因的293T(RIG-I-/-293T)细胞系。Western blotting检测发现,IBV或仙台病毒感染后该细胞系中RIG-I不再表达,说明该敲除细胞系构建成功。IBV感染RIG-I-/-293T细胞后,干扰素、炎性因子及干扰素刺激基因的转录水平与野生型293T细胞相比明显下降,并且在RIG-I-/-293T细胞中检测不到p65和IRF3磷酸化,表明IBV感染早期细胞因子的表达主要依赖于RIG-I信号通路的激活。IBV在野生型及RIG-I-/-293T细胞中的多步生长曲线表明,RIG-I可抑制IBV的复制。以上结果表明,RIG-I敲除的293T细胞系构建成功,RIG-I是IBV激活下游抗病毒天然免疫信号通路的主要受体之一,且对IBV的复制具有负调控作用,该研究为探索IBV的感染机制奠定了基础。     

Establishment and maintenance of the innate antiviral response to West Nile Virus involves both RIG-I and MDA5 signaling through IPS-1

RIG-I and MDA5, two related pathogen recognition receptors (PRRs), are known to be required for sensing various RNA viruses. Here we investigated the roles that RIG-I and MDA5 play in eliciting the antiviral response to West Nile virus (WNV). Functional genomics analysis of WNV-infected fibroblasts from wild-type mice and RIG-I null mice revealed that the normal antiviral response to this virus occurs in two distinct waves. The initial response to WNV resulted in the expression of interferon (IFN) regulatory factor 3 target genes and IFN-stimulated genes, including several subtypes of alpha IFN. Subsequently, a second phase of IFN-dependent antiviral gene expression occurred very late in infection. In cells lacking RIG-I, both the initial and the secondary responses to WNV were delayed, indicating that RIG-I plays a critical role in initiating innate immunity against WNV. However, another PRR(s) was able to trigger a response to WNV in the absence of RIG-I. Disruption of both MDA5 and RIG-I pathways abrogated activation of the antiviral response to WNV, suggesting that MDA5 is involved in the host's defense against WNV infection. In addition, ablation of the function of IPS-1, an essential RIG-I and MDA5 adaptor molecule, completely disabled the innate antiviral response to WNV. Our data indicate that RIG-I and MDA5 are responsible for triggering downstream gene expression in response to WNV infection by signaling through IPS-1. We propose a model in which RIG-I and MDA5 operate cooperatively to establish an antiviral state and mediate an IFN amplification loop that supports immune effector gene expression during WNV infection.     

Induction of interferon gamma in human gingival fibroblasts challenged with phytohaemagglutinin

Interferon gamma (IFN-gamma) is a potential immunoregulatory cytokine, which is secreted mainly by cells of immune origin. In this study, we examined the capacity of human gingival fibroblasts as non-professional immune cells to express IFN-gamma messenger RNA (mRNA) and to produce the protein. Cultures of fibroblast cells were established from gingival biopsies from three children. The expression of mRNA for IFN-gamma was studied by in situ hybridization, and the level of IFN-gamma was determined by cell-released capturing ELISA. Treatment of the cells with phytohaemagglutinin (PHA) (2.5, 5.0, and 10 microg/ml) increased the number of IFN-gamma mRNA expressing cells and the protein production at 1, 6, and 24 h. Non-stimulated cells did not reveal measurable levels of IFN-gamma mRNA or the protein. The inflammatory cytokines interleukin 1beta (IL-1beta) (100 microg/ml) and tumour necrosis factor alpha (TNFalpha) (10 ng/ml) did not affect IFN-gamma mRNA expression or protein production. Treatment of the cells with 1 microM phorbol 12-myristate-13-acetate (PMA) stimulated IFN-gamma mRNA expression but had no effect on IFN-gamma protein production. We conclude that human gingival fibroblasts not only transcribe IFN-gamma mRNA but also produce the IFN-gamma protein in response to PHA. The finding that human gingival fibroblasts, produce the cytokine IFN-gamma, further support the concept that these cells take an active part in the modulation of the inflammatory and immune response in the periodontal tissue.     

Effect of peroxisome proliferator-activated receptor-gamma ligands on the expression of retinoic acid-inducible gene-I in endothelial cells stimulated with lipopolysaccharide

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box protein family and designated as a putative RNA helicase. RIG-I is implicated in host defense and inflammatory reactions by regulating the expression of various genes. RIG-I is expressed in endothelial cells and upregulated with lipopolysaccharide (LPS). Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear hormone receptor and regulates gene expressions in response to its specific ligands. In the present study, we examined the effect of PPAR-gamma ligands on the LPS-induced RIG-I expression in cultured human umbilical vein endothelial cells (HUVEC). 15-Deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), a metabolite of PGD2, is a natural ligand for PPAR-gamma and known to modulate inflammatory reactions by regulating the expression of various genes in PPAR-gamma-dependent and -independent manners. LPS-induced RIG-I expression in HUVEC was inhibited by pretreatment of the cells with 15d-PGJ2 in time-and concentration-dependent manners. However, ciglitazone and bisphenol A diglycide ether, authentic and specific ligands for PPAR-gamma, did not affect the RIG-I expression. These results suggest that 15d-PGJ2 inhibits LPS-induced RIG-I expression through a mechanism independent on PPAR-gamma. 15d-PGJ2 may regulate inflammatory reactions, at least in part, by inhibiting the expression of RIG-I.     

Sensing of viral nucleic acids by RIG-I: from translocation to translation

The innate immune system is a first layer of defense against infection by pathogens. It responds to pathogens by activating host defense mechanisms via interferon and inflammatory cytokine expression. Pathogen associated molecular patterns (PAMPs) are sensed by specific pattern recognition receptors. Among those, the ATP dependent helicase related RIG-I like receptors RIG-I, MDA5 and LGP2 sense the presence of viral RNA in the cytoplasm of host cells. While the precise PAMPs and functions of MDA5 or LGP2 are still unclear, RIG-I senses predominantly viral RNA containing a 5′-triphosphate along with dsRNA regions. Here we review our current knowledge of how these PAMPs are sensed and integrated by RIG-I, and how RIG-I's innate immune function can be used in translational medical approaches.     

Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-alpha, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-gamma-inducing cytokines, IL-12 and IFN-alpha, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-gamma production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-alpha, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-alpha and IL-18 play a significant role in enhancing IFN-gamma synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.     

Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.

Fractalkine/CX3C-chemokine ligand 1 is expressed as a membrane-spanning adhesion molecule that can be cleaved from the cell surface to produce a soluble chemoattractant. Within the vasculature, fractalkine is known to be generated by endothelial cells, but to date there are no reports describing its expression by smooth muscle cells (SMC). In this study we demonstrate that IFN-gamma and TNF-alpha, but not IL-1beta, cooperate synergistically to induce fractalkine mRNA and protein expression in cultured aortic SMC. We also report the release of functional, soluble fractalkine from the membranes of stimulated SMC. This release is inhibited by the zinc metalloproteinase inhibitor batimastat, resulting in the accumulation of membrane-associated fractalkine on the SMC surface. Therefore, an SMC-derived metalloproteinase activity is involved in fractalkine shedding. While soluble fractalkine present in SMC-conditioned medium is capable of inducing calcium transients in cells expressing the fractalkine receptor (CX3CR1), blocking experiments using neutralizing Abs reveal that it can be inactivated without affecting the chemotactic activity of SMC-conditioned media on monocytes. However, membrane-bound fractalkine plays a major role in promoting adhesion of monocytic cells to activated SMC. This fractalkine-mediated adhesion is further enhanced in the presence of batimastat, indicating that shedding of fractalkine from the cell surface down-regulates the adhesive properties of SMC. Hence, during vascular inflammation, the synergistic induction of fractalkine by IFN-gamma and TNF-alpha together with its metalloproteinase-mediated cleavage may finely control the recruitment of monocytes to SMC within the blood vessel wall.     

SRSF1 Facilitates Cytosolic DNA-Induced Production of Type I Interferons Recognized by RIG-I

BackgroundEvidence has shown that psoriasis is closely associated with infection; however, the mechanism of this association remains unclear. In mammalian cells, viral or bacterial infection is accompanied by the release of cytosolic DNA, which in turn triggers the production of type-I interferons (IFNs). Type I IFNs and their associated genes are significantly upregulated in psoriatic lesions. RIG-I is also highly upregulated in psoriatic lesions and is responsible for IFN production. However, RIG-I mediated regulatory signaling in psoriasis is poorly understood.MethodsWe screened a cDNA library and identified potential RIG-I interacting partners that may play a role in psoriasis.ResultsWe found that serine/arginine-rich splicing factor 1 (SRSF1) could specifically interact with RIG-I to facilitate RIG-I mediated production of type-I IFN that is triggered by cytosolic DNA. We found SRSF1 associates with RNA polymerase III and RIG-I in a DNA-dependent manner. In addition, treatment with a TNFα inhibitor downregulated SRSF1 expression in peripheral blood mononuclear cells (PBMCs) from psoriasis vulgaris patients.DiscussionBased on the abundance of pathogenic cytosolic DNA that is detected in psoriatic lesions, our finding that RIG-I interacts with SRSF1 to regulate type-I IFN production reveals a critical link regarding how cytosolic DNA specifically activates aberrant IFN expression. These data may provide new therapeutic targets for the treatment of psoriasis.     

Cigarette smoke extract suppresses the RIG-I-initiated innate immune response to influenza virus in the human lung

Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and predisposes subjects to severe respiratory tract infections. Epidemiological studies have shown that cigarette smokers are seven times more likely to contract influenza infection than nonsmokers. The mechanisms underlying this increased susceptibility are poorly characterized. Retinoic acid-inducible gene (RIG)-I is believed to play an important role in the recognition of, and response to, influenza virus and other RNA viruses. Our study focused on how cigarette smoke extract (CSE) alters the influenza-induced proinflammatory response and suppresses host antiviral activity in the human lung using a unique lung organ culture model. We first determined that treatment with 2-20% CSE did not induce cytotoxicity as assessed by LDH release. However, CSE treatment inhibited influenza-induced IFN-inducible protein 10 protein and mRNA expression. Induction of the major antiviral cytokine IFN-β mRNA was also decreased by CSE. CSE also blunted viral-mediated RIG-I mRNA and protein expression. Inhibition of viral-mediated RIG-I induction by CSE was prevented by the antioxidants N-acetyl-cysteine and glutathione. These findings show that CSE suppresses antiviral and innate immune responses in influenza virus-infected human lungs through oxidative inhibition of viral-mediated induction of the pattern recognition receptor RIG-I. This immunosuppressive effect of CSE may play a role in the enhanced susceptibility of smokers to serious influenza infection in the lung.     

Genomic HIV RNA induces innate immune responses through RIG-I-dependent sensing of secondary-structured RNA

Background

Innate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs responsible for inducing innate immune responses remains sparse.

Methods/Principal Findings

Here we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor α were produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-κB, MAP kinases, and IRF3 and co-localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA.

Conclusions/Significance

These results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune system.     

Avian influenza virus H5N1 induces rapid interferon-beta production but shows more potent inhibition to retinoic acid-inducible gene I expression than H1N1 in vitro

ABSTRACT: BACKGROUND: The mechanisms through which the avian influenza virus H5N1 modulate the host's innate immune defense during invasion, remains incompletely understood. RIG-I as a pattern recognition receptor plays an important role in mediating innate immune response induced by influenza virus. So, modulating RIG-I might be adopted as a strategy by influenza virus to antagonize the host's innate immune defense. Methods: Here we chose an avian influenza virus A/tree sparrow/Henan/1/04 (H5N1) directly isolated from a free-living tree sparrow in Mainland China which is amplified in egg allantoic cavity, and researched its interferon induction and manipulation of RIG-I expression compared with influenza virus A/WSN/1933(H1N1), a well charaterized mouse adapted strain, in human lung epithelial A549 cells and human embryonic kidney 293T cells. Results: Although the avian influenza virus H5N1 infection initiated a rapid IFN-beta production early on, it eventually presented a more potent inhibition to IFN-beta production than H1N1. Correspondingly, the H5N1 infection induced low level expression of endogenous RIG-I, an Interferon Stimulating Gene (ISG), and showed more potent inhibition to the expression of endogenous RIG-I triggered by exogenous interferon than H1N1. Conclusions: Manipulating endogenous RIG-I expression might constitute one of the mechanisms through which avian influenza virus H5N1 control the host's innate immune response during infection.     

Antiviral immune responses in H5N1-infected human lung tissue and possible mechanisms underlying the hyperproduction of interferon-inducible protein IP-10

Information on the immune response against H5N1 within the lung is lacking. Here we describe the sustained antiviral immune responses, as indicated by the expression of MxA protein and IFN-α mRNA, in autopsy lung tissue from an H5N1-infected patient. H5N1 infection of primary bronchial/tracheal epithelial cells and lung microvascular endothelial cells induced IP-10, and also up-regulated the retinoic acid-inducible gene-I (RIG-I). Down-regulation of RIG-I gene expression decreased IP-10 response. Co-culturing of H5N1-infected pulmonary cells with TNF-α led to synergistically enhanced production of IP-10. In the absence of viral infection, TNF-α and IFN-α also synergistically enhanced IP-10 response. Methylprednisolone showed only a partial inhibitory effect on this chemokine response. Our findings strongly suggest that both the H5N1 virus and the locally produced antiviral cytokines; IFN-α and TNF-α may have an important role in inducing IP-10 hyperresponse, leading to inflammatory damage in infected lung.     

RIG-I is required for the inhibition of measles virus by retinoids

Vitamin A can significantly decrease measles-associated morbidity and mortality. Vitamin A can inhibit the replication of measles virus (MeV) in vitro through an RARα- and type I interferon (IFN)-dependent mechanism. Retinoid-induced gene I (RIG-I) expression is induced by retinoids, activated by MeV RNA and is important for IFN signaling. We hypothesized that RIG-I is central to retinoid-mediated inhibition of MeV in vitro. We demonstrate that RIG-I expression is increased in cells treated with retinoids and infected with MeV. The central role of RIG-I in the retinoid-anti-MeV effect was demonstrated in the Huh-7/7.5 model; the latter cells having non-functional RIG-I. RAR-dependent retinoid signaling was required for the induction of RIG-I by retinoids and MeV. Retinoid signaling was also found to act in combination with IFN to induce high levels of RIG-I expression. RIG-I promoter activation required both retinoids and MeV, as indicated by markers of active chromatin. IRF-1 is known to be regulated by retinoids and MeV, but we found recruitment of IRF-1 to the RIG-I promoter by retinoids alone. Using luciferase expression constructs, we further demonstrated that the IRF-1 response element of RIG-I was required for RIG-I activation by retinoids or IFN. These results reveal that retinoid treatment and MeV infection induces significant RIG-I. RIG-I is required for the retinoid-MeV antiviral response. The induction is dependent on IFN, retinoids and IRF-1.