Expression Of RIG-I Research Articles

RIG-I promotes IFN/JAK2 expression and the endoplasmic reticulum stress response to inhibit chemoradiation resistance in nasopharyngeal carcinoma.

RIG‐I is associated with the occurrence and development of many tumors. However, the role of RIG‐I in radiotherapy and chemotherapy in NPC has not been reported to date. In our study, RIG‐I expression was significantly reduced in chemoradiotherapy‐resistant NPC tissues and cells compared with that in therapy‐sensitive tissues and cells. RIG‐I expression increased in nonresistant NPC cells, including CNE1 and CNE2, in a dose‐dependent manner with increasing chemotherapy drug concentration or radiotherapy dose. RIG‐I overexpression promoted radiotherapy and chemotherapy sensitivity in NPC cells, leading to cellular apoptosis and increased expression of the proapoptotic factors BAX and caspase‐3. Similarly, RIG‐I knockdown in NPC cells promoted chemoradiotherapy resistance and reduced apoptosis. Analysis of microarray data indicated that the expression of IFN/JAK2 and endoplasmic reticulum (ER) stress response markers, such as JAK2, STAT1, IRF9, IFNB1, IRF3, p‐IRF3, XBP1, ATF6, IFIT2, and ISG15, was inhibited in chemoradiotherapy‐resistant cells compared with that in sensitive cells. Conversely, activation of IFN/JAK2 and ER stress response pathways in NPC cells reduced paclitaxel resistance and increased apoptosis. RIG‐I promotes IFN/JAK2 and ER stress response‐mediated apoptosis to inhibit chemoradiation resistance in nasopharyngeal carcinoma.

The expression profile of RNA sensors in colorectal cancer and its correlation with cancer stages

Background: RNA sensors represent the most important pattern recognition receptors (PRRs) to defend against RNA pathogens, such as RNA viruses. Recent studies revealed their critical roles in inflammatory and autoimmune diseases. Furthermore, more recent evidences indicated that RNA sensors mediate the development of colitis or colorectal cancer (CRC). However, a systematic understanding of RNA sensors in CRC is still lacking, especially the expression patterns in CRC. Methods: Here, we analyzed RNA sensor expression, clinical significance, and possible mechanisms in CRC by combining bioinformatic analysis and the analysis on pre-cancerous animal model and clinical tissue samples. Results: We found that most of the members of RNA sensors, including RNA-sensing Toll-like receptors ( TLR3, TLR7 , and TLR8 ) and RIG-I-like receptors ( MDA5 and RIG-I ), were down-regulated in CRC, while the expression of DDX21 were up-regulated in human CRC. In addition, we also analyzed the correlation between gene expression and cancer stages. We found that the expression of RNA-sensing TLRs, RIG-I , and DDX21 in CRC were associated with cancer stages. Finally, in order to explore the possible mechanisms, the correlation between RNA sensors and the main downstream signaling molecules were analyzed. A positive correlation was observed in TLR7/8 and MyD88, RIG-I/MDA5 and LGP2 , while a negative correlation was observed in RIG-I/MDA5 and MAVS . Conclusions: This study reveals the potential values of RNA-sensing genes including TLRs, RIG-I and DDX21 as biomarkers of CRC formation, progression and therapy.

Immune-Related Gene Expression in Ducks Infected With Waterfowl-Origin H5N6 Highly Pathogenic Avian Influenza Viruses.

Clade 2.3.4.4 H5 avian influenza viruses (AIVs) are widely prevalent and of significant concern to the poultry industry and public health in China. Nowadays, the clade 2.3.4.4 H5N6 virus has become a dominant AIV subtype among domestic ducks in southern China. We found that waterfowl-origin clade 2.3.4.4 H5N6 viruses (A/goose/Guangdong/16568/2016, GS16568 and A/duck/Guangdong/16873/2016, DK16873) isolated from southern China in 2016 could replicate in multiple organs of inoculated ducks. DK16873 virus caused mild infections and killed 2/5 of inoculated ducks, and GS16568 virus did not kill inoculated ducks. In addition, the two viruses could be transmitted via direct contact between ducks. DK16873 and GS16568 viruses killed 2/5 and 1/5 of contact ducks, respectively. Furthermore, ducks inoculated with the two H5N6 viruses exhibited different expressions of immune-related genes in their lungs. The expression of RIG-I, TLR3 and IL6 was significantly upregulated at 12 h post-inoculation (HPI) and most of the tested immune-related genes were significantly upregulated at 3 days post-inoculation (DPI). Notably, the expression of RIG-I and IL-6 in response to DK16873 virus was significantly higher than for GS16568 virus at 12 HPI and 3 DPI. Our research have provided helpful information about the pathogenicity, transmission and immune-related genes expression in ducks infected with new H5N6 AIVs.

The expression profile of RNA sensors in colorectal cancer and its correlation with cancer stages.

BackgroundRNA sensors represent the most important pattern recognition receptors (PRRs) to defend against RNA pathogens, such as RNA viruses. Recent studies revealed their critical roles in inflammatory and autoimmune diseases. Furthermore, more recent evidences indicated that RNA sensors mediate the development of colitis or colorectal cancer (CRC). However, a systematic understanding of RNA sensors in CRC is still lacking, especially the expression patterns in CRC.MethodsHere, we analyzed RNA sensor expression, clinical significance, and possible mechanisms in CRC by combining bioinformatic analysis and the analysis on pre-cancerous animal model and clinical tissue samples.ResultsWe found that most of the members of RNA sensors, including RNA-sensing Toll-like receptors (TLR3, TLR7, and TLR8) and RIG-I-like receptors (MDA5 and RIG-I), were down-regulated in CRC, while the expression of DDX21 were up-regulated in human CRC. In addition, we also analyzed the correlation between gene expression and cancer stages. We found that the expression of RNA-sensing TLRs, RIG-I, and DDX21 in CRC were associated with cancer stages. Finally, in order to explore the possible mechanisms, the correlation between RNA sensors and the main downstream signaling molecules were analyzed. A positive correlation was observed in TLR7/8 and MyD88, RIG-I/MDA5 and LGP2, while a negative correlation was observed in RIG-I/MDA5 and MAVS.ConclusionsThis study reveals the potential values of RNA-sensing genes including TLRs, RIG-I and DDX21 as biomarkers of CRC formation, progression and therapy.

Ergosterol peroxide suppresses influenza A virus-induced pro-inflammatory response and apoptosis by blocking RIG-I signaling

Ergosterol peroxide has been shown to exhibit anti-tumor, antioxidant and anti-bacterial properties. However, the effects of ergosterol peroxide isolated from the herbal Baphicacanthus cusia root on influenza virus infection remain poorly understood. In the present study, ergosterol peroxide (compound 22) was obtained from the B. cusia root and subjected to investigation regarding its immunoregulatory effect on influenza A virus (IAV)-induced inflammation in A549 human alveolar epithelial cells. The structure of compound 22 isolated from B. cusia root. was elucidated by NMR analyses. Structure determination showed that the chemical structure of compound 22 closely resembles that of ergosterol peroxide. We observed that ergosterol peroxide treatment significantly suppressed IAV-induced upregulation of RIG-I expression. Additionally, ergosterol peroxide inhibited the activation of RIG-I downstream signaling pathways, including p38 MAP kinase and NF-κB, which ultimately resulted in the reduced production of an array of pro-inflammatory mediators and interferons (IFN-β and IFN-λ1). Interestingly, inhibitory effects of ergosterol peroxide on the expression of IFNs did not affect the expression of antiviral effectors or enhance viral replication. On the other hand, ergosterol peroxide effectively abolished the amplified production of pro-inflammatory mediators in cells pretreated with IFN-β (500 ng/ml) prior to IAV infection. Moreover, Annexin V and Hoechst 33258 staining revealed that increased apoptosis of IAV-infected cells was reversed by the presence of ergosterol peroxide. Our findings suggest that ergosterol peroxide from the B. cusia root suppressed IAV-associated inflammation and apoptosis via blocking RIG-I signaling, which may serve as a supplementary approach to the treatment of influenza.

Abstract 5024: Treatment with synthetic RIG-I agonist triphosphate RNA leads to local and systemic anti-tumor effects in a mouse melanoma tumor model

Abstract RIG-I is a highly important cytosolic pattern recognition receptor (PRR) involved in sensing RNA virus infection and inducing interferon (IFN) production. RIG-I’s natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. This study validates the use of intratumoral treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Firstly, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Secondly, cellular models of human melanoma confirm susceptibility of cancer cells to ppp-RNA treatment, revealing unexpected heterogeneity between cell lines in their selectivity for RNA features, including sequence, secondary structures and presence of triphosphate. Cellular RNA treatment responses (type I IFN, FasR, MHC-I, cytotoxicity) were demonstrated to be RIG-I dependent using RIG-I KO cells. Thirdly, we show that ppp-RNA treatment of a mouse melanoma tumor model, leads to significant local and systemic anti-tumor effects and survival benefits, associated with a type I IFN response, tumor cell apoptosis and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen specific CTLs following treatment with RIG-I agonist. Overall our study demonstrates that ppp-RNA or analogs thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy. However, potential challenges in the generation and formulation of potent RIG-I agonists remain to be solved. Citation Format: Mike W. Helms, Eric Parmantier, Kerstin Jahn-Hofmann, Felix Gnerlich, Lars König, Christiane Metz-Weidmann, Monika Braun, Gabriele Dietert, Kaj Grandien, Joachim Theilhaber, Hui Cao, Tim Wagenaar, Max Schnurr, Stefan Endres, Dmitri Wiederschain, Sabine Scheidler, Simon Rothenfusser, Bodo Brunner. Treatment with synthetic RIG-I agonist triphosphate RNA leads to local and systemic anti-tumor effects in a mouse melanoma tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5024.

Deficient pulmonary IFN‐β expression in COPD patients

COPD patients are prone to acute infectious exacerbations that impair their quality of life and hamper prognosis. The purpose of the present study was to investigate the in situ IFN-β response in the lungs of stable COPD and non-COPD patients. Lung samples from 70 subjects (9 control never smokers, 19 control smokers without COPD, 21 patients with moderate COPD and 21 patients with very severe COPD) were studied for the expression of IFN-β, its main transcription factor, IRF-7, and two products of its autocrine function, namely RIG-I and MDA-5, by immunohistochemical techniques and quantitative real-time PCR. IFN-β, IRF-7, RIG-I and MDA-5 were widely detected in most lung cell types. In epithelial tissues and alveolar macrophages, IFN-β and IRF-7 labeling scores were decreased up to 65% and 74%, respectively, for COPD patients, paralleling an analogous reduction (43% and 65%, respectively) in the amount of their lung mRNA. Moreover, this decreased production of IFN-β in COPD patients correlated with a similar decrease in the expression of RIG-I and MDA-5, two essential members of the innate immune system. Our study indicates that most lung cells from stable COPD patients show a constitutive decreased expression of IFN-β, IRF-7, RIG-I and MDA-5, suggesting that this deficiency is the main cause of their acute viral exacerbations.

Identification of LPS-Activated Endothelial Subpopulations With Distinct Inflammatory Phenotypes and Regulatory Signaling Mechanisms.

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Endothelial cells (EC) are actively involved in sepsis-associated (micro)vascular disturbances and subsequent organ dysfunction. Lipopolysaccharide (LPS), a Gram-negative bacterial product, can activate EC leading to the expression of pro-inflammatory molecules. This process is molecularly regulated by specific receptors and distinct, yet poorly understood intracellular signaling pathways. LPS-induced expression of endothelial adhesion molecules E-selectin and VCAM-1 in mice was previously shown to be organ- and microvascular-specific. Here we report that also within renal microvascular beds the endothelium expresses different extents of E-selectin and VCAM-1. This heterogeneity was recapitulated in vitro in LPS-activated human umbilical vein EC (HUVEC). Within 2 h after LPS exposure, four distinct HUVEC subpopulations were visible by flow cytometric analysis detecting E-selectin and VCAM-1 protein. These encompassed E-selectin−/VCAM-1− (–/–), E-selectin+/VCAM-1− (E-sel+), E-selectin+/VCAM-1+ (+/+), and E-selectin−/VCAM-1+ (VCAM-1+) subpopulations. The formation of subpopulations was a common response of endothelial cells to LPS challenge. Using fluorescence-activated cell sorting (FACS) we demonstrated that the +/+ subpopulation also expressed the highest levels of inflammatory cytokines and chemokines. The differences in responsiveness of EC subpopulations could not be explained by differential expression of LPS receptors TLR4 and RIG-I. Functional studies, however, demonstrated that the formation of the E-sel+ subpopulation was mainly TLR4-mediated, while the formation of the +/+ subpopulation was mediated by both TLR4 and RIG-I. Pharmacological blockade of NF-κB and p38 MAPK furthermore revealed a prominent role of their signaling cascades in E-sel+ and +/+ subpopulation formation. In contrast, the VCAM-1+ subpopulation was not controlled by any of these signaling pathways. Noteworthy is the existence of a “quiescent” subpopulation that was devoid of the two adhesion molecules and did not express cytokines or chemokines despite LPS exposure. Summarizing, our findings suggest that LPS activates different signaling mechanisms in EC that drive heterogeneous expression of EC inflammatory molecules. Further characterization of the signaling pathways involved will enhance our understanding of endothelial heterogeneous responses to sepsis related stimuli and enable the future design of effective therapeutic strategies to interfere in these processes to counteract sepsis-associated organ dysfunction.

Baby’s First Macrophage: How do placental macrophages (Hofbauer Cells, HCs) contribute to immune tolerance and infection response during pregnancy?

Abstract Placental immunity is dichotomous: tolerance of the semiallogenic fetus is balanced with limiting transmission of maternal pathogens. HCs are the major fetal immune cell at the placenta, but mechanisms responsible for maintaining immune homeostasis while preventing infection require elucidation. We determined the phenotype of human HCs throughout gestation and analyzed stimulation response. Activated HCs were present in early pregnancy and reduced in number by term while maintaining similar phenotypes. Tolerant HC numbers were highest in midgestation, after a relatively intolerant phenotype early in gestation. We saw a significant shift in macrophage polarization as gestation progressed. Transcription of ARG2 exceeded iNOS at all points, reaching 10-fold higher at term. Following treatment with IL4+IL13 or IFNγ+LPS midgestation HCs underwent significant phenotype change and activation. Basal expression of antiviral IFN stimulated genes (ISGs) was lowest at midgestation and was enhanced by IFN-α and IFN-λ1 with a 10-fold stronger response to IFN-α. RIG-I agonism induced HC activation, 10-fold iNOS upregulation and enhanced transcription of IFNs, MDA5, RIG-I, and ISGs. Response to stimulation by IFNγ+LPS, IL4+IL13, IL1β+HAGG was limited to loss of tolerance at term. IL-10 treatment increased numbers of CD163+ HCs, and IFNγ+LPS caused loss of discernible polarization patterns. Basal expression of RIG-I, MDA5 and ISGs was highest in term HCs, but IFN-α did not activate them. RIG-I agonism reduced markers of tolerance. HCs are variable macrophages, with phenotype and immune capacity strongly dependent on gestational age. Understanding placental immunobiology is fundamental to addressing key pregnancy morbidities.

Toll-like receptors, long non-coding RNA NEAT1, and RIG-I expression are associated with HBeAg-positive chronic hepatitis B patients in the active phase.

BackgroundInnate immunity plays a crucial role in host‐virus interactions and greatly influences viral replication including HBV infection. However, few studies have investigated the possible antiviral immune roles played by TLRs, RIG‐I, and long no‐coding RNA NEAT1 in chronic HBV infection (CHB) patients in clinical samples and their relationships among immune responses. In this study, we sought to investigate the mRNA expression levels of TLR1‐10, RIG‐I, and NEAT1 expression in HBeAg‐positive CHB treatment‐naïve patients with the active phase.MethodsThe expression levels of TLR1‐10, RIG‐I, and NEAT1 of CHB patients with the active phase and healthy controls were measured by qPCR. Serum HBV DNA and routine liver biochemistry including ALT, etc were also measured to evaluate the impaired physiological function of the liver affected by CHB.ResultsThe expression levels of TLR1 and TLR6 in CHB with active phase were remarkably lower than that in healthy controls. The levels of TLR3 in CHB patients with active phase were remarkably higher than that in healthy controls. The total NEAT1 expression was abnormally decreased in CHB patients as compared with healthy controls. The levels of RIG‐I were significantly decreased in CHB patients in the active phase when compared to healthy controls. The expression of TLR6 and RIG‐I was closely correlated with NEAT1 expression. TLR6 level was positively correlated with RIG‐I level.ConclusionChronic HBV infection can alter the innate immune response by downregulating functional expression of TLR1, TLR6, NEAT1.

Transmembrane Protein 2 Inhibits Zika Virus Replication Through Activation of the Janus Kinase/Signal Transducers and Activators of Transcription Signaling Pathway

Aim: TMEM2 has been demonstrated to suppress HBV infection by activating the Jak/STAT pathway. In this study, we sought to explore the mechanism by which TMEM2 effects on Zika virus (ZIKV) replication. Materials & methods: TMEM2 was overexpressed. Selected gene mRNA, p-STAT1 levels and interferon stimulated response element activity were examined by qRT-PCR, Western blot and luciferase assay respectively. Results: Overexpression of TMEM2 significantly inhibited ZIKV replication, upregulated MDA5 and RIG-I expression, increased IFN-β promoter activity and IFN-β expression. Overexpression of TMEM2 enhanced the Jak/STAT signaling including increased p-STAT1 level, ISRE activity as well as the expression of several antiviral interferon-stimulated genes. Conclusion: TMEM2 inhibited ZIKV replication through increased IFN-β production and enhanced activation of the Jak/STAT signaling pathway.

Clinical significance of retinoic acid-induced protein I expression in human gastric cancer

Objective To investigate the clinical significance and regulation of retinoic acid-induced protein I (RIG-I) in human gastric cancer. Methods Immunohistochemistry was used to study RIG-I expression in gastric cancer and normal tissues. We used Wilcoxon test to analyze the difference of RIG-I expression levels in cancer and normal tissues. Chi-square and Log-rank tests were used to analyze the RIG-I expression in cancer tissues in relation to patients’ clinical parameters and prognoses respectively. The gastric cancer cell lines SGC-7901 and AGS were used to construct the stable knockdown expression of RIG-I using RNA interference (RNAi) method. The cell counting kit-8 (CCK-8), wound healing, and Transwell assays were used to examine the cellular function after RIG-I knockdown of these cells. Results RIG-I expression level in human gastric cancer tissues was significantly lower than that in adjacent normal tissues (U=1 490, P=0.001). The ratio of higher RIG-I expression in cancer patients with pathological stage (Ⅲ-Ⅳ) was significantly lower than that in normal tissues (χ2=4.668, P=0.031). The overall survival of the patients with higher RIG-I expression was significantly longer than that with lower RIG-I expression (P=0.023). The COX model showed that the age (P=0.047), TNM stage (P=0.039) and the RIG-I expression (P=0.007) could be used as independent prognostic predictors for cancer patients. CCK-8 assay showed that at the time points of 48 h and 72 h after culture, the A450 value in LV-RIG-I-short hairpin RNA (shRNA) group was significantly higher than that in LV-NC group (for SGC7901: P=0.000 and P=0.000 respectively; for AGS: P=0.002 and P=0.016 respectively). The wound healing assay showed that the cell migration abilities in LV-RIG-I-shRNA group was significantly stronger than LV-NC group (for SGC7901: P=0.001; for AGS: P=0.004). The Transwell assay showed that the cell invasion abilities in LV-RIG-I-shRNA group was significantly stronger than that in LV-NC group (for SGC7901: P=0.032; for AGS: P=0.016). Conclusion RIG-I plays important roles in gastric cancer progression, its knockdown could promote proliferation, migration and invasion of cancer cells, and thus could be used as potential therapeutic target for gastric cancer. Key words: Retinoic acid-induced protein I; Gastric cancer; RNA interference; Immunohistochemistry

Influenza Virus NS1 Protein-RNA Interactome Reveals Intron Targeting.

The NS1 protein of influenza A virus is a multifunctional virulence factor that inhibits cellular processes to facilitate viral gene expression. While NS1 is known to interact with RNA and proteins to execute these functions, the cellular RNAs that physically interact with NS1 have not been systematically identified. Here we reveal a NS1 protein-RNA interactome and show that NS1 primarily binds intronic sequences. Among this subset of pre-mRNAs is the RIG-I pre-mRNA, which encodes the main cytoplasmic antiviral sensor of influenza virus infection. This suggested that NS1 interferes with the antiviral response at a posttranscriptional level by virtue of its RNA binding properties. Indeed, we show that NS1 is necessary in the context of viral infection and sufficient upon transfection to decrease the rate of RIG-I intron removal. This NS1 function requires a functional RNA binding domain and is independent of the NS1 interaction with the cleavage and polyadenylation specificity factor CPSF30. NS1 has been previously shown to abrogate RIG-I-mediated antiviral immunity by inhibiting its protein function. Our data further suggest that NS1 also posttranscriptionally alters RIG-I pre-mRNA processing by binding to the RIG-I pre-mRNA.IMPORTANCE A key virulence factor of influenza A virus is the NS1 protein, which inhibits various cellular processes to facilitate viral gene expression. The NS1 protein is localized in the nucleus and in the cytoplasm during infection. In the nucleus, NS1 has functions related to inhibition of gene expression that involve protein-protein and protein-RNA interactions. While several studies have elucidated the protein interactome of NS1, we still lack a clear and systematic understanding of the NS1-RNA interactome. Here we reveal a nuclear NS1-RNA interactome and show that NS1 primarily binds intronic sequences within a subset of pre-mRNAs, including the RIG-I pre-mRNA that encodes the main cytoplasmic antiviral sensor of influenza virus infection. Our data here further suggest that NS1 is necessary and sufficient to impair intron processing of the RIG-I pre-mRNA. These findings support a posttranscriptional role for NS1 in the inhibition of RIG-I expression.

RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication

BackgroundRetinoic acid-inducible gene I (RIG-I) is a key cytosolic receptor of the innate immune system. Seneca valley virus (SVV) is a newly emerging RNA virus that infects pigs causing significant economic losses in pig industry. RIG-I plays different roles during different viruses infections. The role of RIG-I in SVV-infected cells remains unknown. Understanding of the role of RIG-I during SVV infection will help to clarify the infection process of SVV in the infected cells.MethodsIn this study, we generated a RIG-I knockout (KO) porcine kidney PK-15 cell line using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing tool. The RIG-I gene sequence of RIG-I KO cells were determined by Sanger sequencing method, and the expression of RIG-I protein in the RIG-I KO cells were detected by Western bloting. The activation status of type I interferon pathway in Sendai virus (SeV)- or SVV-infected RIG-I KO cells was investigated by measuring the mRNA expression levels of interferon (IFN)-β and IFN-stimulated genes (ISGs). The replicative state of SVV in the RIG-I KO cells was evaluated by qPCR, Western bloting, TCID50 assay and indirect immunofluorescence assay.ResultsGene editing of RIG-I in PK-15 cells successfully resulted in the destruction of RIG-I expression. RIG-I KO PK-15 cells had a lower expression of IFN-β and ISGs compared with wildtype (WT) PK-15 cells when stimulated by the model RNA virus SeV. The amounts of viral RNA and viral protein as well as viral yields in SVV-infected RIG-I WT and KO cells were determined and compared, which showed that knockout of RIG-I significantly increased SVV replication and propagation. Meanwhile, the expression of IFN-β and ISGs were considerably decreased in RIG-I KO cells compared with that in RIG-I WT cells during SVV infection.ConclusionAltogether, this study indicated that RIG-I showed an antiviral role against SVV and was essential for activation of type I IFN signaling during SVV infection. In addition, this study suggested that the CRISPR/Cas9 system can be used as an effective tool to modify cell lines to increase viral yields during SVV vaccine development.

ABIN-1 heterozygosity sensitizes to innate immune response in both RIPK1-dependent and RIPK1-independent manner.

ABIN-1 (encoded by the gene Tnip1) is a ubiquitin-binding protein that can interact with ubiquitin-editing enzyme A20 (encoded by the gene TNFAIP3) to restrain the activation of necroptosis and NF-κB activation. Genetic variants in the genes Tnip1 and TNFAIP3 are both strongly associated with susceptibility to autoimmune chronic inflammatory diseases such as psoriasis vulgaris and systemic lupus erythematosus (SLE) in humans. Here we investigated the mechanism by which ABIN-1 regulated innate immune responses. We show that ABIN-1 heterozygosity sensitizes cells to antiviral response by mediating NF-κB-dependent and RIPK1-independent expression of pattern recognition molecules, including TLR3, RIG-I, and MDA5, in MEFs. Furthermore, we demonstrate that increased interaction of ABIN-1 and A20 with prolonged poly(I:C) stimulation of WT cells leads to A20-dependent reduction of ABIN-1 protein. Finally, we show that ABIN-1 heterozygosity sensitizes innate immune response of Abin-1+/- mice in vivo by promoting the production of proinflammatory cytokines, which can be blocked upon inhibition of RIPK1 kinase. Inhibition of RIPK1 kinase activity in vivo partially reduces the expression of MDA5, RIG-I, and caspase-11 in Abin-1+/- mice but not in WT mice. Thus, we conclude that ABIN-1 is a suppressor of innate immune response and the interaction of ABIN-1 with A20 controls innate immunity response through the NF-κB pathway and in both RIPK1 kinase activity-independent and dependent manner.

Decreased RIG-I expression is associated with poor prognosis and promotes cell invasion in human gastric cancer

BackgroundRetinoic acid-induced protein I (RIG-I), known as a cytoplastic pattern recognition receptor, can recognize exogenous viral RNAs, and then initiate immune response. Recently, numerous studies also showed that RIG-I play an important role in oncogenesis and cancer progression as well. As of now, the expression pattern and the role of RIG-I in gastric cancer still remain largely unexplored. In this study, we investigated the clinical associations of RIG-I expression in human gastric cancer tissues and further explore its important contribution in the regulation of malignant phenotype of gastric cancer cells.MethodsImmunohistochemistry was performed to study the correlation between patients’ clinical parameters and RIG-I expression in gastric cancer tissues. Knockdown of RIG-I was achieved by RNAi technology to examine the contribution of RIG-I in the regulation of biological functions in the cell lines of human gastric cancer. The Affymetrix GeneChip was performed to figure out the differential gene expression profile between RIG-I wild type and RIG-I knockdown cell lines of gastric cancer.ResultsImmunohistochemistry result demonstrated that the expression of RIG-I in gastric cancer tissues significantly correlated with pathological stage and patients’ prognoses. Furthermore, decreased RIG-I expression in human gastric cancer cell lines could significantly increase the cell migration, cell viability, and the ratio of cells in G2/M phase. Our microarray analysis also revealed that the differentially expressed gene profiles were enriched in related signal pathways or biological processes in KEGG or GO analysis respectively.ConclusionsOur present findings showed that the decreased RIG-I expression significantly correlated with patients’ prognoses, and such down-regulation could promote the cell invasion in this malignancy.

Enhanced expression of IFI16 and RIG-I in human third-trimester placentas following HSV-1 infection.

The innate immune response in the placenta depends on the ability of maternal immune cells and fetal trophoblast cells to detect and eliminate invading pathogens through germline-encoded pattern recognition receptors (PRRs). In the present study, we analysed the transcripts and protein expression of interferon (IFN)-inducible protein (IFI)16, melanoma differentiation-associated protein 5 (MDA5), RIG-I-like receptor (RIG-I) and Toll-like receptor (TLR)-3 in third-trimester human placentas and investigated cytokine profiles generated during herpes simplex type 1 (HSV-1) infection. Decidual and chorionic villous biopsies (38-42 weeks of gestation) were obtained from healthy women immediately after a caesarean section. The expression of the DDX58 (RIG-I), IFIH1 (MDA5), IFI16 and TLR3 transcripts was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Extracellular cytokine and PRRs levels were then quantified by enzyme-linked immunosorbent assays (ELISAs). All examined PRRs genes, including DDX58, IFIH1, IFI16 and TLR3, were expressed constitutively at the mRNA and protein levels in the placental biopsies. The concentration of the IFI16 protein was increased in HSV-1-infected decidual and chorionic villous explants compared to those of mock-infected tissues (P=0·029). Higher protein expression levels of RIG-I in both the maternal and fetal parts of the placenta were found (P=0·009 and P=0·004, respectively). In addition, increased production of IFN-β by HSV-1-infected tissues was noticed (P=0·004 for decidua, P=0·032 for chorionic villi). No significant differences in the IFN-α, interleukin (IL)-6 and IL-8 levels were found. These results showed that HSV-1 infection can enhance the expression of IFI16 and RIG-I proteins in the human term placenta.

Abstract 3801: Functional relevance of A-to-I RNA editing on the immune response in breast cancer

Abstract INTRODUCTION: RNA editing is a post-transcriptional modification that changes double stranded RNA sequences (dsRNA). The most common way of editing in humans is the Adenosine-to-Inosine type, which is catalyzed by members of the ADAR enzymes family. A-to-I RNA editing has been shown to be essential for normal tissue development but also plays a crucial role in many cancers. We have notably shown that in breast cancer the editing frequency is increased in tumors as compared to normal tissue and correlates with ADAR expression. We also showed that type I IFN response and ADAR DNA copy number together explain 53% of ADAR expression in breast cancers. Moreover, the implication of ADAR-mediated RNA editing in preventing the sensing of endogenous dsRNA leading to inhibition of the immune response has been recently reported. ADAR-mediated RNA editing may play a crucial role in tumor progression by modulating immune response through the dsRNA sensing pathway. Here, we aim to assess the influence of ADAR-mediated RNA editing in the sensing of endogenous dsRNA and the immune response in breast cancer. MATERIAL & METHODS: ADAR expression was modulated in MDAMB231 breast cancer cell line using shRNA lentiviral transduction and lentiviral plasmid transduction for ADAR inhibition and ADAR wild-type and editing-deficient mutant overexpression respectively. ADAR expression was assessed by RTqPCR/Western blotting. dsRNA level was evaluated by immunofluorescence using the dsRNA-specific J2 antibody (Scicons). RESULTS: Poly I:C transfection, mimicking dsRNAs, as well as IFN Type I exposure induced the immune response in BC cell lines, in particular it increases the expression of RIG-I, MDA5 and IRF7 involved in the dsRNA sensing pathway. DNMTi treatment, known as an inducer of dsRNA level, also activated cytosolic dsRNA response in these cells in a dose dependent manner. In order to assess the role of ADAR-mediated RNA editing in the activation of the dsRNA sensing pathway, we exposed MDAMB231 cell lines in which ADAR expression was modulated to DNMTi. A wide range of doses of DNMTi, from 0.3 to 50μM, and time of exposure, from 3h to 72h, were tested. The cytosolic dsRNA pathway was induced in a dose dependent manner after DNMTi exposure. However, no effect of ADAR modulation on the activation of the pathway was observed. No difference in dsRNA levels was observed after activation of the immune pathway by DNMTi or IFN exposure, nor after ADAR modulation. CONCLUSION & PERSPECTIVES: dsRNA pathway activation by DNMTi, poly I:C transfection or IFN Type I treatment is not dependent of ADAR in MDAMB231 cell lines. Moreover, in contrast to data in colorectal cell lines, our data suggest that DNMTi exposure does not increase cytosolic dsRNA level. Citation Format: Floriane Dupont, Florian Clatot, David Venet, Véronique Kruys, Debora Fumagalli, Vincent Detours, Françoise Rothé, Christos Sotiriou. Functional relevance of A-to-I RNA editing on the immune response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3801.

Latent Membrane Protein 1 of Epstein-Barr Virus Promotes RIG-I Degradation Mediated by Proteasome Pathway.

RIG-I signaling is critical to host innate immune response against RNA virus infection, and also can be activated against many kinds of cancer. Oncogene LMP1 of Epstein–Barr virus (EBV) contributes to various tumors progress. In this study, we have provided strong evidence that LMP1 inhibits Sendai virus mediated type I interferon production and downregulates RIG-I signaling pathway by promotion RIG-I degradation dependent on proteasome. Nineteen kinds of E3 ligase are identified by IP-MS as LMP1-interactors, they are candidate E3s, which are possibly recruited by LMP1 to mediate RIG-I degradation. CHIP is among these E3s, which has been reported to lead RIG-I degradation. Notably, we find C666-1, an EBV-positive nasopharyngeal carcinoma cell line, expresses low level of RIG-I, even treated with IFN-α, RIG-I expression could not be induced. This evidence indicates that EBV employs a unique strategy to evade RIG-I mediated immune responses.

SAT-376 - EFTUD2 restricts hepatitis B virus infection by regulating RIG-I expression

SAT-376 - EFTUD2 restricts hepatitis B virus infection by regulating RIG-I expression