Including Interferon Regulatory Factor Research Articles

Abstract B39: Novel dinucleotides that activate STING signaling for immuno-oncology

Abstract Background: Immunotherapy has recently emerged as a transformative approach for the treatment of cancer; nevertheless, many patients remain unresponsive to treatment. It is being recognized that induction of type I interferons (IFN) and interferon-stimulated genes (ISGs) in tumor cells and within the tumor microenvironment (TME) is essential for modulating the host-immune response and inducing apoptosis of tumor cells. Furthermore, the antigen-presenting cells within TME can cause induction of adaptive immune response, through priming of CD8+ T cells and tumor killing. Importantly, the DNA released from damaged cells and cancer cells can be sensed by cyclic GMP-AMP synthase (cGAS) leading to the synthesis of cyclic-GMP-AMP (2',3'-cGAMP), a second messenger that activates Stimulator of Interferon Genes (STING) pathway resulting in the production of type I IFN and ISGs. The cumulative effects of activation of innate and adaptive immune response can result in potent anti-cancer effects. Therefore, therapeutic agents that activate the cGAS-STING signaling pathway in tumor cells and TME are urgently needed. Herein, we describe the discovery of novel potent, first-in-class small molecules for application in immuno-oncology. Methods: Using structure-guided drug design, in conjunction with published crystal structures of cyclic dinucleotides bound to STING, a focused library of dinucleotide compounds was synthesized using phosphoramidite chemistry and evaluated for: (a) Induction of IFN signaling: The compounds were screened for the induction of Interferon regulatory factor (IRF), ISG54, and NF-κB using reporter assays. We used HEK293 cell line (SZ14) stably expressing ISG54 (ISRE)-promoter-driven firefly luciferase reporter gene for screening and the active compounds were further characterized in THP1 cells and human primary PBMCs. The IRF, ISG54, and NF-κB induction was calculated from % fold-change in luminescence compared to DMSO-treated cells and EC50s of the compounds were ascertained to identify active compounds, (b) Expression of IFN-β and IRF7 in THP1 cells: THP1 cells were treated with active compounds or controls for 22hrs. RNA was extracted and the expression of IFN-β, IRF7, was ascertained using semi-quantitative RT-PCR, (c) Induction of pathogen recognition receptors (PRRs) including RIG-I, MDA5, LGP2, and OAS-1 and ISG54: THP1 cells and PBMCs were treated with active compounds, 2',3'-cGAMP (control), or DMSO and the gene expression of different PRRs, ISGs, was determined by quantitative RT-PCR using ΔΔct method, (d) Induction of cGAS-STING signaling using reporter assays: HEK293 cells stably expressing ISG54 were transfected with plasmids encoding human cGAS (wild-type, or K384A, K400A, or K411A mutants) and treated with active compounds, poly (dA:dT) (positive control), or DMSO for 21 hrs. ISG54 induction was calculated as fold-change in luminescence compared to DMSO-treated controls. (e) Cytotoxicity assays: THP1 cells were treated with active compounds or DMSO control with Lipofectamine and cytotoxicity assessed using the CellTiter-Glo® Luminescent assays. Cytotoxicity was calculated from %-fold change in luminescence compared to DMSO-treated sample. Results: Through in vitro assays in conjunction with Structure Activity Relationship studies, we have identified potent compounds that activate cGAS-STING signaling pathway for induction of IRF, IFN, and NF-κB. These compounds also cause induction of expression of PRRs, including RIG-I, MDA5, LGP2, as well as, ISG54 and OAS-1. Conclusion: We have discovered potent, first-in-class agents that cause induction of IFN, NF-κB, ISGs, and PRRs. Further optimization and preclinical evaluation of the compounds for application in immuno-oncology is underway. Citation Format: Shenghua Zhou, Sreerupa Challa, Seetharamaiyer Padmanabhan, Anjaneyulu Sheri, Samantha Delaney, Geeta Meher, Dillon Cleary, Rayomand Gimi, Santosh Khedkar, Radhakrishnan Iyer. Novel dinucleotides that activate STING signaling for immuno-oncology. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B39.

Abstract B40: Nucleotide analogs as novel STING agonists for immuno-oncology

Abstract Immunotherapy has recently emerged as a transformative approach for the treatment of cancer; nevertheless, many patients remain unresponsive to treatment. Recent evidence suggests that the activation of Stimulator of Interferon Genes (STING) pathway in tumor cells and/or antigen presenting cells (APCs) within the tumor microenvironment (TME) can induce type I Interferon production leading to apoptosis of tumor cells, as well as, induction of adaptive immune response (through priming of CD8+ T cells to tumor-associated antigens) thereby providing a powerful anti-cancer strategy. Therefore, therapeutic agents that activate STING signaling pathway in tumor cells and APCs in the TME are urgently needed. Herein, we describe the discovery of highly potent and selective first-in-class STING agonists for application in immuno-oncology. Methods: Using structure-guided drug design, in conjunction with published crystal structures of different cyclic dinucleotides bound to STING, a focused library of nucleotide compounds was prepared using standard phosphoramidite chemistry. The compounds were screened for induction of Interferon regulatory factor (IRF), Interferon-stimulated gene 54 (ISG54), and NF-KB using reporter assays. We used HEK293 cell line stably expressing ISG54 (ISRE)-promoter-driven firefly luciferase reporter gene for initial hit discovery and the actives were further characterized in PBMCs and THP1 cells. The IRF and NF-kB induction was calculated from % fold-change in luminescence compared to DMSO-treated cells and EC50 of the compounds were ascertained using Xlfit. Lead STING agonists were further evaluated for: (a) Binding affinity: Binding assays were conducted by Differential Scanning Fluorimetry (DSF) and Tm was calculated using Thermal Shift software, (b) Induction of pathogen recognition receptors (PRRs), ISGs and Programmed Death Ligands 1 & 2 (PDL1, PDL2) genes: THP1 cells and PBMCs were treated with various concentrations of lead compounds or 2,'3'-cGAMP or DMSO and the gene expression of different PRRs, ISGs, PDL1, and PDL2 was determined by quantitative RT-PCR using ΔΔct method, (c) Apoptosis-inducing activity: PBMCs and THP1 cells were treated with various concentrations of lead compounds, 2',3'-cGAMP, or DMSO control and the apoptotic activity was evaluated using Caspase-Glo® 3/7 Assay (Promega), and (d) In vitro anti-tumor activity: STING-dependent anti-tumor activity of lead compounds in various tumor cell lines was assessed by either high-content imaging or through Cell titer Glo® Cytotoxicity Assay (Promega). Cell survival was calculated based upon % reduction of live cells compared to DMSO control. CC50 of the compounds were generated by curve fit in Xlfit. Results: Through in vitro assays in conjunction with Structure Activity Relationship (SAR) studies, we have identified several highly potent and selective first-in-class STING agonists. A promising lead nucleotide compound SB 11285 caused STING-dependent induction of: (a) IRF with an EC50 of 2 nM that is 1000-fold more potent than the natural STING agonist 2',3'-cGAMP, (b) NF-kB with an EC50 of 200 nM that is >200-fold more potent than 2',3'-cGAMP, (c) selective apoptosis of human monocyte leukemic cell lines (CC50, 500 nM) as compared to normal PBMCs through induction of IFN, and NF-kB signaling, and (d) expression of various PRRs and ISGs including RIG-I, MDA-5, LGP2, ISG54 and OAS-1, as well as, PDL1 and PDL2. Finally, SB 11285 showed potent in vitro anti-tumor activity in multiple tumor cell lines. Conclusion: We have discovered highly potent first-in-class STING agonists that show excellent selectivity in induction of IFN, NF-KB, ISGs, and PRRs, and apoptosis of tumor-derived cell lines. The lead STING agonist SB 11285 has potent immune-modulating, as well as, anti-tumor activities and is being advanced for additional preclinical studies for application in immuno-oncology. Citation Format: Sreerupa Challa, Shenghua Zhou, Anjaneyulu Sheri, Seetharamaiyer Padmanabhan, Samantha Delaney, Geeta Meher, Dillon Cleary, Vishal Nair, Rayomand Gimi, Santosh Khedkar, Radhakrishnan Iyer. Nucleotide analogs as novel STING agonists for immuno-oncology. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B40.

Endothelial Interferon Regulatory Factor 1 Regulates Lipopolysaccharide-Induced VCAM-1 Expression Independent of NFκB

Sepsis is a severe systemic inflammatory response to infection. Endothelial activation and dysfunction play a critical role in the pathophysiology of sepsis and represent an important therapeutic target to reduce sepsis mortality. Interferon regulatory factor 1 (IRF-1) was recently identified as a downstream target of TNF-α-mediated signal transduction in endothelial cells. The aim of this study was to explore the importance of IRF-1 as a regulator of lipopolysaccharide (LPS)-induced endothelial proinflammatory activation. We found that renal IRF-1 was upregulated by LPS in vivo as well as in LPS-stimulated endothelial cells in vitro. Furthermore, we identified intracellular retinoic acid inducible gene-I (RIG-I) as a regulator of LPS-mediated IRF-1 induction. IRF-1 depletion specifically resulted in diminished induction of VCAM-1 in response to LPS, but not of E-selectin or ICAM-1, which was independent of NFκB signaling. When both IRF-1 and the RIG-I adapter protein mitochondrial antiviral signaling (MAVS) were absent, VCAM-1 induction was not additionally inhibited, suggesting that MAVS and IRF-1 reside in the same signaling pathway. Surprisingly, E-selectin and IL-6 induction were no longer inhibited by MAVS knockdown when IRF-1 was also absent, revealing a redundant endothelial activation pathway. In summary, we report an IRF-1-mediated proinflammatory signaling pathway that specifically regulates LPS-mediated VCAM-1 expression, independent of NFκB.

TRIM65-catalized ubiquitination is essential for MDA5-mediated antiviral innate immunity.

MDA5 plays a critical role in antiviral innate immunity by functioning as a cytoplasmic double-stranded RNA sensor that can activate type I interferon signaling pathways, but the mechanism for the activation of MDA5 is poorly understood. Here, we show that TRIM65 specifically interacts with MDA5 and promotes K63-linked ubiquitination of MDA5 at lysine 743, which is critical for MDA5 oligomerization and activation. Trim65 deficiency abolishes MDA5 agonist or encephalomyocarditis virus (EMCV)-induced interferon regulatory factor 3 (IRF3) activation and type I interferon production but has no effect on retinoic acid-inducible I (RIG-I), Toll-like receptor 3 (TLR3), or cyclic GMP-AMP synthase signaling pathways. Importantly, Trim65-/- mice are more susceptible to EMCV infection than controls and cannot produce type I interferon in vivo. Collectively, our results identify TRIM65 as an essential component for the MDA5 signaling pathway and provide physiological evidence showing that ubiquitination is important for MDA5 oligomerization and activation.

Fish TRIM32 functions as a critical antiviral molecule against iridovirus and nodavirus

Tripartite motif-containing 32 (TRIM32) has been demonstrated to pay vital roles in cancer, genetic disorders and antiviral immunity. However, the molecular functions of fish TRIM32 still remained largely unknown. Here, a novel TRIM32 gene from orange spotted grouper (EcTRIM32) was cloned and characterized. EcTRIM32 encoded a 685-aa protein which showed 93%, and 60% identity to large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Amino acid alignment showed that EcTRIM32 contained a conserved RING-finger domain, a BBOX domain and NHL domain. In healthy grouper, the transcript of EcTRIM32 was predominantly detected in brain, liver, intestine, spleen and skin. After injection with Singapore grouper iridovirus (SGIV) and polyI:C, the relative expression of EcTRIM32 in grouper spleen was differently regulated, suggested that EcTRIM32 was involved in antiviral immune response. In transfected grouper spleen (GS) cells, EcTRIM32 displayed bright fluorescence aggregates or spots in the cytoplasm. Notably, the deletion RING domain altered its precise localization and distributed throughout the cytoplasm in GS cells. In EcTRIM32 overexpressing cells, the replication of SGIV or red-spotted grouper nervous necrosis virus (RGNNV) was significantly inhibited compared to the vector control cells. Moreover, the overexpression of EcTRIM32 positively regulated the interferon immune response, evidenced by the significant increase of the expression level of interferon related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon-stimulated gene 15 (ISG15), interferon-induced 35-kDa protein (IFP35), MXI, TIR-domain-containing adaptor-inducing interferon-β (TRIF) and melanoma differentiation-associated protein 5 (MDA5). Further studies showed that overexpression of EcTRIM32 significantly enhanced the MDA5-mediated interferon immune response, but decreased stimulator of interferon genes (STING)-mediated interferon immune response. Meanwhile, the expression levels of pro-inflammation cytokines, including TNFα, IL-6 and IL-8 were up-regulated by the ectopic expression of EcTRIM32. We speculated that the regulation of IRF7, and pro-inflammation cytokines by EcTRIM32 overexpression might contribute critical roles in SGIV infection. In addition, the deletion of RING domain not only significantly weakened the antiviral roles of EcTRIM32, but also obviously affected the regulatory effects of EcTRIM32 on interferon immune and inflammation response. Together, our results firstly demonstrated that fish TRIM32 acted as an antiviral factor against both DNA and RNA virus infection.

Fish TRIM16L exerts negative regulation on antiviral immune response against grouper iridoviruses

Tripartite motif 16 (TRIM16), has been demonstrated to act as a tumor suppressor through affecting cell proliferation and migration or tumorigenicity in carcigenesis. However, the roles of TRIM16 in immune response were unknown up to now. Here, we cloned a TRIM16-like gene (TRIM16L) from orange spotted grouper (EcTRIM16L) and investigated its roles in response to virus infection. EcTRIM16L encoded a 478 amino acid peptide which showed 72% and 29% identity to large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Sequence alignments indicated that EcTRIM16L shared the different gene structures with human TRIM16, evidenced by the presence of RING domain, but absence of the B-box domain. In transfected grouper cells, the green fluorescence mainly distributed in cytoplasm, and the deletion of SPRY domain affected the accurate localization of EcTRIM16L. In response to different stimuli, including infection with Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis (RGNNV), and transfection with b-DNA or poly I:C, the transcript of EcTRIM16L were differently regulated in grouper spleen cells. After incubation with SGIV, the ectopic expression of EcTRIM16L significantly enhanced the viral replication, demonstrated by the increase of cytopathic effect (CPE) severity and viral gene transcriptions. Simultaneously, we also found that overexpression of EcTIRM16L in vitro significantly weakened the expression of interferon related molecules, including interferon regulatory factor 3 (IRF3), IRF7, and melanoma differentiation-associated protein 5 (MDA5). Moreover, the ectopic expression of EcTRIM16L significantly decreased both MDA5-and mediator of IRF3 activation (MITA)-induced interferon immune responses. Further studies showed that the RING domain played more important roles in the molecular action of EcTIRM16L during grouper virus infection. Our data, for the first time, demonstrated that fish TRIM16L exerted negative regulation on the interferon immune response against DNA virus infection.

Yersinia YopJ negatively regulates IRF3-mediated antibacterial response through disruption of STING-mediated cytosolic DNA signaling

The Yersinia outer protein J (YopJ) plays a pivotal role in evading the host immune response and establishes a persistent infection in host cells after bacterial infection. YopJ is a cysteine protease and can act as a deubiquitinating enzyme that deubiquitinates several targets in multiple signaling pathways. Stimulator of interferon genes (STING) is a critical adapter for the induction of interferon regulatory factor 3 (IRF3) phosphorylation and subsequent production of the cytokines in response to nucleic acids in the cytoplasm. Our studies demonstrate that YopJ targets STING to inhibit IRF3 signaling. Specially, YopJ interacts with STING to block its ER-to-Golgi traffic and remove its K63-linked ubiquitination chains. Deubiquited STING perturbs the formation of STING-TBK1 complex and the activation of IRF3. The 172th cysteine of YopJ mediated STING deubiquitination and IRF3 signaling inhibition. Consequently, mice infected with WT and ΔYopJ/YopJ bacteria induced lower levels of IRF3 and IFN-β, decreased inflammation and reduced staining of STING as compared to ΔYopJ and ΔYopJ/YopJ C172A strains infection. The data herein reveal a previously unrecognized mechanism by which YopJ modulates innate immune signaling.

Mumps Virus Induces Protein-Kinase-R-Dependent Stress Granules, Partly Suppressing Type III Interferon Production.

Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-β through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection.

Negative regulation of the innate antiviral immune response by TRIM62 from orange spotted grouper

Increased reports uncovered that mammalian tripartite motif-containing 62 (TRIM62) exerts crucial roles in cancer and innate immune response. However, the roles of fish TRIM62 in antiviral immune response remained uncertain. In this study, a TRIM62 gene was cloned from orange spotted grouper (EcTRIM62) and its roles in grouper RNA virus infection was elucidated in vitro. EcTRIM62 shared 99% and 83% identity to bicolor damselfish (Stegastes partitus) and human (Homo sapiens), respectively. Sequence alignment indicated that EcTRIM62 contained three domains, including a RING-finger domain, a B-box domain and a SPRY domain. In healthy grouper, the transcript of EcTRIM62 was predominantly detected in brain and liver, followed by heart, skin, spleen, fin, gill, intestine, and stomach. Subcellular localization analysis indicated that bright fluorescence spots were observed in the cytoplasm of EcTRIM62-transfected grouper spleen (GS) cells. During red-spotted grouper nervous necrosis (RGNNV) infection, overexpression of EcTRIM62 significantly enhanced the severity of CPE and increased viral gene transcriptions. Furthermore, the ectopic expression of EcTRIM62 significantly decreased the transcription level of interferon signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon-stimulated gene 15 (ISG15), melanoma differentiation-associated protein 5 (MDA5), myxovirus resistance gene MXI, and MXII, suggesting that the negative regulation of interferon immune response by EcTRIM62 might directly contributed to its enhancing effect on RGNNV replication. Furthermore, our results also demonstrated that overexpression of EcTRIM62 was able to differently regulate the expression levels of pro-inflammation cytokines. In addition, we found the ectopic expression of EcTIRM62 negatively regulated MDA5-, but not mediator of IRF3 activation (MITA)-induced interferon immune response. Further studies showed that the deletion of RING domain and SPRY domain significantly affected the action of EcTRIM62, including the enhancing effect on virus replication and regulation of interferon immune response. Thus, our studies firstly demonstrated that EcTRIM62 negatively regulated the innate antiviral immune response against fish RNA viruses.

Potentiation and tolerance of toll-like receptor priming in human endothelial cells

Repeated challenge of lipopolysaccharide (LPS) alters the response to subsequent LPS exposures via modulation of toll-like receptor 4 (TLR4). Whether activation of other TLRs can modulate TLR4 responses, and vice versa, remains unclear. Specifically with regards to endothelial cells, a key component of innate immunity, the impact of TLR cross-modulation is unknown. We postulated that TLR2 priming (via Pam3Csk4) would inhibit TLR4-mediated responses while TLR3 priming (via Poly I:C) would enhance subsequent TLR4-inflammatory signaling. We studied human umbilical vein endothelial cells (HUVECs) and neonatal human dermal microvascular endothelial cells (HMVECs). Cells were primed with a combination of Poly I:C (10μg/ml), Pam3Csk4 (10μg/ml), or LPS (100ng/ml), then washed and allowed to rest. They were then rechallenged with either Poly I:C, Pam3Csk4 or LPS. Endothelial cells showed significant tolerance to repeated LPS challenge. Priming with Pam3Csk4 also reduced the response to secondary LPS challenge in both cell types, despite a reduced proinflammatory response to Pam3Csk4 in HMVECs compared to HUVECs. Poly I:C priming enhanced inflammatory and interferon producing signals upon Poly I:C or LPS rechallenge, respectively. Poly I:C priming induced interferon regulatory factor 7, leading to enhancement of interferon production. Finally, both Poly I:C and LPS priming induced significant changes in receptor-interacting serine/threonine-protein kinase 1 activity. Pharmacological inhibition of receptor-interacting serine/threonine-protein kinase 1 or interferon regulatory factor 7 reduced the potentiated phenotype of TLR3 priming on TLR4 rechallenge. These results demonstrate that in human endothelial cells, prior activation of TLRs can have a significant impact on subsequent exposures and may contribute to the severity of the host response.

Abstract 2788: Inhibition of DNA methylation suppresses intestinal tumor organoids by inducing an anti-viral response

Abstract Background and Aim It is believed that the anti-tumor effect of DNA methylation inhibitors is mediated by re-activation of epigenetically silenced tumor suppressor genes in cancer cells. However, recent studies have proposed that the major anti-tumor effect of DNA methylation inhibitors is induction of interferon-related genes via dsRNAs-containing endogenous retroviruses. These studies used conventional cancer cell lines, which are maintained in 2D culture condition with serum-containing medium and are considered to be relatively artificial models of cancer cells. Recently, 3D culture system for stem cells known as organoid culture has been developed. Lgr5-positive stem cells form organoids that closely recapitulate the properties of original tissues. To investigate the effect of DNA demethylation on tumor organoids, we have established intestinal tumor organoids from tumors of ApcMin/+ (Min) mice and subjected them to 5-aza-2’-deoxycytidine (5-Aza-CdR) treatment and Dnmt1 knockdown. Methods Min mice were treated with 5-Aza-CdR (1 μg/body weight, n = 12) or PBS (n = 11) by subcutaneous injection weekly from 6 weeks of age. At 21 weeks of age, mice were dissected and number of intestinal polyps was counted. Stem cells were isolated from intestinal tumors of Min mice and maintained by organoid culture. Treatment with 5-Aza-CdR and lentivirus-mediated knockdown of Dnmt1 were performed in organoids derived from intestinal tumors. Expression profiles of genes after treatment with 5-Aza-CdR and Dnmt1-knockdown were analyzed. Results Treatment of Min mice with 5-Aza-CdR significantly reduced the average number of intestinal adenomas from 66 to 44 (male) and from 65 to 47 (female). The average number of large adenomas (≧3mm) in Min mice treated with 5-Aza-CdR was significantly decreased from 24 to11, whereas there was no significant difference in the average number of small adenomas (< 3mm). We successfully established organoids derived from intestinal tumors of Min mice by 3D culture with serum-free medium including epidermal growth factor (EGF) and Noggin. DNA demethylation induced by 5-Aza-CdR treatment and Dnmt1 knockdown significantly reduced the cell proliferation of the tumor organoids. Microarray analyses of the tumor organoids after 5-Aza-CdR treatment and Dnmt1 knockdown revealed that interferon-related genes including interferon regulatory factor 7 (Irf7) were activated by DNA demethylation. Gene ontology and pathway analyses clearly demonstrated that these genes activated by DNA demethylation are involved in the anti-viral response. Conclusions DNA demethylation suppresses the proliferation of intestinal tumor organoids by inducing an anti-viral response including activation of interferon-related genes. These findings suggest that treatment of colon cancers with DNA methylation inhibitors such as 5-Aza-CdR may be an effective therapeutic approach inhibiting cancer stem cells by immune response. Citation Format: Yoshimasa Saito, Kasumi Sakai, Toshihide Muramatsu, Toshiaki Nakaoka, Masaki Kimura, Hidetsugu Saito. Inhibition of DNA methylation suppresses intestinal tumor organoids by inducing an anti-viral response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2788.

RING domain is essential for the antiviral activity of TRIM25 from orange spotted grouper.

Tripartite motif-containing 25 (TRIM25) has been demonstrated to exert crucial roles in the regulation of innate immune signaling. However, the roles of fish TRIM25 in antiviral immune response still remained uncertain. Here, a novel fish TRIM25 gene from orange spotted grouper (EcTRIM25) was cloned and its roles in grouper virus infection were elucidated. EcTRIM25 encoded a 734-aa protein which shared 68% identity to large yellow croaker (Larimichthys crocea). Amino acid alignment showed that EcTRIM25 contained three conserved domains, including a RING-finger domain, a B box/coiled-coil domain and a SPRY domain. In healthy grouper, the transcript of EcTRIM25 was predominantly detected in skin, spleen and intestine. After stimulation with Singapore grouper iridovirus (SGIV) or poly I:C, the relative expression of EcTRIM25 in grouper spleen was significantly increased at the early stage of injection. Subcellular localization analysis showed that EcTRIM25 distributed throughout the cytoplasm in grouper cells. Notably, the deletion RING domain affected its accurate localization and displayed microtubule like structures or bright aggregates in GS cells. After incubation with SGIV or red spotted grouper nervous necrosis virus (RGNNV), overexpression of full length of EcTRIM25 invitro significantly decreased the viral gene transcription of SGIV and RGNNV. Consistently, the deletion of RING domain obviously affected the inhibitory effect of EcTRIM25. Furthermore, overexpression of EcTRIM25 significantly increased the expression level of interferon related signaling molecules, including interferon regulatory factor (IRF) 3, interferon-induced 35-kDa protein (IFP35), MXI, IRF7 and myeloid differentiation factor 88 (MyD88), suggesting that the positive regulation of interferon immune response by EcTRIM25 might affected RGNNV replication directly. Meanwhile, the expression levels of pro-inflammation cytokines were differently regulated by the ectopic expression of EcTRIM25. We proposed that the regulation of IRF7, MyD88 and pro-inflammation cytokines might contribute more important roles in SGIV infection. In addition, the RING domain of EcTRIM25 also played critical roles in the regulation of interferon immune and inflammation response. Together, our results will provide new evidences that the RING domain was essential for the antiviral action of fish TRIM25 against iridovirus and nodavirus infection.

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Reports from the 2015 American College of Rheumatology Congress

Systemic sclerosis (SSc) was one of the major topics covered during the 2015 American College of Rheumatology meeting. Selected abstracts related to basic research reported the identification of new relevant targets including interferon regulatory factor 7 (IRF7), long coding RNAs, C-terminal domain of endostatin, M2 macrophages and keratinocytes. Several new compounds, including abatacept, nintedanib and soluble guanylate cyclase stimulators, displayed potent antifibrotic effects in complementary animal models of experimental dermal fibrosis. Clinical aspects of the disease were also largely covered, with a specific focus on cardiac biomarkers and the relationship between cancer and SSc. The results of several clinical trials were also presented, including the comparison between cyclophosphamide and mycophenolate mofetil in the scleroderma lung study-2 and the effects of riociguat in patients with pulmonary arterial hypertension associated with connective tissue diseases.

RIG-I Signaling Is Essential for Influenza B Virus-Induced Rapid Interferon Gene Expression.

Influenza B virus causes annual epidemics and, along with influenza A virus, accounts for substantial disease and economic burden throughout the world. Influenza B virus infects only humans and some marine mammals and is not responsible for pandemics, possibly due to a very low frequency of reassortment and a lower evolutionary rate than that of influenza A virus. Influenza B virus has been less studied than influenza A virus, and thus, a comparison of influenza A and B virus infection mechanisms may provide new insight into virus-host interactions. Here we analyzed the early events in influenza B virus infection and interferon (IFN) gene expression in human monocyte-derived macrophages and dendritic cells. We show that influenza B virus induces IFN regulatory factor 3 (IRF3) activation and IFN-λ1 gene expression with faster kinetics than does influenza A virus, without a requirement for viral protein synthesis or replication. Influenza B virus-induced activation of IRF3 required the fusion of viral and endosomal membranes, and nuclear accumulation of IRF3 and viral NP occurred concurrently. In comparison, immediate early IRF3 activation was not observed in influenza A virus-infected macrophages. Experiments with RIG-I-, MDA5-, and RIG-I/MDA5-deficient mouse fibroblasts showed that RIG-I is the critical pattern recognition receptor needed for the influenza B virus-induced activation of IRF3. Our results show that innate immune mechanisms are activated immediately after influenza B virus entry through the endocytic pathway, whereas influenza A virus avoids early IRF3 activation and IFN gene induction. Recently, a great deal of interest has been paid to identifying the ligands for RIG-I under conditions of natural infection, as many previous studies have been based on transfection of cells with different types of viral or synthetic RNA structures. We shed light on this question by analyzing the earliest step in innate immune recognition of influenza B virus by human macrophages. We show that influenza B virus induces IRF3 activation, leading to IFN gene expression after viral RNPs (vRNPs) are released into the cytosol and are recognized by RIG-I receptor, meaning that the incoming influenza B virus is already able to activate IFN gene expression. In contrast, influenza A (H3N2) virus failed to activate IRF3 at very early times of infection, suggesting that there are differences in innate immune recognition between influenza A and B viruses.

Fine-Tuning of the RIG-I-Like Receptor/Interferon Regulatory Factor 3-Dependent Antiviral Innate Immune Response by the Glycogen Synthase Kinase 3/β-Catenin Pathway.

Induction of an antiviral innate immune response relies on pattern recognition receptors, including retinoic acid-inducible gene 1-like receptors (RLR), to detect invading pathogens, resulting in the activation of multiple latent transcription factors, including interferon regulatory factor 3 (IRF3). Upon sensing of viral RNA and DNA, IRF3 is phosphorylated and recruits coactivators to induce type I interferons (IFNs) and selected sets of IRF3-regulated IFN-stimulated genes (ISGs) such as those for ISG54 (Ifit2), ISG56 (Ifit1), and viperin (Rsad2). Here, we used wild-type, glycogen synthase kinase 3α knockout (GSK-3α(-/-)), GSK-3β(-/-), and GSK-3α/β double-knockout (DKO) embryonic stem (ES) cells, as well as GSK-3β(-/-) mouse embryonic fibroblast cells in which GSK-3α was knocked down to demonstrate that both isoforms of GSK-3, GSK-3α and GSK-3β, are required for this antiviral immune response. Moreover, the use of two selective small-molecule GSK-3 inhibitors (CHIR99021 and BIO-acetoxime) or ES cells reconstituted with the catalytically inactive versions of GSK-3 isoforms showed that GSK-3 activity is required for optimal induction of antiviral innate immunity. Mechanistically, GSK-3 isoform activation following Sendai virus infection results in phosphorylation of β-catenin at S33/S37/T41, promoting IRF3 DNA binding and activation of IRF3-regulated ISGs. This study identifies the role of a GSK-3/β-catenin axis in antiviral innate immunity.

Activation of Toll like receptor 3 induces spermatogonial stem cell apoptosis.

Germ cell apoptosis may be associated with the male infertility. The pathogenesis is to be further understood. Viral infection is one of the causative factors of apoptosis of the body cells. This study aims to investigate the role of activation of Toll like receptor (TLR)3 in the induction of germ cell apoptosis. In this study, spermatogonial stem cells (SSC) were isolated from C57BL/6 mouse testes. The expression of TLR3 on SSC was by RT-qPCR and Western blotting. polyinosinic-polycytidylic acid (Poly I:C) was employed to activate TLR3 on SSCs. The results showed that re-activation by PolyI:C induced SSC apoptosis. Exposure to PolyI:C induced interferon regulatory factor 3 (IRF3) phosphorylation in SSCs. TLR3 and IRF3 formed a complex in the SSCs. The complex of TLR3/IRF3 bound to the promoter of Fas ligand and promoted Fas ligand expression in SSC, and thus induced SSC apoptosis. In conclusion, the results of the present study indicate that activation of TLR3 by PolyI:C induces the SSC apoptosis, which implies that viral infection may interfere with the male germ cell development.

Small molecule inhibits activity of scavenger receptor A: Lead identification and preliminary studies.

Scavenger receptor A (SRA) has been implicated in the processes of tumor invasion and acts as an immunosuppressor during therapeutic cancer vaccination. Pharmacological inhibition of SRA function thus holds a great potential to improve treatment outcome of cancer therapy. Macromolecular natural product sennoside B was recently shown to block SRA function. Here we report the identification and characterization of a small molecule SRA inhibitor rhein. Rhein, a deconstructed analog of sennoside B, reversed the suppressive activity of SRA in dendritic cell-primed T cell activation, indicated by transcription activation of il2 gene and production of IL-2. Rhein also inhibited SRA ligand polyinosinic:polycytidylic acid (poly(I:C)) induced activation of transcriptional factors, including interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 1 (STAT1). Additionally, this newly identified lead compound was docked into the homology models of the SRA cysteine rich domain to gain insights into its interaction with the receptor. It was then found that rhein can favorably interact with SRA cysteine rich domain. Collectively, rhein, being the first identified small molecule inhibitors for SRA, warrants further structure-activity relationship studies, which may lead to development of novel pharmacological intervention for cancer therapy.

Differentiation of neurons restricts Arbovirus replication and increases expression of the alpha isoform of IRF-7.

Susceptibility to alphavirus infection is age dependent, and host maturation is associated with decreased virus replication and less severe encephalitis. To identify factors associated with maturation-dependent restriction of virus replication, we studied AP-7 rat olfactory bulb neuronal cells, which can differentiate in vitro. Differentiation was associated with a 150- to 1,000-fold decrease in replication of the alphaviruses Sindbis virus and Venezuelan equine encephalitis virus, as well as La Crosse bunyavirus. Differentiation delayed synthesis of SINV RNA and protein but did not alter the susceptibility of neurons to infection or virion maturation. Additionally, differentiation slowed virus-induced translation arrest and death of infected cells. Differentiation of uninfected AP-7 neurons was associated with changes in expression of antiviral genes. Expression of key transcription factors was increased, including interferon regulatory factor 3 and 7 (IRF-3 and IRF-7) and STAT-1, suggesting that neuronal maturation may enhance the capacity for antiviral signaling upon infection. IRF-7 produced by undifferentiated AP-7 neurons was exclusively the short dominant negative γ-isoform, while that produced by differentiated neurons was the full-length α-isoform. A similar switch in IRF-7 isoforms also occurred in the brains of maturing C57BL/6J mice. Silencing of IRF expression did not improve virus multiplication in differentiated neurons. Therefore, neuronal differentiation is associated with upregulation of transcription factors that activate antiviral signaling, but this alone does not account for maturation-dependent restriction of virus replication. Viral encephalomyelitis is an important cause of age-dependent morbidity and mortality. Because mature neurons are not readily regenerated, recovery from encephalitis suggests that mature neurons utilize unique antiviral mechanisms to block infection and/or clear virus. To identify maturational changes in neurons that may improve outcome, we compared immature and mature cultured neurons for susceptibility to three encephalitic arboviruses and found that replication of Old World and New World alphaviruses and a bunyavirus was reduced in mature compared to immature neurons. Neuronal maturation was associated with increased baseline expression of interferon regulatory factor 3 and 7 mRNAs and production of distinct isoforms of interferon regulatory factor 7 protein. Overall, our studies identified maturational changes in neurons that likely contribute to assembly of immunoregulatory factors prior to infection, a more rapid antiviral response, increased resistance to virus infection, and improved survival.

Profiling of the embryonic Atlantic halibut (Hippoglossus hippoglossus L.) transcriptome reveals maternal transcripts as potential markers of embryo quality.

BackgroundCommercial Atlantic halibut (Hippoglossus hippoglossus) farming is restricted by variable oocyte quality, slow growth, and early maturation of male fish. Maternally transferred components regulate early developmental processes; therefore, they have an effect on the future viability of the embryo. Using a newly developed Agilent 10 k custom-made oligonucleotide array, we profiled components of the transcriptome involved in immune defence as well as germline and muscle development during early developmental stages: 8-cell embryos (8CS), germ ring stage (GR), 10-somite stage (10SS), and hatched embryos (HT). In addition, we identified differentially expressed transcripts in low (≤9 ± 3% hatching) and high (≥86 ± 3°% hatching) quality eggs at 8CS to identify potential maternal markers for embryo quality.ResultsOut of 2066 differentially expressed transcripts, 160 were identified as maternal transcripts being specifically expressed at 8CS only. Twenty transcripts were differentially expressed in 8-cell embryos between low and high quality egg groups. Several immune-related transcripts were identified as promising molecular markers of hatching success including interferon regulatory factor 7 and mhc class 2A chain. Differential expression was positively validated with quantitative real-time PCR.ConclusionsWe have demonstrated maternal transfer of innate and adaptive immune system transcripts into Atlantic halibut embryos and their relation with future embryo developmental potential. We identified several transcripts as potential molecular markers of embryo quality. The developed microarray represents a useful resource for improving the commercial production of Atlantic halibut.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-829) contains supplementary material, which is available to authorized users.