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Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor characterized by a high metastatic potential with an overall survival rate of ~5%. The transcription factor signal transducer and activator of transcription 3 (STAT3) is overexpressed by >50% of tumors, including SCLC, but its role in SCLC development and metastasis is unclear. Here, we show that, while STAT3 deletion restricts primary tumor growth, it paradoxically enhances metastatic spread by promoting immune evasion. This occurs because STAT3 is crucial for maintaining the immune sensor stimulator of interferon (IFN) genes (STING). Without STAT3, the cyclic adenosine monophosphate-guanosine monophosphate synthase-STING pathway is inactive, resulting in decreased type I IFN secretion and an IFN gene signature. Importantly, restoration of IFN signaling through re-expression of endogenous STING, enforced expression of IFN response factor 7 or administration of recombinant type I IFN re-established antitumor immunity, inhibiting metastatic SCLC in vivo. These data show the potential of augmenting the innate immune response to block metastatic SCLC., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

UNC93B1 is essential for the stability and endosomal trafficking of nucleic-acid sensing Toll-like receptors (TLRs) including TLR7 and TLR8. Increased TLR7 responses are associated with lupus autoimmunity in both mice and humans. In a recent article, Al-Azab et al. demonstrate the role of a variant of UNC93B1 (p.V117L) in the induction of pediatric systemic lupus erythematosus in patients and in mice through TLR7/8 hyperresponsiveness. They also highlight a potential role for the pharmacological inhibition of interleukin-1 receptor-associated kinase (IRAK) 1 and/or 4 in ameliorating disease., (© 2024 the Australian and New Zealand Society for Immunology, Inc.)

Platelets display unexpected roles in immune and coagulation responses. Emerging evidence suggests that STING is implicated in hypercoagulation. STING is an adaptor protein downstream of the DNA sensor cyclic GMP-AMP synthase (cGAS) that is activated by cytosolic microbial and self-DNA during infections, and in the context of loss of cellular integrity, to instigate the production of type-I IFN and pro-inflammatory cytokines. To date, whether the cGAS-STING pathway is present in platelets and contributes to platelet functions is not defined. Using a combination of pharmacological and genetic approaches, we demonstrate here that megakaryocytes and platelets possess a functional cGAS-STING pathway. Our results suggest that in megakaryocytes, STING stimulation activates a type-I IFN response, and during thrombopoiesis, cGAS and STING are transferred to proplatelets. Finally, we show that both murine and human platelets contain cGAS and STING proteins, and the cGAS-STING pathway contributes to potentiation of platelet activation and aggregation. Taken together, these observations establish for the first time a novel role of the cGAS-STING DNA sensing axis in the megakaryocyte and platelet lineage., (© 2023 El-Mortada et al.)

There is a growing appreciation that the direct interaction between bacteriophages and the mammalian host can facilitate diverse and unexplored symbioses. Yet the impact these bacteriophages may have on mammalian cellular and immunological processes is poorly understood. Here, we applied highly purified phage T4, free from bacterial by-products and endotoxins to mammalian cells and analyzed the cellular responses using luciferase reporter and antibody microarray assays. Phage preparations were applied in vitro to either A549 lung epithelial cells, MDCK-I kidney cells, or primary mouse bone marrow derived macrophages with the phage-free supernatant serving as a comparative control. Highly purified T4 phages were rapidly internalized by mammalian cells and accumulated within macropinosomes but did not activate the inflammatory DNA response TLR9 or cGAS-STING pathways. Following 8 hours of incubation with T4 phage, whole cell lysates were analyzed via antibody microarray that detected expression and phosphorylation levels of human signaling proteins. T4 phage application led to the activation of AKT-dependent pathways, resulting in an increase in cell metabolism, survival, and actin reorganization, the last being critical for macropinocytosis and potentially regulating a positive feedback loop to drive further phage internalization. T4 phages additionally down-regulated CDK1 and its downstream effectors, leading to an inhibition of cell cycle progression and an increase in cellular growth through a prolonged G1 phase. These interactions demonstrate that highly purified T4 phages do not activate DNA-mediated inflammatory pathways but do trigger protein phosphorylation cascades that promote cellular growth and survival. We conclude that mammalian cells are internalizing bacteriophages as a resource to promote cellular growth and metabolism., Competing Interests: JJB has a patent application related to this work (WO2018129536A1)., (Copyright: © 2023 Bichet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation., (© 2023. Springer Nature Limited.)

A growing body of preclinical evidence has led to the hypothesis that human Toll-like receptor 8 (hTLR8) activation in the tumor microenvironment (TME) could have potent anticancer effects through its action on monocytes, myeloid dendritic cells (mDCs), and natural killer (NK) cells. This has motivated the initiation of several clinical trials for chemical hTLR8 agonists in a variety of cancers. Concurrently, a growing number of synthetic antisense oligonucleotides (ASOs) are being developed as cancer therapeutics. We have recently reported that 2'-O-methyl (2'OMe)-modified ASOs can potentiate sensing of hTLR8 chemical agonists in a sequence-dependent manner. This suggests that select gene-targeting ASOs with anticancer activity may synergize with low-dose hTLR8 agonists in the TME. Here, we provide a detailed protocol to rapidly screen and identify such synthetic bifunctional oligonucleotides with synergistic activity on hTLR8 sensing., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cytoplasmic detection of DNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) is an essential component of antiviral responses. Upon synthesis, cGAMP binds to the stimulator of interferon (IFN) genes (STING) in infected and adjacent cells through intercellular transfer by connexins forming gap-junctions, eliciting a strong IFN-β-driven antiviral response. We demonstrate here that Genistein, a flavonoid compound naturally occurring in soy-based foods, inhibits cGAS-STING antiviral signaling at two levels. First, Genistein pretreatment of cGAMP-producing cells inhibited gap-junction intercellular communication, resulting in reduced STING responses in adjacent cells. In addition, Genistein directly blocked STING activation by the murine agonist DMXAA, by decreasing the interaction of STING with TBK1 and IKKε. As a result, Genistein attenuated STING signaling in human and mouse cells, dampening antiviral activity against Semliki Forest Virus infection. Collectively, our findings identify a previously unrecognized proviral activity of Genistein mediated via its inhibitory effects at two levels of cGAS-STING signaling. IMPORTANCE Several reports suggest that Genistein exhibits antiviral activities against DNA viruses. Our work uncovers a previously unrecognized proviral effect of Genistein, through inhibition of the cGAS-STING pathway at the level of cGAMP transfer and its sensing by STING. This suggests that the use of Genistein as an antiviral should be taken with caution as it may reduce the protective antiviral effects elicited by host STING activation.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease 1-7 , evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA 8 , 9 and binds to guanosine 10 - 12 . We identified a de novo, previously undescribed missense TLR7 Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7 Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP 10-12 , and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c + age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7 Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition., (© 2022. The Author(s).)

Coatomer complex I (COPI) mediates retrograde vesicular trafficking from Golgi to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency in subunit alpha causes COPA syndrome and is associated with type I IFN signalling, although the upstream innate immune sensor involved was unknown. Using in vitro models we find aberrant activation of the STING pathway due to deficient retrograde but probably not intra-Golgi transport. Further we find the upstream cytosolic DNA sensor cGAS as essentially required to drive type I IFN signalling. Genetic deletion of COPI subunits COPG1 or COPD similarly induces type I IFN activation in vitro, which suggests that inflammatory diseases associated with mutations in other COPI subunit genes may exist. Finally, we demonstrate that inflammation in COPA syndrome patient peripheral blood mononuclear cells and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS/STING pathway as a promising therapeutic approach., (© 2022. The Author(s).)

Activation of the STING pathway upon genotoxic treatment of cancer cells has been shown to lead to anti-tumoral effects, mediated through the acute production of interferon (IFN)-β. Conversely, the pathway also correlates with the expression of NF-κB-driven pro-tumorigenic genes, but these associations are only poorly defined in the context of genotoxic treatment, and are thought to correlate with a chronic engagement of the pathway. We demonstrate here that half of the STING-expressing cancer cells from the NCI60 panel rapidly increased expression of pro-tumorigenic IL-6 upon genotoxic DNA damage, often independent of type-I IFN responses. While preferentially dependent on canonical STING, we demonstrate that genotoxic DNA damage induced by camptothecin (CPT) also drove IL-6 production through non-canonical STING signaling in selected cancer cells. Consequently, pharmacological inhibition of canonical STING failed to broadly inhibit IL-6 production induced by CPT, although this could be achieved through downstream ERK1/2 inhibition. Finally, prolonged inhibition of canonical STING signaling was associated with increased colony formation of MG-63 cells, highlighting the duality of STING signaling in also restraining the growth of selected cancer cells. Collectively, our findings demonstrate that genotoxic-induced DNA damage frequently leads to the rapid production of pro-tumorigenic IL-6 in cancer cells, independent of an IFN signature, through canonical and non-canonical STING activation; this underlines the complexity of STING engagement in human cancer cells, with frequent acute pro-tumorigenic activities induced by DNA damage. We propose that inhibition of ERK1/2 may help curb such pro-tumorigenic responses to DNA-damage, while preserving the anti-proliferative effects of the STING-interferon axis., Competing Interests: MG receives funding from Noxopharm Ltd. to study the activity of STING inhibitors in cancer. MG does not personally own any shares/equity in Noxopharm Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Al-Asmari, Rajapakse, Ullah, Pépin, Croft and Gantier.)

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is an essential innate immune sensor. Remarkably, in addition to its role in the early detection of pathogenic DNA molecules, cGAS also monitors cellular health through the sensing of nuclear and mitochondrial DNA aberrantly localised to the cell cytoplasm. This central position of cGAS requires tight molecular controls which are only starting to be understood. In this issue of EMBO Reports, Zhao and colleagues (Zhao et al, 2021) describe a novel mechanism switching on DNA sensing, relying on the formation of primary condensates of cGAS and GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1)., (© 2021 The Author.)

microRNAs (miRNAs) are important modulators of messenger RNA stability and translation, controlling wide gene networks. Albeit generally modest on individual targets, the regulatory effect of miRNAs translates into meaningful pathway modulation through concurrent targeting of regulons with functional convergence. Identification of miRNA-regulons is therefore essential to understand the function of miRNAs and to help realise their therapeutic potential, but it remains challenging due to the large number of false positive target sites predicted per miRNA. In the current work, we investigated whether genes regulated by a given miRNA were under the transcriptional control of a predominant transcription factor (TF). Strikingly we found that for ~50% of the miRNAs analysed, their targets were significantly enriched in at least one common TF. We leveraged such miRNA-TF co-regulatory networks to identify pathways under miRNA control, and demonstrated that filtering predicted miRNA-target interactions (MTIs) relying on such pathways significantly enriched the proportion of predicted true MTIs. To our knowledge, this is the first description of an in- silico pipeline facilitating the identification of miRNA-regulons, to help understand miRNA function., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Background: Non-alcoholic fatty liver disease is the most common liver disease globally and in its inflammatory form, non-alcoholic steatohepatitis (NASH), can progress to cirrhosis and hepatocellular carcinoma (HCC). Currently, patient education and lifestyle changes are the major tools to prevent the continued progression of NASH. Emerging therapies in NASH target known pathological processes involved in the progression of the disease including inflammation, fibrosis, oxidative stress and hepatocyte apoptosis. Human amniotic epithelial cells (hAECs) were previously shown to be beneficial in experimental models of chronic liver injury, reducing hepatic inflammation and fibrosis. Previous studies have shown that liver progenitor cells (LPCs) response plays a significant role in the development of fibrosis and HCC in mouse models of fatty liver disease. In this study, we examined the effect hAECs have on the LPC response and hepatic oxidative stress in an experimental model of NASH., Methods: Experimental NASH was induced in C57BL/6 J male mice using a high-fat, high fructose diet for 42 weeks. Mice received either a single intraperitoneal injection of 2 × 10 6 hAECs at week 34 or an additional hAEC dose at week 38. Changes to the LPC response and oxidative stress regulators were measured., Results: hAEC administration significantly reduced the expansion of LPCs and their mitogens, IL-6, IFNγ and TWEAK. hAEC administration also reduced neutrophil infiltration and myeloperoxidase production with a concurrent increase in heme oxygenase-1 production. These observations were accompanied by a significant increase in total levels of anti-fibrotic IFNβ in mice treated with a single dose of hAECs, which appeared to be independent of c-GAS-STING activation., Conclusions: Expansion of liver progenitor cells, hepatic inflammation and oxidative stress associated with experimental NASH were attenuated by hAEC administration. Given that repeated doses did not significantly increase efficacy, future studies assessing the impact of dose escalation and/or timing of dose may provide insights into clinical translation., (© 2021. The Author(s).)

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS., Competing Interests: Declaration of Interests S.L.M. declares consultancy with IFM Therapeutics and Quench Bio and received funding from GlaxoSmithKline. S.L.M. and C.-H.Y. are named inventors on International Patent Application No. PCT/AU2019051201. All other authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory molecule with both cytokine and noncytokine activity. MIF is constitutively released from multiple cell types via an unconventional secretory pathway that is not well defined. Here, we looked at MIF release from human and mouse monocytes/macrophages in response to different stimuli. While MIF release was not significantly altered in response to lipopolysaccharide or heat-killed Escherichia coli, cytotoxic stimuli strongly promoted release of MIF. MIF release was highly upregulated in cells undergoing necrosis, necroptosis and NLRP3 inflammasome-dependent pyroptosis. Our data suggest that cell death represents a major route for MIF release from myeloid cells. The functional significance of these findings and their potential importance in the context of autoimmune and inflammatory diseases warrant further investigation., (© 2020 Australian and New Zealand Society for Immunology Inc.)

Signal transduction and the regulation of gene expression are fundamental processes in every cell. RNA-binding proteins (RBPs) play a key role in the post-transcriptional modulation of gene expression in response to both internal and external stimuli. However, how signaling pathways regulate the assembly of RBPs with mRNAs remains largely unknown. Here, we summarize observations showing that the formation and composition of messenger ribonucleoprotein particles (mRNPs) is dynamically remodeled in space and time by specific signaling cascades and the resulting post-translational modifications. The integration of signaling events with gene expression is key to the rapid adaptation of cells to environmental changes and stress. Only a combined approach analyzing the signal transduction pathways and the changes in post-transcriptional gene expression they cause will unravel the mechanisms coordinating these important cellular processes.

Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2'-methoxyethyl (2'MOE) modifications. Through a screen of 192 2'OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2'OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most microRNAs (miRNAs) are expressed as a mix of length isoforms (referred to as isomiRs). IsomiR stoichiometry can be differentially impacted upon cell stimulation, as recently evidenced by our group in the context of immune responses induced by type-I interferon (IFN). Here, we revisit published RNA-seq data sets of human and mouse macrophages stimulated with bacterial products at the isomiR level. We demonstrate that for several miRNAs, macrophage stimulation induces changes in isomiR stoichiometry. Critically, we find that changes in miRNA expression can be misinterpreted when miRNAs are quantified by RT-qPCR, as primers directed against canonical miRNA sequences may not equally target the different isomiRs that are regulated endogenously. Beyond the case of phagocyte stimulation, our analyses reinforce the concept that analysis of miRNA expression at the isoform level should become standard practice., (© 2019 Pillman et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The discovery of microRNAs (miRNAs) in the early 1990's has revolutionized our thinking of post-transcriptional gene control. miRNAs are involved in the regulation of almost every cellular and developmental process in eukaryotic organisms. In the context of infection and immunity, miRNAs play critical roles controlling processes involved in pathogen clearance, while ensuring a rapid return to homeostasis. In this review, we provide a broad overview of the miRNAs that have been implicated in innate immunity and inflammation in mammals over the past decade. We discuss how most miRNAs can have dual activities on inflammation, suggesting that modulation of their activity for therapeutic purposes may be context-dependent., (© 2018 Federation of European Biochemical Societies.)

Murine models of Salmonella enterica serovar Typhimurium infection are one of the commonest tools to study host-pathogen interactions during bacterial infections. Critically, the outcome of S . Typhimurium infection is impacted by the genetic background of the mouse strain used, with macrophages from C57BL/6 and BALB/c mice lacking the capacity to control intracellular bacterial replication. For this reason, the use of congenic strains, which mix the genetic backgrounds of naturally protected mouse strains with those of susceptible strains, has the capacity to significantly alter results and interpretation of S . Typhimurium infection studies. Here, we describe how macrophage knockout cell lines generated by CRISPR/Cas9 gene editing can help determine the contribution of background contaminations in the phenotypes of primary macrophages from congenic mice, on the outcome of S . Typhimurium infection studies. Our own experience illustrates how the CRISPR/Cas9 technology can be used to complement pre-existing knockout models, and shows that there is great merit in performing concurrent studies with both genetic models, to exclude unanticipated side-effects on host-pathogen interactions.

Endogenous microRNAs (miRNAs) often exist as multiple isoforms (known as "isomiRs") with predominant variation around their 3'-end. Increasing evidence suggests that different isomiRs of the same family can have diverse functional roles, as recently demonstrated with the example of miR-222-3p 3'-end variants. While isomiR levels from a same miRNA family can vary between tissues and cell types, change of templated isomiR stoichiometry to stimulation has not been reported to date. Relying on small RNA-sequencing analyses, we demonstrate here that miR-222-3p 3'-end variants >23 nt are specifically decreased upon interferon (IFN) β stimulation of human fibroblasts, while shorter isoforms are spared. This length-dependent dynamic regulation of long miR-222-3p 3'-isoforms and >40 other miRNA families was confirmed in human monocyte-derived dendritic cells following infection with Salmonella Typhimurium, underlining the breadth of 3'-length regulation by infection, beyond the example of miR-222-3p. We further show that stem-loop miRNA Taqman RT-qPCR exhibits selectivity between 3'-isoforms, according to their length, and that this can lead to misinterpretation of results when these isoforms are differentially regulated. Collectively, and to our knowledge, this work constitutes the first demonstration that the stoichiometry of highly abundant templated 3'-isoforms of a same miRNA family can be dynamically regulated by a stimulus. Given that such 3'-isomiRs can have different functions, our study underlines the need to consider isomiRs when investigating miRNA-based regulation., (© 2018 Nejad et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Current studies aiming at identifying single immune markers with prognostic value have limitations in the context of complex antitumor immunity and cancer immune evasion. Here, we show how the integration of several immune markers influences the predictions of prognosis of gastric cancer (GC) patients. We analyzed Tissue Microarray (TMA) by multiplex immunohistochemistry and measured the expression of immune checkpoint molecule PD-L1 together with antitumor CD8 T cells and immune suppressive FOXP3 Treg cells in a cohort of GC patients. Unsupervised hierarchical clustering analysis of these markers was used to define correlations between CD8 T, FOXP3 Treg and PD-L1 cell densities. We found that FOXP3 and PD-L1 densities were elevated while CD8 T cells were decreased in tumor tissues compared to their adjacent normal tissues. However, patient stratification based on each one of these markers individually did not show significant prognostic value on patient survival. Conversely, combination of the ratios of CD8/FOXP3 and CD8/PD-L1 enabled the identification of patient subgroups with different survival outcomes. As such, high densities of PD-L1 in patients with high CD8/FOXP3 and low CD8/PD-L1 ratios correlated with increased survival. Collectively, this work demonstrates the need for the integration of several immune markers to obtain more meaningful survival prognosis and patient stratification. In addition, our work provides insights into the complex tumor immune evasion and immune regulation by the tumor-infiltrating effector and suppressor cells, informing on the best use of immunotherapy options for treating patients.

MicroRNA (miRNA) detection by reverse transcription (RT) quantitative real-time PCR (RT-qPCR) is the most popular method currently used to measure miRNA expression. Although the majority of miRNA families are constituted of several 3'-end length variants ("isomiRs"), little attention has been paid to their differential detection by RT-qPCR. However, recent evidence indicates that 3'-end miRNA isoforms can exhibit 3'-length specific regulatory functions, underlining the need to develop strategies to differentiate 3'-isomiRs by RT-qPCR approaches. We demonstrate here that polyadenylation-based RT-qPCR strategies targeted to 20-21 nt isoforms amplify entire miRNA families, but that primers targeted to >22 nt isoforms were specific to >21 nt isoforms. Based on this observation, we developed a simple method to increase selectivity of polyadenylation-based RT-qPCR assays toward shorter isoforms, and demonstrate its capacity to help distinguish short RNAs from longer ones, using synthetic RNAs and biological samples with altered isomiR stoichiometry. Our approach can be adapted to many polyadenylation-based RT-qPCR technologies already exiting, providing a convenient way to distinguish long and short 3'-isomiRs.

DNA sensing by the STING pathway is emerging to be a crucial component of the antitumor immune response. Although it plays a key role in the activation of tumor immune cells, exactly how STING is activated by tumor cells is not fully understood. Recent evidence suggests that cGAS can be directly engaged and produces 2'3'-cyclic-GMP-AMP (cGAMP) within certain tumor cells upon stimulation with DNA damaging agents. Because cGAMP can transfer between adjacent cells, the capacity of tumor cells to produce cGAMP may activate tumor immune cells, even in the absence of functional STING signaling within the tumor. Here we describe a simple coculture protocol allowing for the functional characterization of cGAS/STING activity in tumor cells, together with cGAMP transfer to adjacent cells. This approach will help define how different tumors engage the STING pathway, and whether synthetic STING agonists should be used to potentiate the antitumor effects of chemotherapies.

Heterogeneity in terms of tumor characteristics, prognosis, and survival among cancer patients is an unsolved issue. Here, we systematically analyzed the aberrant expression patterns of cervical cancer using RNA-Seq data from The Cancer Genome Atlas (TCGA). We incorporated gene profiling, molecular signatures, functional and pathway information with gene set enrichment and protein-protein interaction (PPI) network analysis, to identify sub-networks of genes. Those identified genes relating to DNA replication and DNA repair-mediated signaling pathways associated with systemic lupus erythematosus (SLE). Next, we combined cross-validated prognostic scores to build an integrated prognostic model for survival prediction. The combined approach revealed that the DNA repair-mediated including the functional interaction module of 18 histone genes (Histone cluster 1 H2A, B and H4), were significantly correlated with the survival rate. Furthermore, five of these histone genes were highly expressed in three cervical cancer cohorts from the Oncomine database. Comparison of high and low histone variant-expressing human cervical cancer cell lines revealed different responses to DNA damage, suggesting protective functions of histone genes against DNA damage. Collectively, we provide evidence that two SLE-associated gene sets (HIST1H2BD and HIST1H2BJ; and HIST1H2BD, HIST1H2BJ, HIST1H2BH, HIST1H2AM and HIST1H4K) can be used as prognostic factors for survival prediction among cervical cancer patients.

Deep-sequencing reveals extensive variation in the sequence of endogenously expressed microRNAs (termed 'isomiRs') in human cell lines and tissues, especially in relation to the 3' end. From the immunoprecipitation of the microRNA-binding protein Argonaute and the sequencing of associated small RNAs, we observe extensive 3'-isomiR variation, including for miR-222 where the majority of endogenously expressed miR-222 is extended by 1-5 nt compared to the canonical sequence. We demonstrate this 3' heterogeneity has dramatic implications for the phenotype of miR-222 transfected cells, with longer isoforms promoting apoptosis in a size (but not 3' sequence)-dependent manner. The transfection of longer miR-222 isomiRs did not induce an interferon response, but did downregulate the expression of many components of the pro-survival PI3K-AKT pathway including PIK3R3, a regulatory subunit whose knockdown phenocopied the expression of longer 222 isoforms in terms of apoptosis and the inhibition of other PI3K-AKT genes. As this work demonstrates the capacity for 3' isomiRs to mediate differential functions, we contend more attention needs to be given to 3' variance given the prevalence of this class of isomiR., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development. IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans., (Copyright © 2017 Pépin et al.)

As one of the life-threatening diseases involving multi-step genetic and epigenetic disorders, cancer has long been a dynamic research area for siRNA-based therapy as half of the current siRNA-based clinical trials are involved in oncology. However, despite consistent enthusiasm in the academic world, siRNA-based cancer treatment still faces obstacles and difficulties in clinical development. In this article, we discuss key challenges facing siRNA-based cancer treatment revealed from recent clinical and preclinical studies, including chemical modification, tumour penetration, endosomal escape, target selection and off-target effects. In addition, opportunities and avenues for translating siRNA technology from bench to oncologic clinics are explored., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Acridine dyes, including proflavine and acriflavine, were commonly used as antiseptics before the advent of penicillins in the mid-1940s. While their mode of action on pathogens was originally attributed to their DNA intercalating activity, work in the early 1970s suggested involvement of the host immune responses, characterized by induction of interferon (IFN)-like activities through an unknown mechanism. We demonstrate here that sub-toxic concentrations of a mixture of acriflavine and proflavine instigate a cyclic-GMP-AMP (cGAMP) synthase (cGAS)-dependent type-I IFN antiviral response. This pertains to the capacity of these compounds to induce low level DNA damage and cytoplasmic DNA leakage, resulting in cGAS-dependent cGAMP-like activity. Critically, acriflavine:proflavine pre-treatment of human primary bronchial epithelial cells significantly reduced rhinovirus infection. Collectively, our findings constitute the first evidence that non-toxic DNA binding agents have the capacity to act as indirect agonists of cGAS, to exert potent antiviral effects in mammalian cells., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

MicroRNAs (miRNAs) are involved in most cellular processes and are deregulated in several diseases. Antisense miRNA oligonucleotides (AMOs) therefore present novel therapeutic opportunities. Currently, in vivo delivery of AMOs often relies on high doses of nucleic acids, with nonspecific uptake by most tissues. Critically, AMOs accumulate in phagocytic cells where they can interfere with immune functions, such as the activation of Toll-Like Receptors (TLRs). In this chapter, we describe a method to assess the possible off-target effects of AMOs on TLR7, 8, and 9 sensing.

Stimulator of interferon (IFN) genes (STING) is a key mediator in the immune response to cytoplasmic DNA sensed by cyclic GMP-AMP (cGAMP) synthase (cGAS). After synthesis by cGAS, cGAMP acts as a second messenger activating STING in the cell harboring cytoplasmic DNA but also in adjacent cells through gap junction transfer. While the role of the cGAS-STING pathway in pathogen detection is now well established, its importance in cancer immunity has only recently started to emerge. Nonetheless, STING appears to be an essential component in the recruitment of immune cells to the tumor microenvironment, which is paramount to immune clearance of the tumor. This review presents an overview of the growing literature around the role of the cGAS-STING pathway in the tumor microenvironment, with a specific focus on the role that cancer cells may play in the direct activation of this pathway, and its amplification through cell-cell transfer of cGAMP.

Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

microRNA (miRNA) microarray normalization is a critical step for the identification of truly differentially expressed miRNAs. This is particularly important when dealing with cancer samples that have a global miRNA decrease. In this chapter, we provide a simple step-by-step procedure that can be used to normalize Affymetrix miRNA microarrays, relying on robust normal-exponential background correction with cyclic loess normalization.

Aberrant sensing of self-nucleic acids by Toll-like receptor (TLR) 7, 8, or 9 is associated with several autoimmune disorders, including systemic lupus erythematosus (SLE), rheumatoid arthritis, psoriasis, or systemic sclerosis. In recent years, several classes of synthetic oligonucleotides have been shown to antagonize sensing of immunostimulatory nucleic acids by TLR7/8/9, indicating that these molecules could have therapeutic applications in such autoimmune diseases. Conversely, synthetic oligonucleotides used in therapeutic technologies such as antisense and microRNA inhibitors also have the potential to inhibit TLR7/8/9 sensing, rendering patients more susceptible to viral/bacterial infections. This chapter describes a protocol to define the inhibitory activity of synthetic oligonucleotides on TLR7.