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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 appliedin vitroto 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 eight 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 internalization 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 food source to promote cellular growth and metabolism.

Graphical abstract, 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.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease1–7, 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 RNA8,9 and binds to guanosine10–12. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP10–12, 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 Tlr7Y264H 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.

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).

MicroRNA-101-3p (miR-101-3p) is a tumour suppressor that regulates cancer proliferation and apoptotic signalling. Loss of miR-101-3p increases the expression of the Polycomb Repressive Complex 2 (PRC2) subunit enhancer of zeste homolog 2 (EZH2), resulting in alterations to the epigenome and enhanced tumorigenesis. MiR-101-3p has also been shown to modulate various aspects of cellular metabolism, however little is known about the mechanisms involved. To investigate the metabolic pathways that are regulated by miR-101-3p, we performed transcriptome and functional analyses of osteosarcoma cells transfected with miR-101-3p. We found that miR-101-3p downregulates multiple mitochondrial processes, including oxidative phosphorylation, pyruvate metabolism, the citric acid cycle and phospholipid metabolism. We also found that miR-101-3p transfection disrupts the transcription of mitochondrial DNA (mtDNA) via the downregulation of the mitochondrial transcription initiation complex proteins TFB2M and Mic60. These alterations in transcript expression disrupt mitochondrial function, with significant decreases in both basal (54%) and maximal (67%) mitochondrial respiration rates. Native gel electrophoresis revealed that this diminished respiratory capacity was associated with reduced steady-state levels of mature succinate dehydrogenase (complex II), with a corresponding reduction of complex II enzymatic activity. Furthermore, miR-101-3p transfection reduced the expression of the SDHB subunit, with a concomitant disruption of the assembly of the SDHC subunit into mature complex II. Overall, we describe a new role for miR-101-3p as a modulator of mitochondrial metabolism via its regulation of multiple mitochondrial processes, including mtDNA transcription and complex II biogenesis.

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.

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 × 106 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.

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.

COPA syndrome is caused by loss-of-function mutations in the COP-α subunit of coatomer protein complex I (COPI), which participates in retrograde vesicular trafficking of proteins from the Golgi to the endoplasmic reticulum (ER). Disease manifests early in life with arthritis, lung pathology, kidney dysfunction and systemic inflammation associated with NF-κB activation and type I interferon (IFNαβ) production. Here, we generated in vitro models for COPA syndrome and interrogated inflammatory signalling pathways via a range of biochemical and molecular biological techniques. Results were confirmed with cell lines in which mutant COPA was overexpressed and with COPA syndrome patient PBMCs. We identified Stimulator of Interferon Genes (STING), as a driver of inflammation in COPA syndrome. Furthermore, we found that genetic deletion of COPG1, another COPI subunit protein, induced NF-κB and type I IFN pathways similar to COPA-deficiency. Finally, we demonstrate that in vitro, inflammation due to COPA syndrome mutations was ameliorated by treatment with the small molecule STING inhibitor H-151. Therefore, inflammation induced by deletion of COPI subunits in general suggests a link between retrograde trafficking and STING regulation, and this innate immune sensor represents a novel therapeutic target in COPA syndrome.

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.

Recent studies suggest that extracellular cGAMP can be taken up by macrophages to engage STING through several mechanisms. Our work demonstrates that connexin-dependent communication between epithelial cells and macrophages plays a significant role in the amplification of antiviral responses mediated by cGAMP and suggests that pharmacological strategies aimed at modulating connexins may have therapeutic applications to control antiviral responses in humans., Activation of cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in antiviral responses to many DNA viruses. Sensing of cytosolic DNA by cGAS results in synthesis of the endogenous second messenger cGAMP that activates stimulator of interferon genes (STING) in infected cells. Critically, cGAMP can also propagate antiviral responses to uninfected cells through intercellular transfer, although the modalities of this transfer between epithelial and immune cells remain poorly defined. We demonstrate here that cGAMP-producing epithelial cells can transactivate STING in cocultured macrophages through direct cGAMP transfer. cGAMP transfer was reliant upon connexin expression by epithelial cells and pharmacological inhibition of connexins blunted STING-dependent transactivation of the macrophage compartment. Macrophage transactivation by cGAMP contributed to a positive-feedback loop amplifying antiviral responses, significantly protecting uninfected epithelial cells against viral infection. Collectively, our findings constitute the first direct evidence of a connexin-dependent cGAMP transfer to macrophages by epithelial cells, to amplify antiviral responses.

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. Refereed/Peer-reviewed

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.

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.

MicroRNAs (miRNAs) are short 19-25 nucleotide RNA molecules that impact on most biological processes by regulating the efficiency of messenger RNA (mRNA) translation. To date, most research activities have been focused on the control of miRNA expression and its functional consequences. Nonetheless, much remains unknown about the mechanisms affecting the level of specific miRNAs in the cell, a critical feature impacting their regulatory activity. This review focuses on the factors that regulate the abundance of miRNAs, including synthesis, post-transcriptional modifications, nucleases, target binding, and secretion.

Case details: A 19-year-old girl presented to the emergency department following overdose of 10 g of paracetamol on a background history of cystic fibrosis. Paracetamol concentration was below the nomogram line, but was treated with acetylcysteine seven hours post-overdose given her symptomatology. Nineteen hours following her overdose she developed hepatotoxicity, despite early initiation of acetylcysteine. She was discharged well six days post-ingestion. On presentation, delta miRNA-122-miR483 was 20 times that of control patients, however, alanine aminotransferase was normal.Discussion: Patients with cystic fibrosis are more likely to have glutathione deficiency, and greater susceptibility to liver injury. Delta miRNA may be a better detector of early liver injury than hepatic aminotransferases. Empiric treatment with acetylcysteine and serial biochemical reassessment in this setting should be considered.

Paracetamol overdose is common and microRNA (miR)-122 expression is increased with liver injury. We aimed to measure miR-122 in the setting of an abbreviated paracetamol overdose treatment regimen. We compared miRNA expression in patients treated for paracetamol poisoning with an abbreviated 12-h intravenous acetylcysteine regimen (200 mg/kg over 4 h, 50 mg/kg over 8 h) or a 20-h regimen (200 mg/kg over 4 h, 100 mg/kg over 16 h) (NACSTOP trial). miR-122 expression is increased (decreased cycle threshold (Ct) values) with paracetamol liver injury. We assessed miR-122 expression in patients receiving the two acetylcysteine regimens and in a separate group with acute liver injury (ALI). We examined 121 blood samples in 38 patients. After 20 h of acetylcysteine, median alanine transaminase (ALT) was 12 U/L (18, 14) versus 16 U/L (11, 21) ( p = 0.17) and median miR-122 Ct was 30.1 (interquartile range (IQR): 28.9, 33.3) versus 31.4 (28.9, 33.9) ( p = 0.7) in the NACSTOP abbreviated and control groups, respectively. Median normalized miR-122 Ct after 20 h of acetylcysteine was 2.2 (IQR 1.9, 6.4), 1.1 (0.7, 2.9), 63.9 (2.5, 168), 123.2 (40.9, 207.8) in the NACSTOP-abbreviated, NACSTOP-control, ALI and hepatotoxicity groups, respectively. There was no significant difference in ALT or miRNA between NACSTOP treatment groups and no signal of increased liver injury from an abbreviated 12-h acetylcysteine regimen. These findings suggest that an abbreviated acetylcysteine regimen in low-risk patients who have overdosed on paracetamol is safe. Further study is required to validate this finding utilizing miRNA as a comparative biomarker.

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.

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.

Summary 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., Graphical Abstract, Highlights • TDP-43 enters mitochondria, triggers mtDNA release via the mPTP • TDP-43-induced cytosolic mtDNA accumulation activates the cGAS/STING pathway • Evidence of cytoplasmic mtDNA was found in ALS patient cells and disease models • Blocking STING prevents inflammation and neurodegeneration in vitro and in vivo, TDP-43 causes inflammation in ALS by stimulating mitochondrial DNA release, which is subsequently sensed by the cytosolic cGAS/STING pathway, suggesting that inhibition of cGAS/STING could help alleviate inflammation-related damage in ALS.

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.

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.

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.

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. Refereed/Peer-reviewed

Contains fulltext : 153037.pdf (Publisher’s version ) (Closed access) Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Ralpha. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Ralpha impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-kappaB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Ralpha and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.

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.

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.

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.

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.

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.

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. Refereed/Peer-reviewed

Anti-microRNA (miRNA) oligonucleotides (AMOs) with 2'-O-Methyl (2'OMe) residues are commonly used to study miRNA function and can achieve high potency, with low cytotoxicity. Not withstanding this, we demonstrate the sequence-dependent capacity of 2'OMe AMOs to inhibit Toll-like receptor (TLR) 7 and 8 sensing of immunostimulatory RNA, independent of their miRNA-targeting function. Through a screen of 29 AMOs targeting common miRNAs, we found a subset of sequences highly inhibitory to TLR7 sensing in mouse macrophages. Interspecies conservation of this inhibitory activity was confirmed on TLR7/8 activity in human peripheral blood mononuclear cells. Significantly, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 50 AMOs targeted to human miRNAs in miRBaseV20. DNA/locked nucleic acids (LNA) AMOs synthesized with a phosphorothioate backbone also inhibited TLR7 sensing in a sequence-dependent manner, demonstrating that the off-target effects of AMOs are not restricted to 2'OMe modification. Taken together, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account in their therapeutic development and in vivo application.

Aicda is a critical component of antibody class-switching in B cells. In this work, we study the impact of TLR4 activation and IL-10 stimulation on Aicda expression in B cells. Through the global analysis of miRNAs in response to TLR4 activation, in combination with IL-10 stimulation, we identified that IL-10 can suppress TLR4-induced miR-155 expression, an effect that resulted in enhanced Aicda expression. Furthermore, when preventing miR-155 control of Aicda expression, by genetic mutation of its target site in the Aicda mRNA, IL-10 could further potentiate Aicda expression. Given that miR-155 expression is lost, and expression levels of both Aicda and IL-10 are high in diseases, such as Burkitt’s lymphoma, our results suggest a stringent and sophisticated control of Aicda by a novel IL-10/miR-155 axis, where the imbalance of IL-10 and/or miR-155 may contribute to disease pathogenesis.