Who and how, DNA sensors in NETs-driven inflammation.

During infections, neutrophil extracellular traps act like a meshwork of molecules that captures microbes. In contrast, during sterile inflammation the presence of NETs is usually associated with tissue damage and uncontrolled inflammation. In this context, DNA acts both as activator of NETs formation and immunogenic molecule fueling inflammation within the injured tissue microenvironment. Pattern recognition receptors that specifically bind to and get activated by DNA such as Toll-like receptor-9 (TLR9), cyclic GMP-AMP synthase (cGAS), Nod-like receptor protein 3 (NLRP3) and Absence in Melanoma-2 (AIM2) have been reported to play a role in NETs formation and detection. However, how these DNA sensors contribute to NETs-driven inflammation is not well understood. Whether these DNA sensors have unique roles or on the contrary they are mostly redundant is still elusive. In this review, we summarize the known contribution of the above DNA sensors to the formation and detection of NETs in the context of sterile inflammation. We also highlight scientific gaps needed to be addressed and propose future direction for therapeutic targets.

Type-I Interferon Signaling in Fanconi Anemia.

Fanconi Anemia (FA) is a genome instability syndrome caused by mutations in one of the 23 repair genes of the Fanconi pathway. This heterogenous disease is usually characterized by congenital abnormalities, premature ageing and bone marrow failure. FA patients also show a high predisposition to hematological and solid cancers. The Fanconi pathway ensures the repair of interstrand crosslinks (ICLs) DNA damage. Defect in one of its proteins prevents functional DNA repair, leading to the accumulation of DNA breaks and genome instability. Accumulating evidence has documented a close relationship between genome instability and inflammation, including the production of type-I Interferon. In this context, type-I Interferon is produced upon activation of pattern recognition receptors by nucleic acids including by the cyclic GMP-AMP synthase (cGAS) that detects DNA. In mouse models of diseases displaying genome instability, type-I Interferon response is responsible for an important part of the pathological symptoms, including premature aging, short stature, and neurodegeneration. This is illustrated in mouse models of Ataxia-telangiectasia and Aicardi-Goutières Syndrome in which genetic depletion of either Interferon Receptor IFNAR, cGAS or STING relieves pathological symptoms. FA is also a genetic instability syndrome with symptoms such as premature aging and predisposition to cancer. In this review we will focus on the different molecular mechanisms potentially leading to type-I Interferon activation. A better understanding of the molecular mechanisms engaging type-I Interferon signaling in FA may ultimately lead to the discovery of new therapeutic targets to rescue the pathological inflammation and premature aging associated with Fanconi Anemia.

Biological Investigation of Amaryllidaceae Alkaloid Extracts from the Bulbs of Pancratium trianthum Collected in the Senegalese Flora.

Amaryllidaceae plants are rich in alkaloids with biological properties. Pancratium trianthum is an Amaryllidaceae species widely used in African folk medicine to treat several diseases such as central nervous system disorders, tumors, and microbial infections, and it is used to heal wounds. The current investigation explored the biological properties of alkaloid extracts from bulbs of P. trianthum collected in the Senegalese flora. Alkaloid extracts were analyzed and identified by chromatography and mass spectrometry. Alkaloid extracts from P. trianthum displayed pleiotropic biological properties. Cytotoxic activity of the extracts was determined on hepatocarcinoma Huh7 cells and on acute monocytic leukemia THP-1 cells, while agar diffusion and microdilution assays were used to evaluate antibacterial activity. Antiviral activity was measured by infection of extract-treated cells with dengue virus (DENVGFP) and human immunodeficiency virus-1 (HIV-1GFP) reporter vectors. Cytotoxicity and viral inhibition were the most striking of P. trianthum's extract activities. Importantly, non-cytotoxic concentrations were highly effective in completely preventing DENVGFP replication and in reducing pseudotyped HIV-1GFP infection levels. Our results show that P. trianthum is a rich source of molecules for the potential discovery of new treatments against various diseases. Herein, we provide scientific evidence to rationalize the traditional uses of P. trianthum for wound treatment as an anti-dermatosis and antiseptic agent.

The cost of Medicare-funded medical and pharmaceutical services for mental disorders in children and adolescents in Australia.

OBJECTIVE: To examine the health care costs associated with mental disorders and subthreshold mental disorders within a nationally representative sample of children and adolescents in Australia. METHOD: Data were derived from the Young Minds Matter Survey (N = 6,310). Mental disorders were classified using the Diagnostic Interview Schedule for Children Version IV. Participant data were linked to administrative data on health care costs. Adjusted generalized linear regression models and two-part models were used to estimate mean differences in costs between those with a mental disorder or subthreshold disorder and those without. RESULTS: Costs associated with health care attendances and medications were higher for children and adolescents with mental disorders and subthreshold mental disorders compared to those without a mental disorder. The additional population health care costs due to mental disorders amounted to AUD$234 million annually in children and adolescents, of which approximately 16% was attributed to out-of-pocket costs. Findings showed that those with subthreshold mental disorders or comorbid mental disorders have substantial additional costs of Medicare-funded medical and pharmaceutical services. CONCLUSION AND IMPLICATION: Mental disorders in children and adolescents are associated with significant health care costs. Further research is needed to ensure that this population is receiving effective and efficient care.

Pharmacological Targeting of STING-Dependent IL-6 Production in Cancer Cells.

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)-ß. 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.

Connexin-Dependent Transfer of cGAMP to Phagocytes Modulates Antiviral Responses.

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.IMPORTANCE 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.

The Use of CRISPR/Cas9 Gene Editing to Confirm Congenic Contaminations in Host-Pathogen Interaction Studies.

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.

Assessing the cGAS-cGAMP-STING Activity of Cancer Cells.

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.

Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses.

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.

cGAS-STING Activation in the Tumor Microenvironment and Its Role in Cancer Immunity.

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.

Activation of cGAS-dependent antiviral responses by DNA intercalating agents.

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.

Cre-dependent DNA recombination activates a STING-dependent innate immune response.

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.

TWEAK Negatively Regulates Human Dicer.

The ribonuclease Dicer plays a central role in the microRNA pathway by processing microRNA precursors (pre-microRNAs) into microRNAs, a class of 19- to 24-nucleotide non-coding RNAs that regulate expression of ≈60% of the genes in humans. To gain further insights into the function and regulation of Dicer in human cells, we performed a yeast two-hybrid (Y2HB) screen using human Dicer double-stranded RNA-binding domain (dsRBD) as bait. This approach identified tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) as a Dicer-interacting protein candidate. Confocal immunofluorescence microscopy revealed the colocalization of Dicer and TWEAK proteins at the perinuclear region of HeLa cells. The Dicer-TWEAK protein interaction was confirmed by coimmunoprecipitation and found not likely to be mediated by RNA. TWEAK dose-dependently reduced pre-microRNA conversion into mature microRNA in Dicer activity assays using extracts of transfected human HEK 293 cells. TWEAK expression also impaired microRNA-guided RNA silencing of a reporter gene induced by a pre-microRNA. These findings suggest a role for TWEAK-a pro-inflammatory cytokine-in regulating Dicer function and microRNA biogenesis, and its possible involvement in regulating gene expression during inflammatory processes and diseases.

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain.

Dicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) in the vast majority of eukaryotes. In human, Dicer has been shown to interact with cellular proteins via its N-terminal domain. Here, we demonstrate the ability of Dicer C-terminus to interact with 5-lipoxygenase (5LO), an enzyme involved in the biosynthesis of inflammatory mediators, in vitro and in cultured human cells. Yeast two-hybrid and GST binding assays delineated the smallest 5-lipoxygenase binding domain (5LObd) of Dicer to its C-terminal 140 amino acids comprising the double-stranded RNA (dsRNA) binding domain (dsRBD). The Dicer 5LObd-5LO association was disrupted upon Ala substitution of Trp residues 13, 75 and 102 in 5LO, suggesting that the Dicer 5LObd may recognize 5LO via its N-terminal C2-like domain. Whereas a catalytically active 5LObd-containing Dicer fragment was found to enhance 5LO enzymatic activity in vitro, human 5LO modified the miRNA precursor processing activity of Dicer. Providing a link between miRNA-mediated regulation of gene expression and inflammation, our results suggest that the formation of miRNAs may be regulated by 5LO in leukocytes and cancer cells expressing this lipoxygenase.

Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites.

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots). These hypervariable sites were located at strategic positions on the protein: on each side of the conserved glycine residues in the N-terminal trunk, within the first and second inhibitory loops entering the active site of target enzymes, and surrounding the larfav motif, a sequence of unknown function conserved among plant cystatins. Supporting the assumption that positively selected, hypervariable sites are indicative of amino acid sites implicated in functional diversity, mutants of the 8th cystatin unit of tomato multicystatin including alternative residues at positively selected sites in the N-terminal trunk exhibited highly variable affinities for the cysteine proteases papain, cathepsin B and cathepsin H. Overall, these observations support the hypothesis that plant cystatins have been under selective pressure to evolve in response to predatory challenges by herbivorous enemies. They also indicate the potential of site-directed mutagenesis at positively selected sites for the generation of cystatins with improved binding properties.