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Human bone marrow failure (BMF) syndromes result from the loss of hematopoietic stem and progenitor cells (HSPC), and this loss has been attributed to cell death; however, the cell death triggers, and mechanisms remain unknown. During BMF, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) increase. These ligands are known to induce necroptosis, an inflammatory form of cell death mediated by RIPK1, RIPK3, and MLKL. We previously discovered that mice with a hematopoietic RIPK1 deficiency (Ripk1HEM KO) exhibit inflammation, HSPC loss, and BMF, which is partially ameliorated by a RIPK3 deficiency; however, whether RIPK3 exerts its effects through its function in mediating necroptosis or other forms of cell death remains unclear. Here, we demonstrate that similar to a RIPK3 deficiency, an MLKL deficiency significantly extends survival and like Ripk3 deficiency partially restores hematopoiesis in Ripk1HEM KO mice revealing that both necroptosis and apoptosis contribute to BMF in these mice. Using mouse models, we show that the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) is up-regulated in mouse RIPK1-deficient bone marrow cells and that ZBPl's function in endogenous nucleic acid sensing is necessary for HSPC death and contributes to BMF. We also provide evidence that IFNγ mediates HSPC death in Ripk1HEM KO mice, as ablation of IFNγ but not TNFα receptor signaling significantly extends survival of these mice. Together, these data suggest that RIPK1 maintains hematopoietic homeostasis by preventing ZBP1 activation and induction of HSPC death. [ABSTRACT FROM AUTHOR]

ZBP1 is an interferon‐induced cytosolic nucleic acid sensor that facilitates antiviral responses via RIPK3. Although ZBP1‐mediated programmed cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1‐induced inflammatory signaling pathway mediated by K63‐ and M1‐linked ubiquitin chains, which depends on RIPK1 and RIPK3 as scaffolds independently of cell death. In human HT29 cells, ZBP1 associated with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. ZBP1‐induced K63‐ and M1‐linked ubiquitination of RIPK1 and ZBP1 to promote TAK1‐ and IKK‐mediated inflammatory signaling and cytokine production. Inhibition of caspase activity suppressed ZBP1‐induced cell death but enhanced cytokine production in a RIPK1‐ and RIPK3 kinase activity‐dependent manner. Lastly, we provide evidence that ZBP1 signaling contributes to SARS‐CoV‐2‐induced cytokine production. Taken together, we describe a ZBP1‐RIPK3‐RIPK1‐mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspases, which may induce inflammation when ZBP1 is activated below the threshold needed to trigger a cell death response. Synopsis: ZBP1 is a nucleic acid sensor that induces cell death via RIPK3. This study describes a ZBP1‐induced inflammatory signaling pathway that is independent of cell death and is mediated by non‐degradative ubiquitin chains, and RIPK1 and RIPK3 as scaffolds. ZBP1 stimulates signaling and cytokine production independently of cell death.ZBP1 signaling relies on the scaffolding functions of RIPK3 and RIPK1, but not their kinase activities.ZBP1 signaling is mediated by K63‐ and M1‐linked ubiquitin chains generated by cIAPs and LUBAC.ZBP1 contributes to cytokine production after SARS‐CoV‐2 infection. [ABSTRACT FROM AUTHOR]

Varicella‐Zoster virus (VZV) causes chickenpox and shingles. Although the infection is associated with severe morbidity in some individuals, molecular mechanisms that determine innate immune responses remain poorly defined. We found that the cGAS/STING DNA sensing pathway was required for type I interferon (IFN) induction during VZV infection and that recognition of VZV by cGAS restricted its replication. Screening of a VZV ORF expression library identified the essential VZV tegument protein ORF9 as a cGAS antagonist. Ectopically or virally expressed ORF9 bound to endogenous cGAS leading to reduced type I IFN responses to transfected DNA. Confocal microscopy revealed co‐localisation of cGAS and ORF9. ORF9 and cGAS also interacted directly in a cell‐free system and phase‐separated together with DNA. Furthermore, ORF9 inhibited cGAMP production by cGAS. Taken together, these results reveal the importance of the cGAS/STING DNA sensing pathway for VZV recognition and identify a VZV immune antagonist that partially but directly interferes with DNA sensing via cGAS. Synopsis: Molecular mechanisms that determine innate immune responses to Varicella‐Zoster Virus (VZV) infection remain poorly defined. This work reveals a key role for the cGAS/STING DNA sensing pathway, which is countered by a viral immunosuppressor. The DNA sensor cGAS and its adaptor STING detect VZV infection and induce type I interferons in infected THP1 cells.The VZV protein ORF9 blocks DNA sensing by cGAS.ORF9 interacts with cGAS and inhibits the synthesis of the second messenger cGAMP.ORF9 and cGAS phase‐separate together with DNA in vitro. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2006.09.005 Byline: Jan Rehwinkel (1), Jeroen Raes (1), Elisa Izaurralde (1)(2) Abstract: Recent genome-wide identification of nonsense-mediated mRNA decay (NMD) targets in yeast, fruitfly and human cells has provided insight into the biological functions and evolution of this mRNA quality control mechanism, revealing that NMD post-transcriptionally regulates an important fraction of the transcriptome. NMD targets are associated with a broad range of biological processes, but most of these targets are not encoded by orthologous genes across different species. Yeast and fruitfly NMD effectors regulate common targets in concert, but parallel pathways have evolved in humans, whereby NMD effectors have acquired additional functions. Thus, the phenotypic differences observed across species after inhibition of NMD are driven not only by the functional diversification of NMD effectors but also by changes in the repertoire of regulated genes. Author Affiliation: (1) EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany (2) MPI for Developmental Biology, Spemannstrasse 35, D-72076 Tubingen, Germany

Hypoxia is a common phenomenon in solid tumours strongly linked to the hallmarks of cancer. Hypoxia promotes local immunosuppression and downregulates type I interferon (IFN) expression and signalling, which contribute to the success of many cancer therapies. Double-stranded RNA (dsRNA), transiently generated during mitochondrial transcription, endogenously activates the type I IFN pathway. We report the effects of hypoxia on the generation of mitochondrial dsRNA (mtdsRNA) in breast cancer. We found a significant decrease in dsRNA production in different cell lines under hypoxia. This effect was HIF1α/2α-independent. mtdsRNA was responsible for induction of type I IFN and significantly decreased after hypoxia. Mitochondrially encoded gene expression was downregulated and mtdsRNA bound by the dsRNA-specific J2 antibody was decreased during hypoxia. These findings reveal a new mechanism of hypoxia-induced immunosuppression that could be targeted by hypoxia-activated therapies. [ABSTRACT FROM AUTHOR]

Cyclic GMP‐AMP (cGAMP) is an immunostimulatory molecule produced by cGAS that activates STING. cGAMP is an adjuvant when administered alongside antigens. cGAMP is also incorporated into enveloped virus particles during budding. Here, we investigate whether inclusion of cGAMP within viral vaccine vectors enhances their immunogenicity. We immunise mice with virus‐like particles (VLPs) containing HIV‐1 Gag and the vesicular stomatitis virus envelope glycoprotein G (VSV‐G). cGAMP loading of VLPs augments CD4 and CD8 T‐cell responses. It also increases VLP‐ and VSV‐G‐specific antibody titres in a STING‐dependent manner and enhances virus neutralisation, accompanied by increased numbers of T follicular helper cells. Vaccination with cGAMP‐loaded VLPs containing haemagglutinin induces high titres of influenza A virus neutralising antibodies and confers protection upon virus challenge. This requires cGAMP inclusion within VLPs and is achieved at markedly reduced cGAMP doses. Similarly, cGAMP loading of VLPs containing the SARS‐CoV‐2 Spike protein enhances Spike‐specific antibody titres. cGAMP‐loaded VLPs are thus an attractive platform for vaccination. SYNOPSIS: cGAMP, a cyclic di‐nucleotide, is an adjuvant and activates STING. This study shows that cGAMP‐loaded virus‐like particles, designed to deliver antigen and cGAMP to the same antigen‐presenting cell, are an attractive platform for vaccination. cGAMP‐loading of virus‐like particles (VLPs) enhances both humoral and cellular immune responses upon immunisation.cGAMP‐VLPs induce protective responses to viral antigens like influenza A virus haemagglutinin and SARS‐CoV‐2 Spike.cGAMP‐loading of viral‐vectored vaccines may allow dose‐sparing. [ABSTRACT FROM AUTHOR]

Virus infection in mammals elicits a variety of defense responses that are initiated by signals from virus-sensing receptors expressed by the host. These receptors include the ubiquitously expressed RIG-I--like receptor (RLR) family of RNA helicases. RLRs are cytoplasmic proteins that act in cell-intrinsic antiviral defense by recognizing RNAs indicative of virus presence. Here, we highlight recent progress in understanding how RLRs discriminate between the RNA content of healthy versus virus-infected ceils, functioning as accurate sensors of virus invasion. 10.1126/science.1185068

Human cells respond to infection by SARS-CoV-2, the virus that causes COVID-19, by producing cytokines including type I and III interferons (IFNs) and proinflammatory factors such as IL6 and TNF. IFNs can limit SARS-CoV-2 replication but cytokine imbalance contributes to severe COVID-19. We studied how cells detect SARS-CoV-2 infection. We report that the cytosolic RNA sensor MDA5 was required for type I and III IFN induction in the lung cancer cell line Calu-3 upon SARS-CoV-2 infection. Type I and III IFN induction further required MAVS and IRF3. In contrast, induction of IL6 and TNF was independent of the MDA5-MAVS-IRF3 axis in this setting. We further found that SARS-CoV-2 infection inhibited the ability of cells to respond to IFNs. In sum, we identified MDA5 as a cellular sensor for SARS-CoV-2 infection that induced type I and III IFNs. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is the most severe form of chronic lung fibrosis. Circulating monocytes have been implicated in immune pathology in IPF but their phenotype is unknown. In this work, we determined the immune phenotype of monocytes in IPF using multi-colour flow cytometry, RNA sequencing and corresponding serum factors, and mapped the main findings to amount of lung fibrosis and single cell transcriptomic landscape of myeloid cells in IPF lungs. We show that monocytes from IPF patients displayed increased expression of CD64 (FcγR1) which correlated with amount of lung fibrosis, and an amplified type I IFN response ex vivo. These were accompanied by markedly raised CSF-1 levels, IL-6, and CCL-2 in serum of IPF patients. Interrogation of single cell transcriptomic data from human IPF lungs revealed increased proportion of CD64hi monocytes and "transitional macrophages" with higher expression of CCL-2 and type I IFN genes. Our study shows that monocytes in IPF patients are phenotypically distinct from age-matched controls, with a primed type I IFN pathway that may contribute to driving chronic inflammation and fibrosis. These findings strengthen the potential role of monocytes in the pathogenesis of IPF. [ABSTRACT FROM AUTHOR]

Cell‐autonomous sensing of nucleic acids is essential for host defence against invading pathogens by inducing antiviral and inflammatory cytokines. cGAS has emerged in recent years as a non‐redundant DNA sensor important for detection of many viruses and bacteria. Upon binding to DNA, cGAS synthesises the cyclic dinucleotide 2′3′‐cGAMP that binds to the adaptor protein STING and thereby triggers IRF3‐ and NFκB‐dependent transcription. In addition to infection, the pathophysiology of an ever‐increasing number of sterile inflammatory conditions in humans involves the recognition of DNA through cGAS. Consequently, the cGAS/STING signalling axis has emerged as an attractive target for pharmacological modulation. However, the development of cGAS and STING inhibitors has just begun and a need for specific and effective compounds persists. In this review, we focus on cGAS and explore how its activation by immunostimulatory DNA is regulated by cellular mechanisms, viral immune modulators and small molecules. We further use our knowledge of cGAS modulation by cells and viruses to conceptualise potential new ways of pharmacological cGAS targeting. [ABSTRACT FROM AUTHOR]

Sterile alpha motif and histidine‐aspartic acid domain‐containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi–Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c‐Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break‐point junctions is a notable feature in SAMHD1 deficiency during activation‐induced cytidine deaminase‐mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double‐stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1‐mediated dNTP balance regulates dNTP‐sensitive DNA end‐processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway. Synopsis: Sterile alpha motif and histidine‐aspartic acid domain‐containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase (dNTPase) that protects cells from viral infection and is frequently mutated in cancers and Aicardi‐Goutières syndrome. Here, we demonstrate that depletion of nucleotide by SAMHD1 promotes activation induced cytidine deaminase (AID)‐dependent antibody class switch recombination (CSR) and genomic instability in B cells. SAMHD1 associates with immunoglobulin switch regions, and promotes CSR and aberrant IgH/c‐Myc translocations.The dNTPase activity of SAMHD1 but not its interaction with CtIP is required for efficient CSR.The depletion of SAMHD1 impairs end‐joining of AID‐ or Cas9 nickase‐induced staggered DSBs.Accumulation of dNTPs upon SAMHD1 depletion likely modulates the activities of dNTP‐sensitive end‐repair enzymes which interfere with the normal ligation program and leads to end‐joining by pathways that lead to nucleotide insertions. [ABSTRACT FROM AUTHOR]

The coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a world health concern and can cause severe disease and high mortality in susceptible groups. While vaccines offer a chance to treat disease, prophylactic and anti-viral treatments are still of vital importance, especially in context of the mutative ability of this group of viruses. Therefore, it is essential to elucidate the molecular mechanisms of viral entry, innate sensing and immune evasion of SARS-CoV-2, which control the triggers of the subsequent excessive in- flammatory response. Viral evasion strategies directly target anti-viral immunity, counteracting host restriction factors and hijacking signalling pathways to interfere with interferon production. In Part I of this review, we examine SARS-CoV-2 viral entry and the described immune evasion mechanisms to provide a perspective on how the failure in initial viral sensing by infected cells can lead to immune dysregulation causing fatal COVID-19, discussed in Part II. [ABSTRACT FROM AUTHOR]

Toll‐like receptor 7 (TLR7) is an innate immune sensor for single‐strand RNA (ssRNA). Recent structural analysis revealed that TLR7 has an additional binding site for nucleosides such as guanosine, and is activated when both guanosine and ssRNA bind. The nucleoside binding site also accommodates imidazoquinoline derivatives such as R848, which activate TLR7 in the absence of ssRNA. Here, we report that deoxyguanosine (dG) triggered cytokine production in murine bone marrow derived macrophages and plasmacytoid dendritic cells, as well as in human peripheral blood mononuclear cells, including type I interferons and pro‐inflammatory factors such as TNF and IL‐6. This signalling activity of dG was dependent on TLR7 and its adaptor MyD88 and did not require amplification via the type I interferon receptor. dG‐triggered cytokine production required endosomal maturation but did not depend on the concurrent provision of RNA. We conclude that dG induces an inflammatory response through TLR7 and propose that dG is an RNA‐independent TLR7 agonist. [ABSTRACT FROM AUTHOR]

A study was undertaken to observe target-specific requirements for enhancer of decapping in microRNAs (miRNA)-mediated gene silencing. Results revealed that in the absence of active translation certain miRNA targets were stabilized, some continued to be degraded in a miRNA-dependent manner besides suggesting that miRNAs mediate gene silencing through more than one mechanism.