Sensing of endogenous nucleic acids by ZBP1 induces keratinocyte necroptosis and skin inflammation

Aberrant detection of endogenous nucleic acids by the immune system can cause inflammatory disease. The scaffold function of the signaling kinase RIPK1 limits spontaneous activation of the nucleic acid sensor ZBP1. Consequently, loss of RIPK1 in keratinocytes induces ZBP1-dependent necroptosis and skin inflammation. Whether nucleic acid sensing is required to activate ZBP1 in RIPK1-deficient conditions and which immune pathways are associated with skin disease remained open questions. Using knock-in mice with disrupted ZBP1 nucleic acid–binding activity, we report that sensing of endogenous nucleic acids by ZBP1 is critical in driving skin pathology characterized by antiviral and IL-17 immune responses. Inducing ZBP1 expression by interferons triggers necroptosis in RIPK1-deficient keratinocytes, and epidermis-specific deletion of MLKL prevents disease, demonstrating that cell-intrinsic events cause inflammation. These findings indicate that dysregulated sensing of endogenous nucleic acid by ZBP1 can drive inflammation and may contribute to the pathogenesis of IL-17–driven inflammatory skin conditions such as psoriasis

Activation and viral escape of ZBP1-mediated host cell death

Nucleïnezuursensoren zijn cruciaal om virale infecties te herkennen. Om herkenning van zowel DNA als RNA virussen te garanderen kunnen we terugvallen op een scala aan sensoren. In mijn proefschrift richtte ik me op de karakterisatie van Z-DNA Binding Proteïne 1 (ZBP1), een nucleïnezuursensor die geen onderscheid maakt tussen RNA of DNA, maar nucleïnezuren herkent op basis van hun conformatie. In tegenstelling tot de meeste andere sensoren kan ZBP1 efficiënt celdood gaan induceren en daarmee geïnfecteerde cellen verwijderen. Het onderzoek gepresenteerd in deze thesis toont aan dat ZBP1 na virale infectie RNA liganden herkent, ongeacht het genoom van het virus. Interactie tussen ZBP1 en zijn liganden resulteert in de vorming van stabiele ZBP1-aggregaten. Deze aggregaten faciliteren interactie met kinase eiwitten RIPK1 en RIPK3 waarmee ZBP1 verschillende celdoodmodaliteiten kan aansturen, zoals apoptose en necroptose. Om virale replicatie te verzekeren blokkeert Herpes Simplex Virus 1 ZBP1-afhankelijke celdood via het eiwit ICP6. In deze dissertatie werd de ICP6-afhankelijke necroptose inhibitie verder gekarakteriseerd. Directe interactie van ICP6 met RIPK1, RIPK3 en ZBP1 voorkomt rekrutering van RIPK1 en RIPK3 door ZBP1-aggregaten en kan zo celdood blokkeren. Samenvattend, ZBP1 detectie van nucleïnezuren veroorzaakt ZBP1-aggregaten die efficiënt RIPK1 en RIPK3 kunnen aanwerven, wat kan worden voorkomen door virale eiwit ICP6

Viral manipulation of host cell necroptosis and pyroptosis

Cell death forms an essential component of the antiviral immune response. Viral infection elicits different forms of host cell death, including the lytic and inflammatory cell death modes necroptosis or pyroptosis. The induction of both types of cell death not only eliminates virus-infected cells but also contributes to the development of innate and adaptive immunity through the release of inflammatory mediators. The importance of necroptosis and pyroptosis in host defence is evident from the numerous viral evasion mechanisms that suppress these cell death pathways. Here, we review the emerging principles by which viruses antagonise host cell necroptosis and pyroptosis to promote their spread and block host immunity

Tyramide signal amplification for the immunofluorescent staining of ZBP1-dependent phosphorylation of RIPK3 and MLKL after HSV-1 infection in human cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis

ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)(.) In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutieres syndrome (AGS). In mice, complete loss of ADAR1 activity is embryonically lethal, and mutations similar to those found in patients with AGS cause autoinflammation. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Z alpha domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Z alpha domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function

GTF3A mutations in a patient with herpes simplex encephalitis reveals a novel role in viral immunity by transcribing 5S rRNA pseudogenes serving as RIG-I ligands

Herpes simplex virus type 1 (HSV-1) is one of the most common human pathogens affecting several billion people worldwide. In rare cases, the virus causes herpes simplex encephalitis (HSE). In approximately 5% of patients with HSE monogenic defects of TLR3/IFN-I signaling have been described, emphasising the role of primary immunodeficiencies underlying HSE pathogenesis. Whole exome sequencing in a patient with HSE revealed compound heterozygous loss-of function mutations in GTF3A, located in one of the conserved cysteine residues of the C2H2 zinc finger. GTF3A mutants demonstrated impaired 5S rDNA-binding ability. HSV-1 infection of primary patient fibroblasts and HEK293T GTF3A knock-in clones showed increased viral replication. ChIP-Seq analysis in search of alternative targets of GTF3A identified the recently described RIG-I ligand RNA5SP141. We showed that GTF3A mutations abrogate RNA5SP141 expression with decreased RIG-I activation which ultimately resulted in an impaired type I IFN defense against HSV-1 infection

GTF3A mutations predispose to herpes simplex encephalitis by disrupting biogenesis of the host-derived RIG-I ligand RNA5SP141

Herpes simplex virus 1 (HSV-1) infects several billion people worldwide and can cause life-threatening herpes simplex encephalitis (HSE) in some patients. Monogenic defects in components of the type I interferon system have been identified in patients with HSE, emphasizing the role of inborn errors of immunity underlying HSE pathogenesis. Here, we identify compound heterozygous loss-of-function mutations in the gene GTF3A encoding for transcription factor IIIA (TFIIIA), a component of the RNA polymerase III complex, in a patient with common variable immunodeficiency and HSE. Patient fibroblasts and GTF3A gene–edited cells displayed impaired HSV-1–induced innate immune responses and enhanced HSV-1 replication. Chromatin immunoprecipitation sequencing analysis identified the 5S ribosomal RNA pseudogene 141 (RNA5SP141), an endogenous ligand of the RNA sensor RIG-I, as a transcriptional target of TFIIIA. GTF3A mutant cells exhibited diminished RNA5SP141 expression and abrogated RIG-I activation upon HSV-1 infection. Our work unveils a crucial role for TFIIIA in transcriptional regulation of a cellular RIG-I agonist and shows that GTF3A genetic defects lead to impaired cell-intrinsic anti–HSV-1 responses and can predispose to HSE