ZBP1 induces inflammatory signaling via RIPK3 and promotes SARS-CoV-2-induced cytokine expression

COVID-19 caused by the SARS-CoV-2 virus remains a threat to global health. The disease severity is mediated by cell death and inflammation, which regulate both the antiviral and the pathological innate immune responses. ZBP1, an interferon-induced cytosolic nucleic acid sensor, facilitates antiviral responses via RIPK3. Although ZBP1-mediated cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway that depends on ubiquitination and RIPK3’s scaffolding ability independently of cell death. In human cells, ZBP1 associates with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. RIPK1 and ZBP1 are ubiquitinated to promote TAK1- and IKK-mediated inflammatory signaling. Additionally, RIPK1 recruits the p43/41-caspase-8-p43-FLIP heterodimer to suppress RIPK3 kinase activity, which otherwise promotes inflammatory signaling in a kinase activity-dependent manner. Lastly, we show that ZBP1 contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK1-RIPK3-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspase-8. Our results suggest the ZBP1 pathway contributes to inflammation in response to SARS-CoV-2 infection.

Over-expression of TLR3 in human endometrial cancer.

Gynecologic cancers including cancers of the endometrium are a clinical problem (1, 2, 3, 4). We mined published microarray data (5, 6) to discover genes associated with endometrial cancers by comparing transcriptomes of the normal endometrium and endometrial tumors from humans. We identified the double-stranded ribonucleic acid (RNA) sensor TLR3 (7-9) as among the most differentially expressed genes, transcriptome-wide, in cancers of the endometrium. TLR3 was expressed at significantly higher levels in endometrial tumor tissues as compared to the endometrium. However, higher primary tumor expression of TLR3 was correlated with significantly improved overall survival in patients with endometrial cancer. Together, the data reveal over-expression of TLR3 in human endometrial cancer and indicate activation of a viral nucleic acid sensor in cancers of the endometrium.

Protective Role of the Nucleic Acid Sensor STING in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common and severe type of interstitial lung disease for which current treatments display limited efficacy. IPF is largely driven by host-derived danger signals released upon recurrent local tissue damage. Here we explored the roles of self-DNA and stimulator of interferon genes (STING), a protein belonging to an intracellular DNA sensing pathway that leads to type I and/or type III interferon (IFN) production upon activation. Using a mouse model of IPF, we report that STING deficiency leads to exacerbated pulmonary fibrosis with increased collagen deposition in the lungs and excessive remodeling factors expression. We further show that STING-mediated protection does not rely on type I IFN signaling nor on IL-17A or TGF-β modulation but is associated with dysregulated neutrophils. Together, our data support an unprecedented immunoregulatory function of STING in lung fibrosis.

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.

Zα2 domain of ZBP1 is a molecular switch regulating Influenza-induced PANoptosis and perinatal lethality during development

ABSTRACTZ-DNA-binding protein 1 (ZBP1) is an innate nucleic acid sensor which regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating RIPK3, caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we have generated Zbp1ΔZα2/ΔZα2 mice that lack the Zα2 domain of ZBP1 and demonstrate that this domain is critical for influenza A virus (IAV)-induced PANoptosis and perinatal lethality in RIPK1-RHIM mutated (Ripk1RHIM/RHIM) mice. Deletion of the Zα2 domain in ZBP1 abolished IAV-induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue Ripk1RHIM/RHIM mice from the perinatal lethality which is caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in physiological functions and establish a link between sensing of Z-RNAs via the Zα2 domain and the promotion of influenza-induced PANoptosis and perinatal lethality.

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 signalling 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 characterised 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.SummaryDevos, Tanghe et al. find that the recognition of endogenous nucleic acids by the nucleic acid sensor ZBP1 causes necroptosis of RIPK1-deficient keratinocytes. This process drives the development of an inflammatory skin disease characterised by an IL-17 immune response.

ABCF1 regulates dsDNA-induced immune responses in human airway epithelial cells

BackgroundThe airway epithelium represents a critical component of the human lung that helps orchestrate defences against respiratory tract viral infections, which are responsible for more than 2.5 million deaths/year globally. Innate immune activities of the airway epithelium rely Toll-like receptors (TLRs), nucleotide binding and leucine-rich-repeat pyrin domain containing (NLRP) receptors, and cytosolic nucleic acid sensors. ATP Binding Cassette (ABC) transporters are ubiquitous across all three domains of life – Archaea, Bacteria, and Eukarya – and expressed in the human airway epithelium. ABCF1, a unique ABC family member that lacks a transmembrane domain, has been defined as a cytosolic nucleic acid sensor that regulates CXCL10, interferon-β expression, and downstream type I interferon responses. We tested the hypothesis that ABCF1 functions as a dsDNA nucleic acid sensor in human airway epithelial cells important in regulating antiviral responses.MethodsExpression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression was performed in human airway epithelial cells. Functional experiments were performed with siRNA methods in human airway epithelial cells. Complementary transcriptomic analyses were performed to explore the contributions of ABCF1 to gene expression patterns.ResultsUsing archived human lung and human airway epithelial cells, we confirm expression of ABCF1 gene and protein expression in these tissue samples, with a role for mediating CXCL10 production in response to dsDNA viral mimic challenge. Although, ABCF1 knockdown was associated with an attenuation of select genes involved in the antiviral responses, Gene Ontology analyses revealed a greater interaction of ABCF1 with TLR signaling suggesting a multifactorial role for ABCF1 in innate immunity in human airway epithelial cells.ConclusionABCF1 is a candidate cytosolic nucleic acid sensor and modulator of TLR signaling that is expressed at gene and protein levels in human airway epithelial cells. The precise level where ABCF1 protein functions to modulate immune responses to pathogens remains to be determined but is anticipated to involve IRF-3 and CXCL10 production.