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Inherited deficiency of the RNA lariat-debranching enzyme 1 (DBR1) is a rare etiology of brainstem viral encephalitis. The cellular basis of disease and the range of viral predisposition are unclear. We report inherited DBR1 deficiency in a 14-year-old boy who suffered from isolated SARS-CoV-2 brainstem encephalitis. The patient is homozygous for a previously reported hypomorphic and pathogenic DBR1 variant (I120T). Consistently, DBR1 I120T/I120T fibroblasts from affected individuals from this and another unrelated kindred have similarly low levels of DBR1 protein and high levels of RNA lariats. DBR1 I120T/I120T human pluripotent stem cell (hPSC)-derived hindbrain neurons are highly susceptible to SARS-CoV-2 infection. Exogenous WT DBR1 expression in DBR1 I120T/I120T fibroblasts and hindbrain neurons rescued the RNA lariat accumulation phenotype. Moreover, expression of exogenous RNA lariats, mimicking DBR1 deficiency, increased the susceptibility of WT hindbrain neurons to SARS-CoV-2 infection. Inborn errors of DBR1 impair hindbrain neuron-intrinsic antiviral immunity, predisposing to viral infections of the brainstem, including that by SARS-CoV-2., (© 2024 Chan et al.)

Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5' exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2',3'-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here, we show that IKKβ, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a phospholipid enriched on the trans-Golgi network. NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKβ recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKβ-mediated NEK7-independent pathway constitutes the predominant NLRP3 priming mechanism in human myeloid cells. Our results suggest that PI4P binding represents a primed state into which NLRP3 is brought by IKKβ activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Necroptosis is a form of regulated cell death that can occur downstream of several immune pathways. While previous studies have shown that dysregulated necroptosis can lead to strong inflammatory responses, little is known about the identity of the endogenous molecules that trigger these responses. Using a reductionist in vitro model, we found that soluble TNF is strongly released in the context of necroptosis. On the one hand, necroptosis promotes TNF translation by inhibiting negative regulatory mechanisms acting at the post-transcriptional level. On the other hand, necroptosis markedly enhances TNF release by activating ADAM proteases. In studying TNF release at single-cell resolution, we found that TNF release triggered by necroptosis is activated in a switch-like manner that exceeds steady-state TNF processing in magnitude and speed. Although this shedding response precedes massive membrane damage, it is closely associated with lytic cell death. Further, we found that lytic cell death induction using a pore-forming toxin also triggers TNF shedding, indicating that the activation of ADAM proteases is not strictly related to the necroptotic pathway but likely associated with biophysical changes of the cell membrane upon lytic cell death. These results demonstrate that lytic cell death, particularly necroptosis, is a critical trigger for TNF release and thus qualify TNF as a necroptosis-associated alarmin., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pinci, Gaidt, Jung, Nagl, Kuut and Hornung.)

Approximately 70 million humans worldwide are affected by chronic hepatitis D, which rapidly leads to liver cirrhosis and hepatocellular carcinoma due to chronic inflammation. The triggers and consequences of this chronic inflammation, induced by co-infection with the hepatitis D virus (HDV) and the hepatitis B virus (HBV), are poorly understood. Using CRISPR technology, we characterized the recognition of HDV mono- and co-infection by intracellular innate immunity and determined its influence on the viral life cycle and effector T-cell responses using different HBV and HDV permissive hepatoma cell lines. We showed that HDV infection is detected by MDA5 and -after a lag phase -induces a profound type I interferon response in the infected cells. The type I interferon response, however, was not able to suppress HDV replication or spread, thus providing a persistent trigger. Using engineered T-cells directed against the envelope proteins commonly used by HBV and HDV, we found that HDV immune recognition enhanced T-cell cytotoxicity. Interestingly, the T-cell effector function was enhanced independently of antigen presentation. These findings help to explain immune mediated tissue damage in chronic hepatitis D patients and indicate that combining innate triggers with T-cell activating therapies might allow for a curative approach.

TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Pinci et al.)

Detection of cytosolic DNA constitutes a central event in the context of numerous infectious and sterile inflammatory conditions. Recent studies have uncovered a bipartite mode of cytosolic DNA recognition, in which the cGAS-STING axis triggers antiviral immunity, whereas AIM2 triggers inflammasome activation. Here, we show that AIM2 is dispensable for DNA-mediated inflammasome activation in human myeloid cells. Instead, detection of cytosolic DNA by the cGAS-STING axis induces a cell death program initiating potassium efflux upstream of NLRP3. Forward genetics identified regulators of lysosomal trafficking to modulate this cell death program, and subsequent studies revealed that activated STING traffics to the lysosome, where it triggers membrane permeabilization and thus lysosomal cell death (LCD). Importantly, the cGAS-STING-NLRP3 pathway constitutes the default inflammasome response during viral and bacterial infections in human myeloid cells. We conclude that targeting the cGAS-STING-LCD-NLRP3 pathway will ameliorate pathology in inflammatory conditions that are associated with cytosolic DNA sensing., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Legionella pneumophila is a pathogen widespread in aquatic environment, able to multiply both within amoebae and human macrophages. The aim of this study was to identify genes differently expressed in a spontaneous avirulent Legionella pneumophila serogroup 6 mutant, named Vir-, respect the parental strain (Vir+), and to determine their role in the loss of virulence. Protein profiles revealed some differences in Vir- proteomic maps, and among the identified proteins the undetectable 3-hydroxybutyrate dehydrogenase (BdhA) and a down-produced lipase. Both Legionella enzymes were studied before and were here further characterized at genetic level. A significant down-regulation of both genes was observed in Vir- at the transcriptional level, but the use of defined mutants demonstrated that they did not affect the intracellular multiplication. A mutant (MS1) showed an accumulation of poly-3-hydroxybutyrate (PHB) granules suggesting a role of bdhA gene in its degradation process. The lipase deduced amino acid sequence revealed a catalytic triad, typical of the 'lipase box' characteristic of PHB de-polymerase enzymes, that let us suppose a possible involvement of lipase in the PHB granule degradation process. Our results revealed unexpected alterations in secondary metabolic pathways possibly linking the loss of virulence to Legionella lack of energy sources., (© 2014 APMIS. Published by John Wiley & Sons Ltd.)