Eva Bartok

Cutting Edge: Reactive Oxygen Species Inhibitors Block Priming, but Not Activation, of the NLRP3 Inflammasome

A common denominator among the multiple damage-inducing agents that ultimately lead to activation of NLRP3 has not yet been identified. Recently, production of reactive oxygen species (ROS) has been suggested to act as a common event upstream of the NLRP3 inflammasome machinery. Because de novo translation of NLRP3 is an essential step in the activation of NLRP3, we investigated the role of substances that inhibit either ROS production or its oxidative activity. Although we observe that NLRP3 inflammasome activation is unique among other known inflammasomes in its sensitivity to ROS inhibition, we have found that this phenomenon is attributable to the fact that NLRP3 strictly requires priming by a proinflammatory signal, a step that is blocked by ROS inhibitors. Although these data do not exclude a general role for ROS production in the process of NLRP3-triggered inflammation, they would put ROS upstream of NLRP3 induction, but not activation.

Inflammasomes: current understanding and open questions

The innate immune system relies on its capability to detect invading microbes, tissue damage, or stress via evolutionarily conserved receptors. The nucleotide-binding domain leucine-rich repeat (NLR)-containing family of pattern recognition receptors includes several proteins that drive inflammation in response to a wide variety of molecular patterns. In particular, the NLRs that participate in the formation of a molecular scaffold termed the “inflammasome” have been intensively studied in past years. Inflammasome activation by multiple types of tissue damage or by pathogen-associated signatures results in the autocatalytic cleavage of caspase-1 and ultimately leads to the processing and thus secretion of pro-inflammatory cytokines, most importantly interleukin (IL)-1β and IL-18. Here, we review the current knowledge of mechanisms leading to the activation of inflammasomes. In particular, we focus on the controversial molecular mechanisms that regulate NLRP3 signaling and highlight recent advancements in DNA sensing by the inflammasome receptor AIM2.

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon–inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells

Inflammasome activation culminates in activation of caspase‐1, which leads to the maturation and subsequent release of cytokines of the interleukin 1 (IL‐1) family and results in a particular form of cell death known as pyroptosis. In addition, in the murine system, a so‐called non‐canonical inflammasome involving caspase‐11 has been described that directly responds to cytosolic LPS. Here, we show that the human monocytic cell line THP1 activates the inflammasome in response to cytosolic LPS in a TLR4‐independent fashion. This response is mediated by caspase‐4 and accompanied by caspase‐1 activation, pyroptosis, and IL‐1β maturation. In addition to caspase‐4, efficient IL‐1β conversion upon intracellular LPS delivery relies on potassium efflux, NLRP3, ASC, and caspase‐1, indicating that although caspase‐4 activation alone is sufficient to induce pyroptosis, this process depends on the NLRP3 inflammasome activation to drive IL‐1β maturation. Altogether, this study provides evidence for the presence of a non‐canonical inflammasome in humans and its dependence on caspase‐4.

iGLuc: a luciferase-based inflammasome and protease activity reporter

Measurement of protease activity in living cells or organisms remains a challenging task. We here present a transgene-encoded biosensor that reports the proteolytic activity of caspase-1 in the course of inflammasome activation and that of other proteases in a highly sensitive and specific manner. This protease reporter is based on the biological activity of a pro–interleukin (IL)-1β–Gaussia luciferase (iGLuc) fusion construct, in which pro–IL-1β–dependent formation of protein aggregates renders GLuc enzyme inactive. Cleavage leads to monomerization of this biosensor protein, resulting in a strong gain in luciferase activity. Exchange of the canonical caspase-1 cleavage site in this reporter construct allows the generation of protease biosensors with additional specificities. The high sensitivity, signal-to-background ratio and specificity of the iGLuc system renders it a useful tool to study proteolytic events in mouse and human cells at high throughput and to monitor protease activity in mice in vivo.

Immunoblotting for Active Caspase-1

Immunoblotting for caspase-1 is the gold-standard method of detecting inflammasome activation. In contrast to IL-1β-based readouts, it can be used in an experimental setup independent of de novo gene expression. Here, we present protocols for the preparation and precipitation of supernatant samples containing activated caspase-1 as well as protocols for polyacrylamide gel electrophoresis (PAGE) and protein immunoblotting.

Type I interferon mediated autoinflammation due to DNase II deficiency

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans.Nucleic acid sensing is important to ensure that an innate immune response is only mounted against microbial nucleic acid. Here, the authors identify loss-of-function mutations in the DNASE2 gene that cause type I interferon-mediated autoinflammation due to enhanced systemic interferon signaling.

An unexpected role for RNA in the recognition of DNA by the innate immune system

A central function of our innate immune system is to sense microbial pathogens by the presence of their nucleic acid genomes or their transcriptional or replicative activity. In mammals, a receptor-‐based system is mainly responsible for the detection of these “non-‐ self” nucleic acids. Tremendous progress has been made in the past years to identify host constituents that are required for this intricate task. With regard to the sensing of RNA genome based pathogens by our innate immune system, a picture is emerging that includes certain families of the toll-‐like receptor family (TLR3, TLR7, TLR8) and the RIG-‐ I like helicases (RIG-‐I, MDA5 and LGP2). Genetic loss of function studies implicate that the absence of these pathways can lead to a complete lack of recognition of certain RNA viruses. At the same time, intracellular DNA can also trigger potent innate immune responses, yet the players in this field are less clear. We and another group recently identified a role for RNA polymerase III in the conversion of AT-‐rich DNA into an RNA ligand that is sensed by the RIG-‐I pathway. In this review article, we will discuss the mechanistics and implications of this novel pathway.

Critical role of nucleotide-binding oligomerization domain-like receptor 3 in vascular repair

Vascular remodeling characterized by hyperproliferative neointima formation is an unfavorable repair process that is triggered by vascular damage. This process is characterized by an increased local inflammatory and proliferative response that critically involves the pro-inflammatory cytokine interleukin-1β (IL-1β). IL-1β is expressed and cytosolically retained as a procytokine that requires additional processing prior to exerting its pro-inflammatory function. Maturation and release of pro IL-1β is governed by a cytosolic protein scaffold that is known as the inflammasome. Here we show that NLRP3 (NOD-like receptor family, pryin domain containing 3), an important activating component of the inflammasome, is involved in neointima formation after vascular injury. NLRP3 deficiency itself does not affect the functional cardiovascular phenotype and does not alter peripheral differential blood counts. However, neointima development following wire injury of the carotid artery was significantly decreased in NLRP3-deficient mice as compared to wild-type controls. In all, NLRP3 plays a non-redundant role in vascular damage mediated neointima formation. Our data establish NLRP3 as a key player in the response to vascular damage, which could open new avenues to therapeutic intervention.

G-rich DNA-induced stress response blocks type-I-IFN but not CXCL10 secretion in monocytes

Excessive inflammation can cause damage to host cells and tissues. Thus, the secretion of inflammatory cytokines is tightly regulated at transcriptional, post-transcriptional and post-translational levels and influenced by cellular stress responses, such as endoplasmic reticulum (ER) stress or apoptosis. Here, we describe a novel type of post-transcriptional regulation of the type-I-IFN response that was induced in monocytes by cytosolic transfection of a short immunomodulatory DNA (imDNA), a G-tetrad forming CpG-free derivative of the TLR9 agonist ODN2216. When co-transfected with cytosolic nucleic acid stimuli (DNA or 3P-dsRNA), imDNA induced caspase-3 activation, translational shutdown and upregulation of stress-induced genes. This stress response inhibited the type-I-IFN induction at the translational level. By contrast, the induction of most type-I-IFN-associated chemokines, including Chemokine (C-X-C Motif) Ligand (CXCL)10 was not affected, suggesting a differential translational regulation of chemokines and type-I-IFN. Pan-caspase inhibitors could restore IFN-β secretion, yet, strikingly, caspase inhibition did not restore global translation but instead induced a compensatory increase in the transcription of IFN-β but not CXCL10. Altogether, our data provide evidence for a differential regulation of cytokine release at both transcriptional and post-transcriptional levels which suppresses type-I-IFN induction yet allows for CXCL10 secretion during imDNA-induced cellular stress.