Retroviral Danger from Within: TLR7 Is in Control

In this issue of Immunity, Yu et al. (2012) outline a fascinating model in which TLR7-mediated antibody production acts as a dominant immunosurveillance mechanism against endogenous retroviruses (ERVs), with additional support of TLR3 and TLR9 that function to prevent ERV-mediated malignancy

DNA sensing unchained

In two recent reports in Science, James Chen and colleagues provide compelling evidence that detection of cytosolic DNA triggers the production of a novel second messenger, cyclic GMP-AMP (cGAMP), which in turn activates a signaling pathway that induces type I interferons (IFNs) in a STING-dependent manner. They further unravel a key role for a so far uncharacterized murine protein E330016A19 (human homolog: C6ORF150), now termed cGAMP synthetase (cGAS), to act as the DNA sensor that generates cGAMP

CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism

The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled CRISPaint (CRISPR-assisted insertion tagging) that allows precise and efficient integration of large heterologous DNA cassettes into eukaryotic genomes. CRISPaint makes use of the CRISPR-Cas9 system to introduce a double-strand break (DSB) at a user-defined genomic location. A universal donor DNA, optionally provided as minicircle DNA, is cleaved simultaneously to be integrated at the genomic DSB, while processing the donor plasmid at three possible positions allows flexible reading-frame selection. Applying this system allows to create C-terminal tag fusions of endogenously encoded proteins in human cells with high efficiencies. Knocking out known DSB repair components reveals that site-specific insertion is completely dependent on canonical NHEJ (DNA-PKcs, XLF and ligase-4). A large repertoire of modular donor vectors renders CRISPaint compatible with a wide array of applications

Tumor necrosis factor is a necroptosis-associated alarmin

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

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 through 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 in identifying the 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 have 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 mechanisms and implications of this novel pathway

AIM2 inflammasome-derived IL-1 beta induces postoperative ileus in mice

Postoperative ileus (POI) is an intestinal dysmotility frequently occurring after abdominal surgery. An orchestrated neuroimmune response within the muscularis externa (ME) involves activation of resident macrophages, enteric glia and infiltration of blood-derived leukocytes. Interleukin-1 receptor type-I (IL1R1) signalling on enteric glia has been shown to be involved in POI development. Herein we investigated the distinct role of the IL1R1 ligands interleukin (IL)-1 alpha and IL-1 beta and focused on the mechanism of IL-1 beta production. IL-1 alpha and IL-1 beta deficient mice were protected from POI. Bone-marrow transplantation studies indicated that IL-1 alpha originated from radio-resistant cells while IL-1 beta was released from the radio-sensitive infiltrating leukocytes. Mouse strains deficient in inflammasome formation identified the absent in melanoma 2 (AIM2) inflammasome to be crucial for IL-1 beta production in POI. Mechanistically, antibiotic-treated mice revealed a prominent role of the microbiome in IL-1 beta production. Our study provides new insights into distinct roles of IL-1 alpha and IL-1 beta signalling during POI. While IL-1 alpha release is most likely an immediate passive response to the surgical trauma, IL-1 beta production depends on AIM2 inflammasome formation and the microbiome. Selective interaction in this pathway might be a promising target to prevent POI in surgical patients

Listeria monocytogenes is sensed by the NLRP3 and AIM2 inflammasome

The inflammasome pathway functions to regulate caspase-1 activation in response to a broad range of stimuli. Caspase-1 activation is required for the maturation of the pivotal pro-inflammatory cytokines of the pro-IL-1beta family. In addition, caspase-1 activation leads to a certain type of cell death known as pyroptosis. Activation of the inflammasome has been shown to play a critical role in the recognition and containment of various microbial pathogens, including the intracellularly replicating Listeria monocytogenes; however, the inflammasome pathways activated during L. monocytogenes infection are only poorly defined. Here, we demonstrate that L. monocytogenes activates both the NLRP3 and the AIM2 inflammasome, with a predominant involvement of the AIM2 inflammasome. In addition, L. monocytogenes-triggered cell death was diminished in the absence of both AIM2 and NLRP3, and is concomitant with increased intracellular replication of L. monocytogenes. Altogether, these data establish a role for DNA sensing through the AIM2 inflammasome in the detection of intracellularly replicating bacteria

Cre-dependent DNA recombination activates a STING-dependent innate immune response

Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies