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.
CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a ...CRISPR-associated transposase from cyanobacteria
(ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the
genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.
Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve ...specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.
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•Tagmentation-based tag integration site sequencing (TTISS) scalably detects DSBs•TTISS is a rapid and streamlined protocol compatible with multiplexing•Application of TTISS highlights trade-off in Cas9 variant specificity and activity•LZ3 Cas9 variant exhibits a unique +1 insertion profile
Schmid-Burgk et al. develop tagmentation-based tag integration site sequencing (TTISS), a rapid, streamlined protocol for analyzing double-strand breaks such as those created by CRISPR nucleases. Using TTISS, they comprehensively assess Cas9 variants, revealing a trade-off between specificity and activity and identifying LZ3 Cas9, a variant with a unique +1 insertion profile.
Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. ...The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses.
Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a ...special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericin-induced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3.
Cytosolic detection of microbial products is essential for the initiation of an innate immune response against intracellular pathogens such as Mycobacterium tuberculosis (Mtb). During Mtb infection ...of macrophages, activation of cytosolic surveillance pathways is dependent on the mycobacterial ESX-1 secretion system and leads to type I interferon (IFN) and interleukin-1β (IL-1β) production. Whereas the inflammasome regulates IL-1β secretion, the receptor(s) responsible for the activation of type I IFNs has remained elusive. We demonstrate that the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) is essential for initiating an IFN response to Mtb infection. cGAS associates with Mtb DNA in the cytosol to stimulate cyclic GAMP (cGAMP) synthesis. Notably, activation of cGAS-dependent cytosolic host responses can be uncoupled from inflammasome activation by modulating the secretion of ESX-1 substrates. Our findings identify cGAS as an innate sensor of Mtb and provide insight into how ESX-1 controls the activation of specific intracellular recognition pathways.
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•The DNA sensor cGAS is essential for type I IFN induction in response to Mtb infection•cGAS aggregates and colocalizes with DNA in the cytosol of Mtb-infected cells•Mtb ESX-1 secretion system mutants abrogate cGAS-mediated type I IFN responses•Modulation of the ESX-1 system uncouples IFN production from IL-1β responses to Mtb
The receptor(s) responsible for type I IFNs activation in response to Mycobacterium tuberculosis (Mtb) infection has remained elusive. Wassermann et al. find that intracellular Mtb DNA sensing by the cytosolic sensor cGAS drives type I IFN induction upon infection, and mutations in the Mtb ESX-1 secretion system abrogate this mechanism.
The innate immune defence of multicellular organisms against microbial pathogens requires cellular collaboration. Information exchange allowing immune cells to collaborate is generally attributed to ...soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferons (IFNs), serve to alert non-infected cells to the possibility of pathogen challenge. Moreover, in conjunction with chemokines they can instruct specialized immune cells to contain and eradicate microbial infection. Several receptors and signalling pathways exist that couple pathogen sensing to the induction of cytokines, whereas cytosolic recognition of nucleic acids seems to be exquisitely important for the activation of type I IFNs, master regulators of antiviral immunity. Cytosolic DNA is sensed by the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), which catalyses the synthesis of the second messenger cGAMP(2'-5'). This molecule in turn activates the endoplasmic reticulum (ER)-resident receptor STING, thereby inducing an antiviral state and the secretion of type I IFNs. Here we find in murine and human cells that cGAS-synthesized cGAMP(2'-5') is transferred from producing cells to neighbouring cells through gap junctions, where it promotes STING activation and thus antiviral immunity independently of type I IFN signalling. In line with the limited cargo specificity of connexins, the proteins that assemble gap junction channels, most connexins tested were able to confer this bystander immunity, thus indicating a broad physiological relevance of this local immune collaboration. Collectively, these observations identify cGAS-triggered cGAMP(2'-5') transfer as a novel host strategy that serves to rapidly convey antiviral immunity in a transcription-independent, horizontal manner.
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.
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•DNA inflammasome activation in human monocytes depends on NLRP3, but not AIM2•cGAS-STING signals independently of its type I IFN response upstream of NLRP3•STING activation orchestrates a lysosomal cell death program that engages NLRP3•The cGAS-STING-NLRP3 axis is the default DNA inflammasome in human myeloid cells
In humans, a cGAS-STING-lysosomal cell death-NLRP3 pathway is responsible for the inflammasome response to bacterial and viral DNA with AIM2 being dispensable.
Interleukin-1β (IL-1β) is a cytokine whose bioactivity is controlled by activation of the inflammasome. However, in response to lipopolysaccharide, human monocytes secrete IL-1β independently of ...classical inflammasome stimuli. Here, we report that this constituted a species-specific response that is not observed in the murine system. Indeed, in human monocytes, lipopolysaccharide triggered an “alternative inflammasome” that relied on NLRP3-ASC-caspase-1 signaling, yet was devoid of any classical inflammasome characteristics including pyroptosome formation, pyroptosis induction, and K+ efflux dependency. Genetic dissection of the underlying signaling pathway in a monocyte transdifferentiation system revealed that alternative inflammasome activation was propagated by TLR4-TRIF-RIPK1-FADD-CASP8 signaling upstream of NLRP3. Importantly, involvement of this signaling cascade was limited to alternative inflammasome activation and did not extend to classical NLRP3 activation. Because alternative inflammasome activation embraces both sensitivity and promiscuity of TLR4, we propose a pivotal role for this signaling cascade in TLR4-driven, IL-1β-mediated immune responses and immunopathology in humans.
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•LPS by itself triggers IL-1β secretion in human, but not in murine monocytes•Human monocytes activate an alternative inflammasome in response to LPS•This proceeds independently of K+ efflux, pyroptosome formation, and pyroptosis•Alternative but not classical inflammasome signals via TLR4-TRIF-RIPK1-FADD-CASP8
How human monocytes secrete processed IL-1β upon LPS challenge is unknown. Hornung and colleagues report that LPS triggers an alternative NLRP3 inflammasome pathway in human monocytes. Unlike classical NLRP3 inflammasome signaling, alternative inflammasome activation proceeds independently of potassium efflux, pyroptosome formation, and pyroptosis, while it engages TLR4-TRIF-RIPK1-FADD-CASP8 upstream of NLRP3.
Optical Pooled Screens in Human Cells Feldman, David; Singh, Avtar; Schmid-Burgk, Jonathan L. ...
Cell,
10/2019, Letnik:
179, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of ...complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.
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•In situ sequencing of perturbations or barcodes enables image-based pooled screens•p65 translocation is assayed by imaging in fixed and live cell pools•Pooled live-cell screen identifies MED12 and MED24 as negative regulators of NF-κB
A screening approach that combines high-content imaging with in situ sequencing can identify genes that affect spatially and temporally defined phenotypes like morphology and subcellular localization, expanding the list of scientific questions that can be asked with genetic tools.
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