Seed sequences mediate off-target activity in the CRISPR-interference system.

The CRISPR interference (CRISPRi) system is a powerful tool for selectively and efficiently silencing genes in functional genomics research applications. However, its off-target activity has not been systematically investigated. Here, we utilized a genome-wide CRISPRi-Cas9 single-guide RNA (sgRNA) library to investigate the presence of off-target activity and its effects on gene expression.

Epigenetic and transcriptional control of gasdermins.

Cells undergo an inflammatory programmed lytic cell death called 'pyroptosis' (with the Greek roots 'fiery'), often featuring morphological hallmarks such as large ballooning protrusions and subsequent bursting. Originally described as a caspase-1-dependent cell death in response to bacterial infection, pyroptosis has since been re-defined in 2018 as a cell death dependent on plasma membrane pores by a gasdermin (GSDM) family member [1,2]. GSDMs form pores in the plasma membrane as well as organelle membranes, thereby initiating membrane destruction and the rapid and lytic demise of a cell.

Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ1. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death.

Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection.

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation.

NINJ1 mediates plasma membrane rupture during lytic cell death.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response. The underlying mechanism of PMR, however, is unknown.

Correction: MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

[This corrects the article DOI: 10.1371/journal.pbio.

MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases.

IRF2 transcriptionally induces expression for pyroptosis.

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1β (IL-1β) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of A forward genetic screen with -ethyl--nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling.

Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death.

Limited proteolysis of gasdermin D (GSDMD) generates an N-terminal pore-forming fragment that controls pyroptosis in macrophages. GSDMD is processed via inflammasome-activated caspase-1 or -11. It is currently unknown whether macrophage GSDMD can be processed by other mechanisms.

A Map of Toll-like Receptor Expression in the Intestinal Epithelium Reveals Distinct Spatial, Cell Type-Specific, and Temporal Patterns.

Signaling by Toll-like receptors (TLRs) on intestinal epithelial cells (IECs) is critical for intestinal homeostasis. To visualize epithelial expression of individual TLRs in vivo, we generated five strains of reporter mice. These mice revealed that TLR expression varied dramatically along the length of the intestine.

Caspase-11 auto-proteolysis is crucial for noncanonical inflammasome activation.

Intracellular LPS sensing by caspase-4/5/11 triggers proteolytic activation of pore-forming gasdermin D (GSDMD), leading to pyroptotic cell death in Gram-negative bacteria-infected cells. Involvement of caspase-4/5/11 and GSDMD in inflammatory responses, such as lethal sepsis, makes them highly desirable drug targets. Using knock-in (KI) mouse strains, we herein provide genetic evidence to show that caspase-11 auto-cleavage at the inter-subunit linker is essential for optimal catalytic activity and subsequent proteolytic cleavage of GSDMD.

ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages.

The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation.

Tissue-Resident Macrophages Are Locally Programmed for Silent Clearance of Apoptotic Cells.

Although apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. Here we identified macrophage populations that continually engulf ACs in distinct tissues and found that these macrophages share characteristics compatible with immunologically silent clearance of ACs; such characteristics include high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. Removal of the macrophages from tissues resulted in loss of many of these characteristics and the ability to generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment program macrophages for silent AC clearance.

The Yersinia pestis Effector YopM Inhibits Pyrin Inflammasome Activation.

Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1β and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation.

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation.

Caspase-11: arming the guards against bacterial infection.

As a front line of defense against pathogenic microbes, our body employs a primitive, yet highly sophisticated and potent innate immune response pathway collectively referred to as the inflammasome. Innate immune cells, epithelial cells, and many other cell types are capable of detecting infection or tissue injury and mounting a coordinated molecular defense. For example, Gram-negative bacteria are specifically detected via a surveillance mechanism that involves activation of extracellular receptors such as Toll-like receptors (TLRs) followed by intracellular recognition and activation of pathways such as caspase-11 (caspase-4/5 in humans).

Toll-like receptor-deficient mice reveal how innate immune signaling influences Salmonella virulence strategies.

Pathogens utilize features of the host response as cues to regulate virulence gene expression. Salmonella enterica serovar Typhimurium (ST) sense Toll-like receptor (TLR)-dependent signals to induce Salmonella Pathogenicity Island 2 (SPI2), a locus required for intracellular replication. To examine pathogenicity in the absence of such cues, we evaluated ST virulence in mice lacking all TLR function (Tlr2(-/-)xTlr4(-/-)xUnc93b1(3d/3d)).

Trafficking of endosomal Toll-like receptors.

Over the past decade we have learned much about nucleic acid recognition by the innate immune system and in particular by Toll-like receptors (TLRs). These receptors localize to endosomal compartments where they are poised to recognize microbial nucleic acids. Multiple regulatory mechanisms function to limit responses to self DNA or RNA, and breakdowns in these mechanisms can contribute to autoimmune or inflammatory disorders.

UNC93B1 mediates differential trafficking of endosomal TLRs.

UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER.

ABIN-1 is a ubiquitin sensor that restricts cell death and sustains embryonic development.

Proteins that directly regulate tumour necrosis factor receptor (TNFR) signalling have critical roles in regulating cellular activation and survival. ABIN-1 (A20 binding and inhibitor of NF-kappaB) is a novel protein that is thought to inhibit NF-kappaB signalling. Here we show that mice deficient for ABIN-1 die during embryogenesis with fetal liver apoptosis, anaemia and hypoplasia.

The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor.

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells.

The ubiquitin-editing enzyme A20 restricts nucleotide-binding oligomerization domain containing 2-triggered signals.

Muramyl dipeptide (MDP), a product of bacterial cell-wall peptidoglycan, activates innate immune cells by stimulating nucleotide-binding oligomerization domain containing 2 (NOD2) -dependent activation of the transcription factor NFkappaB and transcription of proinflammatory genes. A20 is a ubiquitin-modifying enzyme that restricts tumor necrosis factor (TNF) receptor and Toll-like receptor (TLR) -induced signals. We now show that MDP induces ubiquitylation of receptor- interacting protein 2 (RIP2) in primary macrophages.

Homeostatic MyD88-dependent signals cause lethal inflamMation in the absence of A20.

Toll-like receptors (TLRs) on host cells are chronically engaged by microbial ligands during homeostatic conditions. These signals do not cause inflammatory immune responses in unperturbed mice, even though they drive innate and adaptive immune responses when combating microbial infections. A20 is a ubiquitin-modifying enzyme that restricts exogenous TLR-induced signals.