Nigericin-Triggered Phosphodynamics in Inflammasome Formation and Pyroptosis.

Innate immune signaling relies heavily on phosphorylation cascades to mount effective immune responses. Although traditional innate immune signaling cascades following TLR4 stimulation have been investigated through a temporally quantitative phosphoproteomic lens, far fewer studies have applied these methods to distinct signaling following the inflammasome trigger leading to IL-1β release. Here, we conducted time-resolved phosphoproteomic profiling to investigate kinase signaling downstream of the inflammasome trigger nigericin.

Bactericidal antibiotic treatment induces damaging inflammation via TLR9 sensing of bacterial DNA.

The immunologic consequences of using bactericidal versus bacteriostatic antibiotic treatments are unclear. We observed a bacteriostatic (growth halting) treatment was more protective than a bactericidal (bacteria killing) treatment in a murine peritonitis model. To understand this unexpected difference, we compared macrophage responses to bactericidal treated bacteria or bacteriostatic treated bacteria.

Efficient electroporation in primary cells with PEDOT:PSS electrodes.

Precise and efficient delivery of macromolecules into cells enhances basic biology research and therapeutic applications in cell therapies, drug delivery, and personalized medicine. While pulsed electric field electroporation effectively permeabilizes cell membranes to deliver payloads without the need for toxic chemical or viral transduction agents, conventional bulk electroporation devices face major challenges with cell viability and heterogeneity due to variations in fields generated across cells and electrochemistry at the electrode-electrolyte interface. Here, we introduce the use of microfabricated electrodes based on the conducting polymer poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS), which substantially increases cell viability and transfection efficiency.

Identification of age-specific gene regulators of La Crosse virus neuroinvasion and pathogenesis.

One of the key events in viral encephalitis is the ability of virus to enter the central nervous system (CNS). Several encephalitic viruses, including La Crosse Virus (LACV), primarily induce encephalitis in children, but not adults. This phenomenon is also observed in LACV mouse models, where the virus gains access to the CNS of weanling animals through vascular leakage of brain microvessels, likely through brain capillary endothelial cells (BCECs).

Biphasic JNK signaling reveals distinct MAP3K complexes licensing inflammasome formation and pyroptosis.

Kinase signaling in the tiered activation of inflammasomes and associated pyroptosis is a prime therapeutic target for inflammatory diseases. While MAPKs subsume pivotal roles during inflammasome priming, specifically the MAP3K7/JNK1/NLRP3 licensing axis, their involvement in successive steps of inflammasome activation is poorly defined. Using live-cell MAPK biosensors to focus on the inflammasome triggering event allowed us to identify a subsequent process of biphasic JNK activation.

Small-molecule screening identifies Syk kinase inhibition and rutaecarpine as modulators of macrophage training and SARS-CoV-2 infection.

Biologically active small molecules can impart modulatory effects, in some cases providing extended long-term memory. In a screen of biologically active small molecules for regulators of tumor necrosis factor (TNF) induction, we identify several compounds with the ability to induce training effects on human macrophages. Rutaecarpine shows acute and long-term modulation, enhancing lipopolysaccharide (LPS)-induced pro-inflammatory cytokine secretion and relieving LPS tolerance in human macrophages.

Absolute protein quantitation of the mouse macrophage Toll-like receptor and chemotaxis pathways.

The Toll-like receptor (TLR) and chemotaxis pathways are key components of the innate immune system. Subtle variation in the concentration, timing, and molecular structure of the ligands are known to affect downstream signaling and the resulting immune response. Computational modeling and simulation at the molecular interaction level can be used to study complex biological pathways, but such simulations require protein concentration values as model parameters.

Lentivirus-mediated Conditional Gene Expression.

The ability to identify the role of a particular gene within a system is dependent on control of the expression of that gene. In this protocol, we describe a method for stable, conditional expression of Nod-Like receptors (NLRs) in THP-1 cells using a lentiviral expression system. This system combines all the necessary components for tetracycline-inducible gene expression in a single lentivector with constitutive co-expression of a selection marker, which is an efficient means for controlling gene expression using a single viral infection of cells.

Author Correction: Commensal-driven immune zonation of the liver promotes host defence.

A Correction to this paper has been published: https://doi.org/10.1038/s41586-021-03346-0.

Correction: Type I IFNs facilitate innate immune control of the opportunistic bacteria Burkholderia cenocepacia in the macrophage cytosol.

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

A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation.

Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes.

Species-Specific Endotoxin Stimulus Determines Toll-Like Receptor 4- and Caspase 11-Mediated Pathway Activation Characteristics.

The innate immune system is the body's first line of defense against pathogens and its protection against infectious diseases. On the surface of host myeloid cells, Toll-like receptor 4 (TLR4) senses lipopolysaccharide (LPS), the major outer membrane component of Gram-negative bacteria. Intracellularly, LPS is recognized by caspase 11 through the noncanonical inflammasome to induce pyroptosis-an inflammatory form of lytic cell death.

Lipid regulation of NLRP3 inflammasome activity through organelle stress.

Inflammation driven by the NLRP3 inflammasome in macrophages is an important contributor to chronic metabolic diseases that affect growing numbers of individuals. Many of these diseases involve the pathologic accumulation of endogenous lipids or their oxidation products, which can activate NLRP3. Other endogenous lipids, however, can inhibit the activation of NLRP3.

Age influences susceptibility of brain capillary endothelial cells to La Crosse virus infection and cell death.

Background: A key factor in the development of viral encephalitis is a virus crossing the blood-brain barrier (BBB). We have previously shown that age-related susceptibility of mice to the La Crosse virus (LACV), the leading cause of pediatric arbovirus encephalitis in the USA, was associated with the ability of the virus to cross the BBB. LACV infection in weanling mice (aged around 3 weeks) results in vascular leakage in the olfactory bulb/tract (OB/OT) region of the brain, which is not observed in adult mice aged > 6-8 weeks.

SIGNAL: A web-based iterative analysis platform integrating pathway and network approaches optimizes hit selection from genome-scale assays.

Hit selection from high-throughput assays remains a critical bottleneck in realizing the potential of omic-scale studies in biology. Widely used methods such as setting of cutoffs, prioritizing pathway enrichments, or incorporating predicted network interactions offer divergent solutions yet are associated with critical analytical trade-offs. The specific limitations of these individual approaches and the lack of a systematic way by which to integrate their rankings have contributed to limited overlap in the reported results from comparable genome-wide studies and costly inefficiencies in secondary validation efforts.

Single-tube genotyping for small insertion/deletion mutations: simultaneous identification of wild type, mutant and heterozygous alleles.

Current methods of genotyping small insertion/deletion (indel) mutations are costly, laborious, and can be unreliable. To address this, we have developed a method for small indel genotyping in a single polymerase chain reaction, with wild-type, heterozygous and mutant alleles distinguishable by band pattern in routine agarose gel electrophoresis. We demonstrate this method with multiple genes to distinguish 10 bp, 4 bp and even 1 bp deletions from the wild type.

Type I IFNs facilitate innate immune control of the opportunistic bacteria Burkholderia cenocepacia in the macrophage cytosol.

The mammalian immune system is constantly challenged by signals from both pathogenic and non-pathogenic microbes. Many of these non-pathogenic microbes have pathogenic potential if the immune system is compromised. The importance of type I interferons (IFNs) in orchestrating innate immune responses to pathogenic microbes has become clear in recent years.

A small sustained increase in NOD1 abundance promotes ligand-independent inflammatory and oncogene transcriptional responses.

Small, genetically determined differences in transcription [expression quantitative trait loci (eQTLs)] are implicated in complex diseases through unknown molecular mechanisms. Here, we showed that a small, persistent increase in the abundance of the innate pathogen sensor NOD1 precipitated large changes in the transcriptional state of monocytes. A ~1.

Commensal-driven immune zonation of the liver promotes host defence.

The liver connects the intestinal portal vasculature with the general circulation, using a diverse array of immune cells to protect from pathogens that translocate from the gut. In liver lobules, blood flows from portal triads that are situated in periportal lobular regions to the central vein via a polarized sinusoidal network. Despite this asymmetry, resident immune cells in the liver are considered to be broadly dispersed across the lobule.

The cAMP Pathway Amplifies Early MyD88-Dependent and Type I Interferon-Independent LPS-Induced Interleukin-10 Expression in Mouse Macrophages.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine, secreted by macrophages and other immune cells to attenuate inflammation. Autocrine type I interferons (IFNs) largely mediate the delayed expression of IL-10 by LPS-stimulated macrophages. We have previously shown that IL-10 is synergistically expressed in macrophages following a costimulus of a TLR agonist and cAMP.

NF-κB Signaling in Macrophages: Dynamics, Crosstalk, and Signal Integration.

The nuclear factor-κB (NF-κB) signaling pathway is one of the best understood immune-related pathways thanks to almost four decades of intense research. NF-κB signaling is activated by numerous discrete stimuli and is a master regulator of the inflammatory response to pathogens and cancerous cells, as well as a key regulator of autoimmune diseases. In this regard, the role of NF-κB signaling in immunity is not unlike that of the macrophage.