A complete understanding of phagocytosis requires insight into both its biochemical and physical aspects. One of the ways to explore the physical mechanism of phagocytosis is to probe whether and how ...the target properties (e.g., size, shape, surface states, stiffness, etc.) affect their uptake. Here we report an imaging-based method to explore phagocytosis kinetics, which is compatible with real-time imaging and can be used to validate existing reports using fixed and stained cells. We measure single-event engulfment time from a large number of phagocytosis events to compare how size and shape of targets determine their engulfment. The data shows an increase in the average engulfment time for increased target size, for spherical particles. The uptake time data on nonspherical particles confirms that target shape plays a more dominant role than target size for phagocytosis: Ellipsoids with an eccentricity of 0.954 and much smaller surface areas than spheres were taken up five times more slowly than spherical targets.
Signal transduction by the Toll-like receptors (TLRs) is central to host defence against many pathogenic microorganisms and also underlies a large burden of human disease. Thus, the mechanisms and ...regulation of signalling by TLRs are of considerable interest. In this Review, we discuss the molecular basis for the recognition of pathogen-associated molecular patterns, the nature of the protein complexes that mediate signalling, and the way in which signals are regulated and integrated at the level of allosteric assembly, post-translational modification and subcellular trafficking of the components of the signalling complexes. These fundamental molecular mechanisms determine whether the signalling output leads to a protective immune response or to serious pathologies such as sepsis. A detailed understanding of these processes at the molecular level provides a rational framework for the development of new drugs that can specifically target pathological rather than protective signalling in inflammatory and autoimmune disease.
Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon ...lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.
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•LPS induces mitochondrial repurposing from ATP synthesis to ROS production•Oxidation of succinate and mitochondrial hyperpolarization drive ROS production•Blocking LPS-induced ROS production or hyperpolarization inhibits IL-1β•SDH is critical for the inflammatory response
To support their pro-inflammatory function, activated macrophages repurpose their mitochondria, switching from ATP production to ROS generation.
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•Many bacteria induce gasdermin-driven host cell pyroptosis which can be a mechanism for removal of the pathogen.•Gasdermin D driven pyroptosis may be important in driving the severe ...inflammation seen in sepsis.•Gasdermins have direct and indirect anti-bacterial actions.•The diversity in gasdermin expression between animal species may have important implications for zoonotic pathogen carriage.
The discovery of pyroptosis and its subsequent implications in infection and immunity has uncovered a new angle of host-defence against pathogen assault. At its most simple, gasdermin-mediated pyroptosis in bacterial infection would be expected to remove pathogens from the relative safety of the cytosol or pathogen containing vacuole/phagosome whilst inducing a rapid and effective immune response. Differences in gasdermin-mediated pyroptosis between cell types, stimulation conditions, pathogen and even animal species, however, make things more complex. The excessive inflammation associated with the pathogen-induced gasdermin-mediated pyroptosis contributes to a downward spiral in sepsis. With no currently approved effective treatment options for sepsis understanding how gasdermin-mediated pyroptotic pathways are regulated provides an opportunity to identify novel therapeutic candidates against this complex disease. In this review we cover recent advances in the field of gasdermin-mediated pyroptosis with a focus on bacterial infection and sepsis models in the context of humans and other animal species. Importantly we also consider why there is considerable redundancy set into these ancient immune pathways.
Germline-encoded pattern recognition receptors (PRRs) sense microbial or endogenous products released from damaged or dying cells and trigger innate immunity. In most cases, sensing of these signals ...is coupled to signal transduction pathways that lead to transcription of immune response genes that combat infection or lead to cell death. Members of the NOD-like receptor (NLR) family assemble into large multiprotein complexes, termed inflammasomes. Inflammasomes do not regulate transcription of immune response genes, but activate caspase-1, a proteolytic enzyme that cleaves and activates the secreted cytokines interleukin-1β and interleukin-18. Inflammasomes also regulate pyroptosis, a caspase-1-dependent form of cell death that is highly inflammatory. Here, we review exciting recent developments on the role of inflammasome complexes in host defense and the discovery of a new DNA sensing inflammasome, and describe important progress made in our understanding of how inflammasomes are activated. Additionally, we highlight how dysregulation of inflammasomes contributes to human disease.
Abstract Devices implanted into the body become encapsulated due to a foreign body reaction. In the central nervous system (CNS), this can lead to loss of functionality in electrodes used to treat ...disorders. Around CNS implants, glial cells are activated, undergo gliosis and ultimately encapsulate the electrodes. The primary cause of this reaction is unknown. Here we show that the mechanical mismatch between nervous tissue and electrodes activates glial cells. Both primary rat microglial cells and astrocytes responded to increasing the contact stiffness from physiological values ( G ′ ∼ 100 Pa) to shear moduli G ′ ≥ 10 kPa by changes in morphology and upregulation of inflammatory genes and proteins. Upon implantation of composite foreign bodies into rat brains, foreign body reactions were significantly enhanced around their stiff portions in vivo . Our results indicate that CNS glial cells respond to mechanical cues, and suggest that adapting the surface stiffness of neural implants to that of nervous tissue could minimize adverse reactions and improve biocompatibility.
Pathogen recognition by nucleotide-binding oligomerization domain-like receptor (NLR) results in the formation of a macromolecular protein complex (inflammasome) that drives protective inflammatory ...responses in the host. It is thought that the number of inflammasome complexes forming in a cell is determined by the number of NLRs being activated, with each NLR initiating its own inflammasome assembly independent of one another; however, we show here that the important foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) simultaneously activates at least two NLRs, whereas only a single inflammasome complex is formed in a macrophage. Both nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 are simultaneously present in the same inflammasome, where both NLRs are required to drive IL-1β processing within the Salmonella -infected cell and to regulate the bacterial burden in mice. Superresolution imaging of Salmonella -infected macrophages revealed a macromolecular complex with an outer ring of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain and an inner ring of NLRs, with active caspase effectors containing the pro–IL-1β substrate localized internal to the ring structure. Our data reveal the spatial localization of different components of the inflammasome and how different members of the NLR family cooperate to drive robust IL-1β processing during Salmonella infection.
Abstract
The IUPHAR/BPS Guide to PHARMACOLOGY (GtoPdb, www.guidetopharmacology.org) and its precursor IUPHAR-DB, have captured expert-curated interactions between targets and ligands from selected ...papers in pharmacology and drug discovery since 2003. This resource continues to be developed in conjunction with the International Union of Basic and Clinical Pharmacology (IUPHAR) and the British Pharmacological Society (BPS). As previously described, our unique model of content selection and quality control is based on 96 target-class subcommittees comprising 512 scientists collaborating with in-house curators. This update describes content expansion, new features and interoperability improvements introduced in the 10 releases since August 2015. Our relationship matrix now describes ∼9000 ligands, ∼15 000 binding constants, ∼6000 papers and ∼1700 human proteins. As an important addition, we also introduce our newly funded project for the Guide to IMMUNOPHARMACOLOGY (GtoImmuPdb, www.guidetoimmunopharmacology.org). This has been 'forked' from the well-established GtoPdb data model and expanded into new types of data related to the immune system and inflammatory processes. This includes new ligands, targets, pathways, cell types and diseases for which we are recruiting new IUPHAR expert committees. Designed as an immunopharmacological gateway, it also has an emphasis on potential therapeutic interventions.
Infection of human macrophages with
serovar Typhimurium (
Typhimurium) leads to inflammasome activation. Inflammasomes are multiprotein complexes facilitating caspase-1 activation and subsequent ...gasdermin D-mediated cell death and IL-1β and IL-18 cytokine release. The NAIP/NLRC4 inflammasome is activated by multiple bacterial protein ligands, including flagellin from the flagellum and the needle protein PrgI from the
Typhimurium type III secretion system. In this study, we show that transfected ultrapure flagellin from
Typhimurium induced cell death and cytokine secretion in THP-1 cells and primary human monocyte-derived macrophages. In THP-1 cells, NAIP/NLRC4 and NLRP3 played redundant roles in inflammasome activation during infection with
Typhimurium. Knockout of
or
in THP-1 cells revealed that flagellin, but not PrgI, now activated the NLRP3 inflammasome through a reactive oxygen species- and/or cathepsin-dependent mechanism that was independent of caspase-4/5 activity. In conclusion, our data suggest that NLRP3 can be activated by flagellin to act as a "safety net" to maintain inflammasome activation under conditions of suboptimal NAIP/NLRC4 activation, as observed in THP-1 cells, possibly explaining the redundant role of NLRP3 and NAIP/NLRC4 during
Typhimurium infection.
Streptococcus pneumoniae is an important cause of fatal pneumonia in humans. These bacteria express virulence factors, such as the toxins pneumolysin and autolysin, that drive host inflammatory ...responses. In this study we confirm loss of pneumolysin and autolysin function in a group of clonal pneumococci that have a chromosomal deletion resulting in a pneumolysin-autolysin fusion gene Δ(lytA'-ply')593. The Δ(lytA'-ply')593 pneumococci strains naturally occur in horses and infection is associated with mild clinical signs. Here we use immortalized and primary macrophage in vitro models, which include pattern recognition receptor knock-out cells, and a murine acute pneumonia model to show that a Δ(lytA'-ply')593 strain induces cytokine production by cultured macrophages, however, unlike the serotype-matched ply+lytA+ strain, it induces less tumour necrosis factor α (TNFα) and no interleukin-1β production. The TNFα induced by the Δ(lytA'-ply')593 strain requires MyD88 but, in contrast to the ply+lytA+ strain, is not reduced in cells lacking TLR2, 4 or 9. In comparison to the ply+lytA+ strain in a mouse model of acute pneumonia, infection with the Δ(lytA'-ply')593 strain resulted in less severe lung pathology, comparable levels of interleukin-1α, but minimal release of other pro-inflammatory cytokines, including interferon-γ, interleukin-6 and TNFα. These results suggest a mechanism by which a naturally occurring Δ(lytA'-ply')593 mutant strain of S. pneumoniae that resides in a non-human host has reduced inflammatory and invasive capacity compared to a human S. pneumoniae strain. These data probably explain the relatively mild clinical disease in response to S. pneumoniae infection seen in horses in comparison to humans.
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