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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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.
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.
Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and ...macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.
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•Mice lacking pyroptosis and apoptosis cannot control Salmonella infection•Macrophages lacking pyroptosis and apoptosis resist Salmonella-induced killing•Caspase-1 kills Salmonella-infected cells by activating GSDMD, BID, or other caspases•Caspase-1 and -8 act as cell death executioners when all cell death effectors are lost
The clearance of intracellular pathogens requires the killing of infected cells, but it remains unclear why host cells have so many different means of inducing programmed cell death. Doerflinger et al. demonstrate that interconnectivity between pyroptosis and apoptosis involving flexible deployment of caspases ensures control of Salmonella infection in mice.
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.
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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.
Lipopolysaccharide (LPS), which is produced by Gram-negative bacteria, is a powerful activator of innate immune responses. LPS binds to the proteins Toll-like receptor 4 (TLR4) and MD2 to activate ...pro-inflammatory signalling pathways. The TLR4-MD2 receptor complex is crucial for the host recognition of Gram-negative bacterial infection, and pathogens have devised many strategies to evade or manipulate TLR4-MD2 activity. The TLR4-MD2 signalling pathway is therefore potentially an important therapeutic target. This Progress article focuses on recent exciting data that have revealed the structural basis of TLR4-MD2 recognition of LPS.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Since first being described in the fruit fly Drosophila melanogaster, Toll-like receptors (TLRs) have proven to be of great interest to immunologists and investigators interested in the molecular ...basis to inflammation. They recognize pathogen-derived factors and also products of inflamed tissue, and trigger signaling pathways that lead to activation of transcription factors such as nuclear factor-kappaB and the interferon regulatory factors. These in turn lead to induction of immune and inflammatory genes, including such important cytokines as tumor necrosis factor-alpha and type I interferon. Much evidence points to a role for TLRs in immune and inflammatory diseases and increasingly in cancer. Examples include clear roles for TLR4 in sepsis, rheumatoid arthritis, ischemia/reperfusion injury, and allergy. TLR2 has been implicated in similar pathologic conditions and also in systemic lupus erythematosus (SLE) and tumor metastasis. TLR7 has also been shown to be important in SLE. TLR5 has been shown to be radioprotective. Recent advances in our understanding of signaling pathways activated by TLRs, structural insights into TLRs bound to their ligands and antagonists, and approaches to inhibit TLRs (including antibodies, peptides, and small molecules) are providing possiblemeans by which to interfere with TLRs clinically. Here we review these recent advances and speculate about whether manipulating TLRs is likely to be successful in fighting off different diseases.
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