Despite the wealth of genomic and transcriptomic data in Parkinson’s disease (PD), the initial molecular events are unknown. Using LD score regression analysis, we show significant enrichment in PD ...heritability within regulatory sites for LPS-activated monocytes and that TLR4 expression is highest within human substantia nigra, the most affected brain region, suggesting a role for TLR4 inflammatory responses. We then performed extended incubation of cells with physiological concentrations of small alpha-synuclein oligomers observing the development of a TLR4-dependent sensitized inflammatory response with time, including TNF-α production. ROS and cell death in primary neuronal cultures were significantly reduced by TLR4 antagonists revealing that an indirect inflammatory mechanism involving cytokines produced by glial cells makes a major contribution to neuronal death. Prolonged exposure to low levels of alpha-synuclein oligomers sensitizes TLR4 responsiveness in astrocytes and microglial, explaining how they become pro-inflammatory, and may be an early causative event in PD.
Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that ...sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3β or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies.
Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is ...thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling.
Infection of the bovine endometrium with Gram-negative bacteria commonly causes uterine disease. Toll-like receptor 4 (TLR4) on cells of the immune system bind Gram-negative bacterial ...lipopolysaccharide (LPS), stimulating the secretion of the proinflammatory cytokines interleukin 1B (IL1B) and IL6, and the chemokine IL8. Because the endometrium is the first barrier to infection of the uterus, the signaling cascade triggered by LPS and the subsequent expression of inflammatory mediators were investigated in endometrial epithelial and stromal cells, and the key pathways identified using short interfering RNA (siRNA) and biochemical inhibitors. Treatment of endometrial cells with ultrapure LPS stimulated an inflammatory response characterized by increased IL1B, IL6, and IL8 mRNA expression, and IL6 protein accumulation in epithelial cells, and by increased IL1B and IL8 mRNA expression, and IL6 and IL8 protein accumulation in stromal cells. Treatment of endometrial cells with LPS also induced the degradation of IKB and the nuclear translocation of NFKB, as well as rapid phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) and MAPK14. Knockdown of TLR4 or its signaling adaptor molecule, myeloid differentiation factor 88 (MYD88), using siRNA reduced the inflammatory response to LPS in epithelial and stromal cells. Biochemical inhibition of MAPK3/1, but not JNK or MAPK14, reduced LPS-induced IL1B, IL6, and IL8 expression in endometrial cells. In conclusion, epithelial and stromal cells have an intrinsic role in innate immune surveillance in the endometrium, and in the case of LPS this recognition occurs via TLR4- and MYD88-dependent cell signaling pathways.
Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer's ...disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aβ42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aβ42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aβ42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aβ42 may contribute to the onset and progression of Alzheimer's disease.
The mammalian innate immune system deals with invading pathogens and stress by activating pattern-recognition receptors (PRRs) in the host. Initially proposed to be triggered by the discrimination of ...defined molecular signatures from pathogens rather than from self, it is now clear that PRRs can also be activated by endogenous ligands, bacterial metabolites and, following pathogen-induced alterations of cellular processes, changes in the F-actin cytoskeleton. These processes are collectively referred to as effector-triggered immunity (ETI). Here, we summarize the molecular and conceptual advances in our understanding of cell autonomous innate immune responses against bacterial pathogens, and discuss how classical activation of PRRs and ETI interplay to drive inflammatory responses.
Pathogen-mediated alterations in the cellular environment can contribute to mammalian innate immunity in a process referred to as effector triggered immunity (ETI).Host-derived second messengers and metabolites are emerging as triggers of innate immune pathways.Subcellular receptor localization, the cytoskeleton, and other cellular components can play key roles in mammalian ETI.Coactivation of PRRs and ETI may be required for maximal activation of innate immunity.
NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, ...micron-scale perinuclear punctum. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like or endosomal vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization.
Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express ...inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.
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•SARS-CoV-2 infection activates human NLRP1 inflammasome in epithelial cells•NSP5 protease cleaves NLRP1 at Glutamine 333 and promotes functional degradation•NSP5 inactivates GSDMD by cleaving in its pore-forming domain at Glutamine 193•GSDME triggers alternative epithelial cell death upon GSDMD inactivation
Planès et al. identify human NLRP1 as an immune sensor of SARS-CoV2 3CL protease.
The anti-viral immune response is dependent on the ability of infected cells to sense foreign nucleic acids. In multiple species, the pattern recognition receptor (PRR) cyclic GMP-AMP synthase (cGAS) ...senses viral DNA as an essential component of the innate response. cGAS initiates a range of signaling outputs that are dependent on generation of the second messenger cGAMP that binds to the adaptor protein stimulator of interferon genes (STING). Here we show that in chicken macrophages, the cGAS/STING pathway is essential not only for the production of type-I interferons in response to intracellular DNA stimulation, but also for regulation of macrophage effector functions including the expression of MHC-II and co-stimulatory molecules. In the context of fowlpox, an avian DNA virus infection, the cGAS/STING pathway was found to be responsible for type-I interferon production and MHC-II transcription. The sensing of fowlpox virus DNA is therefore essential for mounting an anti-viral response in chicken cells and for regulation of a specific set of macrophage effector functions.
Inflammasome activation by Salmonella Clare, Bryant
Current opinion in microbiology,
December 2021, 2021-12-00, 20211201, Volume:
64
Journal Article
Peer reviewed
•Salmonella infection activates inflammasomes including NLRC4, NLRP3 and caspase 11/4/5.•Inflammasome-induced cell death contributes to host control of Salmonella infection.•Species differences in ...inflammasomes may contribute to zoonotic immune tolerance.
Host recognition of bacteria such as Salmonella enterica serovar Typhimurium requires multiple host detection systems to generate complex inflammatory responses which can be cell type specific and has the potential for bacterial subversion of the host. Host detection of Salmonella requires Pattern Recognition Receptors (PRRs) sensing Pathogen Associated Molecular Patterns (PAMPs). These bacteria possess a diverse array of PAMPs including lipopolysaccharide, flagellin, proteins, lipoproteins, DNA, RNA and metabolites which can potentially activate multiple PRRs concurrently in different cell types. Salmonella is sensed by the inflammasome forming cytosolic nucleotide oligomerisation domain leucine rich repeat-like receptor (NLR) PRRs NLRC4 and NLRP3 as well as by the non canonical inflammasome formed by caspase 11 in mice, caspase 4 and 5 in humans. This review will discuss the different inflammasomes and how their activity regulates the host response to Salmonella infection.
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