The inflammasome is a cytoplasmic protein complex that processes interleukins (IL)-1β and IL-18, and drives a form of cell death known as pyroptosis. Oligomerization of this complex is actually the ...second step of activation, and a priming step must occur first. This involves transcriptional upregulation of pro-IL-1β, inflammasome sensor NLRP3, or the non-canonical inflammasome sensor caspase-11. An additional aspect of priming is the post-translational modification of particular inflammasome constituents. Priming is typically accomplished in vitro using a microbial Toll-like receptor (TLR) ligand. However, it is now clear that inflammasomes are activated during the progression of sterile inflammatory diseases such as atherosclerosis, metabolic disease, and neuroinflammatory disorders. Therefore, it is time to consider the endogenous factors and mechanisms that may prime the inflammasome in these conditions.
Summary
Caspase‐8 is an apical component of cell death pathways. Activated caspase‐8 can drive classical caspase‐dependent apoptosis and actively inhibits cell death mediated by RIPK3‐driven ...necroptosis. Genetic deletion of Casp8 results in embryonic lethality as a result of uncontrolled necroptosis. This lethality can be rescued by simultaneous deletion of Ripk3. Recently, caspase‐8 has been additionally connected to inflammatory pathways within the cell. In particular, caspase‐8 has been shown to be crucially involved in the induction of pro‐IL‐1β synthesis and processing via both non‐canonical and canonical pathways. In this review, we bring together current knowledge regarding the role of caspase‐8 in cellular inflammation with a particular emphasis on the interplay between caspase‐8 and the classical and non‐canonical inflammasomes.
Signalling by pattern recognition receptors (PRRs) is critical for protecting the host against pathogens. Disruption of these signalling pathways has been implicated in many diseases ranging from ...infection susceptibility to cancer and autoimmune disease. Understanding how PRRs signal is of critical importance due to their potential as therapeutic targets to ameliorate symptoms of inflammatory diseases. The recent advances in microscopy, such as the discovery of fluorescent proteins and the breaking of the diffraction limit of light, offer a unique opportunity to visualise receptor signalling at a single protein level within living cells. Many different microscopy techniques have been developed and used for dissecting different aspects of PRR signalling pathways. This review will provide an overview of the main microscopy techniques used for dissecting these pathways with a focus on Toll-like receptor and NOD-like receptor signalling.
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. The intracellular mechanisms by which these lipids modulate NLRP3 activity are now being identified. This review discusses emerging evidence suggesting that organelle stress, particularly involving mitochondria, lysosomes, and the endoplasmic reticulum, may be key in lipid-induced modification of NLRP3 inflammasome activity.
Multiple classes of endogenous lipids, including oxidized low-density lipoprotein (LDL), saturated fatty acids, and sphingolipids, activate the mammalian NLRP3 inflammasome and can contribute to the progression of certain inflammatory diseases.Lipid activators of NLRP3 often share common features, such as slow-onset kinetics and the ability to provide both priming and triggering signals for inflammasome activation.Organelle stress, especially at mitochondria, lysosomes, and the endoplasmic reticulum, is required for lipid activation of NLRP3, while attenuation of organelle stress by certain lipids can inhibit NLRP3 activity.
Analysis of oxylipins by liquid chromatography mass spectrometry (LC/MS) is challenging because of the small mass range occupied by this diverse lipid class, the presence of numerous structural ...isomers, and their low abundance in biological samples. Although highly sensitive LC/MS/MS methods are commonly used, further separation is achievable by using drift tube ion mobility coupled with high-resolution mass spectrometry (DTIM-MS). Herein, we present a combined analytical and computational method for the identification of oxylipins and fatty acids. We use a reversed-phase LC/DTIM-MS workflow able to profile and quantify (based on chromatographic peak area) the oxylipin and fatty acid content of biological samples while simultaneously acquiring full scan and product ion spectra. The information regarding accurate mass, collision-cross-section values in nitrogen (DTCCSN2), and retention times of the species found are compared to an internal library of lipid standards as well as the LIPID MAPS Structure Database by using specifically developed processing tools. Features detected within the DTCCSN2 and m/z ranges of the analyzed standards are flagged as oxylipin-like species, which can be further characterized using drift-time alignment of product and precursor ions distinctive of DTIM-MS. This not only helps identification by reducing the number of annotations from LIPID MAPS but also guides discovery studies of potentially novel species. Testing the methodology on Salmonella enterica serovar Typhimurium-infected murine bone-marrow-derived macrophages and thrombin activated human platelets yields results in agreement with literature. This workflow has also annotated features as potentially novel oxylipins, confirming its ability in providing further insights into lipid analysis of biological samples.
•The Toll-like receptor signalling proteins MyD88 and IRAK2 form a Myddosome complex.•The Toll-like receptor adaptor protein Tram dimerises and Trif forms oligomers suggesting that a “Trifosome” ...complex analgous to the Myddosome may form.•Inflammasome signalling also generates macromolecular protein complexes.•Macromolecular protein signalling is likely to be a common theme in Pattern Receptor signalling.
The molecular mechanisms by which pattern recognition receptors (PRRs) signal are increasingly well understood. Toll-like receptor 4 (TLR4) signals through two separate pairs of adaptor proteins Mal/MyD88 and Tram/Trif. Structural studies have revealed a common theme for PRR signalling in that their signalling proteins form large macromolecular complexes which are thought to form the active signalling complex. The first of these to be characterised was the MyD88 signalling complex Myddosome. Many questions remain unanswered however. In particular it is unclear whether these signalling complexes form within the living cell, how many of each signalling protein is within the intracellular Myddosome and whether the stoichiometry can vary in a ligand-dependent manner. In this review we will discuss what is known about the macromolecular complexes thought to be important for TLR4 signalling.
The technique of differential dynamic microscopy is extended here, showing that it can provide a powerful and objective method of video analysis for optical microscopy videos of in vitro samples of ...live human bronchial epithelial ciliated cells. These cells are multiciliated, with motile cilia that play key physiological roles. It is shown that the ciliary beat frequency can be recovered to match conventional analysis, but in a fully automated fashion. Furthermore, it is shown that the properties of spatial and temporal coherence of cilia beat can be recovered and distinguished, and that if a collective traveling wave (the metachronal wave) is present, this has a distinct signature and its wavelength and direction can be measured.
The Myddosome is a key innate immune signalling platform. It forms at the cell surface and contains MyD88 and IRAK proteins which ultimately coordinate the production of pro-inflammatory cytokines. ...Toll-like receptor 4 (TLR4) signals via the Myddosome when triggered by lipopolysaccharide (LPS) or amyloid-beta (Aβ) aggregates but the magnitude and time duration of the response are very different for reasons that are unclear. Here, we followed the formation of Myddosomes in live macrophages using local delivery of TLR4 agonist to the cell surface and visualisation with 3D rapid light sheet imaging. This was complemented by super-resolution imaging of Myddosomes in fixed macrophages to determine the size of the signalling complex at different times after triggering. Myddosomes formed more rapidly after LPS than in response to sonicated Aβ 1–42 fibrils (80 vs 372 s). The mean lifetimes of the Myddosomes were also shorter when triggered by LPS compared to sonicated Aβ fibrils (170 and 220 s), respectively. In both cases, a range of Myddosome of different sizes (50–500 nm) were formed. In particular, small round Myddosomes around 100 nm in size formed at early time points, then reduced in proportion over time. Collectively, our data suggest that compared to LPS the multivalency of Aβ fibrils leads to the formation of larger Myddosomes which form more slowly and, due to their size, take longer to disassemble. This explains why sonicated Aβ fibrils results in less efficient triggering of TLR4 signalling and may be a general property of protein aggregates.
Preventing pores and inflammation Pickering, Robert J.; Bryant, Clare E.
Science (American Association for the Advancement of Science),
09/2020, Volume:
369, Issue:
6511
Journal Article
Peer reviewed
Metabolite-directed modification of pore-forming cell death protein limits inflammation
Inflammation is a tightly regulated process that is essential for host protection against infections. When it ...is dysregulated, however, inflammation becomes damaging, causing many common diseases such as sepsis, arthritis, asthma, and diabetes. Some programmed cell death pathways, such as pyroptosis and necroptosis, induce inflammation in response to pathogens or sterile stimuli. The pyroptosis executioner protein gasdermin D is cleaved by the cysteine protease caspases 1, 4, 5, or 11 (
1
,
2
). This produces a toxic amino-terminal fragment that forms pores in membranes, which lyses cells and releases inflammatory mediators (
3
). On page 1633 of this issue, Humphries
et al.
(
4
) reveal a switch whereby dimethyl fumarate (DMF), a drug that is used to treat inflammatory conditions such as multiple sclerosis, succinates critical cysteine residues in gasdermin D to prevent its cleavage, inhibit pyroptosis, and protect against severe inflammation in mice.
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