Studies On The Molecular Mechanisms Of The Late Inflammation Caused By The Internalization Of HMGB1

Systemic inflammatory response syndrome (SIRS) is a serious condition related to systemic inflammation, organ dysfunction, and organ failure. It is one of the most common causes of death in hospitalized patients which lead them to multiple organ dysfunction syndrome (MODS) and death. It is a subset of cytokine storm, in which there is abnormal regulation of various cytokines. Cytokine storm is a potentially fatal immune reaction consisting of a positive feedback loop between cytokines and immune cells, with highly elevated levels of various cytokines. High mobility group box1(HMGB1) is an important "late" mediator in SIRS which is released by inflammatory cells then causes cytokine cascade by promotes the releases of other cytokines.HMGB1, a highly conserved nuclear non-histone protein, is expressed in nearly all cell types. HMGB1was originally described as a DNA-binding protein that functions as a structural co-factor critical for proper transcriptional regulation in somatic cells. Recently, it was found that HMGB1also had many other functions, including promoting the outgrowth of neuritis, tumour invasion and metastasis, differentiation of immune cells, the systemic inflammatory response and injure. The signalling mechanisms by which HMGB1activates the cells are only partially understood. Some HMGB1receptors on mammalian cell membrane have been found, including receptor for advanced glycation end products (RAGE) and toll-like receptor-2/4(TLR-2/4). Although several important receptors have been implicated in HMGB1signaling, many of the functions of HMGB1can not be attributed to the interactions with these receptors. Blocking studies performed with all the receptor-blocking antibodies never completely prevented cellular activation by HMGB1. It is thus likely that there are alternative HMGB1-binding receptors or other pathways. It was reported that HMGB1significantly increases the uptake of exogenous DNA by transporting the DNA through the cell membrane of mammalian cells. Meanwhile, researchers found that more and more cytokines, growth factors or their receptors can be internalized by mammalian cells and this take part in signaling pathways of the cells. All these observations implicate that HMGB1may internalize into mammalian cells and reduce cell responses. There may be the as-yet-unknown receptors or the complex interactions with HMGB1and other signaling molecules or the non-receptor-mediated functions of HMGB1.The innate immune system engages an array of germline-encoded pattern-recognition receptors (PRRs) to detect invariant microbial motifs. PRRs are expressed by cells at the front line of defense against infection. PRRs include the membrane-bound Toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), C-type lectin receptors (CLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). The NLR family, pyrin domain-containing (NLRP) inflammasome is a multiprotein complex that activates caspase1, leading to the processing and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18. The NLRP3inflammasome is activated by a wide range of danger signals that derive not only from microorganisms but also from metabolic dysregulation. Various danger signals activate the NLRP3inflammasome including whole pathogens, pathogen-associated molecular patterns (PAMPs) and other pathogen-associated molecules. Environmental irritants (such as asbestos) and damage-associated molecular patterns (DAMPs), which are host-derived molecules that are indicative of cellular damage (such as extracellular ATP), also activate the NLRP3inflammasome. The mechanisms by which these structurally distinct molecules trigger NLRP3oligomerization and inflammasome activation are currently unclear. Three models for activation of the NLRP3inflammasome have been proposed. They are the channel model, the ROS model and the lysosome rupture model.Caspase1was first recognized as a protease that processes the inactive precursors of IL-1β and IL-18into mature inflammatory cytokines, and was initially called IL-1β-converting enzyme. The mechanisms and regulation of IL-1β production have great relevance to many acute and chronic inflammatory diseases. It is currently hypothesized that two signals are required for IL-1β release from normal human monocytes. The first signal appears to be TLR induction of transcription of pro-IL-1β with subsequent storage in the cell. Lipopolysaccharide (LPS) is a common stimulus for signal1through binding to TLR4. The second signal is NLR-induced IL-1β processing and release through a caspase-1-dependent mechanism. Thus, stimulation through both TLRs and NLRs as well as an intact NALP3inflammasome are all required for processing and release of IL-1β.However, caspase1activation can result not only in the production of activated inflammatory cytokines, but also rapid cell death characterized by plasma-membrane rupture and release of proinflammatory intracellular contents which called pyroptosis. Caspase1dependence is a defining feature of pyroptosis, and it is also the enzyme that mediates this process of cell death. Pyroptosis features rapid plasma-membrane rupture and release of proinflammatory intracellular contents. DNA damage also occurs during pyroptosis. Cell lysis during pyroptosis results from caspase1-mediated processes.Mammalian cells have evolved a variety of mechanisms to internalize small molecules, macromolecules and particles and target them to specific sealed organelles within the cytoplasm. Internalization serves many important cellular functions including the uptake of extracellular nutrients, regulation of cell-surface receptor expression and other cell functions. Endocytosis is one of the most important way by which macromolecules internalize. Endocytosis is a complex process that involves different pathways and a large network of protein-protein, protein-lipid and protein-glycan interactions. The first and best-characterized pathway is clathrin-dependent endocytosis, which starts on the plasma membrane with the formation of clathrin-coated invaginations that pinch off to make up clathrin-coated vesicles. Less defined are the non-classical, clathrin-independent pathways, among which is caveolae-mediated endocytosis. Caveolae are involved in signal transduction and the intracellular transportation.Previous studies have shown that HMGB1is an important mediator mediating inflammation and cytokines release. We have also observed that HMGB1can be internalized into macrophages through a dynamin-dependent pathway. This study is to address whether HMGB1internalization in AM is associated with inflammasome activation and therefore IL-1β release and cell pytoptosis.In our study, we used macrophages isolated from C57BL/6and TLR2/TLR4defective mice to exam the endocytosis of green fluorescent protein (EGFP)-tagged HMGB1and the activations of inflammatory moleculars. Macrophages were stimulated with either EGFP-tagged HMGB1or non-labeled HMGB1for0to24h. HMGB1localization, lysosome rupture, cathepsin B release and activation, as well as caspases-1activation were assessed by confocal microscopy, IL-1β release was measured by ELISA, and cell pyroptosis was measured by Flow CytoMeter.In our research, we found that HMGB1is able to induce caspase-1activation, IL-1β release and cell pyroptosis in macrophages at the late phage after stimulation. Stimulation of macrophage with HMGB1initiates HMGB1internalization that occurred at as early as5min. Visualized GFP-tagged HMGB1localized in endosomes at first, then moved into lysosomes at3h, and caused lysosome rupture at3to6h, which was followed by cathepsin B release and activation as well as caspases-1activation, which caused IL-1β release and cell pyroptosis. Measurement of IL-1βdemonstrated two phases of IL-1β release. The early phase of IL-1β release exhibited before6h, and the later phase occurred at about18h. Furthermore, the results showed that the early phase of IL-1βrelease in response to HMGB1is not an endocytosis-dependent event. We also found that the internalization of HMGB1is a RAGE-depended, TLR2/TLR4-independed and caveolae/clathrin-independed procedure.HMGB1endocytosis-induced inflammasome activation is a novel mechanism responsible for a later phase release of IL-1β from macrophages in HMGB1-mediated inflammation. Our finding indicated that activation of NLRP3inflammasome by HMGB1may represent a critical component of the inflammatory response in severe-ill patients. In addition, we explore a previously unidentified endocytosis-dependent mechanism of cytokine-induced inflammatory response. Thus blocking cell internalization of inflammatory cytokines may serve as a novel therapeutic strategy for the treatment of inflammation. HMGB1is an important proinflammatory cytokine released into the extracellular milieu while its signalling mechanisms are incompletely understood. The insights from our studies on HMGB1may widen the therapeutic window for SIRS and MODS.