Trauma Regulation Of Macrophage Death

Trauma refers to the body suffered tissue organs damage body caused by a strong external force.Trauma is a common surgical disease,manifested as pain and functional activity limitation,the injured area,tissue damaging,swelling,bleeding,and inflammatory responses.Severe trauma leading a lot of inflammatory cytokines release into the blood,leading to excessive inflammation,causing systemic inflammatory response syndrome(systemic inflammatory response syndrome,SIRS),and multiple organ dysfunction syndrome(multiple organ dysfunction syndrome,MODS),etc.,seriously endanger the lives of patients.Severe trauma is common in serious traffic accidents,falls and other combat,which often leads to systemic multiple fractures,so often accompanied by fatal bleeding,hemorrhagic shock,and brain trauma causing disorders of consciousness.Since the area of open wounds,traumatic blow contact with pathogenic microorganisms and other reasons,infection is a common clinical complication following tissue damage resulting from surgery and severe trauma.Infection triggers the innate immune system to release large amounts of proinflammatory mediators that can have deadly consequences,including systemic inflammatory response syndrome(SIRS)and organ failure.Studies have suggested that cell pre-activation caused by antecedent trauma/tissue damage profoundly impacts the response of the innate immune cells to a secondary infectious stimulus,often in a form of enhanced inflammation.Macrophages,important innate immune executive cells,release proinflammatory mediators including cytokines and endogenous danger molecules,and critically regulate the progress of inflammation.The previous studies show that the cell death mainly including apoptosis,necrosis,pyroptosis and necroptosis.The characteristics of apoptosis such as cell shrinkage,DNA fragmentation,cell membrane integrity,and accompanied by caspase activation,such as caspase-3/7/8/9/10.The cell necrosis features as cell swelling,loss of cell membrane integrity.Pyroptosis is an important inflammatory response during infection,characterized as cell swelling,nuclear condensation,DNA fragmentation,and accompanied activation of caspase-1/11.Cell necroptosis,a form of regulated inflammatory cell death,is one mechanism that controls cell release of inflammatory mediators,in the case of non-activation of caspase-8,the phosphorylated RIPK1 is interaction with RIPK3 to form a complex,the RIPK1-RIPK3 complex recruitment and phosphorylation activation of MLKL,the MLKL execution the cell death processes,the RIPK1-RIPK3-MLKL complex so call"necrosome".Necroptosis features RIPK1 activation and necrosome formation,followed by a loss of plasma membrane integrity and the release of cell contents into the extracellular space,which causes increased inflammation.Thus,necroptosis is a regulated inflammatory cell death.Classic necroptosis is initiated by tumor necrosis factor-alpha(TNF-α)through activation of tumor necrosis factor receptor(TNFR),It has been reported that lipopolysaccharide(LPS)acting through TLR4 signaling induces macrophages necroptosis.However,the effect of antecedent trauma on the LPS-induced macrophages necroptosis has yet to be addressed.Tissue and cell damage caused by trauma will directly cause the release of endogenous danger molecules,also well known as endogenous damage-associated molecular patterns(damage associated molecular patterns,DAMPs).DAMPs including extracellular high mobility group protein 1(HMGB1),cold-inducible RNA-binding proteins(CIRP),heat shock protein(HSP),S100 molecular hyaluronic acid fragments,adenosine triphosphate(ATP),adenosine,deoxyribonucleic acid(DNA),ribonucleic acid(RNA)and uric acid,etc.High mobility group box 1(HMGB1),a highly conserved ubiquitous protein present in the nucleus and cytoplasm of nearly all cell types,is the prototypic damage-associated molecular pattern(DAMPs)released by damaged tissues.During tissue injury,HMGB1 is released from cells and serves as a necessary and sufficient mediator of inflammation to induce a variety of cellular responses including cell chemotaxis and the release of pro-inflammatory cytokines.Inflammatory functions of HMGB1 are mediated by binding to cell surface receptors,including the receptor for advanced glycation end products(RAGE),Toll-like receptor(TLR)2,TLR4,and TLR9.The CIRP is a member of cold shock protein family.CIRP plays a critical role in responsing to cold stress.Mouse and human CIRP are 172 amino acid residues(95%identical)from a consensus sequence of the N-terminal RNA-binding domain and a C-terminal glycine-rich domain of nuclear proteins,these proteins as RNA chaperones promote RNA translation.CIRP low level constitutively expressed in various tissues,CIRP mild hypothermia,exposure to ultraviolet(UV)irradiation and hypoxia.Mitochondrial is a cell energy metabolism center.Trauma how to regulate the mitochondrial response seems to be a new research targets.Mitochondria diameter typically between 0.75 to 3 microns,but the size and structure are quite different.Mitochondria are recognized as "core ability of cells" because they generate most of adenosine triphosphate(ATP)supply cells as chemical energy metabolism.In addition to supplying cellular energy,mitochondria are also involved,such as signal transduction,cell differentiation,cell death,and the maintenance of cell cycle and cell growth control process.Mitochondria contain mitochondrial DNA(mtDNA),the human mitochondrial genome is about 16,000 base pairs circular DNA molecule,which encoding 37 genes,including 13 genes for respiratory complex Ⅰ,Ⅲ,Ⅳ and Ⅴ,and 22 subunit mitochondrial tRNA(for 20 standard amino acids,leucine and serine plus an extra gene),and two for the large and small subunits of rRNA.Mitochondrial usually contain two to ten copies of mtDNA.Trauma how to regulate the macrophage mitochondrial DNA response seems to relative with novel therapy target.RAGE is a type I transmembrane protein and a member of the immunoglobulin superfamily expressed in many cell populations including endothelial cells,vascular smooth muscle cells,neurons,neutrophils,and macrophages/monocytes.RAGE has been implicated as a receptor mediating the chemotaxis and cytokine activity of HMGB1 in both macrophages and tumor cells.RAGE engagement by multiple ligands is linked to a range of signaling pathways including activation of NF-κB,PI3K/Akt,MAPKs,Jak/STAT,Rho GTPases,and Cell division control protein 42 homolog,also known as Cdc4226,although how RAGE transduces these signals is not yet fully understood.Autophagy refers to a cell self-regulation process,allowing abnormal cell components orderly degradation and recycling the parts of cell components.In this process,targeted to cytoplasmic constituents isolated from a double membrane vesicle is called autophagy.Then autophagosomes fused with lysosomes,which are degraded and recycled content.In the case of disease,autophagy has been regarded as the adaptive response to stress promotes survival,while it seems to also promote cell death process.TLR4 is a member of toll-like receptor family,is an important receptor responsible for activating the innate immune system.It is well know as recognized the lipopolysaccharide(LPS),a components of Gram-negative bacteria.But its ligands also including several viral proteins such as polysaccharides,and a variety of endogenous proteins such as low density lipoprotein,β-defensins,and heat shock proteins.At present,studies have shown that CIRP also a ligand of TLR4,CIRP through TLR4 signaling pathway plays a role in promoting inflammation.TLR4 internalization has been reported as one of the mechanisms regulating a cell’s responses to TLR4 signaling.Mechanisms of TLR4 internalization can include association with endocytosis proteins such as clathrin,dynamin and caveolin-1.Caveolin-1 is a transmembrane scaffolding protein,and the major structural component of caveolae that contribute to many cell functions including endocytosis,potocytosis,transcytosis,as well as calcium signaling.Caveolin-1-mediated internalization of multiple cell surface receptors is also known to critically regulate receptor signalingThrough research,we have demonstrated that:(1)Pseudo-Fracture induces macrophage cytoplasm DNA fragmentation.We have compared the pseudo-fracture and hemorrhagic shock model,alveolar macrophages and peritoneal macrophages TUNEL staining occurred in different location,TUNEL staining in pseudo-fracture group occurred mainly in the cytoplasm of macrophages,while the hemorrhagic shock group occurs in the nucleus(which is consistent with the typical characteristics of apoptosis),thus,in vivo experiments,we confirmed the bone crushed mixture(BCM),the pseudo-fracture main ingredient,induced DNA fragmentation occur in the cytoplasm of macrophages.(2)Damaged tissue induces macrophage mtDNA fragmentation.Since the mitochondrial DNA is the main source of DNA in cytoplasm,therefore,we confirmed the fragmented DNA derived from damaged mitochondrial DNA through detected mtDNA quantification and used the EtBr induced mtDNA deletion macrophages.And confirmed the CIRP from bone-crush mixture(BCM),which is the main responsible for inducing mtDNA damage.(3)BCM-induced mtDNA fragmentation is mediated by TLR4-MyD88 signaling.It has been reported that CIRP through TLR4-MyD88 signaling pathway inducing inflammation.So we have tested the bone crushed mixture(BCM)has also through TLR4-MyD88 signaling pathway to regulate mtDNA damage by detected the fragemented mtDNA in TLR4 and MyD88 knockout mice.(4)ROS mediates mtDNA damage.Since the ROS is the main intracellular source damage stress of mitochondrial or mtDNA.Therefore,we have demonstrated the ROS mediates bone crushed mixture(BCM)induces mtDNA fragmentation by using the gp91 knockout mice.(5)Fragmented mtDNA induces macrophages autophagy activation.Since the autophagy has an important role for the removal and repair of damaged organelles,when detecting the LC3 activation.We have improved the bone crushed mixture induced mtDNA damage activates autophagy,the strengthen autophagy so that to remove the damaged mtDNA.(6)Autophagy prevents fragmented mtDNA-induced macrophages necroptosis.Due to the presence of autophagy,the cell trend to be in a steady-state equilibrium,so the cells can be kept healthy.We identified the BCM-induced necroptosis,which featured as RIPK1 phospholyation and RIPK1-RIPK3 binding in the lacking of autophagy in macrophages through damaged mtDNA.The fragmented mtDNA induced necroptosis is inhibited by autophagy.(7)Necroptotic macrophages induce the naive macrophages inflammatory response.Trans-well co-culture experiments demonstrated that in BCM-induced autophagy deficient macrophages necroptosis increases normal cells inflammatory response.(8)Tissue damage suppresses LPS-induced monocytes/macrophages necroptosis.The in vivo experiments demonstrated that mice receiving pseudo-fracture or intra-peritoneal injection of bone crushed mixture(BCM)before receiving a second LPS hit,showing tolerance to LPS induced monocyte/macrophage necroptosis.We got the same results in vitro studies.Thus,we demonstrated that tissue damage regulates macrophages to tolerance with the second hit pathogenic microorganisms.(9)BCM decreases cell surface expression of TLR4.Since the TLR4 is the most important receptor for LPS,thus,we detected the cell surface expression of TLR4 through flow cytometry when challenged with BCM,the results showed that BCM induces macrophages cell surface of TLR4 reduction,thereby reducing the sensitivity to LPS.(10)HMGB1-RAGE signaling mediates BCM-suppressed macrophages necroptosis in response to LPS.To identify the role of HMGB1 plays in BCM,we used Tamoxifen-induced HMGB1 knockout mice,and obtained the HMGB 1-lacking BCM,we confirmed the HMGB1 is a major component of BCM,which induced macrophages tolerance of LPS.The RAGE is a major receptor of HMGB 1.We have demonstrated that the HMGB1 in BCM acting through RAGE signaling pathway to play the role of inducing tolerance with LPS.(11)Caveolin-1 mediates RAGE-TLR4 interaction.The above data suggest that BCM through RAGE down regulation of TLR4 cell surface expression,so as to achieve the result of tolerance to LPS.Thus,we have demonstrated that BCM/HMGB1 acting through RAGE inducing caveolin-1 up-regulation in protein and mRNA level by Western Blot and RT-QPCR,and caveolin-1 plays a critical role in the induction of TLR4 internalization,so that the cell surface expression of TLR4 reduced.(12)RAGE-MyD88 signaling mediates translocation of Spl to up-regulate caveolin-1 expression.Previous studies have demonstrated that the transcription factor Spl could regulation of caveolin-1 gene expression.We used a variety of signaling pathway inhibitors and immunoprecipitation to identify the novel signaling pathway which including RAGE-MyD88-Cdc42-Sp1-Caveolin-1.The signaling pathway has revealed the regulation of the caveolin-1 expression.(13)Tissue damage suppresses LPS-induced inflammation.The above experiments prove that tissue damage down regulation of cell surface TLR4,which results of macrophages desensitized for second-hit LPS-induced necroptosis and LPS-induced inflammation correspondingly,including cytokines release and MAPK signaling pathway activation.This result further suggested the necroptosis is to promote the development of inflammation.In this study,we have explored the CIRP in tissue damage,which induces mtDNA damage via TLR4-MyD88 signal to increase the ROS so that ROS-induced mtDNA damage.Also,mtDNA damage induces autophagy activation simultaneously and removes the damaged mtDNA,but in the case of autophagy lacking,mtDNA is not cleared,which can induce macrophages necroptosis,so that the cells process to die and exacerbate the inflammation.These results reveal the novel mechanisms of trauma regulation of macrophages death.We also investigated the mechanisms and role of trauma/tissue damage in the regulation of LPS-induced macrophages necroptosis using a validated mouse model that simulates long bone fracture[pseudo fracture(PF)].We demonstrate that LPS acting through TLR4 promotes macrophages necroptosis,as expected.However,release of HMGB1 from damaged tissue signals via RAGE signaling to up-regulate caveolin-1 expression and so induces macrophages surface TLR4 internalization,which results in amelioration of LPS-TLR4-induced macrophages necroptosis.We also identify a mechanism for up-regulation of caveolin-1 expression via RAGE-MyD88 signaling to activate Cdc42,which leads to nuclear translocation of transcription factor Spl and subsequent change in caveolin-1 expression.These results reveal a previously unexplored negative regulatory effect of tissue damage on macrophages response to a secondary LPS stimulation.