Study On Signaling Mechanism And Function Of Sustained CXCL10 Expression In Monocytes/Macrophages Induced By MRP8/MRP14

Inflammation is the body’s defensive reaction.The immune system is activated to remove harmful factors when the body faced with harmful stimulation.Inflammation is a very common and important pathophysiological process,it’s local manifestations are redness,swelling,heat,pain and organ dysfunction.The general process of inflammation include:harmful stimulus damage tissues and cells;diluting,inflammatory hyperemia and leak to surround and destroy pathogenes;parenchymal and interstitial cells regenerate to repair and heal organizations.Different stages of inflammation have different manifestations and formation,the early pathological changes of inflammatory are metamorphism and seepage,then followed by hyperplasia,but they are not absolutely separated.The causes of inflammation were known as inflammatory factors,including biological inflammatory factors,physical inflammatory factors and chemical inflammatory factors.Under normal conditions,the function of inflammation is to remove harmful stimulus,and it is beneficial.Excessive inflammatory response may lead to out of control of immune system,aggravate the infection or damage.The outcome may be systemic inflammatory response syndrome(SIRS),even the multiple organ dysfunction syndrome(MODS).Inflammation network has a very complex structure,we still need to provide more evidence to understand it.Myeloid-related protein8(MRP8)and myeloid-related protein14(MRP14)are members of the calcium binding protein S100 family,which are normally expressed constitutively on neutrophils,and they are abundant in neutrophils which account for 50%of the soluble cytoplasmic components.MRP8/MRP14 can be released extracellularly by necrotic neutrophils.In the case of inflammation,MRP8/MRP14 can also be secreted by a variety of immune cells,such as macrophages,monocytes,dendritic to the local tissue or blood circulation.MRP8/MRP14 functions as heterodimers usually.MRP8/MRP14 is involved in various physiological and pathological process,such as metabolism and apoptosis.During study in sepsis,Johannes Roth found that after treated with endotoxin,the incidence of fatal shock in MRP8/MRP14 deficient mice are lower than wild-type mice,which earliest revealed the relationship between MRP8/MRP14 and inflammation.Then researchers also found MRP8/MRP14 changes in many autoimmune inflammatory diseases,such as tuberculosis and rheumatoid arthritis.At present a large number of in vitro studies have shown that pathogen associated molecular patterns(PAMPs)or other dangerous signal molecules can induce MRP8/MRP14 secretion,MRP8/MRP14 interacts with receptors on the cell membrane surface like receptor for advanced glycation endproducts(RAGE),CD36 or Toll like receptor 4(TLR4),then MRP8/MRP14 induces the expression of a variety of inflammatory factors,such as TNF-α,IL-8,CXCL10,IL-6,IL-β to promote inflammatory progression.TLR4 is a important pattern recognition receptor(PRR),which is mainly expressed on cell membrane of mononuclear macrophages,dendritic cells(DCs),lymphocytes and natural killer cells(NKs)and other inflammation-associated immune cells.LPS and other inflammatory factors can bind with TLR4,then activate downstream signaling pathway,lead to the release of inflammatory mediators or changes of cell microenvironment.TLR4 plays important roles both in innate immune system and acquired immune system.IFN-α and IFN-β are members of type I interferon(IFN)family,which play important roles in immune disease,such as viral replication and parasitization.They can also inhibit cell proliferation,promote the killing capacity of immune cells,regulate immune response and anti-tumor.Infected and damaged immune cells can synthesize and secrete large amounts of interferon,they bind with interferon-alpha/beta receptorl/2(IFNAR1/2)to phosphorylate downstream kinases,promote related transcription factors to transport into the nucleus,initiate transcription of inflammation-related genes.JAK-STAT signaling pathway is found to be involved in cell proliferation,differentiation,apoptosis immune regulation and many important biological signaling pathways.Many cytokines and growth factors functions through JAK-STAT signaling pathway,including interleukin 2-7(IL-2-7),granulocyte/macrophage colony stimulating factor(GM-CSF),growth hormone(GH),epidermal growth factor(EGF),platelet derived growth factor(PDGF)and interferon.After cytokines combine with respective receptor molecules,receptor molecules dimerize,and then recruit JAK kinases,JAKs are activated by tyrosine phosphorylation.Activated JAK induce tyrosine residue of receptor phosphorylate,then these phosphorylated tyrosine sites and amino acid sequences surrounding it consist of ’docking sites’,while STAT proteins which containing SH2 domains are recruited to this ’docking sites’.Finally,STATs are phosphorylated by JAK kinase and form dimers,then transport into the nucleus and bind with target genes to regulate gene transcription.Our previous study found that MRP 8/MRP14 plays important roles in trauma/hemorrhagic shock,and MRP8/MRP14 functions through the production of CXC chemokine family member CXCL10(also named interferon-y inducible protein-10,IP-10).In our study,we found that MRP8/MRP14 induced sustained expression of CXCL10 from monocytes/macrophages,CXCL10 mRNA increase appeared at 6 h(early)and 12 h(late)respectively after MRP8/MRP14 stimulation.We had already proposed and demonstrated the mechanism of"early CXCL10 production" induced by MRP8/MRP14.However,the mechanism of the production of "secondary CXCL10 production" is not clear.As a member of CXC chemokine family,CXCL10 induce chemotaxis of T lymphocytes,NK and NKT cells by binding to CXCR3 receptors,which play an important role in the regulation of adaptive immune response.Early studies have shown that MRP8/MRP14 can mediate migration of CXCR3+T cells,such as CD4+T lymphocytes,CD8+T lymphocyte and NKT cells.Consistent generation of CXCL10 leading to reinforcement of the body’s acquired immune system,then result in deterioration or regression of the body’s inflammatory response.So,identified the mechanism of sustained CXCL10 expression has great significance.As a class of immune-regulated mature T lymphocyte subsets,regulatory T cells(Tregs)have come across gradually because of negative regulatory function.Studies have shown that Tregs play an important role in the inflammation and sepsis.Clinical studies found that peripheral blood CD4+CD25+Tregs were proliferated and activated rapidly in sepsis and traumatic patients,and then Tregs secreted large amounts of inhibitory cytokines to negatively regulate inflammation,finally leading to immune dysfunction.Local damage can also induce migration of Tregs to the site of injury,and participate in the local inflammatory response.CXCR3 are expressed in Tregs cell surface,so what we concerned is the possibility of sustained CXCL10 migrate Tregs to impact the body’s inflammatory response.Firstly,mice monocytes/macrophage line RAW264.7 was collected for in vitro culture,RT-PCR was used to detect CXCL10 mRNA and type I interferon mRNA express for time gradient effect induced by MRP8/MRP14;Using nucleocytoplasmic separation and western blot were used to detect the possible participative signaling pathway activation;kinase activation and CXCL10 mRNA expression were detected after using type I interferon neutralizing antibody or various pathway inhibitors;Finally airpouch models were built in mice,then flow cytometry was used to detect the chemotaxis of Tregs.Through our study we proved that:(1)MRP8/MRP14 induced CXCL10 sustained expression and the early expression of IFN-β.MRP8/MRP14 induced two CXCL10 sustained expression from RAW264.7 cells in 6 h and 12 h respectively;MRP8/MRP14 induced IFN-β expression,but not IFN-α.(2)MRP8/MRP14 activated JAK-STAT signaling pathway.After MRP8/MRP14 stimulated RAW264.7 cells,phosphorylations of JAK1,STAT1,STAT2 and TYK2 increased,STAT1 and STAT2 translated more into the nucleus.(3)IFN-β neutralizing antibody inhibited JAK-STAT signaling pathway and CXCL10 late expression.After using IFN-β neutralizing antibodies,we found a decrease of JAK1,STAT1,STAT2 and TYK2 phosphorylation activations,translation of STAT1 and STAT2 into the nucleus less than before;as well as the decrease of CXCL10 late expression,but the early expression was not affected.(4)JAK-STAT pathway inhibitors inhibited the CXCL10 late expression.After treated with JAK1 and STAT1 inhibitors,CXCL10 late expression decreased significantly,but the early expression was not affected.(5)The biological significance of MRP8/MRP14 induce CXCL10 expression.MRP8/MRP14 stimulated RAW264.7 cells,then supernatants injected to mice airpouch models,flow cytometry found a significant increase of Tregs percentage.This study found that MRP8/MRP14 induced CXCL10 from monocytes/macrophage line RAW264.7,and expression more in 6 h and 12 h.CXCL10 late expression was a result of synthesis of IFN-β induced by MRP8/MRP14,IFN-β released and combined with it’s specific receptors on the cell membrane surface,then activated JAK-STAT signaling pathway,STAT translated into the nucleus and combined with CXCL10 promoter,induced the late synthesis of CXCL10.At the same time comparing with the control group MRP8/MRP14 induced monocytes/macrophages supernatants recruited more Tregs.MRP8/MRP14 induce sustained expression of CXCL10,leading to the consistent increase of protein concentration,then induce the recruitment of inflammatory cells to local.Inflammatory cells synthesize and release many pro-inflammatory factors,which may cause intensive inflammation,disorder and imbalance of the body’s immune system.Tregs have a negative regulation in the process of immune response,and can directly inhibit inflammatory cells and secrete suppression factors to regulate immune response.Our research provides important knowledge for us to deeply understand the biology function of MRP8/MRP14 and to add more research foundation for further clinical study of immunoregulation.