Effect Of GDF3 On The Phagocytosis Of Mouse Macrophages During Sepsis And Potential Mechanism Behind This Effect

BackgroundSepsis is a major public health problem affecting public health worldwidely,which is a set of abnormal physiological,pathological,and biochemical syndrome caused by infection.Latest international consensus defined sepsis as life-threatening organ dysfunction caused by a dysregulated host response to infection.Sepsis is an infectious disease with high incidence and mortality,heavy medical burden,which often complicates burn,trauma and major surgeries.Recent epidemiological data on sepsis show that the incidence of sepsis is close to 450/100,000,far exceeding the total incidence of myocardial infarction or prostate,breast and lung cancer.It is estimated that there are 15 million to 30 million cases each year in the world,with a mortality rate from 30%to 50%.A survey from the United States demonstrated that more than 20 billion dollars were spent on the treatment of sepsis in 2011,which accounted for 5.2%of total clinical expenses in United States.Sepsis is still a major challenge to human health and economic development.However,to date,the pathogenesis of sepsis remains unclear.Further study on the mechanisms of sepsis and new strategies for the prevention and treatment has become an urgent task of critical care medicine around the world.Sustained infection and inflammation play crucial roles in the development of sepsis.After invading the body,pathogens firstly activate innate immunity system of the host,leading to releases of numerous inflammatory cytokines.Once pathogens were cleared by immune system,the inflammation will be controlled and sepis will be cured.If the pathogens persist,the inflammatory cascades can result in positive feeding back in the release of cytokines,"cytokine storm",which may cause systemic inflammatory response syndrome(SIRS).Previous research focused on inhibiting cytokine storm,because activation and persistence of cytokine storm can cause damage to cells,leading to cell death.During the process,immune cells would be injured,inactivated or even apoptotic,leading to disfunction of the immune system.This would significantly impair the capacity of pathogen clearance and facilitate secondary infection,which ultimately leads to life-threatening organ dysfunction and the poor prognosis of septic patient.Therefore,it is urgent to explore a new way of immunomodulation that can effectively increase pathogen clearance and regulate inflammatory response.Macrophages are innate immune cells that belong to mononuclear phagocytic system which play critical roles in defense,development and homeostasis.They form the first line of defense against the invasion of pathogenic microorganisms.Macrophages exist virtually in every organ or tissue,but are more abundant in the gastrointestinal tract,liver,spleen,upper respiratory tract,and brain tissue.Macrophages play important roles in the progression of sepsis,which mainly reside in two aspects:Firstly,identification,phagocytosis and killing of pathogens.The phagocytosis of pathogens by macrophages initiates innate immune response which subsequently activates the adaptive immune response.When the pathogenic microorganisms invade into the body,macrophages can recognize pathogens through surface receptors and then phagocytize,digest and present to T lymphocytes through antigen presentation,thus triggering large numbers of inflammatory cytokines to regulate adaptive immunity.Phagocytosis of macrophages is pivotal for the uptake and degradation of infectious pathogens and senescent cells,and it is also involved in development,tissue remodeling,immune responses,and inflammation.Secondly,the polarization of macrophage.M1 macrophages(classically activated macrophages)facilitate pro-inflammatory effects and contribute to host defense against infection,whereas M2 macrophages(alternatively activated macrophages)are involved in anti-inflammatory responses and tissue remodeling.Therefore,enhancing the phagocytosis of macrophages and reducing the release of inflammatory cytokines can effectively eliminate pathogen,reduce the occurrence of sepsis,and improve the prognosis of sepsis.Growth differentiation factor 3(GDF3),also known as Vg related gene 2(VGR-2),is encoded by the GDF3 gene.It belongs to the transforming growth factor beta(TGF-beta)superfamily,and is subdivided into the branches of bone morphogenetic protein/growth and differentiation factor(BMP/GDF).GDF3 is expressed in embryonic bone and adult bone marrow,thymus,spleen and adipose tissue.Previous study found that it played an important role in the development of the eye and bone.Previous study also found that GDF3 was a regulator of muscle regeneration that could improve macrophage function in tissue repair in a paracrine manner.As a secreted exogenous effector,GDF3 acted on myoblasts and acted as a specialized macrophage-derived regenerative factor in tissue repair.Previous study showed a significant decrease in the expression of GDF3 in breast cancer tissues.GDF3 inhibited proliferation of MCF-7 and T47D cells.Furthermore,overexpression of GDF3 contributed to paclitaxel-induced cell apoptosis.However,the role of GDF3 in acute inflammation,especially during acute inflammation caused by sepsis,has not been elucidated.Our project focused on the effect and mechanism of GDF3 on phagocytosis of macrophages in septic mice.Objectives1.To explore the protective effects of GDF3 on CLP-induced sepsis mice.2.To study the effect of GDF3 on the phagocytosis of macrophages in septic mice,and to explore its anti-inflammatory protective effect.3.To investigate the mechanism of GDF3 on macrophage phagocytosis stimulation.Methods1.Establish CLP mouse model.Mice were randomly divided into CLP-BSA control group and CLP-GDF3 treatment group,20 mice in each group.0.1%BSA and GDF3 were injected through the tail vein 3 hours before CLP operation respectively.Perform CLP surgery.7-day survival rate of CLP mice was observed postoperatively to investigate the effect of GDF3 on the survival rate of CLP-induced sepsis mice.2.12 hours after CLP surgery,mice were anesthetized,blood samples were collected through cardiac puncture and peritoneal lavage samples were collected by peritoneal lavage.Blood samples and peritoneal lavage samples were diluted and spread on TSA sheep blood medium evenly to determine the bacterial burden and investigate the effects of GDF3 on infection in mice after CLP surgery.3.12 hours after CLP surgery,mice were anesthetized,blood samples were collected through cardiac puncture and peritoneal lavage samples were collected by peritoneal lavage.The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in serum and peritoneal lavage fluid were determined by ELISA to investigate the effects of GDF3 on pro-inflammatory cytokines in CLP-induced sepsis mice.4.RAW264.7 and bone marrow-derived macrophages(BMDMs)were isolated and cultured.Effects of different concentrations of GDF3 on the activity of BMDMs and RAW264.7 cells were detected by MTS assay.5.RAW264.7,bone marrow-derived macrophages(BMDMs)and peritoneal macrophages were cells were isolated cultured.Effects of GDF3 on phagocytosis and bactericidal function of these macrophages were determined by different methods.6.Total RNA extracted from GDF3-treated BMDMs was subjected to RNA sequencing(RNA-seq),gene expression of control group and GDF3 treated group were analyzed to screen for possible candidate genes that may be involved in the enhancement of macrophage phagocytosis.The selected genes were quantitatively analyzed by qPCR to verify their expression.7.Bone marrow-derived macrophages(BMDMs)were isolated and cultured,which were then treated with GDF3 and LXR agonist respectively,immunofluorescence staining was used to detect the nuclear translocation of LXR to explore the effect of GDF3 on LXR.8.Bone marrow-derived macrophages(BMDMs)were isolated and cultured,the phagocytosis of pHrodo Red E.coli Bioparticles in BMDMs treated with GDF3,LXR agonists,LXR antagonists,and GDF3+LXR antagonists respectively was quantified by microplate reader.Phagocytosis of pHrodo Red E.coli Bioparticles in BMDMs treated with LXR agonists and GDF3+LXR antagonists respectively was conducted by confocal microscopy.9.Statistical analysis:Statistical analysis was performed using GraphPad Prism 7.0 software(GraphPad Software,Inc.,USA).The experimental data were expressed as mean ± standard deviation.Differences between two groups were analyzed using unpaired two-tailed t-test to and survival rates were analyzed using log rank test.P<0.05 was considered to be statistically significant.Results1.Effects of GDF3 on the survival rate of septic mice:the 7-day survival rate of septic mice in the GDF3 pretreated group was significantly higher than that of the control group(0.1%BSA pretreated group),indicating that GDF3 could improve the survival rate of clp-induced septic mice.2.Effects of GDF3 on bacterial burden of blood and peritoneal lavage samples of septic mice:both in blood and peritoneal lavage,bacterial burden of GDF3 pretreatment group was significantly lower than that of the control group,suggesting that GDF3 could reduce infection in mice after CLP surgery.3.Effects of GDF3 on pro-inflammatory cytokines levels in serum and peritoneal lavage samples of septic mice:compared with the control group,levels of IL-1 beta,IL-6 and TNF-a in serum and peritoneal lavage of septic mice in the GDF3 group were significantly lower than those in the control group.It indicates that GDF3 can inhibit the release of pro-inflammatory cytokines and thus reduce the inflammatory response in early septic mice.4.Effects of GDF3 at different treatment concentrations on BMDMs and RAW264.7 cell activity:GDF3 at lOng/ml,20ng/ml and 50ng/ml had no significant effect on the activity of the two cells,but at 100ng/ml,the activity of the two cells decreased significantly compared with that of the control group.The results showed that high GDF3 concentration may have potential cytotoxicity to macrophages.5.The ability of different macrophages to phagocytic e.coli Bioparticles was detected by microarray,confocal microscope and flow cytometry,respectively.It was found that GDF3 significantly enhanced the phagocytic function of various macrophages to phagocytic e.coli Bioparticles compared with the control group.Compared with the control group,GDF3 also enhanced the bactericidal effect of macrophages on the live E coli phagocytes.GDF3 can improve the survival rate of septic mice by enhancing phagocytosis and bactericidal action of macrophages.6.RNA-seq analysis showed that GDF3 could promote the expression of CD5L and other genes in BMDMs.QPCR also confirmed that CD5L expression in BMDMs after GDF3 treatment was significantly higher than that in the control group.We speculated that GDF3 may play a role in promoting phagocytosis of macrophages by activating the upstream CD5L regulation genes.7.GDF3 activates the CD5L upstream regulatory gene liver X receptor(LXR):immunofluorescence staining revealed that GDF3 causes nuclear translocation of LXR in BMDMs,as observed in BMDMs treated with LXR receptor agonist GW3965.It is believed that GDF3 activates the CD5L upstream regulatory gene LXR.8.Effects of LXR activation and inhibition on phagocytosis of BMDMs:Quantitative detection by microplate readers showed that LXR agonists significantly enhanced the phagocytosis of BMDMs compared to controls.After the treatment of LXR antagonist,this effect was blocked.The similar result was observed on confocal microscopy.This confirms that GDF3 promotes macrophage phagocytosis by activating the LXR-CD5L signaling pathway.ConclusionWe firstly revealed that GDF3 enhanced phagocytosis and bactericidal effects of macrophages by activating LXR-CD5L signaling pathway,which reduced the inflammatory response of CLP-induced septic mice,thus improving the prognosis of CLP-induced septic mice.GDF3 could be a new potiential medicine for the cure of sepsis.