Role Of Tim-4 In Sepsis And Negatively Regulation Of Tim-4 On The Function Of Murine Macrophage

Sepsis with subsequent septic shock and multiple organ dysfunction syndrome is the most common cause of mortality in the critically ill, including severe trauma, burns, hemorrhage, and major operations, etc. Among the seriously ills in hospital, there is about 25% patients,which grow into sepsis. And there is about 10%?15% patients,which grow into severe sepsis or septic shock. Recently, clinical research certified that the release of endotoxin, LPS, which is caused by gram-negative bacterial infections, is one of the important factors leading to sepsis. The signaling receptor for LPS is TLR4, which initiates a signaling cascade and eventually transduces signaling into nucleus, resulting in the activation of macrophage and the release of varities of inflammatory mediators,such as, TNF-?, IL-1?and IL-6. And then, acting as endogenous medium, these cytokines reach target tissues and target cells by paracrine and distant secretion in circulation, resulting in systemic inflammatory response. Therefore, macrophage plays an important role in LPS-medatied sepsis. And this has great significance for us to make a further study of the activation of macrophage and regulating mechanism in sepsis.The Tim (T-cell immunoglobulin domain and mucin domain) gene family was first discovered in 2001 and received much attention due to its location on mouse chromosome 11B1.1, a genetic region associated with multiple diseases including asthma, allergy and autoimmune diseases. The Tim family consists of eight members in mice, encoding Tim-1 to Tim-4 and putative Tim-5 to Tim-8. Tim-4, unlike the other Tim molecules, is not expressed in T cells, but is expressed in APCs both at the mRNA and protein levels, particularly in mature myeloid-derived lymphoid dendritic cells and macrophages. The recent evidences showed that Tim-4 could inhibit or activate T cell-mediated immune response through binding to Tim-1 or other receptors. Furthermore, Tim-4 also acts as the receptor of phosphatidylserine and is involved in the clearance of apoptotic bodies, which suggests that Tim-4 expression on the surface of macrophage may exert the effector function to these cells and then could be involed in the development of sepsis.To certify regulatory effects of Tim-4 to macrophage, we have established the macrophage line overexpressing Tim-4 molecules, and our previous studies in vitro indicated:Tim-4 negatively regulates the activity of macrophage line RAW264.7 through down-regulating the expression of the co-stimulatory molecules including CD80,CD86 and MHCII. Based on the above experimental results, we aim to confirm Tim-4 may inhibit the activity of macrophage in vivo using autoimmune hepatitis model. Many investigations have shown that macrophages are very important in the murine acute hepatic injury model induced by ConA. So we further evaluate the roles of Tim-4 in vivo with this typical model.This study indicated hepatic injury was alleviated in the group of RAW264.7- Tim-4 transfusion. These results suggest that the molecule of Tim-4 negatively regulated function of murine macrophage.However, it is to be confirmed that the regulation of Tim-4 on macrophage in many other cell systems.Additionally,it has not been reported that what pathway Tim-4 regulates the function of macrophage and whether the regulation of Tim-4 on macrophage has correlations with diseases, which macrophage involves m.In this study, we firstly investigated the role of Tim-4-medatied the regulation of macrophage in sepsis and then explored the effect and mechanism of Tim-4 on the LPS/TLR4-medatied macrophage activation in vitro using of murine macrophage cell line RAW254.7 and murine peritoneal macrophage. On these basis, it has been investigated the effect of Tim-4 on the poly (I:C) and IFN-?-medatied macrophage activation.The study not only provides an experimental basis for further studying the mechanism of pathogenesis in sepsis and the role of Tim-4 in innate immunity but a new target for therpy and preventing of sepsis. I Tim-4 negatively regulated LPS-medatied macrophage activation and inhibited LPS -medatied sepsisThe extert function of macrophage depands on macrophage activation and the release of many kinds of effective molecules. The excessive activation of macrophage caused by LPS results in sepsis. We firstly studied the effect and mechanism of Tim-4 on the LPS/TLR4-medatied macrophage activation. And it was explored that the role of Tim-4-mediated macrophage activation in sepsis. (?) Tim-4 inhibited LPS -medatied sepsis in vivoIn LPS-induced sepsis, LPS activates macrophages and the activated macrophages synthesize and release cytokines, such as,TNF-a, IL-1?and IL-6. These cytokines execute biological effect in process of binding with receptors, and then causes systemic inflammatory response. Consequently, activation of macrophage plays an important role in LPS-induced sepsis. For this, LPS-induced sepsis acts as a model to explore the regulation of Tim-4 on macrophage in vivo. 1. Upregulation of Tim-4 expression in LPS-induced sepsisBALB/c mice were intraperitoneally injected with dose of 15mg/kg LPS. After 8h, bone marrow derived macrophage(BMDM),spleen,lung and kidney were collected. RT-PCR showed that expression of Tim-4 was increased in BMDM,spleen and lung. 2. Establisment of method for tranfection in vivo using PEI loadingTo study the role of Tim-4 in LPS-induced sepsis, we designed overexpression or silence of Tim-4 in vivo. It was applied that plasimids with PEI loading were injected by tail vein and expected to establish the effective method of gene transfection in vivo. To verify the efficiecncy of transfection, the mixture of pEGFP-N1 plasimid expressing green fluorescence with PEI was injected into mice by tail vein. After 3 days and 5 days,green fluorescence in spleen was observed, which indicated that plasmids DNA could be successfully transferred into mice. 3. Overexpression of Tim-4 slow down LPS-induced sepsisTo further invastgate the effect of Tim-4 on LPS-induced sepsis, pEGFP-N1-Tim-4 plasmids were transferred into mice. After 3days, RT-PCR showed that expression of Tim-4 in spleen,liver,lung and kidney were increased,which indicated that Tim-4 was successfully overexpressed in vivo.The mixture of pEGFP-Nl-Tim-4/ pEGFP-N1 plasmid and PEI was transfected into BALB/c mice by tail injection. After 24h,15mg/kg LPS were injected to induce sepsis. Compared with control group(n=10), the survival rate of pEGFP-N1-Tim-4 group(p<0.05, n=10) was increased. The prdduction of serum TNF-a, IL-6 in pEGFP-N1-Tim-4 group(n=6) were decreased compared with control group(TNF-?, 2.22±0.4 ng/mL vs 1.57±0.7ng/mL,p<0.05; IL-1?,1035.5±63.6 pg/mL vs 610.1±56.3 pg/mL, p<0.05).These results indicated overexpression of Tim-4 slow down LPS-induced sepsis. 4. Silence of Tim-4 by Tim-4 shRNA aggravated LPS-induced sepsisTim-4 shRNA expression vector plasmids and control vector plasmids were transferred into BALB/c mice with PEI loading. After 24h,15mg/kg LPS was injected. The survival rate of mice and production of cytokines were observed. RT-PCR showed that expression of Tim-4 in spleen was downregulated in mice with injection of Tim-4 shRNA expression vector plasmids.,suggesting Tim-4 shRNA could effectively inhibit the expression of Tim-4. Compared with control group(n=10), the survival rate of Tim-4 shRNA group (p<0.05, n=10) was dreased. In addition, ELISA showed that serum TNF-a, IL-6 in Tim-4 shRNA group(n=6) were increased compared with control group(n=6) (TNF-?,2.62±0.33ng/mL vs 3.47±0.6pg/mL, p<0.05;IL-1?,985.3±43.6 pg/mL vs 1510.1±44.5 pg/mL,p<0.05). These results showed that Tim-4 shRNA aggravated LPS-induced sepsis.Above results indicated that inhibition of Tim-4 in vivo aggravated LPS-induced sepsis, promoted the production of inflammatory factors and degraded the rate of survival.(?) The effect and mechanism of Tim-4 on the LPS-medatied macrophage activation in vitroLPS is a strong activator of macrophage,which evokes production of cytokines and results in systemic inflammatory response. To futher study the function of Tim-4 on macrophage,we firstly detected expression of Tim-4 in activated macrophages. 1. LPS promoted the expression of Tim-4 in the cultivated-macrophage in vitro After stimulation with end concentration of 0,10,50 and 100ng/mL LPS, expression of Tim-4 mRNA was increased in murine macrophage cell line RAW264.7 with 10ng/mLPS stimulation. And expression of Tim-4 mRNA was further increased with 50 and 100ng/mL LPS stimulation. In addition, expression of Tim-4 mRNA reached the peak at 24h when RAW264.7 was stimulated with 100ng/mL LPS at time point of 6h,12h and 24h.Western bolt showed there was a low expression of Tim-4 in resting macrophage cell line RAW264.7. Expression of Tim-4 mRNA was induced by 50 ng/mL and 100 ng/mL LPS stimulation. After exposure to different time points of LPS, Tim-4 protein expression in RAW264.7 increased with LPS in a time-dependant manne.After exposure to 0,10,50 and 100ng/mL LPS, Tim-4 mRNA accumulated in murine peritoneal macrophage at 24h. RT-PCR showed that expression of Tim-4 was higher with the stimulation of 50 and 100ng/mL LPS. When murine peritoneal macrophage was stimulated with 100ng/mL LPS at time point of 6 h,12h and 24h, expression of Tim-4 was increased at 12h and reached the peak with the stimulation of LPS for 24 hours. These results indicated that Tim-4 could be induced when macrophages were activated and Tim-4 expression was in dose- and time- dependent manner 2. Tim-4 inhibited the production of NO, TNF-?, IL-1?, IL-6 and IFN-?in LPS-mediated imacrophage activationTo study the regulation of Tim-4 to LPS/TLR4-mediated macrophage activation, we used overexpression of Tim-4, Tim-4 shRNA and Tim-4 blocking antibody to investigate the effect of Tim-4 on the production of NO and cytokines in LPS-activated macrophage in both positive and negative aspects. 2.1. Tim-4 overexpression inhibited production of NO, TNF-?, IL-1?, IL-6 and IFN-?in LPS-activated macrophage cellsStatistic analysis showed that the secretion of NO and iNOS protein in RAW264.7-pcDNA3-Tim-4 cells was lower than RAW264.7-pcDNA3 cells with LPS stimulation. Supernants were collected in 0.2?g,0.6?g and 1?g pcDNA3-Tim-4 and pcDNA3-transfected peritoneal macrophage without or with LPS stimulation. Griess assays showed that secretion of NO in 0.6?g and 1?g pcDNA3-Tim-4 group were down-regulated compared with pcDNA3 group (23.15.23±0.72?M vs 19.23±0.69?M,p<0.05). ELISA showed that compared with pcDNA3 group, production of TNF-?in 1?g pcDNA3-Tim-4 group was down-regulated (3002.6±58.1pg/mL vs 2102±49.63pg/mL,p<0.05). In addition, Real Time PCR showed that expression of IFN-P mRNA was upregulated in pcDNA3-Tim-4 group compared with pcDNA3 group(p<0.05). 2.2 Tim-4 blocking antibody promotes production NO, TNF-?, IL-1?,IL-6 and IFN-?in activated macrophage cellsTo further study the effect of Tim-4 blocking on macrophage, supernants were collected with treatment of different concentrations of anti-Tim-4 (final concentrations,1?g/mL?2.5?g/mL?5?g/mL?10?g/mL). Results showed that compared to control IgG, the secretion of NO and the expression of iNOS protein in 5?g/mL anti-Tim-4 blocking group was significantly higher with LPS stimulation (LPS,32.73±0.98?M vs 37.43±0.81?M,p<0.05). ELISA showed that compared to control IgG, IL-6 production in 2.5 u g/mL anti-Tim-4 group was higher (682.7±44.73 pg/mL vs 543.3±20.87 pg/mL,p<0.05);production of TNF-??IL-1?and IL-6 in 5?g/mL anti-Tim-4 group was higher than control IgG group (TNF-a,2503.6±53.42pg/mL vs 3407.2±48.96pg/mL, p<0.05; IL-1?,1125±63.31 pg/mL vs 1945.3±46.8 pg/mL, p<0.05; IL-6,654.4±25.11 vs 1007.7±21.8,p<0.005);production of TNF-a in 10?g/mL anti-Tim-4 group was lower than control IgG group(2850.3±63.12pg/mL vs 2003±58.16pg/mL,p<0.05). Real Time PCR showed that expression of IFN-?mRNA was decreased in 5?g/mL anti-Tim-4 group compared with control IgG group(p<0.05). 2.3 Tim-4 shRNA promotes production NO, TNF-a, IL-1?and IL-6 in activated macrophage cellsGriess assays showed that compared to controls, the secretion of NO in Tim-4 shRNA-transfected group was significantly higher (25.6±2.18?M vs 38.5±1.16?M, p<0.05) with LPS stimulation. ELISA showed that compared to control group, IL-6 production in 0.6?g Tim-4 shRNA -transfected group was upregulated (641.7± 40.83pg/mL vs 504.3±13.86pg/mL,p<0.05),the production of TNF-?, IL-1?and IL-6 in 1?g Tim-4 shRNA -transfected group was upregulated (TNF-a, 2012.5±60.42pg/mL vs 2809.7±40.86pg/mL,p<0.05; IL-1?,1247±53.39 pg/mL vs 845.3±66.03 pg/mL,p<0.05;IL-6,785.3±29.21 vs 477.7±29.19 p<0.005.)These results indicate that Tim-4 blocking upregulated NO production and proinflammation cytokines secretion in macrophages.Above results showed expression of Tim-4 was upregulated in LPS-activated macrophages.And upregulated Tim-4 inhibited the production of NO and cytokines in LPS-activated macrophages,suggesting that Tim-4 inhibited the activation of macrophage induced by LPS. 3. Study of the molecular mechanism of the inhibitory of LPS-mediated macrophage activation by Tim-4LPS-initiated MyD88-dependent signaling pathways can activate MAPK and NF-?B via sequential activation of MyD88/IL-1R-associated kinase/TRAF6, resulting in production of NO and proinflammatory cytokines (IL-1, IL-6, TNF-a, etc), whereas LPS can also initiate MyD88-independent and TRIF-dependent activation of IRFs to regulate the IFN-inducible genes (IFN-?etc). To study the mechanism of the inhibitory of LPS/TLR4-mediated macrophage activation by Tim-4, key proteins in MyD88-dependent and MyD88-independent signaling pathways were detected in Tim-4 overexpression or Tim-4 blocked macrophages. 3.1 Tim-4 inhibited the activation of macrophage via MyD88-dependent signaling pathways and participated in the production of NO and cytokines 3.1.1 Overexpression of Tim-4 inhibited activation of NF-?B signaling pathway and involved in the regulation of production of NO and pro-inflammatory cytokines in macrophageTo futher study the signaling pathways in Tim-4-mediated macrophages regulation, RAW264.7-pcDNA3-Tim-4 and RAW264.7-pcDNA3 cells were stimulated with LPS and western blot was used to detect expression of NF-?B pathway. Western Blot showed dreased phosphorylation of NF-?B p65 at 30min and 60min time points, compared to the control group. NF-?B inhibitory ligand (final concentration was 83.3?g/mL) was used before LPS stimulation in RAW264.7-pcDNA3-Tim-4 and RAW264.7-pcDNA3 cells. Results showed that down-regulation of NO induced by Tim-4 overexpression was reversed by NF-?B inhibitory ligand in LPS stimulated macrophages activation. 3.1.2 Tim-4 blocking antibody promoted activation of NF-?B and then participated in the activation of macrophage and the production of NO and cytokinesNF-?B is the key transcription factor in LPS-initiated MyD88-dependent signaling pathways. To study the signaling pathways in Tim-4-mediated macrophages regulation, Tim-4 blocking antibody pretreated peritoneal macrophage were stimulated with LPS and western blot was used to detect expression of critical molecules in NF-?B pathway, the classical pathway involved in cytokines and NO secretion.Tim-4 blockade peritoneal macrophage cell were stimulated with LPS, and cells were collected at indicated time points. Western Blot showed enhanced phosphorylation of NF-?B p65 at 30min and 60min time points, compared to the control group.To further confirm the the regulation of anti-Tim-4 blocking in NF-?B pathway. NF-?B inhibitory ligand (final concentration was 30?M) was used before anti-Tim-4 treatment. Results showed that up-regulation of NO induced by Tim-4 blockade was reversed by NF-?B inhibitory ligand in LPS stimulated macrophages activation, suggesting Tim-4 blockade promotes NF-?B pathway. Above results showed that Tim-4 inhibited activation of NF-?B signaling pathway and then inhibited the production of NO and pro-inflammatory cytokines in macrophage. 3.2 Tim-4 inhibited activation of LPS/MAPK signaling pathway in macrophage 3.2.1 Overexpression of Tim-4 inhibited the activation of LPS/MAPK signaling pathway To futher understand the effect of Tim-4 on MAPK(ERKl/2,JNKand p38) signaling pathway in macrophage, RAW264.7-pcDNA3-Tim-4 and RAW264.7-pcDNA3 cells were stimulated with LPS and western blot was used to detect expression of MAPK pathway. Western Blot showed dreased phosphorylation of ERK1/2 and p38 at 15min,30min and 60min time points, compared to the control group,which indicated overexpression of Tim-4 inhibits activation of LPS/MAPK signaling pathway in macrophage. 3.2.2 Tim-4 blocking antibody promoted the activation of LPS/MAPK signaling pathwayTo understand the effect of blockade of Tim-4 on MAPK(ERK1/2,JNKand p38) signaling pathway in macrophage, Tim-4 blocking antibody pretreating peritoneal macrophages were stimulated with LPS and western blot was used to detect expression of critical molecules in MAPK pathway. Western Blot showed enhanced phosphorylation of ERK1/2 and p38 at 30min and 60min time points, compared to the control group.These indicated blockade of Tim-4 upregulated activation of MAPK signaling pathway in macrophage.Above results indicated that Tim-4 inhibited the activation of LPS/MAPK signaling pathway in macrophage. 3.3. Tim-4 inhibited activation of IRF3 in macrophage. 3.3.1 Overexpression of Tim-4 inhibited activation of IRF3 in RAW264.7 RAW264.7-pcDNA3-Tim-4 and RAW264.7-pcDNA3 cells were stimulated with LPS for 0min,15min,30 min and 60min. Western blot was used to detect expression of phosphorylation of IRF3. It showed dreased phosphorylation of phosphorylation of IRF3 at 30min and 60min time points, compared to the control group,which indicated overexpression of Tim-4 inhibits activation of IRF3 in macrophage. 3.3.2 Tim-4 blocking antibody promoted activation of IRF3 in macrophage.To research the effect of Tim-4 on LPS/MyD88 independent pathway, western blot showed that enhanced phosphorylation of IRF3 at 30min and 60min time points, compared to the control group.These indicated blockade of Tim-4 enhanced activation of LPS/MyD88 independent pathway.Above results indicated that Tim-4 inhibited the activation of IRF3 in macrophage.All above results indicated that Tim-4 inhibited the activation of LPS/TLR4 pathway in macrophage through inhibition of the activation of NF-?B, ERK1/2, p38, JNK and IRF3 and then inhibited the activation of macrophage induced by LPS.?The negative effect and mechanism of Tim-4 on the secretion of NO inpoly(I:C)-induced macrophage activationTo further investigate the regulation of Tim-4 on the function of macrophage, we initially explored the regulation and mechanism of Tim-4 on the secretion of NO in poly(I:C))-induced macrophage activation. 1.Expression of Tim-4 mRNA was increased in macrophage by poly(I:C)RT-PCR showed that expression of Tim-4 mRNA was increased murine peritoneal macrophage with treatment of 20?g/mL and 50?g/mL poly(I:C) for 24 h (p<0.05). 2. Tim-4 inhibited the secretion of NO in poly(I:C)-activated macrophage 2.1 Overexpression of Tim-4 inhibited secretion of NO in poly(I:C)-activated RAW264.7Griess assays showed that secretion of NO in pcDNA3-Tim-4 group was down-regulated compared with pcDNA3 group (16.45±1.9?M vs 25.13±1.6?M, p<0.05) 2.2 Blockade of Tim-4 promoted the secretion of NO in poly(I:C)-activated peritoneal macrophageGriess assays showed that secretion of NO in pretreatment of Tim-4 blocking antibody group was up-regulated compared with control group (20.47±1.85?M vs26.73±1.19?M,p<0.05) 3 NF-?B signaling pathway was involved in the inhibition of Tim-4 on the secretion of NO in poly(I:C)-activated macrophagePoly(I:C) binds with TLR3,which causes the activation of NF-?B signaling pathway. Then a series of genes were transcribed,such as, iNOS. 3.1 NF-?B signaling pathway was involved in the inhibition of Tim-4 overexpression on secretion of NO in poly(I:C)-activated RAW264.7 NF-?B inhibitory ligand (final concentration was 83.3?g/mL) was used before poly(I:C) stimulation in RAW264.7-pcDNA3-Tim-4 and RAW264.7-pcDNA3 cells. Results showed that down-regulation of NO induced by Tim-4 overexpression was reversed by NF-?B inhibitory ligand in poly(I:C) stimulated macrophages activation. 3.2 NF-?B signaling pathway was involved in the promotion of Tim-4 antibody on the secretion of NO in poly(I:C)-activated peritoneal macrophageTo further confirm the the regulation of anti-Tim-4 blocking in NF-?B pathway. NF-?B inhibitory ligand (final concentration was 30?M) was used before anti-Tim-4 treatment. Results showed that up-regulation of NO induced by Tim-4 blockade was reversed by NF-?B inhibitory ligand in poly(I:C) stimulated macrophages activation.These results suggested that Tim-4 inhibits secretion of NO in macrophage through NF-?B signaling pathway.?Tim-4 negatively regulated the secretion of NO in IFN-?-mediated macrophage activationIt is known that IFN-?constitutes one of the most potent macrophage-activatied factors. We used IFN-?-activated macrophage to further study the regulation of Tim-4 on the function of macrophage. 1. Tim-4 was upregulated in murine peritoneal macrophage by IFN-yRT-PCR showed that expression of Tim-4 mRNA was increased in murine peritoneal macrophage with stimulation of 100U/mL IFN-?for 24h (p<0.001) 2. Tim-4 inhibited production of NO and expression of iNOS in IFN-?activated macrophage 2.1 Overexpression of Tim-4 inhibited production of NO and expression of iNOS in IFN-y activated RAW264.7 Griess assays showed that, compared with RAW264.7-pcDNA3 cells,secretion of NO was down-regulated in RAW264.7-pcDNA3-Tim-4 cells with stimulation of IFN-y for 24h. (19.33±?M vs24.93±1.507?M,p<0.05). Western blot showed that the expression of iNOS was decreased in RAW264.7-pcDNA3-Tim-4 cells compared with control cells with stimulation of IFN-y for 12h. 2.2 Blockade of Tim-4 promoted production NO and expression of iNOS in IFN-y activated peritoneal macrophage cellsRT-PCR showed that the secretion of NO in Tim-4 blocking murine peritoneal macrophages was higher than control cells with stimulation of IFN-y for 24h (21.6±1.78±M vs28.13±0.96?M,p<0.05).In addition, compared with control cells, the expression of iNOS protein in Tim-4 blocking murine peritoneal macrophages was enhanced. 3. Blockade of Tim-4 promoted the activation of Jak2/Statl signaling pathwayBinding of IFN-y to its receptor induces the assembly of an active receptor complex and consequent transphosphorylation of the receptor-associated Janus tyrosine kinases Jakl and Jak2. The activation of these kinases induces phosphorylation of the cytoplasmic tail of the receptor itself, which lacks intrinsic kinase activity. The cytosolic protein Statl is then recruited to the activated IFN-y-receptor complex and phosphorylated. Upon phosphorylation, Statl translocate to the nucleus where they bind to the IFN-y-activated site,found in the promoter of many IFN-y-induced genes.,including the inducible NO synthase (iNOS).To further confirm the the regulation of anti-Tim-4 blocking in Jak2/Statl pathway,5?g/mL Tim-4 blocking antibody pretreating murine peritoneal macrophages were stimulated with IFN-y. Western Blot showed enhanced phosphorylation of p-Jak2 and p-statl at 30min and 60min time points compared to the control group. Results suggested that Tim-4 blockade promoted the activation of Jak2/Statl pathway.Taken together, Tim-4 expression was upregulated in avtivated- macropgage. The upregulated Tim-4 inhibited the production of proinflammatory cytokines and NO in macrophage in vitro. In vivo, Tim-4 negatively regulated LPS-induced sepsis.Thus, Tim-4 negatively regulated macrophage activation.The study provides a basis for further studying biological activity of macrophage and provides a new target for inflammation that macrophages involved in.