Research On Association Of Macrophage Polarization Mediated By CTRP9 With Sepsis And Atherosclerosis

BackgroundSepsis is a serious systemic inflammatory disease that endangers multiple organs and systems throughout the body.It can rapidly progress from systemic inflammatory response syndrome(SIRS)to systemic multiple organ failure and even cause septic shock.Sepsis is a health problem that requires attention worldwide and is one of the main causes of death in ICU patient.More than 45%of the total cost of the ICU is used to treat this infectious disease,which brings a heavy burden to the global economy.Therefore,understanding its pathogenesis to make early diagnosis and intervention is the most effective way to reduce mortality and improve patient prognosis.Inflammatory factors play an important role in the pathogenesis of sepsis.when the body was infected by bacteria or viruses,the systemic immune system will be activated,immune cells released a large number of inflammatory factors,causing inflammatory damage to the body.In addition,the body produces nitric oxide synthase(NOS)under the intervention of toxins,of which inducible nitric oxide synthase(iNOS is also called NOS2)plays an important role.The iNOS is expressed under some stimulations such as LPS.Once expressed,it will digest the substrate and release a large amount of nitric oxide(NO)until the substrate is exhausted.Large amounts of NO continue to act on the body,especially blood vessels,they will cause a decrease in vascular reactivity,thereby lowering blood pressure.The persistent and irreversible hypotension reduced the perfusion of tissues and organs,then the tissue hypoxia eventually led to shock.Macrophages play important role in the immune pathogenesis of sepsis.Macrophages are sentinel cells of the innate immune system with their location varying from peripheral blood to various target organs including lungs,liver,brain,kidneys,skin,testes,vascular endothelium.The expression of NOS2 in macrophages is mainly controlled by cytokines and microbial products and induced by transcription.Human NOS2 is most easily observed in monocytes or macrophages of patients with inflammatory diseases.Therefore,the activation or regulation of macrophage cells in sepsis had tight relationship with the outcome of sepsis.Complement Clq/Tumor Necrosis Factor Related Protein 9(CTRP9)is a new member of CTRPs family,exerts by far the highest expression in the heart and can also be found in serum and adipose tissue.It plays an important role in cardiovascular diseases,such as ischemia-reperfusion diseases and lipid metabolism-related diseases.In addition,studies have shown that CTRP9 also plays a role in inflammatory diseases,it reduced the level of inflammatory factors in macrophages induced by oxidized low-density lipoprotein.However,the effect of CTRP9 on the expression of iNOS in macrophages and in sepsis is still unclear.In the present study,we aimed to investigate whether CTRP9 could induce iNOS expression and to explore the underlying mechanisms.1.Purposes(1)To explore the effect of CTRP9 on iNOS expression in macrophages.(2)To clarify the effect of CTRP9 and LPS on the expression of iNOS in macrophages.(3)To investigate the effect of CTRP9 knockout on the expression of iNOS in the heart and lung tissues of LPS-induced sepsis mice.(4)To study the mechanism of CTRP9 on iNOS expression in macrophages.2.Methods(1)Cell culture:?Raw264.7 cells were cultured as required.?C57BL/6j mice were injected with 6%starch into the peritoneal cavity.Peritoneal macrophages were obtained 72 h after injection by flushing the peritoneal cavity.(2)After intervention of different concentrations of CTRP9 in mouse peritoneal macrophages,CCK8 was used to detect cell cytotoxicity.(3)Peritoneal macrophages were incubated with 0-5mg/ml CTRP9 for 24 h and the iNOS protein expression was measured by western blot and immunofluorescence.(4)Raw 264.7 and peritoneal macrophages were incubated for 0-24 h with 1 ?g/ml CTRP9,western blot and PCR were used to assess the expression of iNOS.(5)The experiment was divided into four groups,namely control group,LPS group,CTRP9 group,LPS+CTRP9 group.The mRNA and protein levels of the iNOS were detected after CTRP9 treatment for 2 h and LPS for another 24 h without moving the CTRP9.(6)Proteinase K experiment:to clarify whether CTRP9 was contaminated by LPS,macrophages were stimulated by CTRP9 and proteinase K,then iNOS expression was detected by western blot.(7)Wild-type and CTRP9-/-mice were injected with saline and LPS respectively,the level of iNOS and NO in the heart and lung tissues of the mice were measured.(8)JAK2/STAT3 signal pathway related protein expression.After the macrophages were stimulated by CTRP9,the expression of JAK2,P-JAK2,STAT3 and P-STAT3 were detected by western blot.After inhibition with JAK2 inhibitor(VX509),the expression of JAK2,P-JAK2,STAT3,P-STAT3,iNOS were detected by western blot.(9)Statistical analysis.3.Results(1)The results of CCK8 showed that cell viability was not affected with different concentrations of CTRP9.(2)The iNOS protein expression increased in a dose-dependent manner,initially increasing at 1 ug/ml after 24 h.The immunofluorescence signal further confirmed that iNOS protein expression was increased in the CTRP9-treated group compared to the control group.(3)A time-dependent rise in iNOS expression was observed,with the iNOS level initially rising when incubation with CTRP9 for 6 h.CTRP9 significantly induce iNOS mRNA expression in Raw 264.7 and peritoneal macrophages as early as 6 h of incubation.The immunofluorescence staining results further showed that CTRP9 incubation triggered iNOS protein expression in time-dependent manners.(4)Both CTRP9 and LPS increased iNOS expression and they show synergistic effect peritoneal macrophage.The induction of iNOS secretion was abrogated by the use of CTRP9 with proteinase K,indicating that the effect was not attributable to contamination of the recombinant protein by LPS.(5)Intraperitoneal injection of LPS could up-regulate the levels of iNOS and NO in WT and CTRP9-/-mice,but the levels of iNOS and NO were decreased in CTRP9-/-group.(6)Both CTRP9 and LPS could up-regulate the phosphorylation levels of JAK2 and STAT3.(7)Inhibiting JAK2/STAT3 pathway by VX509,an inhibition of JAK2,could down-regulate P-JAK2,P-STAT3 and iNOS expression induced by CTRP9 in Raw 264.7 and peritoneal macrophages.4.Conclusion(1)iNOS expression showed a dose-dependent and a time-dependent up-regulation by CTRP9 in peritoneal macrophages.(2)Both CTRP9 and LPS increased iNOS expression and they showed synergistic effect in peritoneal macrophage.(3)CTRP9 knockout could reduce the expression of iNOS in the heart and lung tissues of LPS-induced sepsis mice.(4)CTRP9 induced iNOS expression through JAK2-STAT3 pathway.Background Cardiovascular diseases are the threaten to human health and are the major cause of morbidity and mortality of various cardiovascular diseases.Among them,atherosclerosis(AS)is the most common disease which is characterized by lipids from the blood entering the arterial wall,depositing on the intima,forming atherosclerotic plaques,making the arterial wall thick and hard.The "endothelial injury response theory" shows that macrophages and vascular smooth muscle cells(VSMCs)play an important role in the occurrence and development of atherosclerosis.The main characteristic of macrophages lies on their plasticity.Resting macrophages(MO)can be polarized into two extreme phenotypes,pro-inflammatory(M1)or anti?inflammatory(M2)under different micro-environment.M1 macrophages,induced by LPS or IFN-y and other micro products,are highly expression of key M1 effector molecules,such as the CD86,iNOS,TNF-a? IL-6 and IL-12.M1 macrophages play an important role in the resistance to intracellular pathogens and tumor growth.M2 macrophages,induced by IL-4? IL-13 and IL-10,are highly expression of M2 markers,such as ARG-1,CD206,IL-10,CD163.M2 macrophages are associated with immunosuppression,promotion of tissue remodeling and tumor progression.Study found that M1 and M2 were both enhanced and accumulated in human plaque,M1 macrophages likely increase plaque susceptibility to atherothrombosis by virtue of their preferential allocation to plaque shoulders,while adventitial macrophages are selectively skewed towards an M2 macrophages.Macrophages and their polarization status dominate the process of atheroma formation and progression.VSMCs are major contributor to plaque development at all stages[6],proliferation,migration and apoptosis of VSMCs are associated with formation,development and stability of plaque.Moreover,interaction between macrophages and VSMCs also have impact on plaque progression,especially cytokines and growth factors secreted by macrophages are important for VSMCs migration,apoptosis and proliferation and extracellular matrix production.CTRP9 is highly expression in heart and adipose tissues.Studies have shown its antiinflammatoiy effects in macrophages under negative regulators like ox-LDL and LPS.Furthermore,CTRP9 also plays a central role in cardiovascular disease,especially in atherosclerosis.We have recently shown that CTRP9 induces iNOS expression through JAK2/STAT3 pathway in Raw 264.7 and peritoneal macrophages.As iNOS is generally considered to be one of the markers of M1 macrophages.This study aimed to evaluate the effect and potential pathway of CTRP9 on macrophage phenotype,in AS-related inflammation.In addition,investigating whether CTRP9 have effect on phenotype of macrophages,function of VSMCs induced by CTRP9-treated macrophage supernatant and secretion of MMPs from both the VSMCs and the macrophages might further reveal the underlying mechanism of CTRP9 on AS.1.Purposes(1)To explore and clarify the effect of CTRP9 on the phenotype of macrophages.(2)To investigate the effect of macrophages on the apoptosis and proliferation of VSMCs after stimulated with CTRP9 in a co-culture system of macrophages and VSMCs.(3)To investigate the mechanism of CTRP9 on macrophages polarization*2.Methods(1)Mouse peritoneal macrophages extraction and culture: after intraperitoneal injection of sterile starch in C57BL/6J mice for 72 h5 the peritoneal macrophages were obtained by flushing the peritoneal cavity.(2)THP-1 cell culture: THP-1 cells were maintained in RPMI 1640 Medium supplemented with 10 % fetal bovine serum(FBS),1 % penicillin/streptomycin at 37 °C and 5 % CO2 in a humidified incubator.(3)Detection of macrophage phenotype: Quantitative PCR,immunoblot assays and Immunofluorescence staining were performed to investigate if CTRP9 had effect on macrophage phenotype.Then flow cytometry analysis was applied to explore the ratio of M1(F4/80 +CDllb+CD86+CD206-)and M2(F4/80+CDllb+CD8-CD206+)macrophages.Cell supernatant was used to detect TNF-a and IL-10 concentration by ELISA.(4)Mouse peritoneal macrophages were treated with LPS and different dose of CTRP9 for 24h.PCR,immunoblot assays,flow cytometry analysis and Immunofluorescence staining were performed.(5)To further understand the peritoneal macrophage polarization states specifically in the interference of IL-4,macrophages were incubated with IL-4 or IL-4 plus CTRP9.PCR,immunoblot assays,flow cytometry analysis and Immunofluorescence staining were performed.(6)Established ApoE-/-mouse and ApoE-/-CTRP9-/-mouse plaque models,plaque size and phenotype of macrophages in the plaque were detected by HE staining,oil red O staining,Masson staining and immunohistochemical.(7)Established a co-culture system for macrophages and VSMCs: VSMCs were incubated in culture supernatant with or without CTRP9 or a culture medium containing macrophages or CTRP9-treated macrophages for 24 h,36 h and 48 h.TUNEL staining and western blot was used to assess the apoptosis of VSMCs.(8)Established a co-culture system for macrophages and VSMCs: VSMCs were incubated in culture supernatant with or without CTRP9 or a culture medium containing macrophages or CTRP9-treated macrophages for 24 h,36 h and 48 h.EdU staining and western blot was used to assess the proliferation of VSMCs.(9)In the co-culture system of macrophages and VSMCs,the proteins of macrophages and VSMCs were extracted to detect the expression of MMP2 and MMP9 after incubation of CTRP9.(10)The phosphorylation levels of MAPK signaling,P-JNK,JNK,P-ERK,ERK,PP38,P38 were tested by western blot in the presence of CTRP9.After inhibition of MAPK pathway,M1 macrophages related markers were measured by western blot.(11)After using AdipoRlsi-RNA to interfere AdipoRl expression,the expression of JNK and macrophage phenotype related proteins were detected.(12)Statistical analysis.3.Results(1)CTRP9 induced M1 macrophage polarization in mouse peritoneal macrophages After CTRP9 stimulation in mouse peritoneal macrophages and THP-1 cells,Quantitative PCR and immunoblot assays demonstrated that CTRP9 caused a significantly increased expression of M1 related markers,with a decreased expression of M2 markers compared with control.Immunofluorescence staining also showed high expression of CD86(red)and low expression of CD206(green)after CTRP9 incubation,flow cytometry results showed ratio of M1 was increased and M2 was decreased.TNFa content was increased while IL-10 content has no obvious change after CTRP9 stimulated.(2)CTRP9 accelarated LPS-induced M1 activation LPS and different dose of CTRP9 were treated with mouse peritoneal macrophages,M1 markers were significantly up-regulated while M2 markers were down-regulated compared with LPS stimulates alone.Immunofluorescence staining and Flow cytometry analysis showed the same results.(3)CTRP9 showed antagonism to IL-4 by decreasing M2 macrophages markers M1 related genes including CD86 and iNOS were up-regulated while CD206 and ARG-1 induced by IL-4 were obviously decreased after CTRP9 stimulated.(4)CTRP9 knockout ameliorated inflammatory status in AS plaque The results showed that the areas of the plaque and collagen content were increased,the lipid content was decreased in CTRP9 knockout mice.M2 macrophages were predominant in the plaque of ApoE-/-CTRP9-/-mice.(5)Promotion of VSMCs apoptosis by CTRP9-treated macrophage supernatant CTRP9 did not induce significantly VSMCs apoptosis.But macrophage-VSMCs co?culture system induce apoptosis of VSMCs and CTRP9 accelerated this effect.(6)Suppression of VSMCs proliferation by CTRP9-treated macrophage supernatant EdU assay showed no obvious changes in VSMCs with or without CTRP9 incubation,but the number of EdU-positive VSMCs decreased significantly in co-culture systems,especially in CTRP9-treated macrophages-VSMCs co-culture system.CTRP9-treated macrophages-VSMCs co-culture system obviously inhibited expression of PCNA.(7)Effect of CTRP9 on expression of matrix metalloproteinase in VSMCs and macrophages In the co-culture system,CTRP9 promotes the expression of MMP2 and MMP9 in macrophages.CTRP9 alone has no obvious effect on the expression of MMP2 and MMP9 in VSMCs;In the co-culture system,CTRP9-induced macrophages inhibit the expression of MMP2 in VSMCs and promote the expression of MMP9 in VSMCs.(8)CTRP9 promoted macrophage M1 polarization through the activation of JNK MAPK signaling The phosphorylation levels of MAPK signaling were increased in the presence of CTRP9.And then we found that the JNK inhibitor SP600125 can significantly inhibit the expression of M1 macrophage markers and reduce the secretion of TNF-a.(9)AdipoRl and JNK pathway mediated CTRP9-induced macrophage polarization Suppression of AdipoRl with siRNA blocked the CTRP9-induced JNK signaling pathway.The level of CD86,CD206,ARG-1 and iNOS was significantly decreased with AdipoRl siRNA.The mRNA level of CD86 and IL-6 was also decreased with AdipoRl siRNA compared with NC si-RNA+CTRP9 group.Single staining with CD86 determined by FCM was consistent with that protein and mRNA changes.4.Conclusion(1)CTRP9 induced M1 macrophage polarization and CTRP9 knockout ameliorated inflammatoiy status in AS plaque.(2)CTRP9-treated macrophages promoted the apoptosis of VSMCs in the co-culture system.(3)CTRP9-treated macrophages inhibited the proliferation of VSMCs in the co-culture system.(4)CTRP9 promoted M1 polarization of macrophages by activating the AdipoR1/JNK/MAPK signaling pathway.