The Effects And Potential Mechanisms Of TSLP In Macrophages Polarization And Cardiac Remodeling After Myocardial Infarction

1 BackgroudMyocardial infarction(MI)is one of the leading causes of progression to Heart Failure(HF)among Coronary Artery Disease.MI occurs from coronary artery occlusion,resulting in low blood supply and oxygen deprivation at the downstream myocardium,inducing necrosis and apoptosis of resident cardiomyocytes,infiltration of monocytes/macrophages,initiation of inflammation,and progression of tissue repair.Left untreated,these post-MI healing events lead to cardiac fibrosis and electrophysiological challenge in the myocardium,alter left ventricle(LV)loading,leading to LV dysfunction and ultimately HF.The process maybe affected by molecular abnormalities such as:excessive extracellular matrix(ECM)degradation by matrix metalloproteinases(MMPs),aberrant initiation and prolongation of inflammation,disproportionate fibrosis,and cardiomyocyte hypertrophy.Successfully targeting one or more of these abnormalities has been challenging.Many studies have shown cardiac remodeling is a complex process and that the quality of infarct healing determines the prognosis.Macrophages are the primary responders of the immune system after MI.There are largely two types of macrophages involved in cardiac repair:classically activated M1 macrophages that secrete pro-inflammatory cytokines,such as tumor necrosis factor-α(TNF-α)and interleukin 6(IL-6),and alternatively activated M2 macrophages that produce anti-inflammatory cytokines including interleukin 4(IL-4)and interleukin 10(IL-10).After MI,ischemic injury activates the recruitment of large numbers of peripheral monocytes/macrophages which differentiate into inflammatory M1 macrophages responsible for removal of cell debris and necrotic cell clearance(phase 1).Thereafter,during the inflammation resolution stage(phase 2),cardiac-tissue dominant macrophage populations become reparative M2 macrophages that propagate cardiac repair.Recently,earlier phenotype shift of M1 to M2 macrophages has demonstrated significant improvements in MI wound modeling and cardiac function.Therefore,therapeutic strategies to increase M2 polarization and aid inflammatory resolution within the infarct are needed for MI treatment.Thymic stromal lymphopoietin(TSLP)is a novel IL-7-like cytokine,primarily expressed by epithelial cells,where under inflammatory conditions,some other cells such as macrophages,smooth muscle cells and fibroblasts,are also able to produce TSLP.Under the action of external stimulation,the above cells synthesize and secrete TSLP,and exert biological effects through binding with a heterodimeric TSLP receptor(TSLPR,composed of TSLPR subunit and IL-7Ra subunit)on the cell surface.TSLP/TSLPR has been reported to play a significant role in the amplification of M2 macrophage polarization and anti-inflammatory chemokine production.In this study,we investigated the roles of TSLP on macrophages polarization and cardiac remodeling after MI.2 Objectives2.1 To explore the levels of TSLP in serum and myocardium tissue after MI.2.2 To explore the role of TSLP on macrophages polarization and cardiac remodeling after MI.2.3 To explore the effects of TSLP on cardiac function recovery after MI.3 Methods3.1 Animal modelsEight-week-old C57BL/6J wild-type male mice(weighing 20-25 g)were fed regular chow and maintained under standard conditions.After 1 week of acclimatization,the mice were randomly divided into four groups(n=12 each)for treatments,as follows:control,MI,MI+rTSLP and MI+anti-TSLP.MI was created by permanent left anterior descending coronary artery ligation.Mice were anesthetized for for all procedures(isoflurane,2%;02 2 L/min),intubated and ventilated.Left thoracotomy was performed in the fourth intercostal space and left anterior descending coronary artery was visualized and ligated just distal to its main bifurcation using 7.0 Neuriolon.The success of the coronary occlusion was confrmed by pallor and regional-wall motion abnormality of the left ventricle.After MI surgery,mice were injected subcutaneously in the nuchal area with recombinant mouse TSLP(600 ng/250 μl/mouse/day)or anti-TSLP neutralizing antibody(600 ng/250μl/mouse/day),or PBS for 5 consecutive days.Mice were euthanized 1 week after surgery.3.2 Functional analysisEchocardiography was performed using the Vevo2100 imaging system.Images were obtained from the two-dimensional and M-mode.LV dimensions and wall thickness were made at end-systole and end-diastole using the American Society of Echocardiography criteria.Left ventricular fractional shortening(%FS)and ejection fraction(%EF)were calculated by the echocardiography system.3.3 Enzyme-linked immunosorbent assays(ELISA)The level of TSLP in serum were measured by ELISA kits following the manufacturer’s protocol.3.4 Western blot analysisProteins were separated by 10%SDS-PAGE and transferred onto PVDF.Membranes were blocked and incubated with primary antibodies against TSLP andβ-actin.Membranes were visualized with the chemiluminescence reagent,and the intensities of protein bands were evaluated by ImageJ.Protein expression was assessed relative to p-actin.3.5 ImmunohistochemistryThe mouse hearts were fixed in 4%formalin for a minimum of 24 hours.The fixed tissues were embedded in paraffn and sectioned(5 μm)for staining.Cross-sections of the aortic roots(predilection site of atherosclerosis)were stained with haematoxylin and eosin(H&E)for inflammatory cell infiltration assessments.To evaluate fibrotic tissue formation after infarction,sections were stained with Masson’s trichrome and Picrosirius red staining.Quantitative analysis for H&E staining and Masson’s trichrome staining was performed with densitometry using Image-Pro Plus 5.0 software,and the percentage was further expressed to the total left ventricle.3.6 Immunofluorescence stainingSections were labelled with unconjugated primary antibodies against MOMA-2 and M1 marker CD86,or M2 marker CD206 simultaneously overnight,followed by incubation with a fluorophore-conjugated secondary antibody for 30 min.The stained sections were mounted with DAPI-containing VectorShield mounting medium(Vector)and then viewed using an Olympus BX53 fluorescence microscope.3.7 Statistical analysisAll experiments were repeated at least three times,and data were presented as the mean ± S.E.M.Statistical analysis was carried out using ANOVA followed by Tukey’spost hoc test.P<0.05 was considered significant.4 Results4.1 TSLP expression was increased in reponse to myocardial infarction in vivoTo investigate the changes of TSLP expression after myocardial infarction(MI),we established MI mice models and tested the levels of TSLP in serum and myocardial tissue.ELISA results showed that serum TSLP level rose time-dependently,significant increases were detected as early as day 3 compared with the control group,and the concentration remained quite high through day 7.Western blot results also showed that TSLP expression in myocardial tissue 7 days after MI was significantly higher than the control.4.2 TSLP promoted the early and robust appearance of reparative M2 macrophages in infarcted myocardiumWe then investigated the detailed changes in the macrophage phenotype in infarcted myocardium over the first week of MI.On day 1 post-MI,the infarct tissue of three groups of mice accumulated similar numbers of CD86+inflammatory macrophages.The population of M1 mcrophages dropped at day 3 and were dramatically lower at day 7 in the vehicle-treated hearts,and the trend were even more obvious in rTSLP-treated group.Very few reparative CD206+macrophages were present at day 1 post-M1 but accumulated from day 3 through day 7 in the vehicle-treated mice.Remarkably,rTSLP-treated hearts had dramatically higher numbers of CD206+reparative macrophages as early as day 1 and numbers remained quite high through day 7 in the infarct region when compared to mice treated with the vehicle.By day 7,M2 macrophages remained absolutely predominat in rTSLP-and vehicle-treated mice,though both M1 and M2 cell numbers decreased.However,in anti-TSLP group,the changes in CD86+and CD206+cell numbers were much slight during the first week post-MI,implying that TSLP play a critical role in promoting M1 to M2 macrophage polarization.4.3 TSLP promote inflammation alleviation and cardiac healing after MIHematoxylin and eosin(H&E)staining was performed to quantitatively assess the area of inflammatory cell infiltration at day 7.Cross sections of mouse hearts demonstrated an extensively reduced inflammatory cell population at the border zone in the rTSLP-treated group.Quantitative densitometry analysis indicated that the rTSLP group showed a significant reduction in the inflammatory cell population,while the anti-TSLP group demonstrated aggravated inflammation.Upon analysis of LV collagen volume by Masson’s trichrome and Picrosirius red stainings,the rTSLP+MI group showed greater fibrosis compared with the MI group,while blocking TSLP reduced the collagen deposition.4.4 TSLP improved Recovery of Cardiac FunctionTo assess the functional recovery of the infarcted myocardium,transthoracic echocardiography was performed.One week after the treatments,there is a trend of improvement in ejection fraction(EF)and fractional shortening(FS)in the rTSLP group compared with the vehicle group,while the cardiac function was significantly deteriorative in the anti-TSLP.In addition,the anti-TSLP group demonstrated a increase in the left ventricular volgroupume at end diastole(LVvold)compared to the vehicle,implying an uncontrolled process of cardiac remodeling.The results indicates that TSLP may alleviate post-MI remodeling and improve heart functional recovery.5 Conclusion5.1 The levels of TSLP in mice serum and myocardium tissue were increased after MI.5.2 TSLP promotes phenotype shift of M1 to M2 macrophages,inflammation alleviation and cardiac healing.5.3 TSLP improved recovery of cardiac function.1 BackgroudMyocardial infarction(MI)is one of the leading causes of progression to Heart Failure(HF)among Coronary Artery Disease.MI occurs from coronary artery occlusion,resulting in low blood supply and oxygen deprivation at the downstream myocardium,inducing necrosis and apoptosis of resident cardiomyocytes,infiltration of monocytes/macrophages,initiation of inflammation,and progression of tissue repair.Left untreated,these post-MI healing events lead to cardiac fibrosis and electrophysiological challenge in the myocardium,alter left ventricle(LV)loading,leading to LV dysfunction and ultimately HF.Many studies have shown cardiac remodeling is a complex process and that the quality of infarct healing determines the prognosis.Macrophages are the primary responders of the immune system after MI.There are largely two types of macrophages involved in cardiac repair:classically activated M1 macrophages that secrete pro-inflammatory cytokines,such as tumor necrosis factor-a(TNF-a)and interleukin 6(IL-6),and alternatively activated M2 macrophages that produce anti-inflammatory cytokines including interleukin 4(IL-4)and interleukin 10(IL-10).In addition,macrophages secrete different MMPs(notably MMP-2 and MMP-9),which alter the extracellular matrix,hence participating in cardiac tissue remodeling and predilection to cardiac rupture.M1 macrophages produce high levels of MMPs(e.g.,MMP-1,-2,-3,-10)both in vitro and in atherosclerotic plaques,whereas M2 macrophages produce much lower levels.After MI,ischemic injury activates the recruitment of large numbers of peripheral monocytes/macrophages which differentiate into inflammatory M1 macrophages responsible for removal of cell debris and necrotic cell clearance(phase 1).Thereafter,during the inflammation resolution stage(phase 2),cardiac-tissue dominant macrophage populations become reparative M2 macrophages that propagate cardiac repair.Recently,earlier phenotype shift of Ml to M2 macrophages has demonstrated significant improvements in MI wound modeling and cardiac function.Therefore,therapeutic strategies to increase M2 polarization and aid inflammatory resolution within the infarct are needed for MI treatment.Thymic stromal lymphopoietin(TSLP)is a novel IL-7-like cytokine,primarily expressed by epithelial cells,where under inflammatory conditions,some other cells such as macrophages,smooth muscle cells and fibroblasts,are also able to produce TSLP.Under the action of external stimulation,the above cells synthesize and secrete TSLP,and exert biological effects through binding with a heterodimeric TSLP receptor(TSLPR,composed of TSLPR subunit and IL-7Ra subunit)on the cell surface.TSLP/TSLPR has been reported to play a significant role in the amplification of M2 macrophage polarization and anti-inflammatory chemokine production.Cumulative evidence indicates that the renin-angiotensin system is activated after MI.The central product of the renin-angiotensin system,Ang Ⅱ,is involved in the development of myocardial remodeling following MI.Angll has been reported to induce TSLP production in smooth muscle cells,however,the effects of Angll on TSLP expression in macrophages remains to be explored.In this study,we investigated whether Angll could induce TSLP expression in macrophages,and the roles of TSLP on macrophages polarization and cardiac remodeling after MI.2 Objectives2.1 To explore the effects of TSLP on macrophages polarization and inflammation.2.2 To explore whether Angll could induce TSLP production in macrophages,2.3 To explore whether Angll could promote M2 macrophages polarization and alleviate inflammation by up-regulating TSLP expression.3 Methods3.1 Cell culture and treatmentBone marrow derive macrophage(BMDMs)were isolated from female C57BL/6 mice by flushing the femur and tibia with PBS.These cells were cultured in RPMI 1640 medium containing 10%FBS and 10 ng/ml of murine M-CSF for 5-7 days.BMDMs were incubated with IFN-γ(100 ng/ml)or IL-4(10 ng/mL)to induce M1 or M2 macrophages,respectively.Then cells were stimulated with rTSLP(20ng/ml;RD),AngⅡ(100nM;Sigma-Aldrich Co.)in the presence or absence of anti-TSLP neutralizing antibody(500ng/ml;RD)for 72h.3.2 Western blot analysisProteins were separated by 10%SDS-PAGE and transferred onto PVDF.Membranes were blocked and incubated with primary antibodies against TSLP,iNOS,Arg-1,MMP2,MMP9 and(3-actin.Membranes were visualized with the chemiluminescence reagent,and the intensities of protein bands were evaluated by ImageJ.Protein expression was assessed relative to β-actin.3.3 Enzyme-linked immunosorbent assays(ELISA)The concentrations of TSLP,TNF-a,IL-6,IL-10 and TGF-β in cell culture medium,were measured by ELISA kits following the manufacturer’s protocol.3.4 Gelatin zymographyCell culture supernatants were electrophoresed in a 10%polyacrylamide gel containing 1 mg/ml gelatin.Lytic bands of gelatin digestion represented MMP2 and MMP9 activity.3.5 Flow cytometryBMDMs were washed,blocked and then incubated for 30 min with APC-conjugated anti-mouse CD86(17-0862-82,eBioscience)or PE-conjugated anti-mouse CD206(12-2061-82,eBioscience)using the appropriate isotype controls.The results were acquired using a BD FACS Caliber flow cytometer(BD Biosciences,USA)and analysed with FlowJo v9.0 software(Tree Star,Inc.,FlowJo v9.0,Ashland,Or,USA).3.6 Statistical analysisAll experiments were repeated at least three times,and data were presented as the mean ± S.E.M Statistical analysis was carried out using ANOVA followed by Tukey’spost hoc test.P<0.05 was considered significant4 Results3.1 TSLP promote M2 macrophage polarizationBMDMs were coincubated with IFN-y(100 ng/ml)and IL-4(10 ng/mL)to induce M1 or M2 phenotype,respectively.To investigate the role of TSLP in macrophage polarization,we examined the expression of iNos(Ml marker)and Argl(M2 marker)in rTSLP-stimulated BMDMs.rTSLP dose-dependently decreased iNos expression while increased Argl expression,implying that TSLP promoted M2 polarization of macrophage,while suppressed M1 polarization.In IFN-γ induced M1 macrophages,rTSLP dose-dependently decreased the expression of MMP2 and MMP9,which are engaged in ECM degradation and remodeling.3.2 TSLP could alleviate inflammationTo further investigate the effects of TSLP on inflammation,we measured the levels of inflammatory cytokines in cell supernatants using ELISA.rTSLP treatment markedly decreased the secretion of pro-inflammatory cytokines(IL-6,TNF-a),and promoted that of anti-inflammatory cytokines(IL-10,TGF-β).In addition,the gelatin zymography assay revealed that the activity of MMP2 and MMP9 were decreased by rTSLP treatment.3.3 Angll up-regulated TSLP expression in BMDMsFirst,we examined the effects of Ang Ⅱ on TSLP expression in BMDMs.We tested the AngⅡ dose-response using concentrations of 1,10,and 100 nM for 72 hours,and found that the expression of TSLP increased gradually in a dose-dependent manner.Indeed,signifcant increases were detected as early as 48 hours after Ang Ⅱ incubation,while the maximum effects were observed at 72 hours.3.4 AngⅡ promoted TSLP secrection from BMDMsTo further investigate TSLP protein released from BMDMs,we measured the level of TSLP in cell supernatants using ELISA.In consistent with TSLP expression in cytoplasm,stimulation with AngⅡ induced the release of TSLP in both concentration-dependent and time-dependent manners.Therefore,we treated BMDMs with Ang Ⅱ(100 nM)for 72 hours for the subsequent experiments.3.5 Angll skewed the macrophage phenotype towards M2 by up-regulating TSLP expressionAng Ⅱ-treated group displayed a significant decrease in iNos expression and a marked increase in Argl expression,which were consistent with those changes in rTSLP-treated group.The expression of MMP2 and MMP9 were decreased by AngⅡ,even under IFN-γ stimulation.In addition,the flow cytometry analysis revealed that AngⅡ treatment decreased the population of M1 macrophages and increased that of M2 macrophages.When cells were pre-treated with anti-TSLP neutralizing antibody,the M2 polarizing effects of Angll was abated,suggesting the important role of TSLP in mediating AngⅡ effects in macrophage polarization.5 Conclusion5.1 TSLP promote M2 macrophage polarization and alleviate inflammation.5.2 AngⅡ could induce TSLP production in macrophages.5.3 AngⅡcould promote M2 macrophages polarization and alleviate inflammation by up-regulating TSLP expression.