An Experimental Study Of Anti-Atherosclerotic Role And Regulation Of Tlrs-Mediated Inflammatory Responce Of Complement

BackgroundThe soluble components of complement are present in the blood in precursor forms and can be activated during pathogen invasion or tissue injury via three different pathways,the classical pathway,the lectin pathway and the alternative pathway. Activated complement system promotes inflammation through the generation of leukocyte chemoattractants C3a and C5a,and causes cellular injury by the formation of the membrane attack complex(MAC).To prevent complement-mediated autologous tissue injury,many kinds of host cells,particularly those in the vascular space such as blood cells and endothelial cells,express several membrane-bound complement regulatory proteins.DAF and CD59 are two of them that link to the cell membrane via a glycosylphosphatidylinositol(GPI) anchor.DAF regulates complement activation by promoting the decay of C3 and C5 convertases,whereas CD59 inhibits the formation of the MAC.Recent studies have indicated that atherosclerosis is a chronic inflammatory condition in which many components of innate and adaptive immune systems are involved.As an important part of innate immunity,complement system has been implicated in the pathogenesis of atherosclerosis by several studies.For example,various complement activation products,regulatory proteins,and complement receptors have been detected in human atherosclerotic lesions,particularly in vulnerable and ruptured plaques,and deposition of C5b-9 has been shown to correlate with the disease state. Experimental studies with complement deficient animals have also been performed to elucidate the role of the complement system in atherogenesis.A cholesterol-rich diet was shown to induce less atherosclerosis in rabbits deficient in C6 than in controls with a fully functional complement system.Contrasting results were obtained, however,in C5 deficient ApoE^-/- mice,which showed a similar extent of atherosclerotic lesions compared to complement-competent control mice.In other studies,C3 deficiency was found to increase lipid-positive lesions in the mouse aorta, as well as altering the plasma lipid profile.On the other hand,deficiency of factor B affected neither lipid levels nor lesion size.More recently,Clq deficiency was found to cause larger atherosclerotic lesions in low-density lipoprotein receptor knockout mice,possibly reflecting a role of Clq in the disposal of dying cells.Together,these results suggested a variable and complex role of complement components in atherosclerosis.In contrast to the above studies on individual complement components in atherogenesis,there have been few studies testing the role of complement regulatory proteins in the development of this disease.Previous studies have detected the presence of complement regulators in atherosclerotic lesions and recently,both statins and C-reactive protein,the latter a marker of chronic inflammation and strong predictor of vascular diseases,have been shown to induce the expression of endothelial complement regulators.However,it is still not clear whether complement membrane-bound regulatory proteins are involved in the atherosclerosis.In this study, our hypothesis is that complement membrane-bound regulatory proteins protect agaist atherosclerosis through inhibiting the activation of complement system or the production of complement effectors.Aims1.To produce CD55^-/- ApoE^-/- and CD59^-/- ApoE^-/- mice by crossing CD55^-/-and CD59 ^-/- mice with ApoE^-/- mice respectively.2.To illustrate the anti-atherosclerotic role of membrane-bound regulatory proteins, by analyzing the lesion size and lesion contents of double knockout mice after a high-fat diet. 3.To reveal the mechanism of the anti-atherosclerotic role of membrane-bound regulatory proteins.Methods1.Mice and dietsCD55 knockout mice and CD59 knockout mice were crossed with ApoE^-/- mice respectively to produce CD55^-/- ApoE^-/- and CD59^-/- ApoE^-/- mice.CD55^+/+ ApoE^-/- mice and CD59^+/+ ApoE^-/- mice were used as controls.Starting from 6 weeks of age,they were fed a high fat diet.At 14 or 22 weeks of age,they were fasted overnight and killed by CO2 asphyxiation.Blood was collected for plasma lipid analysis by vena cava nicking and then stored in -80℃refrigerators.The aorta was perfused with cold PBS by inserting a cannula into the left ventricle.The hearts were dissected and embedded in the OCT.Perfused aortas were dissected from aortic arch to iliac bifurcation.All animal experiments were approved by the Institutional Animal Care and Use Committees of the University of Pennsylvania.2.Serum and urine sample analysisPlasma samples were collected after 12 hours of fasting and stored at -80℃.Total cholesterol was measured.Twenty-four hour urine samples were collected in metabolic cages.The nonenzymatic lipid peroxidation product,8,12-iso-iPF2α-â…¥, was measured by HPLC/MS/MS.Metabolite levels were corrected for urinary creatinine.3.Processing and analysis of the aortaAortas were pinned on wax plates and stained with Sudanâ…£.The extent of atherosclerosis was expressed as the percentage of the aorta surface covered by positive staining.Serial 6-μm-thick cryostat sections were prepared from the origin of the aortic valve cusps and cross-sectional analysis for atherosclerotic lesion was performed every 60μm over 360μm by staining with oil-red O.The extent of atherosclerosis was expressed as the percentage of aortic root covered by lesions. Images were captured digitally with a video camera and analyzed by computerized image analysis.4.Histological analysis of aortic lesions The aortic root sections were stained for smooth muscle cells,macrophages,collagen, C3,C9,CD4 and CD8.The ratio of positive area or cells to aortic root area was recorded.5.Statistical AnalysisStatistical analyses were performed with GraphPad software(Prism 3.0).Data were analyzed by two-tailed Student t-test(for data with normal distribution) or Mann-Whitney test(for data with non-parametric distribution).Results are considered to be significant at values of P＜0.05 and are presented as mean±SEM.Results1.CD59 protects against atherosclerosisAfter 8 weeks of HFD,we found that female CD59^-/- ApoE^-/- mice developed significantly larger atherosclerotic lesions in their aortas,as shown by both en face (2.07±0.27%vs.1.34±0.21%,P=0.06) and aortic root section analysis(20.74±1.33% vs.13.12±1.46%,P＜0.005).Interestingly,the increased sensitivity to atherosclerosis development in CD59^-/- ApoE^-/- mice appeared to be gender-biased as we observed no significant difference between male CD59^-/- ApoE^-/- and CD59^+/+ ApoE^-/- mice by either en face(1.35±0.16%vs.1.57±0.19%,P=0.393) or aortic root section analysis (11.23±1.23%vs.12.75±1.41%,P=0.536).After 16 weeks of HFD,a more pronounced difference between female CD59^-/- ApoE^-/- and CD59^+/+ ApoE^-/- mice was observed by en face staining (17.13±1.14%vs.9.72±1.14%,P＜0.001).However,a difference could not be detected between the two groups mice by aortic root section analysis at this advanced stage of lesion development(32.67±1.58%vs.34.44±2.52%,P=0.315).Similar to the finding at 8 weeks of HFD feeding,we detected no significant difference at 16 weeks between male CD59^+/+ ApoE^-/- and CD59^+/+ ApoE^-/- mice by either the en face (12.88±1.49%vs.10.98±0.95%,P=0.779) or aortic root section analysis (26.845±1.97%vs.29.46±2.3%,P=0.408).2.CD55 has no influence on the development of atherosclerosisIn contrast to the effect of CD59 deficiency on atherosclerosis in female ApoE^-/- mice, we observed no significant difference in either gender between CD55^-/- ApoE^-/- and CD55^+/+ ApoE^-/- mice,regardless of the length of HFD feeding.En face analysis showed that the average lesion areas in female CD55^-/- ApoE^-/- and CD55^+/+ ApoE^-/- mice at 8 and 16 weeks of HFD feeding were 0.80±0.17%vs 1.37±0.22%(P=0.07), and 6.39±0.59%vs 5.81±0.51%,(P=0.303),and that for male mice were 1.14±0.17% vs 0.805±0.14%(P=0.207),and 6.09±0.83%vs 6.16±0.69%(P=0.693),respectively. Aortic root section analysis showed the average lesion areas in female CD55^-/- ApoE^-/- and CD55^+/+ ApoE^-/- mice at 8 and 16 weeks of HFD to be 21.45±1.33%vs 23.58±2.10%(P=0.337),and 29.81±2.78%vs 29.11±3.25%(P=0.918),and that for male mice were 15.68±1.58%vs 16.89±1.65%(P=0.921),and 25.25±3.02%vs 30.94±3.22%(P=0.281),respectively.3.The influence of CD59 knockout on the components of atherosclerotic lesions We next performed histological studies in female CD59-deficient and-sufficient mice to evaluate the complexity of the atherosclerotic lesions.Using CD68 as a marker,we measured the lesion areas that were positive for macrophage infiltration.No significant difference was observed between female CD59^-/- ApoE^-/- and CD59^+/+ ApoE^-/- mice in macrophage distribution and content(18.74±2.79%vs. 18.66±2.41%,P=0.983).On the other hand,after 8 weeks of HFD feeding,we detected more collagen(8.14±0.33%vs.6.07±0.82%,P＜0.05) and less smooth muscle cell staining(3.69±0.51%vs.6.42±0.86%,P＜0.05) in the lesions of CD59-deficient mice compared with their littermate controls.However,the difference in smooth muscle cell content was not observed at 16 weeks of HFD feeding (2.25±0.24%vs.2.28±0.54%,P=0.97).4.The anti-atheroslerotic mechanisms of CD59 Plasma lipid analysis showed a moderate increase in total plasma cholesterol levels in female CD59^-/- ApoE^-/- mice compared with their CD59^+/+ ApoE^-/- littermates,but the difference reached statistical significance only at 8 weeks of HFD feeding (1404±43.74 vs.1208±57.78 mg/dL,P＜0.05 ).We observed no significant difference in 8,12-iso-iPF2α-â…¥between female CD59^-/- ApoE^-/- and CD59^+/+ ApoE^-/- mice either at 8 weeks or 16 weeks of HFD feeding(8 weeks:2.98±1.01 vs.3.12±0.96 ng/mg creatinine,P=0.92;16 weeks:1.59±0.38 vs.1.65±0.56 ng/mg creatinine,P=0.92). Staining of aortic root sections with anti-C3 and anti-C9 antibodies showed a similar C3 deposition(28.65±1.69%vs.25.33±3.07%,P=0.371) in female CD59^-/- ApoE^-/- mice and controls,but significantly increased C9 deposition was found in the sections of female CD59^-/- ApoE^-/- mice than those of the controls(17.32±1.52%vs.8.74±3.63%,P＜0.05) after 8 weeks of HFD feeding.This result suggested that CD59 deficiency led to more deposition of the membrane attack complex within the atherosclerotic lesions.We also examined CD4+ and CDS+ T cell infiltration into the lesions but observed no significant difference between female CD59^-/- ApoE^-/- and CD59^+/+ ApoE^-/- mice(for CD4+ T cells:35.89±8.505 vs.40.99±7.091/mm²,P=0.66; for CDS+ T cells:1.622±1.622 vs.5.384±3.946/mm²,P=0.40).Conclusions1.CD59 offers protection against atherosclerosis in the context of ApoE deficiency, most likely through lowering total cholesterol and inhibiting the formation of membrane attack complex.Its anti-atherosclerotic role is independent of oxidative stress and GPI-mediated T cell activation,and gender-biased,which maybe related to the different level of sex hormones.2.CD59 is not involved in the adhesion,migration and proliferation of macrophages; CD59 protect smooth muscle cells against complement attack by inhibiting the formation of MAC3.CD55 shows no obvious influence on atherosclerosis.This maybe because that other complement regulators such as factor H or Crry have compensated for the lack of CD55.Even under the deficiency of CD55,C3 convertase can be inhibited effectively.4.There is a loss of correlation between en face and aortic root analysis.The aortic roots may be particularly sensitive to lesion development caused by ApoE-deficiency, and any additive effect brought upon by other genetic alterations could be difficult to be detected,especially after prolonged HFD feeding.Thus,en face analysis appears to be a more stable method than aortic root analysis. BackgroundInnate immunity is the first line of host defense against pathogens.The Toll-like receptors(TLRs) and complement are 2 critical components of the innate immune system.They play an essential role in host defense by eliciting rapid inflammatory reactions and orchestrating adaptive immune responses to microbial infection. The mammalian TLRs,one kind of the pattern recognition receptors,are horologes of Toll proteins from drosophila.Through recognizing the invading microbes and dead cells,they can activate the innate immunity,and initiate the adaptive immunity by interacting with the antigen-presenting cells.They play critical roles in the regulation of phagocytosis,signal transduetion and cell apoptosis.The complement system consists of soluble proteins and membrane-bound proteins.The soluble components of complement are present in the blood in precursor forms and can be activated during pathogen invasion or tissue injury.Activated complement promotes inflammation through the generation of leukocyte chemoattractants C3a and C5a,and causes cellular injury by the formation of the membrane attack complex(MAC).Many common PAMPs,such as lipopolysaccharide(LPS) from gram-negative bacteria and zymosan,an insoluble carbohydrate from the yeast cell wall,act both as TLR ligands and activators of complement.Whether and how the TLR and the complement systems,when coactivated in vivo,interact with each other and how potential cross talks between the 2 systems might impact the inflammatory and adaptive immune responses of the host has not been well studied.Recently,one of our studies showed that TLRs agonist induced significant inflammatory response in CD55 deficient mice,indicating that complement system upregulates the TLR-mediated inflammatory response.Subsequent analysis showed that this function was mediated by the anaphylatoxins.In vivo study showed that complement upregulated the cytokines production 3 hours after LPS challenge.This means that the function is most likely mediated by the organs or tissues which have direct contaction with circulation system.Endothelial system is the largest endocrinal organ and has direct contaction with blood.This led to our hypothesis that endothelial cells mediate the upregulation of TLRs-mediated inflammatory response by complement.AimTo elucidate whether and how endothelial cedis are involved in the upregulation of the TLR-mediated inflammatory response by complement.Methods1 Cells cultureHUVEC were cultured in Medium 199 containing 10%fetal bovine serum,0.05 mg/ml endothelial cell growth supplement,2mML-glutamine,0.1mg/ml heparin,and 100 U penieillinstreptomycin.H5V,an endothelial cell line derived from mouse heart, was cultured in DMEM supplemented by 10%fetal bovine serum.Endothelial cells were cultured at 37℃in a humidified atmosphere containing.5%CO2 and passaged every two or three days at a split ratio of 1:3.Passages 5-7 of HUVEC were used for experiments.2 Cell toxicity testingThe cell toxicity of C3a,C5a,LPS,Poly I:C,Pam3CKS4 and CPG to endothelial cells was conducted with promega's one solution cell proliferation assay.This test was performed to find the dose range for the stimulation of endothelial cells.3 C5aR expressions in endothelial cellsC5aR expression in HUVEC or H5V was measured by flow cytometry4 Treatment of HUVECEndothelial cells were seed in 24-well plate at 1×105 per well.After 24 hours,the cells were 80%confluent and were stimulated by various concentration of TLRs agonists or for different time to find the way for the joint stimulation. For the joint stimulation,80%confluent endothelial cells were stimulated by anaphylatoxins and different TLRs agonists for 24 hours.5 ELISA analysesELISA for IL-6,IL-8 and chemokine KC was conducted according to the instructions from companies.Results1 The following dose ranges were found with no obvious cell toxicity to endothelial cells:0-100nM for C3a,0-200nM for C5a,0-10000 ng/ml for Pam3CKS4,0-50μg/m for Poly I:C,0-1000 ng/ml for LPS and 0-160ng/ml for CPG.2 A number of C5aR were found in HUVEC and H5V,but not in the negative controls.3 10 ng/ml LPS significantly induced IL-6 expression.More IL-6 was produced along with the increase of LPS concentration until 500ng/ml.Time course analysis showed that significant IL-6 production was detected after 3 hours'LPS stimulation and peaked after 24 hours.According to the concentration and time course analysis,we chose 10 ng/ml and 100 ng/ml LPS for the joint stimulation.Either 50nM C3a or C5a didn't show any influence on the IL-6 or Chemokine KC production induced by LPS. Then this conclusion was confirmed by using different level of C5a.4 In the same way,we analyzed the influence of C3a and C5a on other TLR-mediated inflammatory response.50nM C3a or C5a showed no influence on IL-6,IL-8 or chemokine KC production induced by Pam3CSK4,Poly I:C and CPG.Conclusions1 Pam3CSK4,Poly I:C and LPS but not CPG can activate toll like receptors on the membrane of endothelial cells.Similar to HUVEC,heart endothelial cells can produce a lot of cytokines under the stimulation of Pam3CSK4,Poly I:C and LPS.2 Endothelial cells are not involved in the regulation of TLR-mediated inflammatory response by complement...