Effect Of Heat Shock Protein 65 On The Activity Of Protein Tyrosine Kinase Lck And Reverse Cholesterol Transport And The Related Molecular Mechanisms In T Cells

BackgroundAtherosclerosis (AS), which is a chronic inflammatory disease initiated by vascular endothelial dysfunction and abmormal lipid metabolism, can lead to coronary artery disease(CAD), stroke and other diseases, which threatening human health seriously. In recent years, studies have shown that function of high density lipoprotein cholesterol (HDL) reflects the risk of AS and CAD more accurately than the level of plasma HDL-C. The existing research confirmed there was dysfunctional HDL in patients with CAD, which promoting the development of AS [1]. HDL, as a multifunctional protein complex, plays a role of resisting to AS through promoting reverse cholesterol transport (RCT), anti-inflammatory, antioxidant, anti-thrombosis and enhancing endothelial function, regulating the blood sugar control of diabetes and inhibiting proliferation of hematopoietic stem cell. Cholesterol efflux can be as a better risk predictor of AS, independent of the level of HDL-C and can be used as a gold index of evaluating HDL function according with the publishion in the New England Journal of Medicine in 2014.There are a lot of actived T lymphocytes in AS lesions. These immune cells release enzymes, cytokines, thus inducing vessel wall inflammatory responses. The studys found that T lymphocytes will proliferate like macrophages, smooth muscle cell and increase when injury and plaques exist with the progress of AS.Heat shock protein 65 (HSP65), which is the main antigen of mycobacterium and have strong immunogenicity, expresses highly and identifies by the immune system when body is infectious. Numerous studies have found that HSP65, which contains multiple T cell epitopes, can react cross with own HSP65 antigens, inducing strong cellular immune In addition, T lymphocytes can also identify HSP65 and lead to immune responses, making the function imbalance between Thl and Th2 in the AS plaque. The activated T cells, as effector cells, can secrete chemokines, amounting mast cells, macrophages and smooth muscle cells, and also decide the differentiation and function of B cells and mononuclear cell, promoting the occurrence of AS.Lymphocyte-specific protein tyrosine kinase (Lck) is a tyrosine kinase of Src family expressed in the cytoplasm of T cells, playing a key role in T cell activation process’^. Before the activation of cellular responses, Lck forms covalent bonds wth CD4 and CD8 compound receptor by the end of the N-cystine at first. Interaction between T cells and antigen-presenting cells can lead to Lck activation, and then cause tyrosine phosphorylation of phospholipase C (PLC), which can pyroly cell membrane phospholipids and produce insositol 1,4,5-trisphosphate (IP3) and diacylgycerol (DAG), further link T cell activation through the IP3-Ca2+ and DAG-protein kinase C (PKC)-nuclear factor-κB (NF-κB) the two important signal transduction. Especially, activation of PKC and release of intracellular calcium play the important role in occurrence and development of AS. In addition, Lck can also activate the mitogen activated protein kinase (MAPK) signaling pathways, which working through its downstream molecular:Fos, AP-1,c-Jun.More researches concentrated in reverse cholesterol transport, anti-inflammatory and antioxidant function of HDL. On the other hand, more and more data in recent years confirmed that the immune cells can directly affect HDL-mediated lipid metabolism in turn. The activation of T cells down-regulated expression of cholesterol transport proteins and inhibited cholesterol efflux through promoting oxygen sterone metabolic enzymes, lymph toxins and family members of MAPK pathways. Therefore, combination with domestic and foreign researches shows that immune responses can affect cholesterol efflux in lymphocytes, and HSP65-mediated immune responses could inhibit cholesterol efflux by resulting in HDL dysfunction in macrophages, thus promoting the development of AS. However, Whether HSP65 has influence on the lipid metabolism in T cells has still been unknown. Whether the process performed through Lck and its downstream signaling pathways has not yet been confirmed.ObjectivesWe set out to investigate whether cellular immune response and reverse cholesterol transport are affected in T cells with different concentration of HSP65. What’more, we explore the relative mechanism of RCT influenced by HSP65 through silencing protein tyrosine kinase Lck.Methods1. The effects of cell proliferation, Lck activation and the levels of cytokines with different concentration of HSP65Cells were divided into five groups:1) the normal control group,2) 0.25μg/ml HSP65,3) 0.5μg/ml HSP65 group,4) 0.75μg/ml HSP65 group,5) 1μg/ml HSP65 groupAfter cells were treated for 24h, CCK-8 was performed to detect the proliferation (OD value at 450nm); ELISA was performed to detect the concentration of cytokines IFN-γ and IL-10 in cell supernatant; Western blot was performed to detect the expression of tyrosine kinase Lck protein.2. The effects of cholesterol efflux and relative transport proteins treated by different concentration of HSP65 in Jurkat cellsCells were divided into five groups:1) the normal control group,2) 0.25μg/ml HSP65,3) 0.5μg/ml HSP65 group,4) 0.75μg/ml HSP65 group,5)1μg/ml HSP65 group[3H] labeled cholesterol efflux rate was evaluated by liquid scintillation spectrometry. The total RNA was isolated from cells; Quantitative real-time PCR was performed to detected the levels of ABCA1、 ABCG1、SR-BI、PPAR-γ and LXR-α mRNA; The expression of relative proteins was detected by Western blot.3. Changes in the levels of Lck gene and protein after short hairpin ribonucleic acid (shRNA)-lentiviral constructs targeting Lck (Lck-shRNA) transfected Jurkat cellsCells were divided into four groups:1) the normal control group,2) the negative control group,3) LV1-shRNA interference group (LV1),4) LV2-shRNA interference group (LV2)Two target sequences, containing different genes and packaging with lentivirus carrier, transfected Jurkat cells after determination the drop degree. Screening found the best MOI (50) and the infection condition:The diluents of LV1 and LV2 were prepared and added 200μl each well, then added polybrene diluent by 100 μl/well, making final volume into 1 ml, thus the final concentration of polybrene being 5 μg/ml. After 8 hours, we changed the cultured medium and observed cell growth condition under inverted microscope at 12 h,24 h,48 h,72 h, respectively. We tested transfection efficiency using GFP-labeled fluorescence flow cytometry instrument at 72 h. Then, we collected cells. Gene and protein expression levels of tyrosine kinase Lck were detected by real time PCR and Western blot.4. The effects of shRNA-Lck on TCR further downstream signaling pathways and reverse cholesterol transportCells were divided into four groups:1) the normal control group,2) the negative control group,3) LV1-shRNA interference group (LV1),4) LV2-shRNA interference group (LV2)Concentration of intracellular calcium was detected by Fluo-3/AM labeled laser confocal scanning; [3H] labeled cholesterol efflux rate was evaluated by liquid scintillation spectrometry; Oil red O staining was used to detect the contents of intracellular lipid droplets; High performance liquid chromatography (HPLC) was used to detect the contents of intracellular cholesterol ester.5. The effects of shRNA-Lck on the expression of TCR further downstream signaling pathways proteins and cholesterol transport proteinsCells were divided into four groups:1) the normal control group,2) the negative control group,3) LV1-shRNA interference group (LV1),4) LV2-shRNA interference group (LV2)Western blot was performed to detect TCR downstream signaling pathways, including the expression of PKC-γ, phosphorylation of ERK1/2, JNK proteins, NF-κB protein level and the expression of cholesterol transport related proteins ABCA1、 ABCG1、SR-B1、PPAR-γ and LXR-α.6. The effects of 1μg/ml HSP65 on TCR downstream signaling pathways and RCT after shRNA-Lck transfectionCells were divided into four groups:1) 1μg/ml HSP65 group,2) NC+1μg/ml HSP65 group,3) LV1+1μg/ml HSP65 group,4) LV2+1μg/ml HSP65 groupConcentration of intracellular calcium was detected by Fluo-3/AM labeled laser confocal scanning; Western blot was performed to detect the expression of TCR downstream pathways proteins; [3H] labeled cholesterol efflux was evaluated by liquid scintillation spectrometry; The expression of relative proteins were detected with Western blot; Oil red O staining to was performed to detect the content of intracellular lipid droplets; High performance liquid chromatography (HPLC) was performed to detect the content of intracellular cholesterol ester.7. Statistical analysisAll data are presented as the mean ± SD. The statistical analysis was carried out by Student’s t test or one-way ANOVA coupled with Bonferroni-Dunn post hoc test which are performed using of the SPSS for windows software. P<0.05 was considered to be statistical significant.Results1. HSP6S can promote cell proliferation, active the expression of Lck, increases the level of IFN-γ and reduce IL-10 in the range of 0-1μg/mlHSP65 could stimulate cell proliferation ability in the range of 0-1μg/ml. OD value was significant difference between the different groups (F=90.513, P<0.001). Compared with normal control group,0.25μg/ml,0.5μg/ml,0.75μg/ml and 1μg/ml groups were significantly higher (P<0.001).The expression of Lck protein increased with the concentrations of HSP65 gradually between different groups. Compared with normal control group (0.89±0.22), the expression of Lck protein in 0.25μg/ml HSP65 group had no significant difference (1.17±0.35,P=0.35),0.5μg/ml HSP65 group had no significant difference (1.62±0.50, P=0.50), while the expression of Lck protein in 0.75μg/ml HSP65 group(1.69±0.16, P<0.05) increased; 1μg/ml group (1.90±0.83,P<0.05) significantly increased.HSP65 could increase the levels of IFN-γ and induce levels of IL-10. The concentration of IFN-γ had statistically significant differences (P<0.001) in HSP65 stimulated group compared with normal control group (15.59±0.86). compared to 0.5μg/ml HSP65 group (18.76±1.08), the levels of IFN-γ in 0.25μg/ml HSP65 group (17.58±0.89,P<0.05) was statistically different, while the level of IFN-γ in 0.75μg/ml HSP65 group (19.37±0.81,P>0.81) was not statistically different; And compared with 0.75μg/ml group, the level of IFN-γ in 1μg/ml HSP65 group (25.02±0.98) was statistically significant (P<0.001).The level of IL-10 was decreased in cell supernatant with the increasing concentrations of HSP65. Compared with normal control group (21.86±0.92), these results were statistically significant differences (P<0.05). And compared to 0.5μg/ml HSP65 group (17.07±1.28), the level of IL-10 in 0.25μg/ml group (19.8±1.249, P=1.249), was statistically different, while the level of IL-10 in 0.75μg/ml group (16.67±2.29,P>2.29), was no statistically different; Compared with 0.75μg/ml HSP65 group, the level of IL-10 in 1μg/ml HSP65 group (16.16±1.46) was not significant (P>0.05).2. HSP65 inhibited cholesterol efflux, down-regulated the expression of cholesterol transport proteins and mRNA in the range of 0-1μg/mlCompared with the control group (21.27±2.44%), cholesterol efflux was obviously lower (15.56±2.58%,14.36±1.42%,12.96±2.45% and 12.96±2.03%) in each group. Compared with control group, cholesterol efflux in 1μg/ml HSP65 group was most obvious, therefore our adopted 1μg/ml concentration to intervent cells at following experiments.The above experimental results have confirmed that HSP65 could inhibit cholesterol efflux, therefore in this experiment, we emphatically observed the regulating mechanism of HSP65 mediated cholesterol transport protein:Jurkat cells were treated by different concentration of HSP65 (0,0.25,0.5,0.75,1μg/ml), the expression of ABCA1 ABCG1, SR-BI, PPAR-γ and LXR-α protein had significantly difference between the respective groups (F=15.615, P<0.001; F=18.069, P<0.001; F=5.212, P<0.05; F=8.426, P<0.01; F=40.369, P<0.001). Compared with control group, HSP65 (0.25,0.5,0.75,1μg/ml) showed significantly lower levels of ABCA1, ABCG1, SR-BI, PPAR-γ and LXR-α protein (P<0.05).Consistent with the expression of proteins, the levels of ABCA1, ABCG1, SR-BI, PPAR-γ and LXR-α mRNA were significantly lower (P<0.05) at different concentration of HSP65 groups. Compared with control group,0.25μg/ml HSP65 group showed significantly lower levels of ABCG1, PPAR-y and LXR-a mRNA (P<0.05). The levels of ABCA1 and SR-BI mRNA in 0.25μg/ml HSP65 group had no significant difference (P>0.05). Compared with normal control group,0.5μg/ml HSP65 group showed significantly lower levels of ABCA1,ABCG1, SR-B1, PPAR-γ and LXR-α mRNA (P<0.05). The experiment discovered HSP65 significantly reduced cholesterol efflux, lowered expression of cholesterol transport protein in T cells. However, Which signaling pathways involved in this process needed to be further confirmed.3. shRNA-Lck obviously silenced the level of Lck mRNA and down-regulated expression of Lck protein in Jukat cellsThe experiment proved the expression of tyrosine kinase Lck of protein would gradually increase with the increasing concentration of HSP65, which suggesting that HSP65, as a antigen could activate Lck. Therefore in this experiment, our aim was to study the mechanism of HSP65 mediated reverse cholesterol transport by using lentivirus shRNA-Lck. Compared with normal control group, negative control group showed no significant change of level of Lck mRNA (P>0.05); Compared with negative control group, the levels of Lck mRNA in LV1 group and LV2 group were lower (P<0.001). Similar to the above results, compared with normal control group, the expression of Lck protein in the negative control grout) had no significant change (P>0.05). Compared with negative control group, the expressions of Lck protein in LV1 group and LV2 group were significantly lower (P<0.05).4. shRNA-Lck inhibited TCR downstream signaling pathways and reverse cholesterol transportCompared with the normal control group, concentration of intracellular calcium in negative control group (190.79±5.53 vs.189.36±6.72, P>0.05); Compared with negative control group, concentration of intracellular calcium in LV1 group decreased significantly (99.91 ±7.75, P<0.001), the difference was statistically significant, as well as LV2 group (108.15±11.75, P<0.001). The experimental results hinted:Lck was the important protein in regulating intracellular calciums.In order to understand whether Lck participated in the process of reverse cholesterol transport in immune cells, we detected cholesterol efflux after Lck silencing. Compared with negative control group (19.08±0.63%), cholesterol efflux in LV1 group was obviously higher (23.46±1.53%), cholesterol efflux in LV2 group was obviously higher (25.27±2.01%), both had significant difference (P<0.001). Then, we tested the contents of intracellular lipid droplets and cholesterol ester who works as the indirect index to evaluate cholesterol efflux. The content of intracellular lipid droplets was determined by oil red O staining. Relative quantitative results analysized by Image J software:compared with negative control group (0.15±0.01%), the contents of intracellular lipid droplets in LV1 group had no statistically significant difference (0.09±0.02%, P>0.05); and in LV2 group showed significantly lower (0.07±0.05%, P<0.05). Contents of intracellular cholesterol ester/total cholesterol levels was detected by high performance liquid chromatography in the cell. The results showed that compared with negative control group (0.50±0.02), LV1 group had lower relative content of intracellular cholesterol ester (0.30±0.04, P<0.05), LV2 group had lower content (0.32±0.02, P<0.05). These experimental results hinted:Lck pathways mediated the regulation of cholesterol transport.5. shRNA-Lck down-regulated the expression of TCR further downstream signaling pathways proteins and up-reguated the expression of cholesterol transport proteinsThe above experimental results had confirmed that Lck had influence on cellular immune pathways and reverse cholesterol transport. Therefore in this experiment, we observed which signaling pathways mediated the changes of cholesterol transporters. After cells were transfected with shRNA-Lck, compared with negative control group, LV1 group and LV2 group had no statistically difference of the expression of protein (P>0.05); LV1 group and LV2 group had decreased the expression of PKC-γ and NF-κB protein、phosphorylation levels of ERK1/2 and JNK (F=25.782, P<0.05; F=20.62, P<0.001; F=24.248, P<0.001; F=69.837, P<0.001). Silencing Lck (LV1, LV2) significantly lowered the expression of ERK1/2, JNK, PKC-y and NF-kB proteins.Compared with negative control group, LV1 group showed significantly higher expression of ABCA1 and ABCG1 protein (P<0.05), while SR-BI, PPAR-y and LXR-a had no significant difference between two groups (P>0.05). The expression of ABCA1, ABCG1, SR-BI, PPAR-y and LXR-a protein in LV2 group significantly increased (P<0.05) with significant difference. Based on the experimental results and the overseas studys, we can conclude that Lck may inhibit cholesterol transport proteins through ERK, JNK protein, PKC-γ and NF-κB proteins.6. 1μg/ml HSP65 failed to induce cellular immune responses and inhibited reverse cholesterol transport through Lck-mediated pathways after Lck gene silencedIn order to observe whether Lck and downstream signaling pathways was involved in HSP65 promoting cellular immune, gene silence was done and cells were stimulated with 1μg/ml HSP65 for 24 hours. Compared with NC+1μg/ml HSP65 group (240.47±13.63), LV1+1μg/ml HSP65 group (128.86±10.26) and LV2+1μg/ml HSP65 group (127.20±13.19) showed significantly lower concentration of intracellular calcium (P<0.001). Compared with NC+1μg/ml HSP65 group, LV1+1μg/ml HSP65 group and LV2+1μg/ml HSP65 group phosphorylation levels of ERK 1/2 and JNK and the expression of PKC-y and NF-κB proteins significantly decreased (P<0.05; P<0.001; P<0.05; P<0.001).Compared with NC+1μg/ml HSP65 group (10.83±1.23%), cholesterol efflux in LV1+1μg/ml HSP65 (17.89±2.39%) and LV2+1μg/ml HSP65 group (17.76±1.33%) was significantly higher (P<0.001). We tested the contents of intracellular lipid droplets and cholesterol ester. Compared with NC+1μg/ml HSP65 group (0.22±0.04%), LV1+1μg/ml HSP65 group showed significantly lower contents of intracellular lipid droplets (0.11±0.05%, P<0.05); LV2+1μg/ml HSP65 group also showed lower contents of intracellular lipid droplets (0.12±0.04%) with significant difference (P<0.05). Compared with NC+1μg/ml HSP65 group (0.66±0.01), the content of intracellular cholesterol ester in LV1+1μg/ml HSP65 group (0.40±0.03) and LV2+1μg/ml HSP65 group(0.41±0.01) significantly decreased (P<0.05). Compared with NC+1μg/ml HSP65 group, LV1+1μg/ml HSP65 group showed higher expression of ABCA1, ABCG1, SR-BI, PPAR-γ and LXR-a proteins (P<0.05); The expression of ABCA1 SR-BI and LXR-α proteins in LV2+1μg/ml HSP65 group increased significantly (P<0.05), while ABCG1 and PPAR-γ was no obvious changes between groups (P>0.05).Conclusion1. HSP65 induced T cell proliferation, actived tyrosine kinase Lck, increased levels of proinflammatory factor IFN-γ and reduced anti-flammatory factor IL-10.2. HSP65 impaired reverse cholesterol transport:reducing cholesterol efflux, down-regulating the expression of ABCA1、ABCG1、SR-BI、PPAR and LXR proteins in a concentration-dependent manner.3. Foreign gene carried by lentiviral RNAi vector of Lck gene could be expressed in transfected Jurkat cells. And it could specifically and efficiently suppress Lck gene expression, thus down-regulated Lck expression.4. The expression of Lck gene was blocked by RNAi technology, which could up-regulate the expression of cholesterol transport proteins, improve cholesterol efflux and lower cholesterol ester content in T cells.5. HSP65 could active Lck, which not only promoting TCR-mediated immune signaling pathways, but inibiting RCT in T cells.6. In summary, we can conclude that:HSP65, as an important inflammatory substance, inhibited RCT in T cells, which may contribute to its effect on facilitating Lck-mediated signaling pathways.