TLR4在1型糖尿病肾病及TLR3在自身免疫性肝炎中作用的研究
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摘要
背景
糖尿病肾病是年轻人终末期肾病的主要原因,40%的长期1型糖尿病患者最后会进展成糖尿病肾病。当前的治疗,包括降糖、降压、改善高脂血症都会延缓糖尿病肾病的进展,但最终还是会有一部分糖尿病患者最后发展为慢性肾衰竭。因此,最大限度地发挥当前治疗的效应,以及确立新的治疗策略和找出其他的治疗靶点在糖尿病肾病治疗中显得尤为重要。
很多因素参与了糖尿病肾病的进展。代谢性因素,例如糖基化终末产物的形成和多羟基产物的增加都参与了糖尿病肾病。很多生长因子也在糖尿病肾病中起到了一定作用。虽然糖尿病肾病不是主要由免疫反应介导,然而在1型糖尿病病人肾脏活检中发现了球旁器中T淋巴细胞的浸润。此外,在1型和2型糖尿病肾病患者肾活检切片中都发现了巨噬细胞的浸润。基于这些发现,探讨免疫系统在糖尿病肾病中的作用就尤为重要。
TLR在天然性免疫和获得性免疫系统中都发挥了非常重要的作用。TLR4主要表达在巨噬细胞等免疫细胞表面,同时也表达在气道上皮、脂肪组织、骨骼肌、血管内皮细胞和平滑肌细胞等组织细胞,是革兰氏阴性细菌脂多糖的主要受体。同时TLR4也可以结合包括热休克蛋白、纤维粘素、纤维原、游离脂肪酸、饱和脂肪酸在内的内源性配体。TLR4激活后可以产生炎性细胞因子。近年的研究表明1型糖尿病患者外周血单核细胞表面上的TLR2和TLR4表达增加。TLR4在1型糖尿病的发病、肥胖和2型糖尿病的胰岛素抵抗中都起到重要作用。
研究目的
1.在这项研究中,利用非肥胖性糖尿病(NOD)小鼠,建立1型糖尿病模型和糖尿病肾病模型。
2.利用这个模型,探讨NOD小鼠糖尿病肾病免疫病理的改变。
3.探讨细胞免疫反应和体液免疫反应在糖尿病肾病不同发病病程中的作用。
4.利用TLR4基因敲除(TLR4-/-NOD)小鼠,探讨TLR4在1型糖尿病早期肾病中的作用。
材料与方法
1.动物：雌性NOD/Caj小鼠以及TLR4-/-NOD小鼠
2.糖尿病模型和糖尿病肾病模型的建立：通过筛查尿糖以及测定血糖水平来明确糖尿病,通过筛查尿蛋白以及24小时尿蛋白定量来确定糖尿病肾病。
3.组织学染色：灌注肾脏,4%的多聚甲醛固定,石蜡包埋,切片后进行过碘酸-雪夫(PAS)染色确定肾小球细胞外基质的沉积。
4.免疫荧光染色和共聚焦显微镜检查：肾脏灌注后放入高碘酸盐-赖氨酸-多聚甲醛固定液中(PLP)固定,速冻切片后,加入一抗和二抗进行染色,共聚焦显微镜观察。
5.电子显微镜检查：投射电镜观察肾小球形态,免疫金颗粒标记进一步证实免疫球蛋白的沉积和沉积位置。
6.RNA提取和实时定量PCR技术：TRIzol从小鼠肾脏组织中分离出总的RNA,反转录合成cDNA,进行引物设计后,实时定量PCR测定相关基因的表达。
7.抗胰岛素自身抗体的确定。
8.统计学分析：用GraphPad Prism 4.0软件进行统计学分析。
结果
在糖尿病小鼠的肾小球中有T和B淋巴细胞的浸润,同时有CDllc阳性的树突状细胞浸润,这些CD11c阳性的树突状细胞和CD4和CD8阳性的T细胞有着紧密的接触。在糖尿病小鼠的肾小球中有免疫球蛋白Ig的沉积,同时伴有补体C3的沉积。另外,糖尿病小鼠的血清中含有自身抗体,这些自身抗体能够和正常小鼠的肾小球某些成分起反应,而正常不患病的小鼠血清就不能出现这种反应。随着糖尿病病程的进展,肾脏中免疫改变的出现同时伴随着肾脏的增大和尿白蛋白排泄的增加。TLR4-／-的糖尿病小鼠肾脏组织中也观察到Ig和补体C3的沉积,但比在野生型的糖尿病NOD小鼠肾脏中的沉积减轻。
结论
1.淋巴细胞和自身抗体参与了1型糖尿病肾病。
2.TLR4基因敲除后可以减轻患糖尿病小鼠肾脏中的Ig和补体C3的沉积,表明TLR4在糖尿病早期肾病中起到一定的作用。
3.深刻了解免疫系统在糖尿病肾病中的作用可以更好地确立新的糖尿病治疗方案,对糖尿病肾病的预防起到积极的作用。
背景
TLR通过识别病原体相关分子模式(PAMPs)对外界致病原进行防御,除了在天然性免疫系统中的重要作用,TLR在炎症调节中包括各种无菌性炎症,例如损伤和伤口愈合中也起到了主要作用。TLR在炎症调节中的作用部分取决于TLR可以识别一些被称做损伤相关分子模式(DAMPs)的内源性配体。肝脏不但是代表许多疾病致病细菌PAMPs的主要靶点,而且也是损伤后DAMPs上调的主要器官。
研究表明TLR3能够对来自病毒的双链RNA (dsRNA)来自凋亡或坏死细胞的双链RNA产生反应。死亡的细胞中含有丰富的可以激活TLR(包括TLR3)的各种配体。表达在肝脏中的TLR3很可能是激活天然性免疫和炎症反应的一个介导者。TLR3在急性肝炎中的这些重要作用在以前的研究中没有被详细探讨过。
研究目的
1.建立Con A诱导的小鼠肝炎模型,模拟人类急性爆发性肝炎。
2.探讨了TLR3在没有外源性病毒激活的情况下,在Con A诱导的肝炎中对肝细胞损害的作用。
材料与方法
1.动物：NOD/Caj小鼠,C57BL／6(B6)小鼠,TLR3-／-NOD小鼠,TLR3-／-B6小鼠。
2.肝炎的诱导：ConA被溶于无菌的PBS中,以15mg／kg的剂量静脉注射给小鼠。ConA注射后0、8、24小时给小鼠取血。对照组小鼠注射等剂量的PBS。注射24小时后小鼠被处死。
3.肝功能的评价：血清丙氨酸氨基转移酶用试剂盒测定。
4.肝脏单个核细胞的分离：灌注的肝脏通过200-gauge的不锈钢网后,细胞被悬浮在30%的percoll中,70%percoll被轻轻地打到悬浮液底。密度梯度离心后,收集中间层为肝脏单个核细胞,PBS洗一遍。
5.流式细胞仪分析：除非特别标明,所有用于流式细胞仪分析的抗体购于eBioscience。Fc receptor封闭后,用特定的结合不同荧光素的单克隆抗体进行细胞表面标记；细胞首先被固定,透化处理后,用Cytofix/Cytoperm试剂盒进行染色分析细胞内蛋白；用FACSCalibur的流式细胞仪收集细胞,结果用Flowjo软件分析。
6.组织学检查和免疫染色：按照常规方法对肝脏组织进行H&E染色。灌注过的肝脏组织被放在高碘酸盐-赖氨酸-多聚甲醛固定液中(PLP)中过夜固定,用OCTTissue-Tek包埋,然后速冻,切成10μm的切片,2%的山羊血清封闭,加入一抗和二抗进行免疫荧光染色,用META510共聚焦显微镜进行观察拍照。
7.肝脏细胞凋亡分析：肝细胞核DNA双链断裂用TUNEL试剂盒分析。
8.血清细胞因子测定：血清中IL-6、IFN-γ、TNF-α和IL-17用Luminex测定。IFN-α的水平用ELISA试剂盒按照操作步骤进行检测。
9.骨髓嵌合体小鼠的生成：通过PBS灌洗小鼠胫骨和腓骨,分别从WT和TLR3-／-供体小鼠获得骨髓细胞。接受骨髓移植的受体小鼠进行照射后,将供体骨髓细胞注射到被照射的小鼠中,建立骨髓嵌合体小鼠模型。
10.受损肝脏组织RNA对Con A刺激的脾淋巴细胞反应：从WT和TLR3-／-小鼠分别获取脾淋巴细胞,加入96孔培养板中,然后加入1μg／ml ConA刺激,并加入通过反复冻融导致的坏死肝脏组织提取的RNA、Con A阴性对照进行刺激。11.统计学分析：所有的数据表示为means±SD或means±SE。统计学分析用GraphPad Prism软件4.0进行分析。
结果
Con A注射后,肝脏单个核细胞和窦状内皮细胞的TLR3表达上升；TLR3-/-小鼠在Con A诱导的肝炎中肝脏损害比野生型小鼠明显减轻。而且,TLR3-/-小鼠的脾淋巴细胞对坏死肝脏组织RNA和Con A刺激的增殖反应比正常小鼠的脾淋巴细胞要弱。为了确定在Con A诱导的肝炎中表达在造血系细胞还是非造血系细胞的TLR3在肝脏损害中的作用,我们生成了骨髓嵌合体小鼠。移植正常小鼠造血系后的TLR3-／-小鼠和移植TLR3-／-小鼠造血系后的正常小鼠在Con A诱导的肝脏损害中都得到相似的保护作用。这表明在非造血系细胞和造血系细胞的TLR3信号通路在介导肝脏损害中都起到重要作用。
结论
1.TLR3信号通路在Con A诱导的肝炎损害中是必要的。
2.在没有病毒感染的情况下,TLR3可以调节炎症反应和适应性T细胞免疫反应。
Background
Diabetic nephropathy is the leading cause of end-stage renal failure in young people and affects about 40%of individuals with long-standing type 1 diabetes. Current therapy, which includes treatment for hyperglycemia, hypertension and dyslipidemia can slow the rate of progression of diabetic nephropathy, but eventually end-stage renal failure will still occur in a proportion of patients. Therefore, the effects of currently used treatments must be maximized and identification of new strategies and additional therapeutic targets for treating diabetic nephropathy will be important.
Many factors are involved in the pathogenesis of diabetic nephropathy. Metabolic factors, such as the formation of advanced glycation end products and increased flux through the polyol pathways, have been implicated, In addition, growth factors may also play a role. Although the pathogenesis of diabetic nephropathy is not considered to be primarily immune mediated, nevertheless studies examining renal biopsies in patients with type 1 diabetes have noted T cell infiltration of the juxtaglomerular apparatus. Macrophages have also been detected in kidney sections from patients with diabetic nephropathy although no distinction was made between patients with type 1 or type 2 diabetes.Therefore, it's very important to investigate the role of immune reponses in the diabetic nephropathy. TLR play an important role in the innate immune system. TLR4 is expressed on macrophages, airway epithelia, adipose tissue, skeletal muscle and vascular endothelial and smooth muscle cells and is the key receptor for the lipopolysaccharide (LPS) component of Gram-negative bacteria. TLR4 also interacts with other endogenous and exogenous substances, including heat-shock proteins, fibronectin, fibrinogen, free fatty acids and saturated fatty acids. TLR4 links immune stimulation to the induction of the innate immune response. Activated TLR4 induces expression of a spectrum proinflammatory cytokines. Recent studies have shown increased Toll-like receptor 2 (TLR2) and TLR4 expression and signalling in monocytes from type 1 diabetic patients.TLR4 is one of important receptor in the development of type 1 diabetes, obesity and insulin resistance.
Research Objectives
1. In the present study, we focused on the non-obese diabetic (NOD) mouse model that develops spontaneous autoimmune diabetes similar to human type 1 diabetes.
2. To test the hypothesis that immune responses are involved in the development of diabetic nephropathy.
3. To investigate cellular and humoral immune parameters in spontaneous diabetic NOD mice.
4. To investigate the role of TLR4 in the early stage of diabetic nephropaty.
Materials and Methods
1. Animals
Female NOD/Caj mice and TLR4-/-NOD mice
2. Determination of diabetes and diabetic nephropathy
NOD mice were observed for diabetes development by weekly screening for glycosuria. Mouse urine was screened for proteinuria by Albustix. Twenty-four hour urine collections were obtained using metabolic cages and quantitation of urine albumin was determined using the Protein Assay Kit from Bio-Rad according to the manufacturer's instructions.
3. Histology
Mice were perfused with ice-cold PBS and then with buffer containing 10%formalin. Tissues were further fixed in 4%buffered paraformaldehyde for 2 days, embedded in paraffin and processed for sectioning. Extracellular matrix deposition in glomeruli was assessed by periodic acid/Schiff (PAS) staining.
4. Immunofluorescent labeling and confocal microscopy
Perfused kidney was fixed overnight in periodate-lysine-paraformaldehyde (PLP) fixative, embedded in OCT compound from Tissue-Tek, and snap frozen. Cryosections were rehydrated with PBS followed by blocking with 2%goat serum. The following primary antibodies were used for staining the kidney sections:Kidney sections were examined and photographed using a META510 confocal microscope.
3. Electron microscopy
Thin kidney sections were cut, and then were observed in a Philips CM 10 transmission EM. We performed immunogold labeling to confirm Ig deposition and location of Ig deposition.
4. RNA extraction and real-time quantitative PCR
Total RNA was isolated from mouse kidney tissues using TRIzol. Equal amounts of RNA, measured by spectrophotometer and RNA gel, were used for first-strand cDNA synthesis with SuperScriptⅢ-RT kit. cDNA product was then subject to the quantitative PCR (qPCR) amplification. The primers were designed using the Primer Bank and synthesized by Sigma. Real-time quantitative PCR was performed using an iCycler system.
5. Dertermination of insulin antibody
6. Statistical analysis
Statistical analysis was performed using GraphPad Prism software 4.0
Results
We found that the glomeruli of diabetic NOD mice were infiltrated with T and B cells, as well as CD11c+dendritic cells, which had close contact with CD4+and CD8+T cells in the infiltrates. We also found that Ig deposits in the glomeruli of diabetic NOD mice were accompanied by the presence of complement C3. Moreover, the serum from diabetic mice contained autoantibodies directed towards components of the glomeruli and these antibodies were not present in non-diabetic NOD mice. The immune changes in the kidney occurred together with increasing kidney weight and urinary albumin excretion along with duration of diabetes. We also found Ig depositon and the presence of complement C3 in TLR4-/- mice, which was much less than those in WT mice.
Conclusions
We provide evidence that infiltrating lymphocytes and anti-kidney autoantibodies may be involved in diabetic nephropathy in autoimmune diabetes in the NOD mouse. The deficiency of TLR4 can decrease the deposition of Ig and complement C3, which showed that TLR4 plays a role in the early stage of diabetic nephropathy. Understanding the role that the immune system plays in the pathogenesis of diabetic nephropathy could lead to identification of new strategies and/or additional therapeutic targets for prevention and treatment of diabetic nephropathy.
Backgrounds
Toll-like receptors (TLR) recognise pathogen-associated molecular patterns (PAMPs) to detect the presence of pathogens. In addition to their role in innate immunity, TLR also play a major role in the regulation of inflammation, even under sterile conditions such as injury and wound healing. This involvement has been suggested to depend, at least in part, on the ability of TLR to recognise several endogenous TLR ligands termed damage-associated molecular patterns (DAMPs). The liver not only represents a major target of bacterial PAMPs in many disease states but also upregulates several DAMPs following injury.
Toll-like receptor 3 (TLR3) is known to respond to double-stranded RNA from viruses, apoptotic and/or necrotic cells. Dying cells are a rich source of ligands that can activate TLR, such as TLR3. TLR3 expressed in the liver is likely to be a mediator of innate activation and inflammation in the liver. The importance of this function of TLR3 during acute hepatitis has not previously been fully explored.
Research Objectives
We used the mouse model of concanavalin A (ConA)-induced hepatitis and observed a novel role for TLR3 in hepatocyte damage in the absence of an exogenous viral stimulus.
Materials and Methods
1. Animals:NOD/Caj mice, C57BL/6 (B6) mice, TLR3-/- B6 mice and TLR3-/- NOD mice.
2. Induction of hepatitis:ConA was dissolved in sterile PBS and injected intravenously to mice at a dose of 15mg/kg. Mice were bled 0,8 and 24h following ConA administration. Control mice were injected with PBS. Mice were sacrificed 24h after ConA injection.
3. Assessment of liver function:Serum alanine aminotransferase (ALT) was measured using a standard kit.
4. Isolation of liver mononuclear cells:Perfused livers were passed through a 200-gauge stainless steel mesh. The cells were resuspended in 30%percoll and 70%percoll was gently placed under the suspension. After gradient centrifugation, liver MNCs were collected from the interface and washed with PBS.
5. Flow cytometric analysis:All fluorescence-conjugated antibodies used in this study were purchased from eBioscience unless otherwise specified. After blocking with anti-Fc receptor, surface markers were identified by specific mAbs conjugated with different fluorochromes. To analyze intracellular proteins, cells were first fixed and permeabilized, and then stained with appropriate mAbs using a Cytofix/Cytoperm plus kit. Flow cytometric analysis was performed using Flowjo software.
6. Histological examination and immunostaining:Livers were stained with H&E by standard methods. For immunostaining, perfused liver was fixed overnight in periodate-lysine-paraformaldehyde (PLP) fixative. After embedding in OCT compound, the liver tissue was snap frozen. Cryosections were rehydrated with PBS followed by blocking with 2%goat serum. The primary and secondary antibodies were used to stain the liver sections. Liver sections were examined and photographed using a META510 confocal microscope.
7. Analysis of apoptosis of hepatocytes:Hepatic cell nuclei positive for DNA strand breaks was determined by TUNEL assay using a fluorescence detection kit.
8. Measurement of serum cytokine levels:Serum cytokine levels for IL-6, IFN-y, TNF-a and IL-17 were detected using Luminex technology. IFN-a was measured using an ELISA kit according to the manufacturer's instructions.
9. Generation of bone marrow chimeric mice:Bone marrow cells were harvested from WT or TLR3-/- mice by flushing femurs and tibiae with PBS. Chimeric mice were generated by transferring donor BM cells into irradiated recipients.
10. Effect of RNA from damaged liver tissue on splenocyte response to ConA stimulation: Splenocytes from WT or TLR3-/- mice were stimulated with ConA in the presence or absence of total cellular RNA isolated from liver tissue damaged by repeated freezing and thawing (DT, damaged tissue).
11. Statistical analysis:Data were mostly presented as mean±SEM. Differences between groups were assessed using analysis of variance (ANOVA) followed by Bonferroni post hoc test or student's t-test wherever appropriate. Ap value less than 0.05 was considered statistically significant.
Results
Interestingly, TLR3 expression in liver mononuclear cells and sinus endothelial cells was upregulated after ConA injection and TLR3-/- mice were protected from ConA-induced hepatitis. Moreover, splenocytes from TLR3-/- mice proliferated less to ConA stimulation in the presence of RNA derived from damaged liver tissue compared with wild type (WT) mice. To determine the relative contribution of TLR3 expression by hematopoietic cells or non-hematopoietic to liver damage during ConA-induced hepatitis, we generated bone marrow (BM) chimeric mice. TLR3-/- mice engrafted with WT hematopoietic cells were protected in a similar manner to WT mice reconstituted with TLR3-/- bone marrow, indicating that TLR3 signaling in both non-hematopoietic and hematopoietic cells plays an important role in mediating liver damage.
Conclusions
In summary, our data suggest that TLR3 signaling is necessary for ConA-induced liver damage in vivo and that TLR3 regulates inflammation and the adaptive T cell immune response in the absence of viral infection.
糖尿病肾病是年轻人终末期肾病的主要原因,40%的长期1型糖尿病患者最后会进展成糖尿病肾病。当前的治疗,包括降糖、降压、改善高脂血症都会延缓糖尿病肾病的进展,但最终还是会有一部分糖尿病患者最后发展为慢性肾衰竭。因此,最大限度地发挥当前治疗的效应,以及确立新的治疗策略和找出其他的治疗靶点在糖尿病肾病治疗中显得尤为重要。
很多因素参与了糖尿病肾病的进展。代谢性因素,例如糖基化终末产物的形成和多羟基产物的增加都参与了糖尿病肾病。很多生长因子也在糖尿病肾病中起到了一定作用。虽然糖尿病肾病不是主要由免疫反应介导,然而在1型糖尿病病人肾脏活检中发现了球旁器中T淋巴细胞的浸润。此外,在1型和2型糖尿病肾病患者肾活检切片中都发现了巨噬细胞的浸润。基于这些发现,探讨免疫系统在糖尿病肾病中的作用就尤为重要。
TLR在天然性免疫和获得性免疫系统中都发挥了非常重要的作用。TLR4主要表达在巨噬细胞等免疫细胞表面,同时也表达在气道上皮、脂肪组织、骨骼肌、血管内皮细胞和平滑肌细胞等组织细胞,是革兰氏阴性细菌脂多糖的主要受体。同时TLR4也可以结合包括热休克蛋白、纤维粘素、纤维原、游离脂肪酸、饱和脂肪酸在内的内源性配体。TLR4激活后可以产生炎性细胞因子。近年的研究表明1型糖尿病患者外周血单核细胞表面上的TLR2和TLR4表达增加。TLR4在1型糖尿病的发病、肥胖和2型糖尿病的胰岛素抵抗中都起到重要作用。
研究目的
1.在这项研究中,利用非肥胖性糖尿病(NOD)小鼠,建立1型糖尿病模型和糖尿病肾病模型。
2.利用这个模型,探讨NOD小鼠糖尿病肾病免疫病理的改变。
3.探讨细胞免疫反应和体液免疫反应在糖尿病肾病不同发病病程中的作用。
4.利用TLR4基因敲除(TLR4-/-NOD)小鼠,探讨TLR4在1型糖尿病早期肾病中的作用。
材料与方法
1.动物：雌性NOD/Caj小鼠以及TLR4-/-NOD小鼠
2.糖尿病模型和糖尿病肾病模型的建立：通过筛查尿糖以及测定血糖水平来明确糖尿病,通过筛查尿蛋白以及24小时尿蛋白定量来确定糖尿病肾病。
3.组织学染色：灌注肾脏,4%的多聚甲醛固定,石蜡包埋,切片后进行过碘酸-雪夫(PAS)染色确定肾小球细胞外基质的沉积。
4.免疫荧光染色和共聚焦显微镜检查：肾脏灌注后放入高碘酸盐-赖氨酸-多聚甲醛固定液中(PLP)固定,速冻切片后,加入一抗和二抗进行染色,共聚焦显微镜观察。
5.电子显微镜检查：投射电镜观察肾小球形态,免疫金颗粒标记进一步证实免疫球蛋白的沉积和沉积位置。
6.RNA提取和实时定量PCR技术：TRIzol从小鼠肾脏组织中分离出总的RNA,反转录合成cDNA,进行引物设计后,实时定量PCR测定相关基因的表达。
7.抗胰岛素自身抗体的确定。
8.统计学分析：用GraphPad Prism 4.0软件进行统计学分析。
结果
在糖尿病小鼠的肾小球中有T和B淋巴细胞的浸润,同时有CDllc阳性的树突状细胞浸润,这些CD11c阳性的树突状细胞和CD4和CD8阳性的T细胞有着紧密的接触。在糖尿病小鼠的肾小球中有免疫球蛋白Ig的沉积,同时伴有补体C3的沉积。另外,糖尿病小鼠的血清中含有自身抗体,这些自身抗体能够和正常小鼠的肾小球某些成分起反应,而正常不患病的小鼠血清就不能出现这种反应。随着糖尿病病程的进展,肾脏中免疫改变的出现同时伴随着肾脏的增大和尿白蛋白排泄的增加。TLR4-／-的糖尿病小鼠肾脏组织中也观察到Ig和补体C3的沉积,但比在野生型的糖尿病NOD小鼠肾脏中的沉积减轻。
结论
1.淋巴细胞和自身抗体参与了1型糖尿病肾病。
2.TLR4基因敲除后可以减轻患糖尿病小鼠肾脏中的Ig和补体C3的沉积,表明TLR4在糖尿病早期肾病中起到一定的作用。
3.深刻了解免疫系统在糖尿病肾病中的作用可以更好地确立新的糖尿病治疗方案,对糖尿病肾病的预防起到积极的作用。
背景
TLR通过识别病原体相关分子模式(PAMPs)对外界致病原进行防御,除了在天然性免疫系统中的重要作用,TLR在炎症调节中包括各种无菌性炎症,例如损伤和伤口愈合中也起到了主要作用。TLR在炎症调节中的作用部分取决于TLR可以识别一些被称做损伤相关分子模式(DAMPs)的内源性配体。肝脏不但是代表许多疾病致病细菌PAMPs的主要靶点,而且也是损伤后DAMPs上调的主要器官。
研究表明TLR3能够对来自病毒的双链RNA (dsRNA)来自凋亡或坏死细胞的双链RNA产生反应。死亡的细胞中含有丰富的可以激活TLR(包括TLR3)的各种配体。表达在肝脏中的TLR3很可能是激活天然性免疫和炎症反应的一个介导者。TLR3在急性肝炎中的这些重要作用在以前的研究中没有被详细探讨过。
研究目的
1.建立Con A诱导的小鼠肝炎模型,模拟人类急性爆发性肝炎。
2.探讨了TLR3在没有外源性病毒激活的情况下,在Con A诱导的肝炎中对肝细胞损害的作用。
材料与方法
1.动物：NOD/Caj小鼠,C57BL／6(B6)小鼠,TLR3-／-NOD小鼠,TLR3-／-B6小鼠。
2.肝炎的诱导：ConA被溶于无菌的PBS中,以15mg／kg的剂量静脉注射给小鼠。ConA注射后0、8、24小时给小鼠取血。对照组小鼠注射等剂量的PBS。注射24小时后小鼠被处死。
3.肝功能的评价：血清丙氨酸氨基转移酶用试剂盒测定。
4.肝脏单个核细胞的分离：灌注的肝脏通过200-gauge的不锈钢网后,细胞被悬浮在30%的percoll中,70%percoll被轻轻地打到悬浮液底。密度梯度离心后,收集中间层为肝脏单个核细胞,PBS洗一遍。
5.流式细胞仪分析：除非特别标明,所有用于流式细胞仪分析的抗体购于eBioscience。Fc receptor封闭后,用特定的结合不同荧光素的单克隆抗体进行细胞表面标记；细胞首先被固定,透化处理后,用Cytofix/Cytoperm试剂盒进行染色分析细胞内蛋白；用FACSCalibur的流式细胞仪收集细胞,结果用Flowjo软件分析。
6.组织学检查和免疫染色：按照常规方法对肝脏组织进行H&E染色。灌注过的肝脏组织被放在高碘酸盐-赖氨酸-多聚甲醛固定液中(PLP)中过夜固定,用OCTTissue-Tek包埋,然后速冻,切成10μm的切片,2%的山羊血清封闭,加入一抗和二抗进行免疫荧光染色,用META510共聚焦显微镜进行观察拍照。
7.肝脏细胞凋亡分析：肝细胞核DNA双链断裂用TUNEL试剂盒分析。
8.血清细胞因子测定：血清中IL-6、IFN-γ、TNF-α和IL-17用Luminex测定。IFN-α的水平用ELISA试剂盒按照操作步骤进行检测。
9.骨髓嵌合体小鼠的生成：通过PBS灌洗小鼠胫骨和腓骨,分别从WT和TLR3-／-供体小鼠获得骨髓细胞。接受骨髓移植的受体小鼠进行照射后,将供体骨髓细胞注射到被照射的小鼠中,建立骨髓嵌合体小鼠模型。
10.受损肝脏组织RNA对Con A刺激的脾淋巴细胞反应：从WT和TLR3-／-小鼠分别获取脾淋巴细胞,加入96孔培养板中,然后加入1μg／ml ConA刺激,并加入通过反复冻融导致的坏死肝脏组织提取的RNA、Con A阴性对照进行刺激。11.统计学分析：所有的数据表示为means±SD或means±SE。统计学分析用GraphPad Prism软件4.0进行分析。
结果
Con A注射后,肝脏单个核细胞和窦状内皮细胞的TLR3表达上升；TLR3-/-小鼠在Con A诱导的肝炎中肝脏损害比野生型小鼠明显减轻。而且,TLR3-/-小鼠的脾淋巴细胞对坏死肝脏组织RNA和Con A刺激的增殖反应比正常小鼠的脾淋巴细胞要弱。为了确定在Con A诱导的肝炎中表达在造血系细胞还是非造血系细胞的TLR3在肝脏损害中的作用,我们生成了骨髓嵌合体小鼠。移植正常小鼠造血系后的TLR3-／-小鼠和移植TLR3-／-小鼠造血系后的正常小鼠在Con A诱导的肝脏损害中都得到相似的保护作用。这表明在非造血系细胞和造血系细胞的TLR3信号通路在介导肝脏损害中都起到重要作用。
结论
1.TLR3信号通路在Con A诱导的肝炎损害中是必要的。
2.在没有病毒感染的情况下,TLR3可以调节炎症反应和适应性T细胞免疫反应。
Background
Diabetic nephropathy is the leading cause of end-stage renal failure in young people and affects about 40%of individuals with long-standing type 1 diabetes. Current therapy, which includes treatment for hyperglycemia, hypertension and dyslipidemia can slow the rate of progression of diabetic nephropathy, but eventually end-stage renal failure will still occur in a proportion of patients. Therefore, the effects of currently used treatments must be maximized and identification of new strategies and additional therapeutic targets for treating diabetic nephropathy will be important.
Many factors are involved in the pathogenesis of diabetic nephropathy. Metabolic factors, such as the formation of advanced glycation end products and increased flux through the polyol pathways, have been implicated, In addition, growth factors may also play a role. Although the pathogenesis of diabetic nephropathy is not considered to be primarily immune mediated, nevertheless studies examining renal biopsies in patients with type 1 diabetes have noted T cell infiltration of the juxtaglomerular apparatus. Macrophages have also been detected in kidney sections from patients with diabetic nephropathy although no distinction was made between patients with type 1 or type 2 diabetes.Therefore, it's very important to investigate the role of immune reponses in the diabetic nephropathy. TLR play an important role in the innate immune system. TLR4 is expressed on macrophages, airway epithelia, adipose tissue, skeletal muscle and vascular endothelial and smooth muscle cells and is the key receptor for the lipopolysaccharide (LPS) component of Gram-negative bacteria. TLR4 also interacts with other endogenous and exogenous substances, including heat-shock proteins, fibronectin, fibrinogen, free fatty acids and saturated fatty acids. TLR4 links immune stimulation to the induction of the innate immune response. Activated TLR4 induces expression of a spectrum proinflammatory cytokines. Recent studies have shown increased Toll-like receptor 2 (TLR2) and TLR4 expression and signalling in monocytes from type 1 diabetic patients.TLR4 is one of important receptor in the development of type 1 diabetes, obesity and insulin resistance.
Research Objectives
1. In the present study, we focused on the non-obese diabetic (NOD) mouse model that develops spontaneous autoimmune diabetes similar to human type 1 diabetes.
2. To test the hypothesis that immune responses are involved in the development of diabetic nephropathy.
3. To investigate cellular and humoral immune parameters in spontaneous diabetic NOD mice.
4. To investigate the role of TLR4 in the early stage of diabetic nephropaty.
Materials and Methods
1. Animals
Female NOD/Caj mice and TLR4-/-NOD mice
2. Determination of diabetes and diabetic nephropathy
NOD mice were observed for diabetes development by weekly screening for glycosuria. Mouse urine was screened for proteinuria by Albustix. Twenty-four hour urine collections were obtained using metabolic cages and quantitation of urine albumin was determined using the Protein Assay Kit from Bio-Rad according to the manufacturer's instructions.
3. Histology
Mice were perfused with ice-cold PBS and then with buffer containing 10%formalin. Tissues were further fixed in 4%buffered paraformaldehyde for 2 days, embedded in paraffin and processed for sectioning. Extracellular matrix deposition in glomeruli was assessed by periodic acid/Schiff (PAS) staining.
4. Immunofluorescent labeling and confocal microscopy
Perfused kidney was fixed overnight in periodate-lysine-paraformaldehyde (PLP) fixative, embedded in OCT compound from Tissue-Tek, and snap frozen. Cryosections were rehydrated with PBS followed by blocking with 2%goat serum. The following primary antibodies were used for staining the kidney sections:Kidney sections were examined and photographed using a META510 confocal microscope.
3. Electron microscopy
Thin kidney sections were cut, and then were observed in a Philips CM 10 transmission EM. We performed immunogold labeling to confirm Ig deposition and location of Ig deposition.
4. RNA extraction and real-time quantitative PCR
Total RNA was isolated from mouse kidney tissues using TRIzol. Equal amounts of RNA, measured by spectrophotometer and RNA gel, were used for first-strand cDNA synthesis with SuperScriptⅢ-RT kit. cDNA product was then subject to the quantitative PCR (qPCR) amplification. The primers were designed using the Primer Bank and synthesized by Sigma. Real-time quantitative PCR was performed using an iCycler system.
5. Dertermination of insulin antibody
6. Statistical analysis
Statistical analysis was performed using GraphPad Prism software 4.0
Results
We found that the glomeruli of diabetic NOD mice were infiltrated with T and B cells, as well as CD11c+dendritic cells, which had close contact with CD4+and CD8+T cells in the infiltrates. We also found that Ig deposits in the glomeruli of diabetic NOD mice were accompanied by the presence of complement C3. Moreover, the serum from diabetic mice contained autoantibodies directed towards components of the glomeruli and these antibodies were not present in non-diabetic NOD mice. The immune changes in the kidney occurred together with increasing kidney weight and urinary albumin excretion along with duration of diabetes. We also found Ig depositon and the presence of complement C3 in TLR4-/- mice, which was much less than those in WT mice.
Conclusions
We provide evidence that infiltrating lymphocytes and anti-kidney autoantibodies may be involved in diabetic nephropathy in autoimmune diabetes in the NOD mouse. The deficiency of TLR4 can decrease the deposition of Ig and complement C3, which showed that TLR4 plays a role in the early stage of diabetic nephropathy. Understanding the role that the immune system plays in the pathogenesis of diabetic nephropathy could lead to identification of new strategies and/or additional therapeutic targets for prevention and treatment of diabetic nephropathy.
Backgrounds
Toll-like receptors (TLR) recognise pathogen-associated molecular patterns (PAMPs) to detect the presence of pathogens. In addition to their role in innate immunity, TLR also play a major role in the regulation of inflammation, even under sterile conditions such as injury and wound healing. This involvement has been suggested to depend, at least in part, on the ability of TLR to recognise several endogenous TLR ligands termed damage-associated molecular patterns (DAMPs). The liver not only represents a major target of bacterial PAMPs in many disease states but also upregulates several DAMPs following injury.
Toll-like receptor 3 (TLR3) is known to respond to double-stranded RNA from viruses, apoptotic and/or necrotic cells. Dying cells are a rich source of ligands that can activate TLR, such as TLR3. TLR3 expressed in the liver is likely to be a mediator of innate activation and inflammation in the liver. The importance of this function of TLR3 during acute hepatitis has not previously been fully explored.
Research Objectives
We used the mouse model of concanavalin A (ConA)-induced hepatitis and observed a novel role for TLR3 in hepatocyte damage in the absence of an exogenous viral stimulus.
Materials and Methods
1. Animals:NOD/Caj mice, C57BL/6 (B6) mice, TLR3-/- B6 mice and TLR3-/- NOD mice.
2. Induction of hepatitis:ConA was dissolved in sterile PBS and injected intravenously to mice at a dose of 15mg/kg. Mice were bled 0,8 and 24h following ConA administration. Control mice were injected with PBS. Mice were sacrificed 24h after ConA injection.
3. Assessment of liver function:Serum alanine aminotransferase (ALT) was measured using a standard kit.
4. Isolation of liver mononuclear cells:Perfused livers were passed through a 200-gauge stainless steel mesh. The cells were resuspended in 30%percoll and 70%percoll was gently placed under the suspension. After gradient centrifugation, liver MNCs were collected from the interface and washed with PBS.
5. Flow cytometric analysis:All fluorescence-conjugated antibodies used in this study were purchased from eBioscience unless otherwise specified. After blocking with anti-Fc receptor, surface markers were identified by specific mAbs conjugated with different fluorochromes. To analyze intracellular proteins, cells were first fixed and permeabilized, and then stained with appropriate mAbs using a Cytofix/Cytoperm plus kit. Flow cytometric analysis was performed using Flowjo software.
6. Histological examination and immunostaining:Livers were stained with H&E by standard methods. For immunostaining, perfused liver was fixed overnight in periodate-lysine-paraformaldehyde (PLP) fixative. After embedding in OCT compound, the liver tissue was snap frozen. Cryosections were rehydrated with PBS followed by blocking with 2%goat serum. The primary and secondary antibodies were used to stain the liver sections. Liver sections were examined and photographed using a META510 confocal microscope.
7. Analysis of apoptosis of hepatocytes:Hepatic cell nuclei positive for DNA strand breaks was determined by TUNEL assay using a fluorescence detection kit.
8. Measurement of serum cytokine levels:Serum cytokine levels for IL-6, IFN-y, TNF-a and IL-17 were detected using Luminex technology. IFN-a was measured using an ELISA kit according to the manufacturer's instructions.
9. Generation of bone marrow chimeric mice:Bone marrow cells were harvested from WT or TLR3-/- mice by flushing femurs and tibiae with PBS. Chimeric mice were generated by transferring donor BM cells into irradiated recipients.
10. Effect of RNA from damaged liver tissue on splenocyte response to ConA stimulation: Splenocytes from WT or TLR3-/- mice were stimulated with ConA in the presence or absence of total cellular RNA isolated from liver tissue damaged by repeated freezing and thawing (DT, damaged tissue).
11. Statistical analysis:Data were mostly presented as mean±SEM. Differences between groups were assessed using analysis of variance (ANOVA) followed by Bonferroni post hoc test or student's t-test wherever appropriate. Ap value less than 0.05 was considered statistically significant.
Results
Interestingly, TLR3 expression in liver mononuclear cells and sinus endothelial cells was upregulated after ConA injection and TLR3-/- mice were protected from ConA-induced hepatitis. Moreover, splenocytes from TLR3-/- mice proliferated less to ConA stimulation in the presence of RNA derived from damaged liver tissue compared with wild type (WT) mice. To determine the relative contribution of TLR3 expression by hematopoietic cells or non-hematopoietic to liver damage during ConA-induced hepatitis, we generated bone marrow (BM) chimeric mice. TLR3-/- mice engrafted with WT hematopoietic cells were protected in a similar manner to WT mice reconstituted with TLR3-/- bone marrow, indicating that TLR3 signaling in both non-hematopoietic and hematopoietic cells plays an important role in mediating liver damage.
Conclusions
In summary, our data suggest that TLR3 signaling is necessary for ConA-induced liver damage in vivo and that TLR3 regulates inflammation and the adaptive T cell immune response in the absence of viral infection.
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44. Nguyen, C.Q., Kim, H., Cornelius, J.G., Peck, A.B.2007. Development of Sjogren's syndrome in nonobese diabetic-derived autoimmune-prone C57BL/6.NOD-AeclAec2 mice is dependent on complement component-3. J Immunol,179:2318-29.
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46. Zarember, K.A., Godowski, P.J.2002. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol,168:554-61.
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4. Thobe, B.M., Frink, M., Hildebrand, F., Schwacha, M.G, Hubbard, W.J., Choudhry, M.A., Chaudry, I.H.2007. The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage. J Cell Physiol,210:667-75.
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13. Xu, D., Liu, H., Komai-Koma, M.2004. Direct and indirect role of Toll-like receptors in T cell mediated immunity. Cell Mol Immunol,1:239-46.
14. Schiodt, F.V., Lee, W.M.2003. Fulminant liver disease. Clin Liver Dis,7:331-49, V1.
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17. Tiegs, G, Hentschel, J., Wendel, A.1992. A T cell-dependent experimental liver injury in mice inducible by concanavalin A.J Clin Invest,90:196-203.
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19. Akira, S., Takeda, K.2004. Toll-like receptor signalling. Nat Rev Immunol,4: 499-511.
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