外周Th17/Treg失衡在肺腺癌及肺鳞状上皮细胞癌中的作用及相关机制探讨
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摘要
研究背景
肺癌无论是发病率还是死亡率，均居全球癌症首位。按组织病理学分类，分为小细胞肺癌和非小细胞肺癌，据估计，非小细胞肺癌（NSCLC）约占所有肺癌类型的80%，其中肺腺癌和肺鳞状上皮细胞癌占据了NSCLC的绝大多数。近二十年来，虽然以含铂类化疗为主的综合治疗使肺癌的生存率有所延长，但是五年生存率仍然低于15%，并且由于化疗的附加损害，肺癌患者的生活质量和实际生存期并未显著改善，因此，探索新的治疗手段，寻找更为有效的治疗模式成为研究热点。肺癌的抗肿瘤免疫失衡研究可能为治疗提供一种极具前景的治疗模式。
CD4~+CD25~+Foxp3~+调节性T细胞（Treg）和辅助性T细胞17（Th17）是两类不同于Th1和Th2的CD4~+T淋巴细胞。Treg细胞是CD4~+T细胞的一类特殊分支，在维持免疫稳态和免疫耐受上发挥重要作用，可以避免免疫炎症反应的扩大和减轻免疫机体损伤，在机体免疫系统中发挥负向调节作用。在动物和人研究中发现，调节性T细胞的缺失可以导致严重的自身免疫性炎症；相反，Treg细胞的增多也不利于机体，在许多癌症中，增加的Treg细胞通过与自身免疫监控系统的相互作用，抑制抗肿瘤免疫活性细胞，促进了癌症的进展。Foxp3是Treg细胞特异的标记物，Foxp3属转录因子Forkhead家族，它可以控制Treg细胞的发生发育，剔除Foxp3基因的小鼠，因产生大量慢性炎症因子，导致小鼠产生致死性炎症疾病。在肿瘤微环境中，Tregs细胞抑制CD4~+T细胞和CD8+T细胞的活化，抑制抗癌细胞发挥抗肿瘤作用。产生IL-17的T辅助细胞（Th17）是近来发现的效应性T细胞的新亚型，转录因子孤核受体(RORγt)是Th17细胞分化的特异性转录调控因子。研究发现T17细胞在固有免疫，适应性免疫和自身免疫中发挥重要作用。Th17细胞作为炎症反应的一个重要组成部分，不仅在炎症性疾病中促进炎症反应，而且在肿瘤发生、发展过程中也发挥重要作用。Th17细胞可以在肿瘤动物模型和癌症患者中经常发现：在RORγt缺陷的肿瘤小鼠模型中，由于肿瘤微环境中Th17细胞比率下降，导致肿瘤增殖，但是，这种增殖可以被输入的Th17细胞逆转，在肾癌、卵巢癌及胰腺癌患者外周血中也有Th17细胞比例的显著降低。Th17细胞和Treg细胞不仅在分化上而且在功能上都存在紧密联系，转化生长因子-beta(TGF-β)诱导Treg细胞特异性转录因子Foxp3，但是，如果增加了IL-6则抑制Treg细胞的分化，转而诱导Th17细胞的分化。Treg细胞和Th17细胞在免疫稳态中可能发挥着相互拮抗的平衡作用，既然Treg细胞在肿瘤中发挥重要作用，理所当然Th17细胞也参与其中。因此，进一步探讨二者在肿瘤免疫环境中的作用机制对肿瘤的免疫治疗具有重要意义。
综上所述，Treg细胞和Th17细胞之间存在复杂的相互关系，二者在分化上相互联系，在功能上相互拮抗，二者的平衡在肿瘤的发生发展中具有重要作用。目前，Th17/Treg的平衡紊乱已经成为恶性肿瘤研究领域中新的热点，但是，在非小细胞肺癌中的研究尚少，是否存在比较显著的Th17/Treg紊乱尚不清楚，与不同分期NSCLC的关系有待进一步探讨。
目的
通过分析Th17/Treg失衡与不同分期NSCLC的关系，分析相关转录因子及细胞因子的水平变化，探讨Th17/Treg失衡在NSCLC发病中的分子机制，明确Th17/Treg失衡在NSCLC中的意义。
方法
1.收集59例非小细胞肺癌患者及健康对照外周血标本
临床病理确诊患者30例鳞癌、29例腺癌和10名健康对照的外周血标本，标本分为两份，一份用于细胞分型、mRNA、蛋白质检测；另一份冻存以备细胞因子检测。
2.多参数流式细胞术检测肺腺癌和肺鳞癌外周血单个核细胞中Treg细胞和Th17细胞表达率
分别检测59例NSCLC患者外周血单个核细胞中Treg细胞和Th17细胞在T细胞中的表达率。
3. Real time quantitative PCR检测非小细胞肺癌外周血单个核细胞中RORγt及FoxP3mRNA水平
分别提取非小细胞肺癌外周血有核细胞总RNA，用RT-PCR法检测Th17细胞转录因子RORγt和Treg细胞FoxP3mRNA表达水平。
4. Western-blotting检测非小细胞肺癌外周血单个核细胞中RORγt和Foxp3蛋白表达水平
分别提取分别提取非小细胞肺癌外周血单个核细胞总蛋白，采用western-blotting方法分别检测RORγt和Foxp3蛋白表达情况。
5.酶联免疫吸附法检测非小细胞肺癌外周血清中细胞因子的水平
用ELISA试剂盒分别检测非小细胞肺癌外周血清细胞因子IL-6、IL-17、IL-10和TGF-β1的水平。
结果
1.59例非小细胞肺癌患者的临床资料
59例非小细胞肺癌患者，其中鳞癌30例，男性18例，女性12例，年龄56-81岁，平均年龄64.7岁；腺癌29例，男性18例，女性11例，年龄55-80岁，平均年龄63.0岁，所有患者均符合WHO诊断与分期标准，Ⅰ期7人，Ⅱ期13人，Ⅲ期19人，Ⅳ20人。同期选择10名健康志愿者作为对照。
2.非小细胞肺癌外周血中Treg细胞表达率升高
流式细胞学分析结果表明：与对照组相比，鳞癌组外周血中CD4~+CD25~+CD127lowTreg细胞表达率显著升高（5.598±0.227%vs.3.444±0.2974%，P <0.05）；腺癌组也显著升高（7.237±0.377%vs.3.444±0.2974%，P <0.05）。
3.非小细胞肺癌外周血中Th17细胞表达率下降
流式细胞学分析结果表明：与对照组相比，鳞癌患者外周血中Th17细胞表达率（0.4313±0.0209%vs.0.6090±0.0558%， P <0.05）明显下降，腺癌组（0.3624±0.0273%vs.0.6090±0.0558%, P <0.05）也显著下降。
4.非小细胞肺癌外周血中Th17/Treg比率下降，且与分期呈负相关
比较分析Th17/Treg比率表明：与健康对照组相比，腺癌（0.1865±0.0212vs.0.1865±0.0212，P <0.05）及鳞癌（0.0547±0.0054vs.0.1865±0.0212， P <0.05）Th17/Treg比率明显下降，且分期越差，比例下降越明显，Th17/Treg比例与NSCLC分期呈负相关。
5.非小细胞肺癌外周FoxP3mRNA表达显著升高，但RORγtmRNA表达降低
RT-PCR结果表明：与健康对照组相比，鳞癌Treg细胞相关转录因子FoxP3mRNA表达显著升高（0.9957±0.1799vs.0.07382±0.01382，P <0.05），腺癌FoxP3mRNA表达显著升高（1.681±0.2401vs.0.07382±0.01382，P <0.05），鳞癌Th17细胞相关转录因子RORγt表达降低（2.101±0.2759vs.4.202±0.9663，P <0.05），腺癌RORγt表达降低（1.126±0.1725vs.4.202±0.9663，P <0.05），鳞癌组与腺癌组相比FoxP3mRNA （0.9957±0.1799vs.1.681±0.2401, P>0.05）,RORγtmRNA(2.101±0.2759vs.1.126±0.1725P>0.05）无明显差异。
6.非小细胞肺癌Foxp3蛋白表达显著升高，但RORγt蛋白表达下降
Western-blotting结果表明：肺鳞癌和腺癌患者的Foxp3蛋白表达分别是健康对照组的3.245（P <0.05）,5.119（P <0.05），腺癌组Foxp3表达水平高于鳞癌组（5.119vs.3.245，P <0.05）；鳞癌及腺癌组RORγt蛋白表达水平分别是健康对照组的0.6693（P <0.05），0.5456（P <0.05），两者RORγt蛋白表达水平无差异（0.6693vs.0.5456，P>0.05）。
7.非小细胞肺癌外周血清中IL-10、TGF-β1显著升高，IL-6、IL-17显著降低
ELISA法结果表明：与健康对照比较，鳞癌外周血清Treg细胞相关因子IL-10（898±52.45ng/mL vs.362.00±53.25ng/mL，P <0.05）、TGF-β1（408.9±25.5ng/mLvs.189.4±27.51ng/mL，P <0.05）显著升高；鳞癌组Th17细胞相关因子IL-6（327.8±44.44ng/mL vs.2139±787.7ng/mL，P <0.05）、IL-17（588.3±71.67ng/m vs.1557±375.8ng/mL，P <0.05）显著下降；与健康对照比较，腺癌IL-10（1458±145.8ng/mL vs.362.00±53.25ng/mL， P <0.05）、 TGF-β1（541.5±32.9ng/mL vs.189.4±27.51ng/mL，P <0.05）升高，IL-6（369.5±61.5ng/mL vs.2139±787.7ng/mL，P <0.05）、IL-17（613.6±92.06ng/m vs.1557±375.8ng/mL，P <0.05）显著下降。
结论
1.非小细胞肺癌常见病理类型腺癌和鳞癌外周Th17/Treg平衡紊乱，Treg细胞表达显著增强，Th17细胞表达率下降，相关转录因子RORγt和Foxp3，相应主要细胞因子IL-6、IL-17、IL-10、TGF-β1等均发生对应变化，提示Th17/Treg平衡紊乱在NSCLC的发生、发展中产生重要影响。
2.肺腺癌较鳞癌的免疫抑制明显，可能与腺癌较易发生血行转移有关。
3.非小细胞肺癌常见病理类型腺癌和鳞癌Th17/Treg平衡与分期呈负相关。
Background
Lung cancer is a leading cause of cancer-related morbidity and mortality in theworld.It has been estimated that non-small cell lung cancer (NSCLC) makes up to80%of all cases of lung cancer, and that adenocarcinoma and squamous cell carcinomaaccount for most cases of non-small cell lung cancer. Despite improvements in thedetection and treatment of lung cancer in the past two decades, the5-year survival rateremains less than15%, and chemotherapy side effects have limited the patients’s qualityof life and actual survival time,highlighting the need for new treatment method andmode.Therapies targeting the immune system may represent a promising strategy forthe treatment of lung cancer.
Distinguished from Th1and Th2cells, CD4~+CD25~+regulatory T (Treg) cells andT-helper17(Th17) cells are two original subsets of CD4~+T leukomonocyte.Regulatory T cells (Treg) are a specialized subset of CD4T cells that have anindispensable role in maintaining immune homeostasis and tolerance. Emerging asnegative regulation in immune system, Treg cells can prevent expansion ofimmunoreaction and alleviate immunologic body damage.Studies in mice and humanshave clearly highlighted that the absence of these cells results in severe autoimmunityand inflammation, and increased Treg numbers and/or function is not always beneficial.This is best exemplified in certain cancers where increased Tregs promote cancerprogression by interfering with immune surveillance. Transcriptional factor Foxp3serves as a lineage specification factor of Treg cells. Depletion of the Foxp3~+Tregpopulation in adult mice was found to result in severe and fatal full-body autoimmunity.Tregs can inhibit the activation of both CD4~+and CD8+T cells, and within the tumormicroenvironment may serve to suppress anti-cancer cell immunity. T helper cells thatproduce interleukin-17(IL-17)(Th17cells) have recently been identified as the newdistinct subset of effector T cells, the differentiation of which depends on specifictranscription nuclear factor retinoic acid-related orphan nuclear receptor-gammat.Emerging data have suggested that Th17cells play an important role in innate immunity,adaptive immunity and autoimmunity.Th17cells act as not only an importantinflammatory component in inflammation diseases but also play an important role inanti-tumorigenic processes. T helper type17(Th17) lymphocytes are found in highfrequency in tumour-burdened animals and cancer patients. A reduced percentage ofTh17cells in the tumour microenvironment in RORγt-deficient mice led to enhancedtumour growth, that could be reverted by adoptive transfer of Th17cells. Previous studyhas shown that Th17cells were significantly decreased in peripheral blood in humanovarian, renal, and pancreatic malignancies. There is a reciprocal relationship betweenTh17cells and regulatory T cells (Treg), not only in development, but also in their effector function. Transforming growth factor (TGF)-beta induces Treg-specifictranscription factor Forkhead box P3(Foxp3), while the addition of IL-6to TGF-betainhibits the generation of Treg cells and induces Th17cells. It is proposed that the finebalance between Th17and Treg cells is crucial for maintenance of immune homeostasis.Since Treg cells play an important role in tumor, it is reasonable to assume that Th17cells may play a reciprocal role in tumor. Thus, future research on the Treg/Th17balance may provide an opportunity to illustrate the pathogenesis of NSCLC and toexplore new therapeutic targets for immune-related lung cancer.
In sum, above reports show that the reciprocal balance of Th17/Treg plays animportant role in the occurrence and development of tumor. At present, the imbalanceof Th17/Treg has become a new hotspot in the field of oncology. But, whether theimbalance exists in NSCLC and it’s relation with different stage are still unclear.
Objective
To evaluate the relationship between Th17/Treg ratio and pathological types,stages and cytokine levels. To further understand the role of the Th17and Treg cells inthe pathogenesis of NSCLC.
Methods
Subjects. The current study involved59patients with NSCLC who were seen atthe Respiratory Department of the Second Hospital of AnHui Medical University,between October2008and December2010. Peripheral blood (PB;8mL) was collectedfrom59patients and10healthy control samples. Of the PB,6mL was anti-coagulatedwith EDTA-2K for the isolation of peripheral blood mononuclear cells (PBMCs) andprocessed within4h, while the remaining2mL was used for the preparation of serum.PBMCs were isolated for analysis by flow cytometry and real-time polymerase chainreaction (PCR) using Ficoll-Hypaque (Lymphoprep, Norway) density gradientcentrifugation (840g,20min,20°C). Trypan blue staining revealed the viability offreshly isolated cells to be>95%. Serum was separated from the specimens and storedat-70°C until used for cytokine determination.
Flow cytometry. Analysis of Th17cells was conducted as described previously.Briefly, about100μL of PBMC were suspended at a density of2×106cells/mL incomplete culture medium. Cultures were stimulated for4h using2u/mL leukocyteactivation cocktail at37°C and5%CO2. Cells were then washed twice with PBS andsurface-labeled with PE-Cy5-conjugated anti-CD3and FITC-conjugated anti-CD8for20min at room temperature in the dark. Following surface staining, cells were fixed andpermeabilized using the IntraPrep Permeabilization Reagent and then incubated withPE-conjugated anti-IL-17A, washed with PBS and analyzed using a Coulter Epics XLflow cytometer with System II software. For the analysis of Tregs.EDTA-2K-anticoagulated blood (100μL) was incubated with PE-conjugated anti-CD127, FITC-conjugated anti-CD25, and PE-Cy5-conjugated anti-CD4at25°C for15min. After incubation, red blood cells were lysed and washed twice in PBS. Labeledcells were assessed by FACS and analyzed using the System II software. In each case,staining was compared with that of the appropriately labeled isotype control antibody.The frequency of CD4~+CD25~+CD127lowT cells was expressed as a percentage of CD4~+T cells by sequential gating on lymphocytes and CD4~+T cells.
Quantitative PCR. Total RNA was extracted from individual PBMC preparationsamples using the Trizol reagent and reversed transcribed, according to themanufacturer’s instructions. Quantitative real-time polymerase chain reaction wasperformed using the SYBR Green PCR Mix. Cycling reactions were performed using anABI7900Sequence Detection System. The PCR thermal cycle was95°C for10min,40cycles of95°C for15s,60°C for30s, and72°C for30s, followed by a5-min extensionat72°C and holding at4°C. For each sample, mRNA expression level was normalizedto the level of the β-actin housekeeping gene. The comparative threshold cycle (2－ΔΔCT)method was used to enable quantification of the mRNA of these genes.
Western-blotting. Frozen cells were lysed in buffer and the lysates were separatedby10%SDS–PAGE. The proteins were transferred onto a polyvinylidenedifluoridemembranes. The membranes were blocked with5%bovine serum albumin intris-buffered saline/tween20for1h. Primary antibodies specific for RORγt,FoxP3, orβ-actin were incubated with the membranes overnight at4℃. Membranes were washedwith PBS and incubated with the secondary antibody horseradish peroxidase-linkedanti-rabbit IgG. Chemiluminescent signals were generated by the addition of theSuperSignal West Pico Chemiluminescent Substrate and detected byenhancedchemiluminescence detection system.
ELISA. Levels of IL-6, IL-17, IL-10, and TGF-β1proteins were measured withhuman IL-6, IL-17, IL-10, and TGF-β1enzyme-linked immunosorbent assay kitsaccording to the manufacturer’s protocol.
Statistics. The SPSS v11.5software was used in statistical calculations. The resultswere expressed as the means±SE. A one-way ANOVA test was used to determinesignificant differences between groups. Spearman’s test was used for correlationanalysis. P-values <0.05was considered to indicate statistical significance.
Results
Clinical chararcteristics: Of the59patients with NSCLC,30with squamous cellcarcinoma(male18, female12, age range from56to81years old, median age64.7years old)， and29with adenocarcinoma(male18, female11, age range from55to80years old, median age63.0years old), and10healthy people as controls(male5, female5, age range from57to80years old, median age62.2years old), Histological type andTNM staging were determined according to the WHO classification. According to thestaging criteria of NSCLC,7patients were at stageⅠ,13at stageⅡ,19at stage Ⅲ and20at stage Ⅳ.
Increased percentages of Tregs in peripheral blood of NSCLC patients：The percentages of Tregs in peripheral blood of NSCLC patients and healthy controlswere determined by flow cytometry. The level of Tregs (CD4~+CD25~+CD127low) inadenocarcinoma and squamous cell carcinoma patients were7.237±0.377%and5.598±0.227%, respectively. The prevalence in healthy controls (3.444±0.2974%) wassignificantly lower than that in adenocarcinoma and squamous cell carcinoma patients(both P <0.05). Moreover, adenocarcinoma patients had a higher percentage of Tregsthan squamous cell carcinoma patients(P <0.05).
Decreased frequencies of Th17cells in peripheral blood of NSCLC patients：The frequencies of Th17cells in peripheral blood of adenocarcinoma patients,squamous cell carcinoma patients, and healthy controls were also determined by flowcytometry. There was a decreased prevalence of Th17cells (CD3~+CD8-IL-17A+) ofadenocarcinoma and squamous cell carcinoma patients than those in healthy controls(0.3624±0.0273%vs.0.6090±0.0558%,0.4313±0.0209%vs.0.6090±0.0558%; both P<0.05), while there was no obvious difference between adenocarcinoma and squamouscell carcinoma patients (P>0.05).
Expression of FoxP3and RORγt in PBMCs of NSCLC patients:To confirm these observations, we also determined the specific transcription factor ofboth T subsets in three groups by real-time PCR and western blotting. Increased mRNAand protein expression of the Treg-specific transcription factor FoxP3was observed inNSCLC patients compared with healthy controls (both P <0.05), while there was also asignificant difference between adenocarcinoma and squamous cell carcinoma patients(P <0.05). Regarding the Th17-specific transcription factor, we found decreased RORγtmRNA and protein expression in NSCLC patients compared with healthy controls (P<0.05), while there was no obvious difference between adenocarcinoma and squamouscell carcinoma patients (P>0.05). These results were consistent with the flowcytometric analyses of Th17cells and Tregs.
Increased Treg-related cytokines and decreased Th17-related cytokines inserum from NSCLC patients:Treg-related cytokines (IL-10, TGF-β1) and Th17-related cytokines (IL-6, IL-17) weremeasured by ELISA. Basal levels of IL-10and TGF-β1were362.00±53.25ng/mL and189.4±27.51ng/mL, respectively, in healthy controls, whereas the highest levels ofIL-10and TGF-β1were1458±145.8ng/mL and541.5±32.9ng/mL, respectively, inadenocarcinoma patients. Basal level of IL-6and IL-17were2139±787.7ng/mL and1557±375.8ng/mL, respectively, in healthy controls, whereas the lowest levels of IL-6and IL-17were327.8±44.44ng/mL and588.3±71.67ng/mL, respectively, in squamouscell carcinoma patients. Adenocarcinoma and squamous cell carcinoma patientsexhibited higher levels of serum IL-10and TGF-β1than healthy controls (P <0.05),but lower levels of IL-6and IL-17(P <0.05).
Imbalance of circulating Th17cells/Tregs in NSCLC patients:The significance of increased Tregs and decreased Th17cells was further explored by calculating theTh17/Treg percentage ratio of individual NSCLC patient and healthy control. The Th17/Treg ratio was compared among three groups. The ratios were0.1865±0.0212,0.0547±0.0054, and0.0818±0.0060in healthy controls, adenocarcinoma, and squamous cell carcinoma patients,respectively. The adenocarcinoma and squamous cell carcinoma patients all exhibited lowerTh17/Treg ratios than healthy controls (P <0.05), while there was no obvious difference betweenadenocarcinoma and squamous cell carcinoma patients (P>0.05). In addition, Th17/Treg ratio wascompared in different stages of NSCLC. It is remarkably clear that the Th17/Treg ratio was lowest instage Ⅳpatients, and Th17/Treg ratio was negatively correlated with the stages.
Conclusion
1. The imbalance of Th17/Treg ratio, and corresponding alterations of specifictranscription factor Foxp3and RORγt mRNA expression and relatedcytokines(IL-6、IL-17、IL-10、TGF-β1) implied that imbalance of Th17/Tregplayed an important role the in the pathogenesis of NSCLC.
2. More apparent immune-suppressed status was found in lung adenocarcinomapatients, this is why adenocarcinoma is more prone to progress and metastasize.
3. Th17/Treg ratio was compared in different stages of NSCLC. It is remarkably clearthat the Th17/Treg ratio are lowest in stage Ⅳpatients, and Th17/Treg ratio wasnegatively correlated with the stages.
肺癌无论是发病率还是死亡率，均居全球癌症首位。按组织病理学分类，分为小细胞肺癌和非小细胞肺癌，据估计，非小细胞肺癌（NSCLC）约占所有肺癌类型的80%，其中肺腺癌和肺鳞状上皮细胞癌占据了NSCLC的绝大多数。近二十年来，虽然以含铂类化疗为主的综合治疗使肺癌的生存率有所延长，但是五年生存率仍然低于15%，并且由于化疗的附加损害，肺癌患者的生活质量和实际生存期并未显著改善，因此，探索新的治疗手段，寻找更为有效的治疗模式成为研究热点。肺癌的抗肿瘤免疫失衡研究可能为治疗提供一种极具前景的治疗模式。
CD4~+CD25~+Foxp3~+调节性T细胞（Treg）和辅助性T细胞17（Th17）是两类不同于Th1和Th2的CD4~+T淋巴细胞。Treg细胞是CD4~+T细胞的一类特殊分支，在维持免疫稳态和免疫耐受上发挥重要作用，可以避免免疫炎症反应的扩大和减轻免疫机体损伤，在机体免疫系统中发挥负向调节作用。在动物和人研究中发现，调节性T细胞的缺失可以导致严重的自身免疫性炎症；相反，Treg细胞的增多也不利于机体，在许多癌症中，增加的Treg细胞通过与自身免疫监控系统的相互作用，抑制抗肿瘤免疫活性细胞，促进了癌症的进展。Foxp3是Treg细胞特异的标记物，Foxp3属转录因子Forkhead家族，它可以控制Treg细胞的发生发育，剔除Foxp3基因的小鼠，因产生大量慢性炎症因子，导致小鼠产生致死性炎症疾病。在肿瘤微环境中，Tregs细胞抑制CD4~+T细胞和CD8+T细胞的活化，抑制抗癌细胞发挥抗肿瘤作用。产生IL-17的T辅助细胞（Th17）是近来发现的效应性T细胞的新亚型，转录因子孤核受体(RORγt)是Th17细胞分化的特异性转录调控因子。研究发现T17细胞在固有免疫，适应性免疫和自身免疫中发挥重要作用。Th17细胞作为炎症反应的一个重要组成部分，不仅在炎症性疾病中促进炎症反应，而且在肿瘤发生、发展过程中也发挥重要作用。Th17细胞可以在肿瘤动物模型和癌症患者中经常发现：在RORγt缺陷的肿瘤小鼠模型中，由于肿瘤微环境中Th17细胞比率下降，导致肿瘤增殖，但是，这种增殖可以被输入的Th17细胞逆转，在肾癌、卵巢癌及胰腺癌患者外周血中也有Th17细胞比例的显著降低。Th17细胞和Treg细胞不仅在分化上而且在功能上都存在紧密联系，转化生长因子-beta(TGF-β)诱导Treg细胞特异性转录因子Foxp3，但是，如果增加了IL-6则抑制Treg细胞的分化，转而诱导Th17细胞的分化。Treg细胞和Th17细胞在免疫稳态中可能发挥着相互拮抗的平衡作用，既然Treg细胞在肿瘤中发挥重要作用，理所当然Th17细胞也参与其中。因此，进一步探讨二者在肿瘤免疫环境中的作用机制对肿瘤的免疫治疗具有重要意义。
综上所述，Treg细胞和Th17细胞之间存在复杂的相互关系，二者在分化上相互联系，在功能上相互拮抗，二者的平衡在肿瘤的发生发展中具有重要作用。目前，Th17/Treg的平衡紊乱已经成为恶性肿瘤研究领域中新的热点，但是，在非小细胞肺癌中的研究尚少，是否存在比较显著的Th17/Treg紊乱尚不清楚，与不同分期NSCLC的关系有待进一步探讨。
目的
通过分析Th17/Treg失衡与不同分期NSCLC的关系，分析相关转录因子及细胞因子的水平变化，探讨Th17/Treg失衡在NSCLC发病中的分子机制，明确Th17/Treg失衡在NSCLC中的意义。
方法
1.收集59例非小细胞肺癌患者及健康对照外周血标本
临床病理确诊患者30例鳞癌、29例腺癌和10名健康对照的外周血标本，标本分为两份，一份用于细胞分型、mRNA、蛋白质检测；另一份冻存以备细胞因子检测。
2.多参数流式细胞术检测肺腺癌和肺鳞癌外周血单个核细胞中Treg细胞和Th17细胞表达率
分别检测59例NSCLC患者外周血单个核细胞中Treg细胞和Th17细胞在T细胞中的表达率。
3. Real time quantitative PCR检测非小细胞肺癌外周血单个核细胞中RORγt及FoxP3mRNA水平
分别提取非小细胞肺癌外周血有核细胞总RNA，用RT-PCR法检测Th17细胞转录因子RORγt和Treg细胞FoxP3mRNA表达水平。
4. Western-blotting检测非小细胞肺癌外周血单个核细胞中RORγt和Foxp3蛋白表达水平
分别提取分别提取非小细胞肺癌外周血单个核细胞总蛋白，采用western-blotting方法分别检测RORγt和Foxp3蛋白表达情况。
5.酶联免疫吸附法检测非小细胞肺癌外周血清中细胞因子的水平
用ELISA试剂盒分别检测非小细胞肺癌外周血清细胞因子IL-6、IL-17、IL-10和TGF-β1的水平。
结果
1.59例非小细胞肺癌患者的临床资料
59例非小细胞肺癌患者，其中鳞癌30例，男性18例，女性12例，年龄56-81岁，平均年龄64.7岁；腺癌29例，男性18例，女性11例，年龄55-80岁，平均年龄63.0岁，所有患者均符合WHO诊断与分期标准，Ⅰ期7人，Ⅱ期13人，Ⅲ期19人，Ⅳ20人。同期选择10名健康志愿者作为对照。
2.非小细胞肺癌外周血中Treg细胞表达率升高
流式细胞学分析结果表明：与对照组相比，鳞癌组外周血中CD4~+CD25~+CD127lowTreg细胞表达率显著升高（5.598±0.227%vs.3.444±0.2974%，P <0.05）；腺癌组也显著升高（7.237±0.377%vs.3.444±0.2974%，P <0.05）。
3.非小细胞肺癌外周血中Th17细胞表达率下降
流式细胞学分析结果表明：与对照组相比，鳞癌患者外周血中Th17细胞表达率（0.4313±0.0209%vs.0.6090±0.0558%， P <0.05）明显下降，腺癌组（0.3624±0.0273%vs.0.6090±0.0558%, P <0.05）也显著下降。
4.非小细胞肺癌外周血中Th17/Treg比率下降，且与分期呈负相关
比较分析Th17/Treg比率表明：与健康对照组相比，腺癌（0.1865±0.0212vs.0.1865±0.0212，P <0.05）及鳞癌（0.0547±0.0054vs.0.1865±0.0212， P <0.05）Th17/Treg比率明显下降，且分期越差，比例下降越明显，Th17/Treg比例与NSCLC分期呈负相关。
5.非小细胞肺癌外周FoxP3mRNA表达显著升高，但RORγtmRNA表达降低
RT-PCR结果表明：与健康对照组相比，鳞癌Treg细胞相关转录因子FoxP3mRNA表达显著升高（0.9957±0.1799vs.0.07382±0.01382，P <0.05），腺癌FoxP3mRNA表达显著升高（1.681±0.2401vs.0.07382±0.01382，P <0.05），鳞癌Th17细胞相关转录因子RORγt表达降低（2.101±0.2759vs.4.202±0.9663，P <0.05），腺癌RORγt表达降低（1.126±0.1725vs.4.202±0.9663，P <0.05），鳞癌组与腺癌组相比FoxP3mRNA （0.9957±0.1799vs.1.681±0.2401, P>0.05）,RORγtmRNA(2.101±0.2759vs.1.126±0.1725P>0.05）无明显差异。
6.非小细胞肺癌Foxp3蛋白表达显著升高，但RORγt蛋白表达下降
Western-blotting结果表明：肺鳞癌和腺癌患者的Foxp3蛋白表达分别是健康对照组的3.245（P <0.05）,5.119（P <0.05），腺癌组Foxp3表达水平高于鳞癌组（5.119vs.3.245，P <0.05）；鳞癌及腺癌组RORγt蛋白表达水平分别是健康对照组的0.6693（P <0.05），0.5456（P <0.05），两者RORγt蛋白表达水平无差异（0.6693vs.0.5456，P>0.05）。
7.非小细胞肺癌外周血清中IL-10、TGF-β1显著升高，IL-6、IL-17显著降低
ELISA法结果表明：与健康对照比较，鳞癌外周血清Treg细胞相关因子IL-10（898±52.45ng/mL vs.362.00±53.25ng/mL，P <0.05）、TGF-β1（408.9±25.5ng/mLvs.189.4±27.51ng/mL，P <0.05）显著升高；鳞癌组Th17细胞相关因子IL-6（327.8±44.44ng/mL vs.2139±787.7ng/mL，P <0.05）、IL-17（588.3±71.67ng/m vs.1557±375.8ng/mL，P <0.05）显著下降；与健康对照比较，腺癌IL-10（1458±145.8ng/mL vs.362.00±53.25ng/mL， P <0.05）、 TGF-β1（541.5±32.9ng/mL vs.189.4±27.51ng/mL，P <0.05）升高，IL-6（369.5±61.5ng/mL vs.2139±787.7ng/mL，P <0.05）、IL-17（613.6±92.06ng/m vs.1557±375.8ng/mL，P <0.05）显著下降。
结论
1.非小细胞肺癌常见病理类型腺癌和鳞癌外周Th17/Treg平衡紊乱，Treg细胞表达显著增强，Th17细胞表达率下降，相关转录因子RORγt和Foxp3，相应主要细胞因子IL-6、IL-17、IL-10、TGF-β1等均发生对应变化，提示Th17/Treg平衡紊乱在NSCLC的发生、发展中产生重要影响。
2.肺腺癌较鳞癌的免疫抑制明显，可能与腺癌较易发生血行转移有关。
3.非小细胞肺癌常见病理类型腺癌和鳞癌Th17/Treg平衡与分期呈负相关。
Background
Lung cancer is a leading cause of cancer-related morbidity and mortality in theworld.It has been estimated that non-small cell lung cancer (NSCLC) makes up to80%of all cases of lung cancer, and that adenocarcinoma and squamous cell carcinomaaccount for most cases of non-small cell lung cancer. Despite improvements in thedetection and treatment of lung cancer in the past two decades, the5-year survival rateremains less than15%, and chemotherapy side effects have limited the patients’s qualityof life and actual survival time,highlighting the need for new treatment method andmode.Therapies targeting the immune system may represent a promising strategy forthe treatment of lung cancer.
Distinguished from Th1and Th2cells, CD4~+CD25~+regulatory T (Treg) cells andT-helper17(Th17) cells are two original subsets of CD4~+T leukomonocyte.Regulatory T cells (Treg) are a specialized subset of CD4T cells that have anindispensable role in maintaining immune homeostasis and tolerance. Emerging asnegative regulation in immune system, Treg cells can prevent expansion ofimmunoreaction and alleviate immunologic body damage.Studies in mice and humanshave clearly highlighted that the absence of these cells results in severe autoimmunityand inflammation, and increased Treg numbers and/or function is not always beneficial.This is best exemplified in certain cancers where increased Tregs promote cancerprogression by interfering with immune surveillance. Transcriptional factor Foxp3serves as a lineage specification factor of Treg cells. Depletion of the Foxp3~+Tregpopulation in adult mice was found to result in severe and fatal full-body autoimmunity.Tregs can inhibit the activation of both CD4~+and CD8+T cells, and within the tumormicroenvironment may serve to suppress anti-cancer cell immunity. T helper cells thatproduce interleukin-17(IL-17)(Th17cells) have recently been identified as the newdistinct subset of effector T cells, the differentiation of which depends on specifictranscription nuclear factor retinoic acid-related orphan nuclear receptor-gammat.Emerging data have suggested that Th17cells play an important role in innate immunity,adaptive immunity and autoimmunity.Th17cells act as not only an importantinflammatory component in inflammation diseases but also play an important role inanti-tumorigenic processes. T helper type17(Th17) lymphocytes are found in highfrequency in tumour-burdened animals and cancer patients. A reduced percentage ofTh17cells in the tumour microenvironment in RORγt-deficient mice led to enhancedtumour growth, that could be reverted by adoptive transfer of Th17cells. Previous studyhas shown that Th17cells were significantly decreased in peripheral blood in humanovarian, renal, and pancreatic malignancies. There is a reciprocal relationship betweenTh17cells and regulatory T cells (Treg), not only in development, but also in their effector function. Transforming growth factor (TGF)-beta induces Treg-specifictranscription factor Forkhead box P3(Foxp3), while the addition of IL-6to TGF-betainhibits the generation of Treg cells and induces Th17cells. It is proposed that the finebalance between Th17and Treg cells is crucial for maintenance of immune homeostasis.Since Treg cells play an important role in tumor, it is reasonable to assume that Th17cells may play a reciprocal role in tumor. Thus, future research on the Treg/Th17balance may provide an opportunity to illustrate the pathogenesis of NSCLC and toexplore new therapeutic targets for immune-related lung cancer.
In sum, above reports show that the reciprocal balance of Th17/Treg plays animportant role in the occurrence and development of tumor. At present, the imbalanceof Th17/Treg has become a new hotspot in the field of oncology. But, whether theimbalance exists in NSCLC and it’s relation with different stage are still unclear.
Objective
To evaluate the relationship between Th17/Treg ratio and pathological types,stages and cytokine levels. To further understand the role of the Th17and Treg cells inthe pathogenesis of NSCLC.
Methods
Subjects. The current study involved59patients with NSCLC who were seen atthe Respiratory Department of the Second Hospital of AnHui Medical University,between October2008and December2010. Peripheral blood (PB;8mL) was collectedfrom59patients and10healthy control samples. Of the PB,6mL was anti-coagulatedwith EDTA-2K for the isolation of peripheral blood mononuclear cells (PBMCs) andprocessed within4h, while the remaining2mL was used for the preparation of serum.PBMCs were isolated for analysis by flow cytometry and real-time polymerase chainreaction (PCR) using Ficoll-Hypaque (Lymphoprep, Norway) density gradientcentrifugation (840g,20min,20°C). Trypan blue staining revealed the viability offreshly isolated cells to be>95%. Serum was separated from the specimens and storedat-70°C until used for cytokine determination.
Flow cytometry. Analysis of Th17cells was conducted as described previously.Briefly, about100μL of PBMC were suspended at a density of2×106cells/mL incomplete culture medium. Cultures were stimulated for4h using2u/mL leukocyteactivation cocktail at37°C and5%CO2. Cells were then washed twice with PBS andsurface-labeled with PE-Cy5-conjugated anti-CD3and FITC-conjugated anti-CD8for20min at room temperature in the dark. Following surface staining, cells were fixed andpermeabilized using the IntraPrep Permeabilization Reagent and then incubated withPE-conjugated anti-IL-17A, washed with PBS and analyzed using a Coulter Epics XLflow cytometer with System II software. For the analysis of Tregs.EDTA-2K-anticoagulated blood (100μL) was incubated with PE-conjugated anti-CD127, FITC-conjugated anti-CD25, and PE-Cy5-conjugated anti-CD4at25°C for15min. After incubation, red blood cells were lysed and washed twice in PBS. Labeledcells were assessed by FACS and analyzed using the System II software. In each case,staining was compared with that of the appropriately labeled isotype control antibody.The frequency of CD4~+CD25~+CD127lowT cells was expressed as a percentage of CD4~+T cells by sequential gating on lymphocytes and CD4~+T cells.
Quantitative PCR. Total RNA was extracted from individual PBMC preparationsamples using the Trizol reagent and reversed transcribed, according to themanufacturer’s instructions. Quantitative real-time polymerase chain reaction wasperformed using the SYBR Green PCR Mix. Cycling reactions were performed using anABI7900Sequence Detection System. The PCR thermal cycle was95°C for10min,40cycles of95°C for15s,60°C for30s, and72°C for30s, followed by a5-min extensionat72°C and holding at4°C. For each sample, mRNA expression level was normalizedto the level of the β-actin housekeeping gene. The comparative threshold cycle (2－ΔΔCT)method was used to enable quantification of the mRNA of these genes.
Western-blotting. Frozen cells were lysed in buffer and the lysates were separatedby10%SDS–PAGE. The proteins were transferred onto a polyvinylidenedifluoridemembranes. The membranes were blocked with5%bovine serum albumin intris-buffered saline/tween20for1h. Primary antibodies specific for RORγt,FoxP3, orβ-actin were incubated with the membranes overnight at4℃. Membranes were washedwith PBS and incubated with the secondary antibody horseradish peroxidase-linkedanti-rabbit IgG. Chemiluminescent signals were generated by the addition of theSuperSignal West Pico Chemiluminescent Substrate and detected byenhancedchemiluminescence detection system.
ELISA. Levels of IL-6, IL-17, IL-10, and TGF-β1proteins were measured withhuman IL-6, IL-17, IL-10, and TGF-β1enzyme-linked immunosorbent assay kitsaccording to the manufacturer’s protocol.
Statistics. The SPSS v11.5software was used in statistical calculations. The resultswere expressed as the means±SE. A one-way ANOVA test was used to determinesignificant differences between groups. Spearman’s test was used for correlationanalysis. P-values <0.05was considered to indicate statistical significance.
Results
Clinical chararcteristics: Of the59patients with NSCLC,30with squamous cellcarcinoma(male18, female12, age range from56to81years old, median age64.7years old)， and29with adenocarcinoma(male18, female11, age range from55to80years old, median age63.0years old), and10healthy people as controls(male5, female5, age range from57to80years old, median age62.2years old), Histological type andTNM staging were determined according to the WHO classification. According to thestaging criteria of NSCLC,7patients were at stageⅠ,13at stageⅡ,19at stage Ⅲ and20at stage Ⅳ.
Increased percentages of Tregs in peripheral blood of NSCLC patients：The percentages of Tregs in peripheral blood of NSCLC patients and healthy controlswere determined by flow cytometry. The level of Tregs (CD4~+CD25~+CD127low) inadenocarcinoma and squamous cell carcinoma patients were7.237±0.377%and5.598±0.227%, respectively. The prevalence in healthy controls (3.444±0.2974%) wassignificantly lower than that in adenocarcinoma and squamous cell carcinoma patients(both P <0.05). Moreover, adenocarcinoma patients had a higher percentage of Tregsthan squamous cell carcinoma patients(P <0.05).
Decreased frequencies of Th17cells in peripheral blood of NSCLC patients：The frequencies of Th17cells in peripheral blood of adenocarcinoma patients,squamous cell carcinoma patients, and healthy controls were also determined by flowcytometry. There was a decreased prevalence of Th17cells (CD3~+CD8-IL-17A+) ofadenocarcinoma and squamous cell carcinoma patients than those in healthy controls(0.3624±0.0273%vs.0.6090±0.0558%,0.4313±0.0209%vs.0.6090±0.0558%; both P<0.05), while there was no obvious difference between adenocarcinoma and squamouscell carcinoma patients (P>0.05).
Expression of FoxP3and RORγt in PBMCs of NSCLC patients:To confirm these observations, we also determined the specific transcription factor ofboth T subsets in three groups by real-time PCR and western blotting. Increased mRNAand protein expression of the Treg-specific transcription factor FoxP3was observed inNSCLC patients compared with healthy controls (both P <0.05), while there was also asignificant difference between adenocarcinoma and squamous cell carcinoma patients(P <0.05). Regarding the Th17-specific transcription factor, we found decreased RORγtmRNA and protein expression in NSCLC patients compared with healthy controls (P<0.05), while there was no obvious difference between adenocarcinoma and squamouscell carcinoma patients (P>0.05). These results were consistent with the flowcytometric analyses of Th17cells and Tregs.
Increased Treg-related cytokines and decreased Th17-related cytokines inserum from NSCLC patients:Treg-related cytokines (IL-10, TGF-β1) and Th17-related cytokines (IL-6, IL-17) weremeasured by ELISA. Basal levels of IL-10and TGF-β1were362.00±53.25ng/mL and189.4±27.51ng/mL, respectively, in healthy controls, whereas the highest levels ofIL-10and TGF-β1were1458±145.8ng/mL and541.5±32.9ng/mL, respectively, inadenocarcinoma patients. Basal level of IL-6and IL-17were2139±787.7ng/mL and1557±375.8ng/mL, respectively, in healthy controls, whereas the lowest levels of IL-6and IL-17were327.8±44.44ng/mL and588.3±71.67ng/mL, respectively, in squamouscell carcinoma patients. Adenocarcinoma and squamous cell carcinoma patientsexhibited higher levels of serum IL-10and TGF-β1than healthy controls (P <0.05),but lower levels of IL-6and IL-17(P <0.05).
Imbalance of circulating Th17cells/Tregs in NSCLC patients:The significance of increased Tregs and decreased Th17cells was further explored by calculating theTh17/Treg percentage ratio of individual NSCLC patient and healthy control. The Th17/Treg ratio was compared among three groups. The ratios were0.1865±0.0212,0.0547±0.0054, and0.0818±0.0060in healthy controls, adenocarcinoma, and squamous cell carcinoma patients,respectively. The adenocarcinoma and squamous cell carcinoma patients all exhibited lowerTh17/Treg ratios than healthy controls (P <0.05), while there was no obvious difference betweenadenocarcinoma and squamous cell carcinoma patients (P>0.05). In addition, Th17/Treg ratio wascompared in different stages of NSCLC. It is remarkably clear that the Th17/Treg ratio was lowest instage Ⅳpatients, and Th17/Treg ratio was negatively correlated with the stages.
Conclusion
1. The imbalance of Th17/Treg ratio, and corresponding alterations of specifictranscription factor Foxp3and RORγt mRNA expression and relatedcytokines(IL-6、IL-17、IL-10、TGF-β1) implied that imbalance of Th17/Tregplayed an important role the in the pathogenesis of NSCLC.
2. More apparent immune-suppressed status was found in lung adenocarcinomapatients, this is why adenocarcinoma is more prone to progress and metastasize.
3. Th17/Treg ratio was compared in different stages of NSCLC. It is remarkably clearthat the Th17/Treg ratio are lowest in stage Ⅳpatients, and Th17/Treg ratio wasnegatively correlated with the stages.
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