Imbalance In The Th17/Treg And Cytokine Environment In Peripheral Blood Of Patients With Adenocarcinoma And Squamous Cell Carcinoma

BackgroundLung 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.ObjectiveTo 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.MethodsSubjects. 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.ResultsClinical 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.Conclusion1. 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.