辅助性CD4~+ T细胞亚群失衡在实验性自身免疫性重症肌无力大鼠发病机制中的作用

目的研究实验性自身免疫性重症肌无力(Experimental autoimmune myasthenia gravis,EAMG)大鼠发病过程中辅助性CD4+T细胞亚型的变化。方法将Lewis大鼠分为完全弗氏佐剂(CFA)早、晚期发病时对照相和EAMG早期(初次免疫后13d)、高峰期(初次免疫后50d)发病时相4组,各组分别给予相应的药物后,于发病早期和高峰期分离淋巴细胞,采用流式细胞术检测各组大鼠淋巴细胞中Th1、Th2、Th17和Treg细胞的比例,ELISA法检测淋巴细胞培养上清中各种细胞因子水平。结果在EAMG发病的早期时相,与CFA组比较,EAMG组Th1、Th2、Th17细胞的比例下降,而Treg细胞比例上升;淋巴细胞培养上清中IFNγ和IL-4水平显著下降(P<0.05),IL-6水平显著升高(P<0.05),IL-17和TGF-β水平略升高,但差异无统计学意义(P>0.05)。在EAMG发病的高峰期,与CFA组比较,EAMG组Th1和Th17细胞的比例显著上升(P<0.05),而Th2和Treg细胞的比例显著下降(P<0.05),淋巴细胞培养上清中IFNγ和IL-17水平显著上升(P<0.05),IL-4和TGF-β水平显著下降(P<0.01),IL-6水平略上升,但差异无统计学意义(P>0.05)。结论在EAMG发生过程中,4种辅助性CD4+T细胞亚型之间的平衡被打破,晚期时相中Th1和Th17细胞比例上升,而Th2和Treg细胞比例下降。     

Th17及IL-17与实验性自身免疫性重症肌无力发病过程的相关性

目的探讨Th17及其相关因子IL-17与实验性自身免疫性重症肌无力(EAMG)发病过程的相关性。方法建立EAMG大鼠模型及CFA对照组,分别于两发病时相(早期发病高峰和晚期发病高峰),采用ELISA法检测血清以及淋巴细胞培养上清中IL-17的含量;流式细胞仪检测CD4+IL-17+淋巴细胞含量;3H增殖试验检测淋巴细胞的增殖能力;B-ELISPOT法检测B细胞的抗体分泌情况。结果与CFA组相比,EAMG组大鼠血清、淋巴细胞培养上清中IL-17的表达以及CD4+IL-17+淋巴细胞含量在早期发病时相差异均无统计学意义;而在晚期发病时相则均明显增多。与非刺激组相比,IL-17的刺激对CFA和EAMG组淋巴细胞的增殖能力及B细胞抗体分泌水平,在早期发病时相均无明显影响;而在晚期发病时相,EAMG组均明显升高。结论Th17及其相关因子IL-17参与大鼠EAMG的晚期发病时相,并促进疾病的发展。     

LFA-1基因对胶原诱导小鼠关节炎关节局部引流淋巴结T细胞活性的影响

目的探讨淋巴细胞功能相关抗原1(LFA-1)在类风湿性关节炎形成中的作用。方法采用Ⅱ型胶原诱导小鼠关节炎模型,观察LFA-1基因敲除小鼠引流淋巴结T细胞增殖和细胞因子水平。结果LFA-1基因敲除小鼠在应用Ⅱ型胶原免疫后,引流淋巴结和关节局部Th1型细胞因子IFN-γ和IL-12p40mRNA水平明显低于野生型对照小鼠,Th2型细胞因子IL-4mRNA水平无明显变化。LFA-1基因敲除小鼠引流淋巴结来源的CD4+T细胞在体外受Ⅱ型胶原刺激后,其特异性增殖和细胞因子IFN-γ的产生也明显低于野生型对照小鼠。结论LFA-1基因缺失抑制了辅助性T细胞的活化和向Th1方向的分化,进而抑制关节炎的发生。     

人细胞内Ca2+对CD4+T细胞免疫功能调控作用的研究进展

CD4⁺T淋巴细胞是人体免疫系统中重要的免疫细胞之一,主要分为Th1、Th2、Th17、滤泡辅助T细胞(T f ollicular helper cells,Tfh)及调节性T细胞(regulatory T cells,Tregs)。CD4⁺T淋巴细胞功能取决于T细胞受体(T cell receptor,TCR),TCR的激活触发了内质网释放Ca²⁺,Ca²⁺作为细胞的第二信使,通过改变其在细胞内外的浓度来参与细胞周期进展和增殖等多个生物活动过程,以维持免疫细胞的正常功能。CD4⁺T淋巴细胞与脂肪肝、肝脂沉积、肝炎、肿瘤等多种疾病的发生发展有关。因此,本文就细胞内Ca²⁺对CD4⁺T细胞免疫功能调控作用的研究进展作一综述,以期为某些疾病新临床治疗方案的研发提供参考。     

IL-17对髓鞘碱性蛋白诱导鼻黏膜免疫耐受治疗实验性自身免疫性脑脊髓炎的影响

目的探讨IL-17对髓鞘碱性蛋白(MBP)68-86诱导鼻黏膜免疫耐受治疗实验性自身免疫性脑脊髓炎(EAE)的影响。方法将大鼠分为5组,分别经鼻黏膜滴注PBS、MBP68-86、MBP68-86+IL-17(0.01μg/d)、MBP68-86+IL-17(0.05μg/d)和MBP68-86+IL-17(0.1μg/d)诱导其发生免疫耐受,并在此基础上建立EAE动物模型,观察各组大鼠发病情况;通过3H掺入试验检测特异性抗原MBP68-86多肽诱导T淋巴细胞增殖活性;HE染色观察脊髓淋巴细胞浸润情况,免疫组化方法检测脊髓中单位面积IL-17+细胞数。结果PBS组和IL-170.1μg/d组与MBP组相比,大鼠免疫后出现进食减少、体重减轻、尾瘫、后肢瘫痪等临床症状,IL-170.01μg/d组和0.05μg/d组大鼠临床症状较轻或无症状。淋巴细胞增殖试验结果显示,PBS组和IL-170.1μg/d组与MBP组相比,特异性淋巴细胞增殖反应显著增高,IL-170.01μg/d组和0.05μg/d组与MBP组相比,差异无显著意义,与IL-170.1μg/d组相比差异有显著意义;PBS组、IL-170.1μg/d组与MBP组、IL-170.01μg/d组和0.05μg/d组相比,脊髓切片中淋巴细胞浸润面积较大且细胞数量较多,IL-17+细胞数也显著增多。结论MBP68-86特异性肽段可诱导EAE免疫耐受的形成,预防EAE的发生;鼻黏膜给予IL-17可以打破MBP诱导的特异性免疫耐受,且存在剂量依赖性。     

小鼠白细胞介素-17及其shRNA重组表达质粒的构建

目的构建小鼠白细胞介素-17(interleukin-17,IL-17)及其shRNA重组表达质粒。方法以小鼠脾脏细胞总RNA为模板,PCR扩增IL-17基因,并亚克隆至质粒pLVX-IRES-ZsGreen1,构建重组表达质粒pLVX-IL-17-IRESZsGreen1;设计并合成3对针对IL-17基因的shRNA序列和1对阴性对照shRNA序列,分别插入质粒pLVX-shRNA,构建重组干扰质粒pLVX-shRNA1、pLVX-shRNA2、pLVX-shRNA3和阴性对照质粒shRNAC,进行测序;用脂质体法将各重组干扰质粒分别与表达质粒共转染293T细胞,分为空白对照组、过表达组、shRNA1组、shRNA2组、shRNA3组和shRNAC组,经荧光定量PCR和ELISA法分别检测各组细胞中IL-17基因mRNA的转录水平和蛋白的表达水平。结果 PCR扩增获得500 bp的目的基因,IL-17重组表达质粒及其shRNA重组质粒经酶切及测序鉴定证明构建正确;重组表达质粒转染293T细胞48 h后可见绿色荧光表达;过表达组IL-17基因mRNA及蛋白表达水平均明显高于空白对照组(P均<0.05),shRNA1组、shRNA2组和shRNA3组IL-17基因mRNA和蛋白表达水平均明显低于shRNAC组(P均<0.05)。结论成功构建了小鼠IL-17基因重组表达质粒及其特异性shRNA表达质粒,并筛选出shRNA1,其对IL-17的表达具有最佳的抑制效应。     

CTLA4-Ig治疗自身免疫性疾病的机理

B7－1、B7－2是B7家族的两个重要成员，它们介导的共刺激信号诱导Th细胞分化为Th1和Th2细胞。Th1和Th2细胞分别分泌两种不同类型的细胞因子，它们在自身免疫性疾病的发生发展中起着非常重要的作用。CTLA4Ig通过封闭B7－1和B7－2调节Th细胞的分化方向，从而达到治疗自身免疫性疾病的作用。因而是一种很有前途的免疫调节药物。     

水油微球/BCG复合佐剂对结核杆菌融合蛋白PstS1-LEP免疫原性的影响

目的探讨水油微球与热灭活BCG(heat-killed BCG,HKBCG)或无细胞BCG(acellular BCG,ABCG)复合佐剂对结核杆菌融合蛋白PstS1-LEP免疫原性的影响。方法制备水油微球/HKBCG、水油微球/ABCG复合佐剂PstS1-LEP疫苗,经皮下注射免疫BALB/c小鼠3次,间隔2周,末次免疫后2周采血,处死小鼠并无菌摘脾;ELISA法检测小鼠血清抗Pst S1-LEP的IgG、IgG1和IgG2a抗体,ELISPOT法检测小鼠脾淋巴细胞经PstS1-LEP刺激分泌IFNγ、IL-4和IL-17的斑点形成细胞数(spots forming cell,SFC)。结果水油微球/HKBCG、水油微球/ABCG复合佐剂PstS1-LEP疫苗免疫小鼠诱导Pst S1-LEP特异性IgG、IgG1和IgG2a水平比较,差异无统计学意义(P0.05);水油微球/ABCG复合佐剂PstS1-LEP疫苗免疫小鼠的IFNγ-SFC、IL-17-SFC及IL-17-SFC/IL-4-SFC均高于水油微球/HKBCG复合佐剂PstS1-LEP疫苗,而两种疫苗免疫小鼠的IL-4-SFC、IFNγ-SFC/IL-4-SFC和IFNγ-SFC/IL-17-SFC比较,差异均无统计学意义(P0.05)。结论水油微球/ABCG复合佐剂PstS1-LEP疫苗诱导偏向Th1和Th17型细胞免疫,IFNγ-SFC、IL-4-SFC和IL-17-SFC的两两比值显示,水油微球/ABCG复合佐剂PstS1-LEP疫苗更有利于诱导Th17型细胞免疫漂移。     

白介素-17对骨髓瘤细胞增殖和凋亡的影响

目的探讨白介素-17(interleukin-17,IL-17)对骨髓瘤细胞株RPMI8226增殖和凋亡的影响及骨髓瘤细胞株IL-17R的表达。方法体外培养RPMI8226细胞,取对数生长期的细胞,分别加入浓度为25、50、100、200、500 ng/ml的rh IL-17(另设不加rh IL-17的对照组),共同培养72 h后,采用MTT法检测IL-17对RPMI8226细胞增殖的影响;TUNEL法检测IL-17对RPMI8226细胞凋亡的影响。将BALB/c雌性小鼠随机分为试验组和对照组,分别经腹部皮下注射含100 ng/ml IL-17和不含IL-17的RPMI8226细胞(5×106个),观察肿瘤生长情况。应用流式细胞术检测RPMI8226细胞IL-17R的表达。结果不同浓度的IL-17与对照组相比,均可促进RPMI8226细胞的增殖(P﹤0.05),抑制细胞凋亡(P﹤0.05)。经IL-17处理的RPMI8226细胞荷瘤小鼠与对照组相比,成瘤时间缩短(P﹤0.05),肿瘤体积增大(P﹤0.05)。RPMI8226细胞显著表达IL-17R。结论 IL-17在体内外均可促进RPMI8226细胞增殖,抑制其凋亡;RPMI8226细胞显著表达IL-17R。     

Low-Dose Interleukin-2 Therapy: A Driver of an Imbalance between Immune Tolerance and Autoimmunity

For many years, the role of interleukin-2 (IL-2) in autoimmune responses was established as a cytokine possessing strong pro-inflammatory activity. Studies of the past few years have changed our knowledge on IL-2 in autoimmune chronic inflammation, suggesting its protective role, when administered at low-doses. The disrupted balance between regulatory and effector T cells (Tregs and Teffs, respectively) is a characteristic of autoimmune diseases, and is dependent on homeostatic cytokines, including IL-2. Actually, inherent defects in the IL-2 signaling pathway and/or levels leading to Treg compromised function and numbers as well as Th17 expansion have been attributed to autoimmune disorders. In this review, we discuss the role of IL-2 in the pathogenesis of autoimmune diseases. In particular, we highlight the impact of the dysregulated IL-2 pathway on disruption of the Treg/Th17 balance, reversal of which appears to be a possible mechanism of the low-dose IL-2 treatment. The negative effects of IL-2 on the differentiation of follicular helper T cells (Tfh) and pathogenic Th17 cells, both of which contribute to autoimmunity, is emphasized in the paper as well. We also compare the current IL-2-based therapies of animal and human subjects with immune-mediated diseases aimed at boosting the Treg population, which is the most IL-2-dependent cell subset desirable for sufficient control of autoimmunity. New perspectives of therapeutic approaches focused on selective delivery of IL-2 to inflamed tissues, thus allowing local activity of IL-2 to be combined with its reduced systemic and pleiotropic toxicity, are also proposed in this paper.     

Th17 Cells as Potential Probiotic Therapeutic Targets in Inflammatory Bowel Diseases

Inflammatory bowel diseases (IBD) are characterized by wasting and chronic intestinal inflammation triggered by various cytokine-mediated pathways. In recent years, it was shown that T helper 17 (Th17) cells are involved in the pathogenesis of IBD, which makes them an attractive therapeutic target. Th17 cells preferentially produce interleukin (IL)-17A–F as signature cytokines. The role of the interplay between host genetics and intestinal microbiota in the pathogenesis of IBD was demonstrated. Probiotics are live microorganisms that when orally ingested in adequate amounts, confer a health benefit to the host by modulating the enteric flora or by stimulating the local immune system. Several studies indicated the effectiveness of probiotics in preventing and treating IBD (ulcerative colitis, and Crohn’s disease). Furthermore, there is mounting evidence of probiotics selectively targeting the Th17 lineage in the prevention and management of inflammatory and autoimmune diseases such as IBD. This review highlights critical roles of Th17 cells in the pathogenesis of IBD and the rationale for using probiotics as a novel therapeutic approach for IBD through manipulation of Th17 cells. The potential molecular mechanisms by which probiotics modulate Th17 cells differentiation and production are also discussed.     

CD4+ T-Cell Plasticity in Non-Infectious Retinal Inflammatory Disease

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4⁺ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4⁺FoxP3⁺CD25⁺ regulatory T cells (Tregs) were known to suppress function of effector CD4⁺ T cells and contribute to resolution of disease. It has been recently reported that some CD4⁺ T-cell subsets demonstrate shared phenotypes with another CD4⁺ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4⁺ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.     

Deficiency of IL-27 Signaling Exacerbates Experimental Autoimmune Uveitis with Elevated Uveitogenic Th1 and Th17 Responses

Human uveitis is an autoimmune disease of the central nervous system that is characterized by ocular inflammation with the involvement of uveitogenic Th1 and Th17 responses. In experimental autoimmune uveitis (EAU), the animal model for human uveitis, both responses are proven to be critical in disease development. Therefore, targeting both Th1 and Th17 cells has therapeutic implication for disease resolution. IL-27 is a multifunctional cytokine that can either promote or inhibit T cell responses and is implicated in both autoimmune and infectious diseases. The aim of this study is to characterize the role of IL-27/IL-27R signaling in regulating uveitogenic Th1/Th17 responses in EAU. By immunizing IL-27Rα^−/− mice and their wild-type (WT) littermates for EAU, we demonstrated that IL-27 signaling deficiency exacerbated EAU with severe ocular inflammation and impairment of visual function. Furthermore, there was a significant increase in the eye-infiltrating Th1 and Th17 cells in IL-27Rα^−/− EAU mice compared to WT. Their retinal antigen-specific Th1 and Th17 responses were also significantly increased, as represented by the elevation of their signature cytokines, IFN-γ and IL-17A, respectively. We also observed the upregulation of another pathogenic cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), from effector T cells in IL-27Rα^−/− EAU mice. Mechanistic studies confirmed that IL-27 inhibited GM-CSF production from Th17 cells. In addition, the induction of IL-10 producing type 1 regulatory T (Tr1) cells was impaired in IL-27Rα^−/− EAU mice. These results identified that IL-27 signaling plays a suppressive role in EAU by regulating multiple CD4⁺ cell subsets, including the effector Th1 and Th17 cells and the regulatory Tr1 cells. Our findings provide new insights for therapeutic potential in controlling uveitis by enhancing IL-27 signaling.     

Elevated Th22 Cells Correlated with Th17 Cells in Peripheral Blood of Patients with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematological tumor in which progress T helper (Th) subsets including Th22, Th17, and Th1 cells play a pivotal role. However, the role of T helper (Th) subsets in the immune pathogenesis of AML remains unclear. Here, we investigated frequencies of Th22, Th17, pure Th17, and Th1 cells in the peripheral blood (PB) of AML patients. We demonstrated that Th22, Th17, and pure Th17 in newly-diagnosed (ND) and non-complete remission (Non-CR) AML patients and plasma IL-22 in ND AML patients were significantly increased. Retinoid-related orphan receptor C (RORC) expression was significantly elevated in CR and Non-CR AML patients. However, Th1 in ND AML patients and IL-17 in ND, Non-CR or CR AML patients was significantly decreased compared with controls. Moreover, Th22 and IL-22 showed positive correlation with pure Th17, but Th22 showed negative correlation with Th1 in ND AML patients. RORC showed positive correlation with Th22 and approximately positive correlation with pure Th17 in Non-CR patients. PB blast cell showed positive correlation with Th22 and negative correlation with Th1 in ND AML patients. Our results indicate that Th22 and pure Th17 cells conjointly contribute to the pathogenesis of AML and might be promising novel clinical index for AML.     

Interleukin-17 and Th17 Lymphocytes Directly Impair Motoneuron Survival of Wildtype and FUS-ALS Mutant Human iPSCs

Amyotrophic lateral sclerosis (ALS) is a progressive disease leading to the degeneration of motor neurons (MNs). Neuroinflammation is involved in the pathogenesis of ALS; however, interactions of specific immune cell types and MNs are not well studied. We recently found a shift toward T helper (Th)1/Th17 cell-mediated, pro-inflammatory immune responses in the peripheral immune system of ALS patients, which positively correlated with disease severity and progression. Whether Th17 cells or their central mediator, Interleukin-17 (IL-17), directly affects human motor neuron survival is currently unknown. Here, we evaluated the contribution of Th17 cells and IL-17 on MN degeneration using the co-culture of iPSC-derived MNs of fused in sarcoma (FUS)-ALS patients and isogenic controls with Th17 lymphocytes derived from ALS patients, healthy controls, and multiple sclerosis (MS) patients (positive control). Only Th17 cells from MS patients induced severe MN degeneration in FUS-ALS as well as in wildtype MNs. Their main effector, IL-17A, yielded in a dose-dependent decline of the viability and neurite length of MNs. Surprisingly, IL-17F did not influence MNs. Importantly, neutralizing IL-17A and anti-IL-17 receptor A treatment reverted all effects of IL-17A. Our results offer compelling evidence that Th17 cells and IL-17A do directly contribute to MN degeneration.     

Targeting the Tumor Microenvironment: The Protumor Effects of IL-17 Related to Cancer Type

The inflammatory process contributes to immune tolerance as well as to tumor progression and metastasis. By releasing extracellular signals, cancerous cells constantly shape their surrounding microenvironment through their interactions with infiltrating immune cells, stromal cells and components of extracellular matrix. Recently, the pro-inflammatory interleukin 17 (IL-17)-producing T helper lymphocytes, the Th17 cells, and the IL-17/IL-17 receptor (IL-17R) axis gained special attention. The IL-17 family comprises at least six members, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F. Secreted as disulfide-linked homo- or heterodimers, the IL-17 bind to the IL-17R, a type I cell surface receptor, of which there are five variants, IL-17RA to IL-17RE. This review focuses on the current advances identifying the promoting role of IL-17 in carcinogenesis, tumor metastasis and resistance to chemotherapy of diverse solid cancers. While underscoring the IL-17/IL-17R axis as promising immunotherapeutic target in the context of cancer managing, this knowledge calls upon further in vitro and in vivo studies that would allow the development and implementation of novel strategies to combat tumors.     

Identification of Corosolic and Oleanolic Acids as Molecules Antagonizing the Human RORγT Nuclear Receptor Using the Calculated Fingerprints of the Molecular Similarity

RORγT is a protein product of the RORC gene belonging to the nuclear receptor subfamily of retinoic-acid-receptor-related orphan receptors (RORs). RORγT is preferentially expressed in Th17 lymphocytes and drives their differentiation from naive CD4+ cells and is involved in the regulation of the expression of numerous Th17-specific cytokines, such as IL-17. Because Th17 cells are implicated in the pathology of autoimmune diseases (e.g., psoriasis, inflammatory bowel disease, multiple sclerosis), RORγT, whose activity is regulated by ligands, has been recognized as a drug target in potential therapies against these diseases. The identification of such ligands is time-consuming and usually requires the screening of chemical libraries. Herein, using a Tanimoto similarity search, we found corosolic acid and other pentacyclic tritepenes in the library we previously screened as compounds highly similar to the RORγT inverse agonist ursolic acid. Furthermore, using gene reporter assays and Th17 lymphocytes, we distinguished compounds that exert stronger biological effects (ursolic, corosolic, and oleanolic acid) from those that are ineffective (asiatic and maslinic acids), providing evidence that such combinatorial methodology (in silico and experimental) might help wet screenings to achieve more accurate results, eliminating false negatives.