| BackgroundMultiple sclerosis （MS） is the principal inflammatory disease of the central nervous system （CNS） in which the immune system reacts with myelin peptides of CNS and mediates the destruction of CNS tissue, resulting in demyelination, axonal damage and subsequent neurological disability. According to population-based studies, the incidence of MS ranges from 60-200 per 105 in Northern Europe and North America, and 6-20 per 105 in low-risk areas such as japan. MS is a heterogeneous disease clinically, pathologically and radiologically. The most common form of the disease is relapsing-remitting MS （RR-MS） that affects up to 85% of patients. A majority of RR-MS will go on to develop secondary progressive MS （SP-MS） characterized by worsening neurological disability with or without superimposed attacks. About 10% of patients exhibit primary progressive MS （PP-MS） which involves continuous disease progression from onset without relapse or remission. The pathophysiology of MS is heterogeneous and complex. It has been well known that autoreactive T cells against myelin antigens play important roles in the progression of MS. It was initially suggested that T helper （Th） 1 cells were responsible for the pathogenesis of MS.More recently, a new subset of Th cells that secrete IL-17, termed Th17 cells,have been shown to be associated with the pathogenesis of several inflammatory diseases, including experimental autoimmune encephalomyelitis （EAE）,psoriasis,rheumatoid arthritis,and RR-MS.The precise roles of Thl and Th17 cells in autoimmunity still remain to be clarified.Interleukin 27 （IL-27）, a member of IL-12 family, is a heterodimeric cytokine composed of Epstein-Barr virus-induced gene protein 3 （EBI3） and p28 subunits,and is mainly produced by activated antigen-presenting cells （APCs）. By signaling through a receptor complex consisting of the unique IL-27 receptor （IL-27R, also known as WSX-1 and TCCR） and gp130 subunit, IL-27 can promote Thl differentiation from naive T cells in a signal transducer and activator of transcription 1 （STAT1）-dependent manner, inducing the production of interferon γ （IFN-γ）.More recent studies revealed that IL-27 might play a regulatory role by expanding the inducible regulatory T cells to produce IL-10. IL-27 also suppresses inflammation via inhibiting the differentiation and function of Thl7 cells. Through secreting a group of pro-inflammatory cytokines, particular IL-17 and IL-21, Th17 cells are implicated in a variety of autoimmune diseases.A series of studies have carried out to investigate the pathophysiological significance of IL-27 in Thl/Thl7-mediated inflammatory disorders, such as system lupus erythematosus （SLE）,rheumatoid arthritis （RA）and primary immune thrombocytopenia （ITP）, and most of which suggested a protective role of IL-27 in autoimmunity.The discovery of T helper 17 cells was based on the researches of EAE （experimentalautoimmuneencephalomyelitis） and CIA（collagen-induced arthritis）, in which were previously believed that Thl cells were pathogenic Tcells. Th1 cells were inducedby IL-12. Blocking IL-12 signaling was expected to amelio rate EAE. IL-12 is a heterodimeric cytokine composed of p35 and p40 subunit. However,it was shown that p35 - deficient mice were susceptible, but p40-deficient mice were resistant to EAE. IL-23 is a heterodimeric cytokine that shares IL-12p40 subuni twith IL-12 and possesses aunique p19 subunit. They demonstrated that IL-23p19 and IL-12p40, but notIL-12p35, were essential for the development of EAE. Researching the mechanism underlying the essential role of IL-23 has revealed that CD4+Tcells producing IL-17 were not induced in IL-23-deficient mice in EAE and CIA. Thi sdemonstrates that producing IL - 17cells rather than Thl cells are important for the development of EAE and CIA. In 2005, Harrington identified these producing IL-17 cells as Thelper 17cells, anewlinage of CD4+Tcells, based on cytokineproduction.Thl7 were a distinct lineage cells from Thland Th2 cells in differentiation commitment, It has shown that combination of transforming growth Il-6induces the differentiation of the Th17cells Although Il-24 plays mo apparent role in Th17 lineage commitment it seeMS to be required for promoting, survival and proliferation of these cells. IL-21 amplifies th17 pathway in autocrine fashion. CD4+Tcelllineage commitment isregulated by specific transcription factors. Namely,Thl differentiation requires STAT1, STAT4, andT-bet, whereas STAT6, c-maf,and GArA-3act to promote Th2 cytokine production. Regarding to Th17 cell differentiation retinoicacid-related orphan nuclear receptorrt（RORyt） is the key transcription factor that orchestrates the differentiation of Th17 celllineage. At the same time RORyt å’Œ STAT3, ale also an essential transcription factor in T17 cell differentiationTh 17 cells can produce several cytokines, suchas IL-17Aã€IL-17Fã€IL-22ã€IL-21 and SO on. IL-17 nowreferred to as IL-1 is the founding member of the IL-17 family,which includes IL-17Ato F. IL-17 is the main effctor of the T17 cells. IL-17 has an important function as an inflammatory mediator. It can increase elL-6 and other Proinflammatory cytokine production from a variety of celltypes, induce and strengthen the immune response. It coordinates the recruitment of myeloid cellslike Monocytes and neutrophils to the site of an inflammation, thereby furthering the local Inflammatory environment. Nissuggested that IL-17 Can have acritical role in inflammatory conditionsin general.The abnormality of T cells is implicated in the pathogenesis of many autoimmune diseases, and many autoimmune diseases, especially arthritis, were considered to be mainly driven by Thl cells. A new IL-17-producing T cell subset, termed Th17 cells, has been described in recent years. It has been established that Th17 cells play critical roles in several animal models of autoimmunity, such as experimental allergic encephalomyelitis （EAE） and murine arthritis models. Besides, Th17 cells are considered to be involved in many human inflammatory diseases, including primary immune thrombocytopenia psoriasis and inflammatory arthritis 。Thelper.17（Th1 7）cells, have been shown to be crucial for the development of certain autoimmunediseases. IL-17 functionsasa proinflammatory cytokine and drives the production of inflammatory mediators such as TNF, IL-1, IL-6, IL-8, granulocyte-macrophage colony stimulatory factor（GM-CSF） by a variety of cell types。Recent studies suggested that the expression of IL-23 is increased in psoriasis patients. The expression of IL-23 and I L-17 were also found upregulated in certain autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis and Inflammatory boweldisease.Although MS is speculated to be a T cell-mediated autoimmune disease directed against myelin proteins, the cause of the disease is not well illuminated. Up to date, the roles of IL-27 and its relation with Th1 and Th17 cells in MS remain unsettled. In the present study, plasma levels of IL-27, IFN-y and IL-17 in progressive MS were measured by enzyme-linked immunosorbent assays （ELISA）. mRNA expression of IL-27, T-bet, RORyt, IL-17 and frequencies of Thl,Th17 were also determined by real-time polymerase chain reaction （PCR） analysis and flow cytometry （FCM）, respectively. All of these might provide reference data for further investigation of IL-27 in the development of progressive MSObjectiveTo examine the levels of IL-27, IFN-y and IL-17 in the plasma of progressive MS patients and healthy people; To examine frequencies of Th1, Th17 and mRNA expression of IL-27, T-bet, RORyt, IL-17 in the peripheral blood of progressive MS patients and healthy people; To analyze the correlations between IL-27 and mRNA expression of IL-27, T-bet, RORyt, IL-17 in progressive MS patients. To investigate the roles of IL-27 and Th cells in the pathogenesis of MS。Materials and Methods（1） forty-five adult patients with progressive MS （25 females and 20 males, age range 18-63 years, median 35 years） were enrolled in this study. Among them, there were 29 patients with SP-MS （16 females and 13 males, age range 18-62 years, median 34 years）, and 16 patients with PP-MS （9 females and 7 males, age range 25-64 years, median 36 years）. Twenty-five healthy control subjects （14 females and 11 males, median 32 years, range 19-63 years） without any history of autoimmune disease were included.. Peripheral blood of thirty millilitres was collected into heparin-anticoagulant vacutainer tubes..（2） Peripheral blood was collected into heparin-anticoagulant vacutainer tubes. Plasma was obtained from all subjects by centrifugation and stored at -80℃ for determination of cytokines. PBMCs were isolated from heparinized blood by gradient centrifugation using 1.077 g/ml Ficoll-Paque （Pharmacia Diagnostic, Uppsala, Sweden）. The isolated PBMCs were stored at -80℃ for RNA isolation.（3） Plasma IL-27, IFN-γ, and IL-17 from MS patients and healthy controls were determined using commercial ELISA kits （eBioscience） according to the manufacturer’s instructions. The detection limit or these three cytokines was 9.5 pg/ml,3.2 pg/ml and 0.5 pg/ml, respectively.（4） Peripheral blood was collected and cultured under stimulation conditions before flow cytometric analysis. Briefly, heparinized peripheral whole blood （400 μL） in an equal volume of Roswell Park Memorial Institute （RPMI） 1640 medium was incubated for 4 hours at 37℃ with 5% CO2 in the presence of 25 ng/mL of phorbol myristate acetate （PMA）,1 μg/ml of ionomycin, and 1.7 μg/mL of Golgiplug （Monensin; all from Alexis Biochemicals, San Diego, CA）. PMA and ionomycin are pharmacological T-cell activating agents that mimic signals generated by the T-cell receptor （TCR） complex and have the advantage of stimulating T cells of any antigen specificity. Monensin was used to block intracellular transport mechanisMS, thereby leading to an accumulation of cytokines in the cells. After incubation, cells were stained with PE-Cy5-conjugated anti-CD4 monoclonal antibodies at room temperature in the dark for 20 min. After staining, cells were fixed and permeabilized, and next stained with fluorescein isothiocyanate （FITC）-conjugated anti-IFN-y monoclonal antibodies and PE-conjugated anti-IL-17 monoclonal antibodies. All the antibodies were from eBioscience （San Diego, CA）. Isotype controls were included to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FACScan cytometer equipped with CellQuest software （BD Bioscience PharMingen）. Th1, Th17 cells were defined as CD4+IFN-γ+ and CD4+IL-17+IFN-γ- T cells, respectively..（5） TRlzol reagent （Invitrogen） was used to isolate total RNA of PBMCs. RNA was converted into cDNA using the PrimeScript RT reagent kit （Perfect Real Time; Takara） according to the manufacturer’s instructions. Multiplex real-time RT-PCR was performed for mRNA expression of IL-27, IFN-y, T-bet, IL-17, RORyt and GAPDH （endogenous control） on a PRISM7500 Sequence Detection System （Applied Bio-system, Foster City, CA, USA）. The primers for all mRNA assays were intron spanning. Fluorescence was acquired at extension 72℃. ABI Sequence Detection System software version 1.0 （PE Applied BiosysteMS, Warrington, UK） was used to determine the cycle number at which fluorescence emission crossed the automatically determined Ct value..Results（1） In consistence with a previous report,plasma levels of IL-27 in progressive MS patients were significantly lower than that in healthy controls （119.16 ± 29.86 vs. 140.01 ± 15.23 pg/mL; P<0.05）, while no statistical difference was found in plasma levels of IFN-γ between progressive MS patients and healthy controls （43.05 ±7.13 vs.40.02 ± 5.72 pg/ml; P=0.074）. Significantly higher plasma IL-17 levels were found in progressive MS patients compared with healthy controls （33.68 ± 9.07 vs. 20.17 ± 4.97 pg/ml; P<0.001）. However, no significant difference was found in plasma levels of IL-27, IFN-y or IL-17 between SP-MS and PP-MS patients （IL-27: 117.5 ± 31.36 vs.122.16 ± 27.66 pg/ml, P=0.564; IFN-γ:42.99 ±7.18 vs.43.13 ± 7.26 pg/ml, P=0.949; and IL-17:34.39 ± 8.26 vs.32.40 ± 10.56 pg/ml, P=0.42, respectively）.（2） It has been reported that IL-27 could enhance IFN-γ production in T cells, antagonize the effect of IL-4 in Th1 differentiating process and suppress the production of IL-17 in T cells. Hence correlation analysis was performed to evaluate if there was a latent relationship between plasma levels of IL-27 and IFN-γ or IL-17. Results showed that plasma levels of IL-27 were negatively correlated to concentrations of IL-17 in progressive MS patients （r=-0.371, P=0.012）, while no statistical correlation was found between plasma levels of IL-27 and IFN-γ （r=-0.07, P=0.649）..（3） Frequencies of Th1 cells and Th17 cells in peripheral blood of progressive MS patients and healthy controls were examined by flow cytometry. Percentages of circulating Th1 cells （CD4+IFN-γ+） in progressive MS patients were slightly higher than that in healthy controls （11.54 ± 3.38% vs.10.06 ± 3.04%）, but this difference did not achieve statistical significance （P=0.075）. In consistence with plasma levels of IL-17, frequencies of peripheral blood Th17 cells in progressive MS patients were significantly increased compared with healthy controls （2.01 ± 0.58% vs.1.33 ± 0.36%; P<0.001）. Additionally, no statistical difference was found in circulating Thl or Th17 cells between SP-MS and PP-MS patients （Thl:11.70 ± 3.46% vs.11.25 ± 3.32%, P=0.662; Th17:2.02± 0.56% vs.1.99 ± 0.63, P=0.82, respectively）.（4） Correlation analysis was performed to assess the relationship between plasma IL-27 concentrations and circulating Th17, Thl levels in progressive MS patients. The data indicated that plasma levels of IL-27 were negatively correlated to circulating Thl7 levels in progressive MS patients （r=-0.301, P=0.044; Fig 4）, whereas percentages of Thl cells failed to show a correlation with IL-27 levels （r=-0.164, P= 0.281）..（5） To further define the relationship between IL-27 and Th1, Th17 cells in progressive MS patients, mRNA expression levels of IL-27, IFN-y, IL-17 and the Thl-specific transcription factor T-bet as well as Thl7-specific transcription factor RORyt were determined by real-time RT-PCR. the relative amount IL-27 mRNA in progressive MS patients was 0.69-fold of that in healthy controls （P<0.001）. By contrast, mRNA levels of IFN-y and T bet showed no significant difference between progressive MS patients and healthy controls （P>0.05）. In line with plasma levels of IL-17, considerably elevated IL-17 and RORyt mRNA levels were also found in progressive MS patients compared with healthy controls （4.39-fold, P<0.001; and 9.58-fold, P<0.001, respectively）.Conclusions（1） Our data demonstrated that decreased expression of IL-27 was involved in the pathophysiology of MS.（2） Our data demonstrated that the level of IL-17 in the plasma of progressive MS patients and Th17-specific transcription factor RORyt in the Peripheral blood was significantly higher than healthy controls. In addition, the percentage of Th17 cells in peripheral blood and level of IL-17 in plasma were elevated consistently.;（3） plasma levels of IL-27 were found to be negatively correlated to the frequencies of circulating Th17 or plasma IL-17 concentrations in patients with progressive MS, providing further evidence about the anti-inflammatory role of IL-27 in MS.In summary, MS had decreased plasma and mRNA expression levels of IL-27, and increased plasma and mRNA levels of IL-17 as well as circulating Th17 cells, suggesting their involvement in the pathophysiological process of the disease. Additionally, plasma IL-27 levels were negatively correlated to percentages of circulating Th17 cells and concentrations of plasma IL-17. Modulation of IL-27 might be a reasonable therapeutic strategy for progressive MS. |
