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@#Introduction: Cytokine immunotherapy such as Interleukin-27 (IL-27) has been foreseen as a promising alternative anti-cancer treatment. Thus, this study aimed to investigate whether IL-27 gene therapy regulates crosstalk between breast cancer cells and macrophages in the sense of pro-apoptotic activities. Methods: This study has led to the development of recombinant pcDNA3.4-IL27. The recombinant pcDNA3.4-IL27 was transfected into 4T1 murine mammary carcinoma cells alone and co-culture of 4T1 with M2 macrophages. The successful expression of IL-27 in the cells were determine through the immunofluorescence staining and detection of CD206, M2 macrophages marker. Apoptotic effects of pcDNA3.4-IL27 were assessed through MTT assay, Annexin V flow cytometer analysis, and AO/PI dual staining. Results: Our findings shows that pcDNA3.4-IL27 has the ability to induce apoptosis in both of the cell group and performs better in the co-culture of 4T1 with M2 macrophages compared to 4T1 cells alone. PcDNA3.4-IL27 induced apoptosis through the altered cell morphology and reduction in the number of viable cells. Conclusion: These data demonstrate that pcDNA3.4-IL27 has the ability to induce apoptosis in both 4T1 cell alone and co-cultured 4T1 with M2 macrophages. Thus, could serve as a potential anti cancer candidate against breast cancer.
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@#[Abstract] Objective: To investigate the effect of IL-27 in combination with IL-15 on the anti-tumor effects of NK92 cells and the possible molecular and signaling mechanisms. Methods: NK92 cells with high IL-15 expression (IL-15-NK92 cells) were cultured in different mass concentrations of IL-27 (0, 10, 20, 30 and 60 ng/ml) for 24 h. The effects of IL-27 on IL-15 secretion, migration and proliferation of IL-15-NK92 cells were detected by ELISA, Transwell and CCK-8 assay, respectively. Flow cytometry was used to detect the expression levels of IL-15-NK92 cell surface receptors NKG2D, NKp30 and NKp46, as well as the secretion levels of perforin and granzyme B. LDH method was used to detect the cytotoxic effect of IL-15-NK92 cells on hematologic tumor cells and solid tumor cells, and WB was used to detect the expressions and phosphorylation level of STATs pathway-related proteins. Results: IL-27 at the concentration of 30 ng/ml promoted IL-15-NK92 cells secreting IL-15 (P<0.01), significantly enhanced the cell migration (P<0.05) but inhibited the proliferation of IL-15-NK92 cells (P<0.05). 30 ng/ml IL-27 could significantly promote the expressions of NKG2D, NKp30 and NKp46 on surface of IL-15-NK 92 cells, as well as elevate the secretion of perforin (all P<0.05), but didn’t affect the secretion of granzyme B (P>0.05); moreover, it also significantly enhanced the cytotoxicity of IL-15-NK92 cells against hematologic malignancies and solid tumor cells (P<0.05 or P<0.01), and up-regulated the phosphorylation levels of STAT1, STAT3 and STAT5 (all P<0.01). Conclusion: IL-27 can enhance the cytotoxicity of IL-15-NK92 cells against hematologic tumor cells and solid tumor cells, which might be related with its upregulation of phosphorylation level of STAT1, STAT3 and STAT5 in JAK-STAT pathway and multiple activating receptors in IL-15-NK92 cells.
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ABSTRACT Objectives Graves' disease (GD) is an autoimmune disease causing the overproduction of the thyroid hormone from thyroid gland. This disease is mainly the result of the production of antibodies against TSH receptors. Cytokines play an important role in orchestrating the pathophysiology in autoimmune thyroid disease. The regulatory role of IL-12 on TH1 cells has been proven. IL-27 and IL-35, members of IL-12 cytokine family, are two cytokines that have been newly discovered. IL-35 has been identified as a novel immunosuppressive and anti-inflammatory cytokine while IL-27 has both inflammatory and anti-inflammatory functions. The objective of the current study was to examine the changes in the serum level of the foregoing cytokines in GD patients in comparison to healthy controls. Materials and methods In this study, serum levels of IL-27 and IL-35 were determined by an ELISA method; anti TPO and anti Tg were measured by an RIA method in 40 new cases of Graves's disease. The findings were compared with 40 healthy controls. Results The results showed a significant difference between IL-27 and IL-35 regarding their serum levels with P values of 0.0001 and 0.024, respectively; anti TPO and anti Tg levels of the cases were also significantly different from controls (p < 0.001). Conclusion The reduction in the serum levels of IL-27 and IL-35 in GD patients compared to normal subjects suggests the possible anti-inflammatory role of these cytokines in GD.
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Humans , Graves Disease , Hashimoto Disease , Receptors, Thyrotropin , Cytokines , InterleukinsABSTRACT
Interleukin-27 (IL-27), a heterodimeric cytokine, plays a protective role in diabetes. Ghrelin, a gastric hormone, provides a hunger signal to the central nervous system to stimulate food intake. The relationship between IL-27 and ghrelin is still unexplored. Here we investigated that signal transducer and activator of transcription 3 (STAT3)-mechanistic target of rapamycin (mTOR) signaling mediates the suppression of ghrelin induced by IL-27. Co-localization of interleukin 27 receptor subunit alpha (WSX-1) and ghrelin was observed in mouse and human gastric mucosa. Intracerebroventricular injection of IL-27 markedly suppressed ghrelin synthesis and secretion while stimulating STAT3-mTOR signaling in both C57BL/6J mice and high-fat diet-induced-obese mice. IL-27 inhibited the production of ghrelin in mHypoE-N42 cells. Inhibition of mTOR activity induced by siRNA or rapamycin blocked the suppression of ghrelin production induced by IL-27 in mHypoE-N42 cells. siRNA also abolished the inhibitory effect of IL-27 on ghrelin. IL-27 increased the interaction between STAT3 and mTOR in mHypoE-N42 cells. In conclusion, IL-27 suppresses ghrelin production through the STAT3-mTOR dependent mechanism.
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@#Objective: To investigate the molecular and signal pathway mechanism of Interleukin-27 affecting the anti-tumor effect of NK92 cells. Methods: NK92 cells were cultured with different concentrations of IL-27 (10, 20, 30 and 60 ng/ml) for 24 hours. The cytotoxicity of NK92 cells to target cells was detected by LDH assay. The expressions of NKG2D, NKp30 and NKp46 on the surface of NK92 cells and the secretion of perforin and granzyme B were detected by Flow cytometry. The expression and phosphorylation level of STATs protein was detected by WB. The DU145 cell transplanted tumor model of prostatic carcinoma in NOD-PrkdcscidIl2rgem1/Smoc mice was established and treated with the combination of NK92 cells and IL-27 to evaluate their anti-tumor efficacy. Results: IL-27 at concentrations of 10, 20 and 30 ng/ml could significantly increase the cytotoxicity of NK92 cells to target cells, and 30 ng/ml exerted the best effect (P<0.05 or P<0.01). 30 ng/ml IL-27 could significantly promote the expressions of NKG2D, NKp30 and NKp46 on surface of NK92 cells, as well as elevate the secretion of perforin (all P<0.05), but didn’t affect the secretion of granzyme B (P>0.05); moreover, it also up-regulated the phosphorylation of STAT1, STAT3 and STAT5 protein (all P<0.01). The combined treatment of IL-27 and NK92 cells obviously extended the survival time of tumor-bearing mice (P<0.05). Conclusions: IL-27 can promote the cytotoxicity of NK92 cells against solid tumor cells and blood tumor cells by promoting expressions of NKG2D, NKp30 and NKp46 on the surface of NK92 cells and the secretion of perforin, which might be related with the phosphorylation of STAT1, STAT3 and STAT5 in JAKSTAT pathway.
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Breast cancer is one of the commonest cancers among women. Conventional therapies cause adverse side effects inpatients. Cytokine immunotherapy such as interleukin-27 (IL-27) has been sought as an alternative cancer treatment inrecent years. IL-27 has been shown to improve anticancer immunity and anti-angiogenesis in cancers, however, its effecton apoptotic and anti-apoptotic gene expression especially in breast cancers is yet to be explored. Cytotoxicity of IL-27in non-cancerous (184b5) and cancerous (MCF-7 and MDA-MB-231) breast cell lines was first determined for 24-72h in this study. The results indicated that IL-27 treatment did not retard 184b5 cell growth, however, did inhibit MCF-7(48 h) and MDA-MB-231 (72 h) cell growth with IC50 at 442 and 457 ng/ml, respectively. Apoptotic (TRAIL, FADD, FAS,caspase-3 and caspase-8) and anti-apoptotic (BCL-2, AKT, and COX-2) genes were then amplified from untreated (control)and treated breast cancer cells and studied. TRAIL, caspase-3, caspase-8 gene expression was significantly (p < 0.05)upregulated in treated MCF-7 (442 ng/ml) and MDA-MB-231 (457 ng/ml) cells. Expression of FADD and FAS genes wasnot detected in both control and treated MCF-7 and MDA-MB-231 cells. COX-2 gene was also not expressed by MCF-7cells, but reduced significantly (p < 0.05) in treated MDA-MB-231 cells. In MDA-MB-231 cells, IL-27 treatment seemedto slightly enhance the expression of AKT and BCL-2 genes which, on the other hand, was downregulated in treatedMCF-7 cells. Conclusively, IL-27 is able to inhibit breast cancer cell growth and regulate apoptotic and anti-apoptoticgene expression in breast cancer cells.
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Objective To analyze the predictive value of serum interleukin-27 (IL-27) for acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) from unrelated donors. Methods Serum samples were collected from 72 patients after receiving allo-HSCT from unrelated donors during January to December 2012. Serum samples collected from 70 patients received allo-HSCT in 2013 were used for confirmation. All patients received myeloablative conditioning regimen prior to allo-HSCT. Cyclosporin A (CsA)+mycophenolate mofetil (MMF)+short-term methotrexate (MTX) were used for GVHD prophylaxis. Serum IL-27 levels in patients with aGVHD were measured by ELISA. The pre-dictive value of IL-27 index,defined as the ratio of serum IL-27 level at neutrophil engraftment to that before pre-conditioning regimen, for allogeneic HSCT was retrospectively analyzed. Results Serum IL-27 index was significantly decreased in patients with gradeⅡ-ⅣaGVHD(grade 0-Ⅰ : 1.89±0.68 vs gradeⅡ-Ⅳ :1.26±0.49;P<0.000 1). IL-27 index had good value for grade Ⅱ-Ⅳ aGVHD (AUC=0.782,95% CI:0.675-0.889,P<0.001). Patients with a lower serum IL-27 index (<1.33) were more likely to have a higher cumulative incidence of grade Ⅱ-Ⅳ aGVHD than those with a higher serum IL-27 index (P<0.001). Multivariate analysis confirmed that low IL-27 index was the most significant risk factor for gradeⅡ-Ⅳ aGVHD (HR=4.50,95% CI:2.1-9.8,P<0.01). These findings were consistent with the results found in the serum samples collected in 2013. Conclusion Low IL-27 index could be used to predict the incidence of grade Ⅱ-Ⅳ acute GVHD after allo-HSCT from unrelated donors.
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Objective To explore whether IL-27 inhibited the pulmonary fibrosis through regulating the expression of TGF-β/Smad signaling pathway in the bleomycin-induced pulmonary fibrosis model. Methods Forty male C57/BL6 mice were randomly divided into normal control group(group A),bleomycin-induced pulmonary fibrosis group(group B),bleomycin+IL-27 group(group C)and bleomycin+IL-27 antibody group(group D) with 10 in each. Five mice in each group were sacrificed on days 7 and 28 after with intratracheal bleomycin. TGF-βR1,Smad1 and Smad3 in right lung tissue were measured by Western Blot. Results 1. In the bleomycin-induced pulmonary fibrosis model,the expression of TGF-βR1 was higher on days 7 and 28,which was inhibited by IL-27. 2. The expressions of p-Smad1 and p-Smad3 were highest in group D on days 7 and 28, but were lower in group C on day 7 than those in group B. Conclusion Exogenous IL-27 might alleviate pulmonary fibrosis through inhibiting the related protein phosphorylation in TGF-β/Smad signaling pathway.
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Objective:To observe chicken typeⅡcollagen-induced arthritis( CIA) in serum of mice with the dynamic changes of IL-27,IL-17,IL-10. Methods: 54 DBA1/J mice were randomly divided into control group(n=6) and model group(n=12), according to the progress of CIA mouse course early,middle and late(7,14,and 35 days after booster immunization) ,taking the eyeball for blood and separating the serum under sterile condition. The dynamic levels of cytokines IL-27,IL-17,IL-10 were detected by flow cy-tometry. Results:The level of IL-27 in the model group was significantly declined in the the progress of CIA mouse course early and middle(P 0. 05);the level of IL-17 in the disease early and late was no significant difference(P> 0. 05) compared with the control group,while in the mid course significantly higher than control group(P 0. 05). Conclusion: IL-27,IL-17,IL-10 paticipate in the pathogenesis of CIA and their alterations at different stages of the disease have a relation to the development of arthritis.
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The interleukin-12 (IL-12) family, including IL-12, IL-23, IL-27,and IL-35, is characterized by unique structures and molecular partners.This is the only family of heterodimeric cytokines, which endows them with a unique set of connections and functional interactions.They not only play an important role in the regulation of inflammation, but are closely related to various autoimmune diseases.Here we discuss the structural aspects of these cytokines and their effect on some autoimmune diseases.
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Objective To investigate the effect of continuous renal replacement therapy on the levels of IL-27 in peripheral blood of patients with chronic heart failure concurrence acute kidney failure. Methods 78 patients with chronic heart failure were enrolled from June 2012 to December 2013 in Zhujiang hospital affiliated Southern medical university. According to patient′s condition, 38 patients with chronic heart failure concurrence acute kidney failure undertaking CRRT, 40 patients with chronic heart failure undertaking conventional therapy. Heart function were determined by cardiac ultrasound, the levels of IL-27 and BNP were determined by ELISA. Results Compared with chronic heart failure patients, the levels of IL-27 were decreasing but BNP were increasing in patients with chronic heart failure concurrence acute kidney failure (P < 0.05). After CRRT, the levels of IL-27 were increasing but BNP were decreasing in patients with chronic heart failure concurrence acute kidney failure. Correlation analysis showed that the levels of IL-27 is negatively correlated with the levels of BNP , but positively correlated with LVEF (P<0.05). Conclusion IL-27 participated in the development of chronic heart failure concurrence acute kidney failure. CRRT regulating and controlling the heart and kidney function may partly through effecting on immune-inflammatory mechanism.
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Objective:To monitor the effects of asarone injections on the expression of IL-25, IL-27 and eosinophils cationic pro-tein ( ECP) in the serum of asthma patients. Methods:Totally 100 patients with bronchial asthma were randomly divided into the ob-servation group ( 55 cases ) and the control group ( 45 cases ) . The control group was treated with the conventional symptomatic treat-ment ( oxygen inhalation, spasmolysis and anti-asthma, and anti-infection, etc) . The observation group was given asarone injections 24 mg in 250 ml 0. 9% sodium chloride infusions, once a day for 14 days on the basis of the conventional therapy. The changes in symp-toms, and pulmonary function in the patients were observed with simultaneous detection of IL-25, ECP and IL-27 levels. Results:To-tal effective rate of the observation group was 92. 7%,which was higher than that of the control group (P<0. 05). Asarone injections could decrease IL-25, ECP and IL-27 levels in the observation group compared with those in the control group with statistically signifi-cant difference (P<0. 05 or 0. 01). Conclusion:Asarone injections can decrease the levels of IL-25, IL-27and ECP in the serum of asthma patients and inhibit the formation of inflammatory cells to alleviate the symptoms of airway inflammation.
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ObjectiveTo investigate mechanisms for IL-27 induced proliferation and differentiation of peripheral blood CD4+ T cells in primary biliary cirrhosis (PBC).MethodsPeriperal blood CD4+ T cells were isolated from patients with PBC,chonic hepatitis B (CHB) and health controls (HCs).After IL-27 stimulation,proliferation ability of CD4+ T cells was evaluated by CCK-8 kit,and cytokines were analyzed by ELISA.Real-time PCR was employed to assay mRNA expression of T-bet and GATA3 in CD4+ T cells.p-STAT-1 and pSTAT-3 expression in CD4+ T cells were detected by Western blot.ResultsEnhanced proliferation of CD4+ T cells was found in all subjects after IL-27 stimulation.However,the proliferation ability in patients with PBC was greater than that in CHB and HCs ( P＜0.001 ).Levels of IL-2 and IFN-γ in supernatant from IL-27-incubated PBC blood CD4+ T cells were higher than that from CHB and HCs (P＜0.001 ).In normal situation,T-bet mRNA of CD4+ T cells in PBC group was higher than that in CHB group (P=0.007).Furthermore,after IL-27 stimulation,elevated T-bet mRNA expression and GATA3 inhibition were found in patients with PBC.High expression of p-STAT-1 and p-STAT-3 in blood CD4+ T cells were found in PBC,CHB and HCs after stimulation by IL-27.But their expression in patients with PBC were higher than those in patients with CHB and HCs.ConclusionProliferation of blood CD4+ T cells could be induced by IL-27 in patients with PBC.The signaling pathways of p-STAT-1,p-STAT-3 were involved to induce Th1 immune response and related cytokines expression.This study implicated that IL-27 may play important roles in early inflammation damage in PBC.
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Objective:To study the inhibitory effect of IL-27 against human tumor angiogenesis and the related mechanisms.Methods: Human esophageal carcinoma cells(Eca109/IL-27) stably transfected with IL-27 gene were injected into nude mice to establish tumor-bearing mouse model.The survival time and tumor growth were observed.IFN-? level secreted by splenocytes was measured by ELISA.Expression of VEGF and CD34 was detected by immunohistochemistry method and MVD was calculated according to CD34 level.RT-PCR was used to detect the expression of IP-10 and MIG mRNA in the tumor tissues.Results: The survival time of mice injected with Eca109/IL-27 cells was significantly longer than those of mice injected with wide type Eca109 or Eca109/LXSN(blank vector) cells(P