| [Background and Objective]Asthma is a chronic inflammatory respiratory disorder of the airways, which isinvolved in a variety of cells and cytokines, whose pathogenesis has not yet been clarified.Asthma is managed mainly through medications in clinical practice. Currently,allergen-specific immunotherapy is the most effective treatment method of immunotherapy.However, there are still drawbacks affect the efficacy, such as hypersensitivity,standardization of allergen, long course of treatment, the poor compliance of patients.Looking for much safer and effective immunotherapy is one of the hot spots of preventionand treatment of allergic diseases.Recent studies show that the pathogenesis of asthma is related with the imbalancebetween the helper T cells the1,2(Thl/Th2), the abnormalities of CD4+CD25+T cells(regulatory T cells, referred to as Tregs) and T helper cell17(T help cell17Th17) Th17/interleukin-17(IL-17). Interleukin-35(IL-35) is a novel discovered cytokine closelyrelated with the immunomodulatory function of Tregs and Th17/IL-17, which participatein the pathogenesis and regulator of asthma through a variety of ways. It is expected tobecome the new target of the treatment of asthma, which is a hot research in recent years.The current studies have demonstrated that IL-35can effectively enhance the function ofTregs, suppress Th17cell differentiation even during acute infection, to prevent the onsetof excessive autoimmune responses. Thus we believe that IL-35may be a potentialimmunomodulatory and therapeutic target for bronchial asthma and other allergic diseases.We constructed the recombinant murine IL-35eukaryotic expression vector, andtransfected the verified plasmid into Chinese hamster ovary cells (CHO cells). The IL-35positive CHO clones were chooesen, and we purified the IL-35protien form the culturesupernatant of the cell by Ni-NTA column. Finally IL-35protein was intraperitoneallyinjected in a murine model of asthma to observe whether it would ameliorate the typicalacute asthma attack and downregulate allergic airway inflammation. We want to partlyclarify the immunomodulatory mechanism of the recombinant IL-35on allergic asthmaand other allergic diseases and the promising application of IL-35in the immunotherapy ofthem. [Contents and Method]Part1Constrct and express of the eukaryotic plasmid pSectag2A-IL-35EBV-induced gene3(EBI3) and interleukin-12(IL-12p35, or p35) were amplifiedfrom the first-strand cDNA of J774cells.Linker was obtained by annealing. After that weinserted them into the eukaryotic expression vector pSectag2A. The pSectag2A-IL-35plasmid was verified by DNA sequencing.Then transfected it into CHO cells. The IL-35positive CHO clones were obtained after repeated screening. And the supernatant wascollected and concentrated by affinity chromatography to obtain the purified recombinantIL-35protein.Part2Study of the immunomodulatory and therapeutic effects of recombinantInte rleukin-35on asthma in mice1. Modeling Allergic Asthma in Mice12C57BL/6asthmatic mice were randomly divided into the control and asthmaticgroup, each containing6mice. Sensitization: the mice in asthmatic group were sensitizedby intraperitoneal injection with10μg OVA (mixed with2.25mg aluminum hydroxideand0.2mL NS)/mouse on day0,7. The control group were injected0.2mL NS with2.25mg aluminum hydroxide instead.Challenge: The asthmatic group was exposed to an aerosol of5%OVA (Sigma) in10mL saline for30minutes on days14-18. The control group was challenged by10mL NSinstead.After the final daily OVA challenge, the mice were sacrified24hours later. Theanimals were anesthetized with1%sodium pentobarbital (60mg/kg) by intraperitonealinjection. We collected bronchoalveolar lavage fluid and lungs to count eosinophilt inBALF, detection the concentrationof various cytokines (IFN-γ, IL-4) in the supernatant ofBALF, routine pathological examination of lung tissue. Compare the difference of theseresults between the groups.2. Study of the immunomodulatory and therapeutic effects of recombinantInterleukin-35on asthma in mice18C57BL/6asthmatic mice were randomly divided into three groups (n=6), group A: control group, group B: asthmatic group, group C: IL-35treated group.Sensitization: group B and C were sensitized by means of intraperitoneal injectionwith10μg OVA (mixed with2.25mg aluminum hydroxide and0.2mL NS)/mouse onday0,7. Group A was injected0.2mL NS with2.25mg aluminum hydroxide instead.Intervention: Group C was intraperitoneal injected10%recombinant IL-35protein in0.2mL saline on days7-13. Group A and B were intraperitoneal injected0.2mL NSinstead.Challenge: Group B and C were exposed to an aerosol of5%OVA (Sigma) in10mLsaline for30minutes on days14-18. Group A was challenged by10mL NS instead.After the final daily OVA challenge, the mice were sacrified24hours later. Theanimals were anesthetized with1%sodium pentobarbital (60mg/kg) by intraperitonealinjection. We collected bronchoalveolar lavage fluid and lungs to count eosinophilt inBALF, detection the concentrationof various cytokines (IFN-γ, IL-4, IL-17, TGF-β) in thesupernatantof BALF, routine pathological examination of lung tissue, detection the forkedhead transcription factor P3(FoxP3), IL-17protein expression levels of lung tissue byimmunohistochemical. Compare the difference of these results among the3groups.[Result]1〠EBI3and p35were amplified from the first-strand cDNA of J774cells byRT-PCR. Agarose gel electrophoresis showed single band of624bp,576bp, whose sizeswere similar to expected. Sequence results were as the same as GenBank sequence.51bpLinker was obtained by annealing, as described previously[19].2〠The PCR products were cloned into the pUcm-TA–cloning vector, and then theyand Linker were resepectively cloned into eukaryotic expression vector pSectag2A toconstruct recombinant the plasmid pSectag2A-IL-35. The constructed plasmid with therecombinant IL-35was verified by restriction enzyme digestion analysis，PCR and DNAsequencing without incomplete reading frame or frameshift mutations. These results allindicated that pSectag2A-IL-35recombinant plasmid was successfully constructed. Theplasmid pSectag2A-IL-35was transfected into CHO cells using Fugene9（Roche）. TheIL-35positive CHO clones were obtained after repeated screening by Zeocin (Invotrigen)on day18. The IL-35protein was purified from the media collected from the positive CHO clones by Ni-NTA affinity chromatography. The SDS-PAGE and Wlestern-blot resultsshowed that one protein band was found to match well with the predicted relativemolecular mass of mouse IL-35(46.3kDa). And this protein could specifically bind toanti-His-mouse monoclonal antibody and anti-IL-12p35-mouse monoclonal antibody,which suggested that CHO cell line stably expressing IL-35protein had been successfullyestablished.3〠There were no significant differences of appearance, behavior, and response tostimuli and body weight between two groups before challenged with OVA. The sensitizedmice appeared obvious scratch nose head facial itching, dysphoria or quiet breathingdeeply and fast, nodding breathing, arched back, forelimbs shrink lift, incontinenceperformance as acute asthma attacks between the challenge process. And the naive miceperformance as nomal. Mice were harvested, and collect the BALF, serum, lung asdescribed previously. The result showed that compared with the na ve, the imflammatorycells infiltrate seriously in the BALF and lung of challenged mice. And the expressionlevel of IL-4was up-regulated in the BALF of challenged mice, at the meanwhile, theexpression level of IFN-γ was down-regulated, which were similar to the the asthmapatients. The results aboved indicated that this kind of asthma model could be used forfurther research.4〠The expression levels of IL-4, IL-17, TGF-β in BALF of asthma group weresignificantly higher than contol group. And those cytokines expression levels of IL-35group were in the middle of asthma group and control group. However, the expression ofIFN-γ in the BALF was just the opposite with those cytokines. The infiltration ofinflammatory cells was most serious in asthma group, which mainly were eosinophils. Theinflammatory cells of IL-35group significantly decreased compared with the asthma group,but still much serious than the control group (P<0.05). Immunohistochemical resultsshowed that: the FoxP3-positive cells expression levevl of IL-35group was the lowset.This indicated that the FoxP3protein didn’t express by iTr35cell, that was consistent withthe reported[17]. But the IL-17positive cells expression levevl of asthma gro up (129.72±18.47) a/mm2was higher than control group (21.37±5.31)/mm2and IL-35group (46.31±17.26)/mm2(P<0.05), asthma group IL-17protein express ion was e levated. [Conclusion]1〠We obtained IL-35cDNA by PCR and succeeded to construct eukaryoticexpression vector pSectag2A-IL-35by molecular cloning techniques.2〠Successfully established an IL-35positive CHO cell line in the best transfectionconditions using Zeocin, which could stably express IL-35protein in supernatant of theculture. Purifiy a sufficient amount of mouse recombinant IL-35protient with biologicalactivity through affinity chromatography.3〠The airway inflammation of asthmatic mice could be significantly reduced byintraperitoneal injections of IL-35we recombined. IL-35will become the new target of theallergic disease immunotherapy. |
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