A Study On The Mechanism Of Th17 Cells In The Pathogenesis Of Asthma

BackgroundBronchial asthma is characterized by airway hyperresponsiveness and chronic inflammation,whereas the pathogenesis has not yet fully understood.It is generally believed that asthma is duo to chronic airway inflammation involving a variety of cells such as epithelial cells,fibroblasts,dendritic cells,eosinophils,mast cells and T lymphocytes.Among those cells,T lymphocytes are most important as a Th2 biased Th1/Th2 imbalance has been proved to play a cruicial role in the pathogenesis of asthma.Th2 cells secret cytokines IL-4,IL-13,IL-5 and IL-9,in which IL-4 and IL-13 promotes B cell to produce IgE;IL-5 is responsible for bone marrow eosinophil differentiation;IL-9 attracts mast cells and induces mast cell differentiation.However, Th1/Th2 imblance can not fully explain all of the mechanisms in the pathogenesis of asthma.Recently,a new Th subset(Th17) different from Th1 and Th2 was identified with IL-17 as its signature cytokine.Th17 derives from natural T cell precursor-Th0 cells and participates in pro-inflammatory responses and autoimmune diseases. Because of its proinfiammatory role,it is also known as inflammatory T cell.The differentiation and functions of Th17 cell subset are subjected to the regulation of the Th1 and Th2 cytokines.A combined treatment of IL-6 and transforming growth factorβ(TGF-β) induces naive T cells to differentiate into Th17 cells.IL-17A is a homodimer of two 155 amino acid glycoprotein monomers with signal peptides at their N-terminals that are linked by disulfides.As a pro-inflammatory factor,IL-17A induces airway epithelial cells and fibroblasts to increase the expression of a variety of cytokines such as IL-6,IL-8,G-CSF,GM-CSF and granulocyte chemoattractant protein-2(GCP-2),and promotes infiltration of many inflammatory cells into the airway.The interactions among those cells further enhance the secretion of many more inflammatory cytokines.Consequently,all those inflammatory cells as well as their cytokines constitute a complex network to prmote chronic inflammation in the respiratory tract.Due to the lack of detailed mechanisms of IL-17A as well as Th17 cells in the pathogenesis of asthma,this study was designed to investigate the effect of Th17 cells and IL-17A in the mouse asthma model and to explore their roles in the pathogenesis of asthma.Part one Establishment of the asthmatic mouse modelObjectiveTo establish a BALB/c mouse asthma model by OVA sensitization and re-challenge.MethodsSPF female BALB/c mice(4 to 6 weeks),18～20g,were purchased from Laboratory Animal Services Center of Southern Medical University,and maintained on an ovalbumin-free diet.The mice were randomly divided into 2 groups:control group and asthmatic group.Mice were sensitized on days 0 and 14 by an intraperitoneal injection of a mixture containing 2 mg of ovalbumin and 5 mg of Al(OH)3 in saline (a total volume of 0.2 ml).At 21 days after the first immunization the animals were firstly challenged with intranasal drop of 1%ovalbumin,then challenged by exposure to an aerosol of 2%ovalbumin generated by an ultrasonic nebulizer for 40 min.The 40-min rechallenge procedure was repeated once each day from day 22 to day 26. The control animals were treated with 5 mg of Al(OH)3 in saline and re-challenged with saline solution.The animals were killed by cervical dislocation 1 h after the last exposure to aerosol re-challenge.Six mice from each guoup were subjected to bronchoalveolar lavage.The bronchoalveolar lavages were stained with HE and eosinophil number counted with a hematocytometer.At least 400 lymphocytes were counted by microscopy before the percentages of various types of cells were calculted. After bronchoalveolar lavage,the animals were sacrificed,their lungs harvested and fixed in 10%phosphate buffered formalin for further analysis.Tissues were sliced and HE stained for histological examination,including morphology,epithelial injury, perivascular and peritracheal infiltration of eosinophils.Results1.The behavior changes after re-challengeMice in asthmatic group displayed various types of allergic responses,such as scratching head and face,dyspnea,oral lip cyanosis,muscle spasm as well as gatism, while control group showed no such symptoms.2.The changes in total cells and different cell types in BALFa.The total BALF cell number in the asthma group was 1.41±0.13×10⁵/ml,that is significantly higher than that in the control group 0.62±0.43×10⁵/ml(P＜0.01). b.The percentages of BALF eosinophils and neutrophils were 25.42%±4.05%and 29.63%±4.91%in the asthma group,that were also significantly higher than those in the control group 0.83%±1.18%and 5.67%±1.42%(P＜0.01 for both comparisons).3.Histological analysisMassive leukocyte infiltration was present in the lungs from the asthmatic mice. Neutrophils,eosinophils and lymphocytes were mainly found in the alveolar, interstitial and peribronchial regions.Goblet cell hypertrophy,epithelial damage, mucus hypersecretion,mucus plugs,as well as collagen deposition were also easily identified.While lungs in the control mice displayed a clear airway structure without any significant inflammatory cell infiltration or collagen deposition.ConclusionsBALB/c mice first sensitized by an intraperitoneal injection of a mixture containing ovalbumin and Al(OH)3,and then re-challenged with intranasal ovalbumin inhalation in combination with exposure to an aerosol of ovalbumin successfully developed asthmatic clinical characteristics and pathological changes,which is consistent with airway pathophysiological changes during asthma. Part two A study on the mechanism of Th17 cells in the pathogenesis of asthmaObjectiveTo investigate the mechanisms of Th17 cells in the pathogenesis of asthma.MethodsThirty mice were randomly divided into control group and asthmatic group.For the establishment of mouse asthma model,the mice were first sensitized to OVA(2mg and aluminum hydroxide emulsion 5mg in 200ul saline) via intra-peritoneal injection on days 0(the day on the beginning of the experiment) and day 14.those mice were firstly challenged with 1%ovalbumin in saline intranasally,then were re-challenged with 2% OVA in saline aerosol in a total volume of 40ml for 40 min every day from day 21 to 26. The control mice received saline alone under the same conditions as used for the asthma group without sensitization and re-challenge of OVA.The changes in the cell proportions in the BALF and the lung histological changes were observed.Th17 cells in the peripheral blood,thymus and lungs were detected by flow cytometry.ResultsThe total cell number and the percentage of neutrophils,eosinophils and lymphocytes in BALF of the asthmatic mice were significantly higher than that in the control mice(P＜0.01).The neutrophil and eosinophil infiltration was dramatically increased in asthmatic mice,but much less in the control mice.The percentages of Th17 cells in the peripheral blood(3.62%±0.58%),spleens(4.22%±0.69%) and thymuses(6.96%±1.35%) of asthmatic mice were significantly higher than that in the control mice (0.68%±0.26%,0.76%±0.31%and 0.80%±0.30%,respectively,P＜0.01 for all the three comparisons between the two groups).While Th17 cells in the lungs of asthmatic mice(0.64%±0.30%) were not different from that of control mice(0.55%±0.23%,P＞0.05).In the asthmatic mice,Th17 cells in the peripheral blood was positively correlated with the percentages of neutrophils(r=0.933,P=0.007) and eosinophils (r=0.867,P=0.028) in the BALF.Conclusions Th17 cells are probably involved in the pathogenesis of asthma.The cells may differentiate and mature in the thymus,and then are released into the blood.Th17 cells in the peripheral blood was positively correlated with the percentages of neutrophils and eosinophils,possible recruiting neutrophils and eosinophils into airways and leading to asthmatic airway inflammation.Part three Expression of interleukin-17A in asthmatic mice and its significanceObjectiveTo study the serum level of interleukin-17A(IL-17) and its expression in the lung,spleen and thymus in asthmatic mice.MethodsIn 14 normal BALB/c female mice and 14 asthmatic mice,the changes in the airway pathology and the cell proportion in the bronchoalveolar lavage fluid(BALF) were observed.After blood collection by eye extraction,lungs,spleen and thymus were harvested and cut into small pieces and smashed on the mesh to make single cell suspension.The cells were incubated at 37℃with 5%CO₂ for 24h and then supernatant collected and stored in -20℃.IL-17A in serum and in the supernatnats fom lung,spleen and thymus tissues of the two groups was determined by sandwich enzyme-linked immunosorbent assay(ELISA).ResultsThe total cell number and the percentage of neutrophils,eosinophils and lymphocytes in BALF of the asthmatic mice were significantly higher than that in the control mice(P＜0.01).The neutrophil and eosinophil infiltration was dramatically increased in asthmatic mice,but much less in the control mice.IL-17A in serum and in the lungs,spleen and thymus was significantly higher in the asthmatic mice than that in the normal control group(P＜0.01 for all comparisons).In the asthmatic mice,IL-17A levels in the lung tissues were positively correlated with the percentages of neutrophils(r=0.832,P=0.040) and eosinophils(r=0.856,P=0.030) in the BALF.ConclusionsHigh amount of IL-17A is secreted in the serum,lungs,spleen,and thymus of asthmatic mice.IL-17A levels in the lung tissues were positively correlated with the percentages of neutrophils and eosinophils in the asthmatic airway.Therefore, IL-17A may be one of the major cytokines involved in exacerbation of bronchial asthma,and is probably associated with the recruitment of neutrophils and eisinophils into the airways.