Repeated Inflammation Promote The Differentation Of Th17-cell Subset In Mice Sensitized With OVA

Background and purpose:Traditionally,it has been shown that the CD4+ T cells can be induced to secrete IFN-γ(Th1) or secrete IL-4,IL-5 and IL-13(Th2) in a mutually exclusive manner.However,recently a new subset of activated CD4+ T cell, reffered to as Th17-cell subset,was identified.Previous studies have indicated that Th17-cell subset plays a vital role in the development of some autoimmune disease such as CIA,EAE,cystic fibrosis or asthma.Th17-cell subset was distinct from the traditional Thl and Th2-cell subset,and its generation from native precursors was not dependent on the cytokines and transcription factors that mediate Thl and Th2-cell development.TGF -βplus IL-6 induced Th17-cell development from native T cells,IL-23 may expand or stabilized previously differentiated Th17 cells.The orphan nuclear receptor RORyt has been identified as the key transcription factors in the regulation of Th17 cell differentiation. The cytokines IL-2,IFN-γ,IL-4 and transcription factors T-bet,Ets-l could inhibit differentiation of Th17 cell.IL-17,a prominent cytokine producted by Th17-cell,might promote recruitment and activation of neutrophils in the airway by the release of chemokine IL-8 or others, play important roles in airway inflammation,or even in airway remodeling particularly in asthma.But the IL-17 mechanism involved in different period of airway inflammation is not clear. In this study,we establish the experimental mouse model sensitized and challenged with OVA.Through comparing the content of the cytokines,cells and transcription factor were analyzed in acute and chronic asthma model(inflammation), and compared with those of control group(PBS group),we observe that repeated inflammation promote the differentation of Th17-cell subset in mice sensitized with OVA.Materials and Methods:Male BALB/c mice,aged 4-6 wk,were sensitized with OVA from d0 to d12.Then,experimental mice were challenged daily with 5%OVA aerosolized for 30min from d18 to d24,termed acute asthma mice model.At the same time,control mice received PBS aerosolized(termed PBS group).Prolonged inflammation was induced by subsequent exposure to aerosolized 5%OVA for 30min three times a week from d26 onwards(OVA group),and control group use PBS as alternative.Mice were then sacrificed on d35 and d55,which represented the chronic phase of asthma.Next,bronchoalveolar lavage fluid(BALF) was performed as described.Cell supernatants were removed and analyzed for cytokines(INF-γ,IL-4,IL-5,IL-13 andIL-17).Differential cell counts were performed on Giemsa-stained cytospins.Percentages of eosinophils,lymphocytes,neutrophils and macrophages were determined.Single-cell suspensions made from lymph node and lung were cultured and restimulated,Then cells were analyzed on a FACSCalibur using CellQuest software to determine the tissue content of Th17 cells.At the same time,the supernatant were collected for cytokine analysis including TGF-β,IL-23 and IL-6. Furthermore,real-time quantitative PCR was performed for transcription factor RORγt.The content of the cytokines,cells and transcription factor were analyzed in acute and chronic asthma model(inflammation),and compared with those of control group(PBS group).Results 1.Among the cytokines secreted by CD4+ T cells,IL-17 level was respectively higher than PBS group through out the period of challenge,which was distinct from the other cytokines product by traditional Th1 and Th2-cell subset.2.The content of Th17 cells from lung tissue and spleen tissue was higher after challenge with OVA, and became highest in chronic inflammation.3.TGF-P and IL-6,the critical cytokines which induced Th17-cell development from native T cells,were promoted early in acute inflammation,and it's level was highest in chronic inflammation.IL-23,which perhaps expand or stabilized previously differentiated Th17 cells.Was promoted only in chronic inflammation.4.The level of orphan nuclear receptor RORyt,identified as the key transcription factors in the regulation of Th17 cell differentiation,was higher step by step during the period of inflammation challenged with OVA.Conclusion:Repeated inflammation promote the differentation of Th17-cell subset in mice sensitized with OVA.