The Effect Of Lnterleukin-23on Memory T Cells In Asthmatic Mice

Background:Asthma is considered as a Th2-dominant disease in which memory T cells play animportant role. After initial immune response, a few memory T cells developed into memoryT cells and exist for a long time. When the body exposes to the same allergy again, memoryT cells will proliferate and be actived to Th2cells by secreting Th2cytokines which induceTh2type airway inflammation. Regulating memory T cells proliferation and activation mayreduce the secretion of a series of cytokines, consequently, reduce Th2airway inflammation.So it is important to understand the mechanism and regular pattern of memory T cells inasthma and find the reagents that can regulate the memory T cells proliferation andactivation so to provide new clues for treating asthma based on memory T cells. IL-23hasbeen reported to promote memory T cells proliferation and activation in normal bodies.Meanwhile, it has been determined that IL-23promotes Th2airway inflammation in asthmawhile the mechanism remains unknown. So this study is going to determine whether IL-23promotes Th2airway inflammation by promoting the proliferation of memory T cells so asto find new targets for asthma therapy.Objective:1. To know the variation of memory T cells with the development of asthma in mice soas to decide an opportune moment to research memory T cells and lay the foundation forfurther studying in memory T cells functions;2. To know whether silencing IL-23p19gene can reduce memory T cells proliferation inasthmatic mice so as to disclose the mechanism of IL-23promoting Th2airwayinflammation in asthma.Method:1. To construct asthmatic mouse model by sensitization and challenge with OVA.Regularly sacrifice3mice every1or2days from1day before the first challenge to30daysafter the last challenge. The spleens and lungs of sacrificed mice were separated and madeinto single cell suspension. The ratio of memory T cells in spleens and lungs were detectedby Flow cytometry. 2. Design interference sequence according to mouse IL-23p19mRNA, Lentiviralpackaging, and screen the effective sequence that can silence the expression of mouseIL-23p19in vitro;3. Asthma mice were divided into4groups: normal control, asthma, empty vector andIL-23p19gene silencing groups. The sensitization and challenge in the latter two groups areas the same as asthma group, but Lentiviral liquid was dropped into the noses of mice1dayadvanced the first challenge.2days after the last challenge, mice were sacrificed and severalindexed were detected including the effect of IL-23p19silencing by RT-PCR and SP, theratio of memory T cells in lungs by FCM, the expression of transcription factors includingT-bet, GATA-3and RORC by RT-PCR, the pathological changes by HE stain and Congored stain, the expression of Th2cytokines by PCR and other inflammatory indexes forasthma.Result:1. The results for memory T cells⑴The ratios of memory T cell in spleens and lungs of asthmatic mice are dramaticallyhigher than that of normal control group (p <0.0001);⑵The regularity of CD4~+memory T cells: The ratios of memory T cell in spleens andlungs of asthmatic mice keep rising during the phase of challenge, even after challengestopping and up to peak value during2~9days after the last challenge with over50%forlungs CD4~+memory T cells and7days after the last challenge with over40%for spleensCD4~+memory T cells. Then both CD4~+memory T cells in lungs and spleens descendgradually to the level before challenge at30days after the last challenge;⑶The regularity of CD4~-memory T cells: The ratios of memory T cells in lungs ofasthmatic mice keep rising during the phase of challenge, even after challenge stopping andup to peak value at2days after the last challenge with over60%, and then descend rapidly tothe level before challenge. CD4~-memory T cells in spleens begin to rise only after challenge7days, keep at the levels of30%, and descend to the level before challenge at14days afterthe last challenge;⑷According to this asthma mice model,2days after the last challenge is the opportunemoment to research memory T cells proliferation in lungs;2. The effect of silencing IL-23p19gene on asthmatic mice⑴Memory T cell: ratios of CD4~+memory T cells in lungs of gene silencing group arelower than that of asthma group (p <0.0001), while CD4~-memory T cells have no difference in two groups (p>0.05);⑵Th2airway inflammation: the levels of Th2cytokines, the inflammatory factors andEos infiltration in lungs of gene silencing group are all lower than that of asthma group (p<0.001, p <0.05);⑶Transcription factors: the expression of Th2transcription factor GATA-3and Th1transcription factor T-bet in lungs of gene silencing group are all lower than that of asthmagroup (p <0.0001), while the expression of Th17transcription factor RORC has nodifference in two groups (p>0.05).Conclusion:1. Memory T cells in asthmatic mice present some regularity. Memory T cells in lungsare always rising earlier than that in spleens and significantly higher than that in spleensduring the whole acute stage. In lungs, CD4~+and CD4~-memory T cells both involve in thepathogenesis of asthma and CD4~+memory T cells are the main subsets to cause asthmaairway inflammation;2. Silencing the expression of IL-23decreases the expression of Th2cytokines and Th2transcription factor, Eos infiltration in lungs and the proliferation of CD4~+memory T cells inlungs in asthmatic mice. This may be one of the mechanisms of IL-23promoting Th2airwayinflammation in asthmatic mice.