Role Of Th17 Cells In The Pathogenesis Of Asthma And The Influence Of TLR2 Pathway On The Differentiation Of Th17 Cells

PartⅠChanges of the Ratio of T helper 17 cells and CD8+IL-17+T cells and the Level of Interleukin-17 in a Mouse Model of Asthma【Objective】To investigate the changes of Th17 cells and Tc17 cells and the level of IL-17 in a mouse model of asthma.【Methods】Twenty-four BABL/c mice were randomly divided into asthma group and control group. Mice were sensitized and challenged with ovalbumin(OVA) as asthmatic mouse models. The ratio of Th17 cells and Tcl7 cells in splenocytes was detected by flow cytometry. The level of IL-17 in bronchoalveolar lavage fluid(BALF) was determined by enzyme-linked immunosorbent assay(ELISA).The expression of Th17 cells and Tc17 cells in lungs was confirmed by double immunofluorescence.【Results】The frequency of Thl7 cells and Tc17 cells in splenocytes from asthmatic mice were significantly higher than those of normal controls(P<0.0001 and P< 0.001, respectively); the level of cytokine IL-17 in BALF of asthmatic mice was more higher than those of normal controls(P<0.0001);the numbers of total cells and eosinophils in BAL were significantly higher than those of normal controls (both P<0.01).Th17 cells and Tc17 cells can be found in lung tissues of asthmatic mice, the numbers of these two kinds of cells were were significantly higher than those of normal controls (both P<0.01).[Conclusion] Th17 cells and Tc17 cells may play an important role in the pathogenesis of asthma. PartⅡToll-like Receptor 2 Signaling in EL4 T Cells Promotes the Expression of Interleukin-17【Objective】To investigate the effect of toll-like receptor 2 signaling on the expression of interleukin-17 in EL4 T cell line.【Methods】The RORγt and IL-17A mRNA expression were determined by real time RT-PCR, the expression of interleukin-17 protein were detected by ELISA.[Results] The real time RT-PCR results suggest that TLR2 ligand treatment can significantly up-regulate expression of RORyt and IL-17A on mRNA level and on protein level.【Conclusion】The toll-like receptor 2 signaling in EL4 cells can promote the expression of interleukin-17, this is probably dependent on up-regulating of the expression of the transcriptional factor RORyt.PartⅢThe Differentiation and Function of T Helper 17 cells Promoted by Toll-like Receptor 2 Signaling in vitro via RORgammat pathway【Objective】To investigate the role of toll-like receptor 2 signaling on the differentiation and function of Th17 cells.【Methods】The CD4+T cells were isolated from spleens of asthmatic mice and purified by negative selection with a magnetic cell sort, then for Th17 differentiation, cells were cultured under Th17 condition(IL-6 and TGF-β1) for three days. Where indicated, LTA, TLR2 antibody and RORc-MR-lentivirus were added to cultures. Intracellular staining of cytokine IL-17A was analyzed by flow cytometry. IL-17A cytokine production was assessed by ELISA kits. For Th17 cell proliferation assays, we conducted MTT. Analyses of Th17-related genes(il-17a,il-17f,il-22 and rorc) transcripts were performed with real-time RT-PCR.【Results】(1) Th17 cells proliferated more intensively where LTA was added in the media than those cultured without LTA(P<0.05,P<0.001 and P<0.01,respectively).(2) Fully differentiated Th17 cells treated with a TLR2 agonist LTA were found to produce increased amount of IL-17 proteins(P<0.05),and the neutralizing antibody for TLR2 can reverse the effects of LTA(P<0.05).(3)The results of intracellular staining of cytokine IL-17A showed that IL-17+cells were more dominant in LTA treated groups(from 11.7% up to 22.5%(P< 0.05).(4)Th17-associated mRNA(IL-17A,IL-17F and IL-22)were upregulated by a TLR2 agonist LTA (P<0.05),however,TLR2 antibody and RORc-MR-lentivirus can cancer out these effects(P< 0.05).【Conclusion】TLR2 signaling can enhance Th17 differentiation and promote Th17 cell development.Part IV The Interleukin-21 and Total Serum IgE Levels in Serum of Asthmatics and Their Correlation Analysis【Objective】In order to investigate levels of interleukin-21 (IL-21) and total serum IgE in asthmatics,and to explore the relationship between IL-21 and total serum IgE in patients with allergic asthma.【Methods】Totally 80 subjects were recruited and were divided into three groups:Control group(n=19),allergic asthma group (n=38) and controlled asthma group (n=23).The levels of IL-21 and total IgE in serum were detected by ELISA.【Results】The IL-21 levels in serum control group,allergic asthma group and controlled asthma group were 17.19,21.99 pg/ml and 17.18 pg/ml, respectively.The levels of IL-21 were significantly different from each other between the control group and allergic asthma group (P<0.05).Negative relationship was found between the levels of IL-21 and total serum IgE in allergic asthma group (r=-0.32,P<0.05).【Conclusion】Our results suggested that IL-21 might be involved in the pathogenesis of allergic asthma in human.