Immunoregulatory Effect Of Transcription Factor ROR? On Bronchial Asthma

BackgroundAsthma is a chronic inflammatory disease involved by a variety of immune cells and may happen at any age. There has been mang receach of the role of ILC2s in asthma which are a novel lymphocyte subfamily that is distinguished from the T cells and B cells of the adaptive immune system by their lack of clonotypic antigen receptors, it means innate lymphoid cells recently. Morphologically, ILC2s do not espress any sign of cell lineage, resemblelymphoid cells and, similarly to T cells,they can be divided in three classes due to the characteristic surface receptorsand effector molecules of differentiated T cell subsets and specific transcription factors.There is a small amount of ILC2s in healthy individuals, but it will be activated and increased under pathological conditions. In asthma, activated ILC2s further secreted many cytokines such as IL-13, IL-5, they maintained the Th2 cell polarization s, and participated the progress of immune damage.Under the effect of these proinflammatory cytokines, the airway then be hyperresponsiveness,and show a typical asthma symptoms.The Retinoic acid receptor-related Orphan Receptor Alphaa belongs to the nuclear hormone-receptor superfamily (NR1) and expressed in many organs in various tissues of the organism. As a characteristic transcription factors of ILC2s, it is of vital importance for ILC2s differentiation, proliferation and function. Recent research found that in RORa-/- mice, the amount of ILC2s was dramatically decreased and the immunity to parasites was weakened. However the immunity was rebuild when the mice received the ILC2s from normal mice ILC2s, the immunity was rebuild.ObjectiveIn order to investigate the role of RORa in the development of bronchial asthma,and proposed new ways for clinical treatment of asthma. This study was established on the basis of mice asthma model, analyzing the distribution of ILC2s in asthmatic mice, and the construction of RORa expression vector to research its regulation function on ILC2s, then discusses the possibility of the intervention for asthma model.Metairls and methods(1) Clinical research: Using flow cytometry detected the ILC2s% in peripheral blood mononuclear cells in human with bronchial asthma, at the same time,the using RT-qPCR analyzedthe mRNA expression level of RORa, T - bet, GATA3,IL - 33, IL - 13, IL - 5, IL - 4 in peripheral blood mononuclear cells, and using ELISA to detected the protein concentrateof IL - 33, IL - 13, IL - 5 in serum. Finally, using the linear correlation analysis, analyzed the linear relationship between RORa mRNA level and ILC2s%. RORa mRNA level and Th1, Th2 cells related molecules, ILC2s related molecules and Th2 cells related molecules.(2) The induce of mice asthma model and the intervention trial with RORa expression vector in mice1) Construction of RORa expression vector: first of all, we need to construct adenovirus vector carrying RORa; Sterile separated the spleen tissue in mice, using RT-PCR cloned the cDNA coding region of mRORa from spleen, and then connect the cDNA to PMD - 18 t plasmid. Then directional cloning into the adenovirus shuttle plasmid pDC316 - mCMV - EGFP and then together with skeleton plasmid PBHGloxdetalEl, 3 cre packed to adenovirus and pur-ified adenovirus by purification kits.2) The induce of mice asthma model and the intervention trial with RORa expression vector: first of all, established OVA induced bronchial asthma model, before challenged, the Ad-RORa was used by tail vein injection. Using H&E staining to observe the pathology changes of lung tissue, using flow cytometry to detect enrichment of ILC2s in peripheral blood in mice; Using cell counting pool to count the infiltration of inflammatory cells in BALF; Using ELISA to detect the protein level of IL - 33, IL - 13, IL - 5 cytokines in serum, using RT - qPCR to detect the mRNA level of RORa, IL - 33, IL - 13, IL - 5, IL - 4 and GATA3 in the lung tissue and bronchoalveolar lavage fluid, using western blot to detect the protein expression of RORa in lung tissues.Results(1) The proportion of ILC2s in the peripheral blood was significantly increased in patients with asthmacompared with normal controls, and IL - 33 which prompted the activation of ILC2s in serum and ILC2s products IL - 13, IL - 5 has subsequently increased; At the same time, at the mRNA level, RORa, IL - 33, IL - 13,IL - 5, IL - 4, GATA3 also showed consistent rising trend, and the transcription factor of Th1 cells-T- bet was correspondingly present a downward trend in peripheral blood mononuclear cells. Correlation analysis showed that RORapositively correlated with ILC2s, IL - 33 and cytokines IL - 13, IL - 5, IL - 4.(2) In the construct of adenovirus vector, we successfully cloned mRORa gene, and the sequence is correct, and successfully clone RORa gene into pDC316-mCMV-EGFP.(3) In the section of adenovirus packing, we first succeeded in mass production to the shuttle plasmid pDC316 - RORa - mCMV - EGFP, skeleton plasmid PBHGIoxdetalE1, 3 cre, then shuttle plasmid pDC316 - RORa - mCMV - EGFP with skeleton plasmid PBHGloxdetalE1, 3 ere were transfected together in HEK293 cells,after successful purified, the high drop degree adenovirus carry mROR? was obtained.(1) The mice bronchial asthma model we successfully established using OVA induction method. Through H&E staining, the lung tissue from OVA induced mouse showed more inflammatory cells infiltration, and the similar condition was found in OVA + Ad - EGFP group. When treated with Ad - ROR?, the inflammation cells infiltrated in lung tissue was obviously increased and mice showed more intense,scratching heads, raise forelimbs, incontinence and shortness of breath in the process of challenge. After Ad-RORa treatment, compared with OVA and Ad- EGFP groups,there was more IgE concentration in the supernatant of bronchoalveolar lavage fluid .(4) The detection results of IL-C2s related factors: The proportion of ILC2s in peripheral blood was significantly increased in OVA induced mice than that in control.however in Ad - RORa mice, the proportion of ILC2s in peripheral blood was more higher. It indicated that ILC2s participate in the process of the onset of asthma, and the treatment of transcription factors RORa further promoted the process. Meanwhile,IL - 33, ILC2s products IL - 13, IL - 5, IL - 4 etc were all significantly increased. At the same time, we also found that the protein ST2 in lung tissue was increased, this might be owing to the function of ILC2s autocrine IL - 33, which further promoted the increase of ILC2s.ConclusionThe number of ILC2s and its related molecules increased in asthmatic patients.Meanwhile, the up-regulated RORa showed a positive correlation with the ratio of ILC2s. Ad-RORa viral vector was successfully constructed and a OVA induced asthma model in mice was successfully established. The in vivo experiments demonstrated the immunological pathological effect of exogenous RORa on the development of asthma.