Immunomodulatory Effects Of CpG Oligodeoxynucleotides On Bronchial Asthma

Experimental purposes and backgroundBronchial asthma can be abbreviated as asthma which is a variety of cells and cellular components involved in chronic inflammatory airway diseases. And asthma is characterized as reversible airflow obstruction in the lungs, airway mucosa inflammation and airway hyperresponsiveness. The main effective cells are mast cells, eosinophils (EOS) and T lymphocytes. A key step in inflammatory response of asthma is the T lymphocytes cells activated by antigen. T lymphocytes are the direct effector cells to activate and expand airway inflammation, and involved in the major pathophysiological changes while the acute asthma attacking. Many studies suggest that chronic airway inflammation responses to the imbalance of Th1 and Th2 lymphocytes and the domination of the Th2-type immune response. Th2 cytokines, i.e., IL-4, IL-5, IL-13, can make the eosinophils and mast cells chemotactic and active, and make the transformation from B cells to IgE producing B cells . But the proliferation of Th2 lymphocytes and cytokines induction can be inhibited by the Th1-type cytokines, such as INFγ.CpG ODN (CpG-oligodeoxynucleotide) is single-stranded oligodeoxynucleotides (oligodeoxynucleotide, ODN) containing non-methylated cytosine-guanine dinucleotide motif. In 1984, the research proved that non-methylated CpG motifs of bacterial DNA have the ability of stimulating innate immune system. CpG ODN consists of three types: A, B and C, all of which can stimulate human PBMC. The mechanism is that CpG ODN can stimulate the body's natural and acquired immune responses through stimulating NK cells, macrophages, anti-T-cell (CTL), which are accomplished by the direct activation of the expression cells of TLR9, i.e., cell-plasma cell-like dendritic cells (pDC) and B cells. The prominent characteristic of A-type CpG ODN is the stimulation to the pDC producing high levels of typeⅠinterferon, and has a strong activation of NK cells. B-type CpG ODN is characterized by stimulating the proliferation of stimulated B cells. C-type CpGODN has both A-type and B-type characteristics. CpG ODN combinated with its receptor Toll-like receptor 9 (Toll-like receptor 9, TLR9) lead to a series of intracellular signal transduction cascade, which ultimately cause the activation of transcription factors of NF-κB (Nuclear Factor kappa B) and AP-1 (activation protein-1). Then the B cells, dendritic cells, macrophages, antigen-presenting cells, indirect activation of T cells and NK cells can be activated and induced the Th1-type immune response is induced. Therefore, CpG ODN is a good immune adjuvant . Meanwhile, it has also been widely used in oncology, infectious diseases and the treatment of allergic diseases. In our thesis, through animal experiments and clinical investigations, the pathogenesis of asthma and asthma oligodeoxynucleotides on immune regulation of diseases are investigated. The detailed processes are discussed in the following :1 animal experimentsExperimental methods(1) BABL / C mice as subjects, which were sensitized by injection of 10μg ova/1μL aluminum hydroxide adjuvant [Al (OH)3] in 0.9% sterile saline on days 0, 7 and 14. After three weeks, sensitized mice were further stimulated by OVA for continuous 5 days to establish the mouse asthma model. Experimental results showed that asthmatic mice had some behavior changes of convulsions, pant, nose scratching and ear grasping. And with the increase in the number of inhalation, the performance will become increasingly evident. Asthmatic mice's lung tissue HE staining shows a large number of eosinophilic cells, lymphocytes and other inflammatory cell infiltration. Compared with the control group, there are significant differences, which indicating that the success of the asthma model.(2) 30 female BALB / C mice were used when they reached 6-8 weeks of age, weighting 17-22mg. They were randomly divided into 5 groups (n = 6), including the model group, CpG 01 groups, CpG 02 groups, ODN 03 group and normal saline group. Each group of mice were injected three times on days 0,7,14 respectively with OVA together with corresponding ODN or saline as negative control. Then on days 21, 22, 23, 24, 25, mice in each group were exposed to 10 ml aerosol inhalation of ovalbumin (concentration of 1%), and the responses of mice in each group were recorded, including irritability, respiratory frequency, the number of torsion nose, muscle contraction and convulsion. The blood should be collected after 48 hours at the end of the shot and separated serum. Meanwhile the lavage fluidand spleen cell culture supernatant liquidcollection of mice should be collected and preserve at -20℃for cytokines detection..(3) Mice were killed by straining, and the lungs of mice were pulled out placing in 20ml bottles which filled with 10% neutral formaldehyde. After 24 hours fixed, they were sent to morphology laboratory to be paraffin-embedded, sliced, HE staining to observe the histopathological changes in lung tissue.(4) The detection of serum, lavage fluid and spleen cell culture supernatant of cytokines IL-4 and IFN-γby ELISA method.The experimental results(1) Through the research of the pre-experiment of mouse asthma model, we discover that the injection of both 50ug OVA and 10ug OVA in sensitization phase could induce acute asthma attacks. There is no significant difference.(2) HE staining of lung tissue shows that the control group of mice were out of thickening of airway epithelium and airway inflammatory cells around the non-invasive with intergral structure of alveolar wall. However, the model group shows obvious inflammatory cells in small bronchial bronchial, submucosal and surrounding lung tissue (dominant in EOS, lymphocytes and macrophages). The model group also has edema in vessel wall and a large number of secretions in bronchial cavity. There were no symptoms above in CpG0800 group, which was normal. The lung tissue of CpG684 and ODNsat05f groups have varying degrees of inflammatory cell infiltration and thickening of the wall.(3) Antigens stimulation and ODN CpG intraperitoneal injection were conducted at the same time which can restrain the allergens Th2 reaction in a certain extent, making immune response drift to Th1 direction and inhibiting the local imbalance of Th1/Th2 in lung tissue.2 Clinical InvestigationExperimental methods(1)The nontreatment of asthma: 6 cases by a clear diagnosis which did not use any hormone therapy. The 6 cases with asthma were the children of ages 2 ~ 10 (4.8±2.5) , four of which were male;The normal control group of children: 6 cases of physical examination without a family history of atopic disease and personal history of allergic diseases, also without respiratory tract infection in the same period. The 6 cases without asthma were the children of ages 3 ~ 14 (6.5±2.1 ) , three of which were male;(2) PBMC obtaining from the nontreatment of asthma and normal control group of children, respectively, stimulated with CpG in vitro;(3) Detection of proliferation of PBMC by MTT ;(4) Detection of cytokines IL-4 and IFN-γof culture supernatant PBMV by ELISA. The experimental resultsCpG 02 stimulated stronger to PBMC in both normal and asthna children. Compared with CpG684 and ODNsat05f, there are statistically significant differences(P<0.05).Conclusion1. The asthma models were successfully established which was judged by the clinical manifestations of quantitative index and pathological and histological.2. C type of CpG ODN can significantly reduce clinical symptoms, the lung inflammation and IL - 4 levels of alveolar lavage in asthma mice, B type of CpG ODN can increase the IFN–γlevel of serum.3. Only C type of ODN CpG of the three types can increase IFN–γlevel of PBMC culture supernate in asthma children.4. Three kinds of ODN have significant stimulation to PBMC hyperplasia in asthma children, but only the C type CpG have stimulation to PBMC hyperplasia for normal children.