The Effect Of Live Mycobacterium Bovis Bacille Calmette-Guérin Vaccination On IL-17 And Th17 Cells In Asthma Mice

BackgroundAsthma is a very common disease in the world that seriously threaten human health.It is now estimated that as many as 300 million people suffer from asthma in the world, and is more than 30 million in our country. We should have a positive attitude to prevent and treat asthma, decrease its morbidity and mortality,and effectively control and reduce symptoms of asthmatic attack.The Pathogenesis of asthma is complicated, whereas it has not yet fully understood. It is generally believed that asthma is due to chronic airway inflammation involving variety of cells especially epithelial cells, fibroblasts, dendritic cells, eosinophils, mast cells and T lymphocytes. The recruitment of inflammatory cells and the release of cytokines and mediators of inflammation are the major reason which cause and keep the airway inflammation of asthma,and further cause the tissue damage and airway disfunction. Among these inflammatory cells, T lymphocytes play a vital role in the pathogenesis of asthma. A long time ago, the imbalance of Thl/Th2 has been believed to be an important immunologic mechanism of asthma, but along with the research, people discovered that can not sufficiently to explain all of the mechanisms in the pathogenesis of asthma. Recently,two new lymphocyte subsets have been discovered—regulatory T cell (Treg) and Th17. Treg cells can not only suppress the Thl cells, but also can suppress the Th2 cells. So Treg cells Can explain the pathogenesis better than the Thl/Th2, and become a focus in asthma reseach.Th17 cells have recently emerged as a new independent T cell subset that different from Thl, Th2 cells. There has a complicated relationship between Th17 and Treg cells, and Th17 cells and IL-17 that secreted by it can lead to the development of asthma.The Live Mycobacterium bovis Bacille Calmette-Guerin is a very strong immune reaction irritants of Thl cells, it can correct the imbalance of Thl/Th2. At 1997, Shirakawa and his colleagues claimed that there was inverse relationship between tuberculin responses and atopic disorder, which cost light on the develepment of BCG vaccine on asthma. Until now, all the animal research demonstrated that the airway eosinophil infiltration aroused by OVA can obviously inhibited by BCGAnd there are many researches showed that the Inflammation made by Th17 cells can also be restrained by BCG in experimental allergic encephalitis(EAE) animal model. And BCG vaccination has been shown to rapidly suppress a Th17 response, which is thought to be due to a strong induction of IL-12p70 and a reduction in IL-23.To investigate the effect of Live Mycobacterium bovis Bacille Calmette-Guerin vaccination on Th17 cells in Peripheral blood and IL-17 in mice asthma model, and the possible mechanism of action, so that we can find some way to prevent and cure asthma.Part one Establishment of the asthmatic mouse model ObjectiveObjectiveTo establish a BALB/c mouse asthma model by OVA sensitization and re-challenge.MethodsSPF female BALB/c mice (6 to 8 weeks) 18-20g, were purchased from Laboratory Animal Services Center of Southern Medical University, and maintained on an ovalbumin-free diet. The mice were randomly divided into 2 groups: control group(group A) and asthmatic group(group B). Mice were immunized by OVA/Alum (containing 100μg of OVA bound to 20mg of aluminum hydroxide), made up in 0.2ml sterile saline, On days 21, the animals were challenged by exposure to an aerosol of 2% ovalbumin generated by an ultrasonic nebulizer for 30 min. The control animals were treated with saline and re-challenged with saline solution. The animals were killed by cervical dislocation 1 h after the last exposure to aerosol re-challenge, open the chest and ligated the left primary bronchus, the right lung were subjected to bronchoalveolar lavage. The bronchoalveolar lavages were stained with Wright's staining and eosinophil number counted with a hematocytometer. At least 400 lymphocytes were counted by microscopy before the percentages of various types of cells were calculated. After bronchoalveolar lavage, the animals were sacrificed, the right lungs harvested and fixed in 4% paraformaldehyde for further analysis. Tissues were sliced and HE stained for histological examination, including morphology, epithelial injury, perivascular and peritracheal infiltration of eosinophils.Results1.The behavior changes after re-challengeMice in group B displayed various types of allergic responses, such as scratching head and face, dyspnea, oral lip cyanosis, muscle spasm as well as gatism, while group A showed no such symptoms.2. The changes in total cells and different cell types in BALFa. The total BALF cell number in the asthmatic group was (7.40±1.71)×104/ml, that is significantly higher than that in the control group (20.30±3.02)×104/ml(t=-11.749, P< 0.01). b.The percentages of BALF eosinophils and neutrophils were 0.23%±0.28% and 2.73%±0.51% in the asthmatic group, that were also significantly higher than those in the control group19.40%±5.27% and 29.48%±3.54%(t=-11.480, t=-23.653, P<0.01 for both comparisons).3. Histological analysisMassive leukocytic infiltration was present in the lungs of the mice in group B, Neutrophils,eosinophils and lymphocytes were mainly found in the alveolar,interstitial and peribronchial regions. Goblet cell hypertrophy,epithelial damage, mucus hypersecretion, mucus plugs,as well as collagen deposition were also easily identified. While lungs of the mice in group A displayed a clear airway structure without any significant inflammatory cell infiltration or collagen deposition.Conclusions BALB/c mice first sensitized by an intraperitoneal injection of a mixture containing ovalbumin and AL(OH)3, and then an aerosol of ovalbumin successfully developed asthmatic clinical characteristics and pathological changes, which is consistent with airway pathophysiological changes during asthma.Part two Effects of Live Mycobacterium bovis Bacille Calmette-Guerin vaccination on IL-17 and Th17 cells in the asthma mouse.ObjectiveTo investigate the effects of Live Mycobacterium bovis Bacille Calmette-Guerin vaccination on Th17 cells in Peripheral blood and IL-17 in mice asthma model.Methods30 female BALB/c mice were randomized into 3 groups:group A(control group), group B(asthmatic group) and group C(BCG group). The mice of group B and C were sensitized by OVA and then stimulated with nebulized OVA, and live BCG were inoculated 14 days before sensitized in group C. We immunized mice with OVA/Alum (containing 100μg of OVA bound to 20mg of aluminum hydroxide), made up in 0.2ml sterile saline, injected intraperitoneally and subcutaneously on days 15 and 29, On days 30-36, mice were exposed to aerosolized OVA(2%)for 30 min.The control group were received an sensitization and stimulation by physiological saline. The airway inflammation was evaluated by HE staining.Inflammatory cell numbers and morphological identification of leucocytes in bronchoalveolar lavage fluid (BALF) were measured by light microscopy. The infects of Live Mycobacterium bovis Bacille Calmette-Guerin vaccination on Th17 cells in Peripheral blood and IL-17 in mice asthma model. The level of interleukin-17 in mice bronchoalveolar lavage fluid (BALF)and serum was measured by enzyme-linked immunosorbent assay (ELISA). The percentage of Thl7 cells in Peripheral blood were assessed by flow cytometric analysis.Results 1.The mice of group B demonstrated symptoms of acute asthma when exposed to OVA, such as dysphoria or standing still,breathing deeply and fast, nodding respiration, bowing the back,and urinary and fecal incontinence, and so on; the mice of group A didn't show these symptoms of acute asthma, and the mice in group C occurred by chance.2.Comparision of the total number of cells and different cell types in the BALFThe total number of group A was (7.40±1.71)×104/ml, group B was (20.30±3.02)×104/ml, and group C was (14.30±2.31)×104/ml, the number in group B and C was dramatically higher than group A, but that was lower in group C than in group B. The percentage of neutrophil in group A was (2.73%±0.51%), (29.48%±3.54%) in group B, (11.98%±2.57%) in group C, the number in group B and C was dramatically higher than group A(P<0.01), but that was lower in group C than in group B(P<0.01). The percentage of eosinophil infiltration in group A was (0.23%±0.28%), (19.40%±5.27%) in group B, (10.55%±2.40%) in group C, the number in group B and C was dramatically higher than group A(P<0.01), but that was lower in group C than in group B(P<0.01). The percentage of lymphocytes in group A was (28.50%±4.45%), (39.63%±3.95%) in group B, (40.23%±3.43%) in group C, the number in group B and C was dramatically higher than group A(P<0.01), but there was no such obvious variability between group B and group C (P>0.05).3.The pathology of lung tissueMassive leukocytic infiltration was present in the lungs of the mice in group B. Neutrophils, eosinophils and lymphocytes were mainly found in the alveolar, interstitial and peribronchial regions. Goblet cell hypertrophy, epithelial damage, mucus hypersecretion, mucus plugs, as well as collagen deposition were also easily identified. While lungs in the control mice displayed a clear airway structure without any significant inflammatory cell infiltration or collagen deposition. Neutrophils, eosinophils and lymphocytes were mainly found in the alveolar, interstitial and peribronchial regions in the BCG group, but was more lighten than in grpup B.4. The levels of IL-17 in BALF and serum were much higher in the group B and C than those group A(P<0.01), and there was also a significant difference between the group B and C (P＜0.01).5. Flow cytometric analysis of Th17 cells in Peripheral bloodThe mice in group B and C had a higher percentage of Th17 cells in peripheral blood compared with the mice in group A(P<0.01), and it was lower in group C was than in group B (P<0.01).Conclusion1. Th17 cells and IL-17 may play an important role in the mechanism asthma.2.Live BCG vaccination can effectively prevent the development of airway inflammation, suppress eosinophil and neutrophils aggregation, may associated with altered the percentages of Thl7 cells in Peripheral blood and IL-17 levels.