| Background and AimThe study of pathogen infection on the pathogenesis of bronchial asthma has received more and more attention. Some clinical observations have showed that the incidence of bronchial asthma appear to be rising after the infection of atypical pathogen, fungal and some kinds of bacteria,which also exacerbate the degree of acute episodes. The most notably is the relationship between bronchial asthma and mycoplasma pneumoniae infection. Mycoplasma pneumonia is generally recognized as an important co-factors in acute attack in children with bronchial asthma. The treatment with macrolides antibiotics to mycoplasma pneumoniae leads to improve the lung function and relieve respiratory symptoms. Dendritic cells and Toll-like receptors and T helper cells play a central role in the pathogenesis of bronchial asthma with mycoplasma pneumoniae infection.The aim of this study is as follows: 1)To observe whether mycoplasma pneumoniae infective rate is higher among Children with bronchial asthma, meanwhile, to analyze the relationship between mycoplasma pneumoniae infection and other clinic allergic indicators. 2) To research the variation of DC and TLRs and analyze their function in the bronchial asthma with mycoplasma pneumoniae infection 3) To measure the cytokines of Th1、Th9、Th17 in asthmatic children with and without mycoplasma pneumoniae infection and animal models of asthma with mycoplasma pneumoniae infection and to analyze the function of these cytokines in the pathogenesis of asthma after mycoplasma pneumoniae infection 4) After detecting some transcription factors of lung tissue in asthmatic models with mycoplasma pneumoniae infection,including T-bet,GATA-3 and NF-κ B, we analyze the relationship between mycoplasma pneumoniae infection and the pathogenesis of asthma on genetic level.Methods1.To measure the anti- mycoplasma pneumonia Ig M 、serum total Ig E, mite specific Ig E, ECP and allergen prick skin tests of asthmatic children, and then analyze the correlation between the mycoplasma pneumoniae infection rate and some allergic parameters.2.The children with bronchial asthma were divided into infected group and non-infected group according to serologic detection of anti-mycoplasma pneumonia Ig M. At the same time, we built the animal models of asthma infected with mycoplasma pneumonia. We measured the expression level of TLR2,TLR4,TLR5,TLR6 in DCs of peripheral blood in asthmatic children and in lung and spleen tissue in asthmatic model by flow cytometry.3.The levels of IL-9,IL-17 and IFN-γ of peripheral blood in asthmatic children and asthmatic models and of BALF in asthmatic models were measured by ELISA. We analyzed the differences between the groups, the change of cytokines network after mycoplasma pneumoniae infection.4. The expressions of T-bet、GATA-3 and NF-κ B of lung tissue in asthmatic model were measured by Fluorescence Semi-quantitative PCR, and then analyzed the change of these transcription factors in asthmatic models with mycoplasma pneumonia infection.Results1.The mycoplasma pneumonia infection rate were 44.74%, 23.08% and 7.14% respectively in asmatic children and children with other respiratory tract infection and the control group. The total Ig E、s Ig E、ECP of athsmatic children with mycoplasma pneumonia infection were 977.4±101.6IU/ml, 39.93±7.14IU/ml,87.83±14.43ug/L respectively; And their result of allergen skin tests to mites were follows: the percent of(+~++)positive is 54.55%, more than(++) positive is 45.45%; The total Ig E、s Ig E、ECP of athsmatics without MP infection were 332.2±69.15IU/ml,9.72±3.65IU/ml, 41.57± 6.41ug/L respectively;the result of allergen skin tests showed(+~++) positive was 68.18%, more than(++)was 31.82%.Our results showed that the MP infection rate in asthmatic children was significantly higher than other groups. There are significant difference of the level of serum total Ig E, s Ig E and ECP between the asthmatic children with MP infection and without MP infection group(P<0.01).2. The expression of TLR2,TLR4,TLR5 and TLR6 of peripheral blood DC in the asthmatic children with MP infection and without MP infection and controls and in lung and spleen tissue of asthmatic model were as follows:(1)The percentage of DC in peripheral blood of clinical groups were 0.82±0.13%,0.79±0.11% and 0.40±0.09% respectively. The difference between asthmatic children with and without MP infection group and controls was statistically significant(P<0.05)(2)DC in peripheral blood of asthmatic children with MP infection group mainly expressed TLR2 and TLR4.This indicated that TLR2 and TLR4 on DC surface may involve in the development of asthma.(3) The expression of TLR2 and TLR4 on DC in MP infection group is significantly higher than normal control group and non MP infection group. The expression level of TLR2 and TLR4 was 70.30±5.84% and 54.97±5.65% in asthmatic children with MP infection, 48.59±7.86% and 54.97±5.65% in asthmatic children without MP infection, 24.35±4.70% and 19.98±5.13% in controls. There was statistically significant difference between asthmatic children with MP infection and without MP infection. But the expression level of TLR5 and TLR6 in these groups showed no differences.(4)The percentage of DC in peripheral blood,lung and spleen tissue of the asthmatic model with and without MP infection and the controls was 0.49±0.02%,0.45±0.05%, 0.14±0.01%;0.61±0.06%,0.79±0.13%,0.37±0.06%;0.97±0.15%,1.09±0.23%,0.26±0.05% respectively. The differences between asthmatic children with and without MP infection and the controls was statistically significant(P<0.01).(5) DC in peripheral blood and lung tissue of the asthmatic models with and without MP infection all mainly expressed TLR2 and TLR4. But there was no difference among the four TLRs expression of peripheral blood, lung tissue and spleen tissue in controls. There was no difference among the four TLRs expression of DC in spleen tissue.(6) The expression of TLR2 on DC in peripheral blood of the asthmatic model with MP infection,without MP infection and the control group was 87.00±3.64%,55.84±3.72%, 31.03±4.15%. The expression of TLR4 was 73.75±4.36%,48.46±3.04%,25.15±1.86%,and the expression of TLR5 was 10.71±0.30%,11.10±0.67%,18.06±3.91% respectively,and the expression of TLR6 was 11.17±0.12%,12.93±1.22%,18.47±3.65%. There was significant differences in the expression of TLR2 and TLR4 between asthmatic with MP infection and without MP infection.(P<0.01) There was significant differences in the expression of TLR2 and TLR4 between asthmatic model with MP infection and controls(P<0.01). But there was no significant difference in the expression of TLR5 and TLR6 among these groups. The result of expression level of DC in lung tissue was the same as thosein peripheral blood. But there was no difference of the four kinds of TLR in spleen tissue of all groups.3. The level of IL-9 in asthmatic children with MP infection, without MP infection and controls were 213.8±46.04pg/ml,105.9±13.50pg/ml,57.65±7.50pg/ml. There is significantly statistical differences among asthmatic children with MP infection and without MP infection and controls(P<0.01).The level of IL-17 in these groups were 42.50±13.52pg/ml,51.90±14.55pg/ml,22.16±4.39pg/ml, and no statistical difference among groups. The level of IFN-γ Were 9.20±4.47pg/ml,22.90±3.85pg/ml,93.05±37.55pg/ml respectively, and there were significantly statistical differences between asthmatic children with MP infection and controls(P<0.05).The level of IL-9 in BALF of the asthmatic model with MP infection,without MP infection and the controls were 567.8 ±23.11 pg/ml,212.8 ±21.70pg/ml, 115.3±5.50pg/ml. There was significantly statistical differences among asthmatic model with MP infection and without MP infection and controls(P<0.01). The level of IL-17 in these groups were 34.51±20.37pg/ml, 23.35±3.19pg/ml, 56.40 ±17.71 pg/ml respectively. There was no statistical difference among these groups. The level of IFN-γ was 9.20±4.47pg/ml,22.90±3.85pg/ml,93.05±37.55pg/ml respectively, There was significantly statistical differences between asthmatic model with MP infection and controls(P<0.01).This indicated that IL-9 Level was increased obviously after MP infection,while the level of IFN-γ decreased. It indicated that the deviationof Th1/Th9 may be happened after MP infection.4. The expression level of T-bet of lung tissue in the asthmatic model with MP infection,without MP infection and the controls were 6.012±1.67, 5.25±0.75,127.4 ±27.04;The expression of GATA-3 were 0.36±0.02, 0.29±0.02, 0.10± 0.00, and the expression of NF-κ B were 9.95±1.36,3.89±1.23,0.58±0.18 respectively. The expression of GATA-3 and NF-κ B of lung tissue in asthmatic model with MP infection was significantly higher than those of controls(P<0.01). The expression of T-bet was obviously lower than control group(P<0.01).Conclusions1.The MP infection rate in asthmatic children is higher than controls and children with other respiratory tract infection.It may certify that the relationship between MP infection and the pathogenesis of asthma, and MP infection may be a co-factors in the development of asthma.2.The study of DC and TOLL receptors in asthmatic children with MP infection and asthmatic mouse model with MP infection showed that TLR2 and TLR4 may play a key role in the process of MP infection in asthma. It may indicate that pathogen may active the signal transduction pathways by TLR2 and TLR4 on DC in the process of MP infection.3.The research on cytokines of T cell subgroups in asthmatic children with MP infection indicated that the level of IL-9 significantly increased and the level of IFN-γ decreased sharply after MP infection. There was no obvious change in the level of IL-17.The results indicated that the deviation of Th1/Th9 may be exist after MP infection; The level of IL-9 of BALF and plasma in asthmatic model with MP infection was significantly increased too. These finding further proved that the core effect of IL-9 in the development of asthma with MP infection4.The study on T cell subgroup related transcription factors of T-bet,GATA-3 and NF-κ B showed that the expression level of GATA-3 and NF-κ B in lung tissue of asthmatic model increased, which manifested MP infection may influence the expression of Th2 related transcription factors after MP infection. |
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