Effect Of Cigarette Smoking On Th1/Th2 Imbalance And Its Mechanisms Of Transcription Regulation In Asthma

Asthma is a serious global health problem. Worldwide prevalence and severity of asthma is increasing in the general population. Although this increase is probably the result of several factors, epidemiological studies have implicated both a decrease in childhood infections and an increase in environmental pollution as risk factors. Cigarette smoking (including passive and active smoking) is the major risk factor for astham. Epidemiological studies have inplicated that almost one third of asthmatic patients are often exposured to cigarette smoking. It is estimated that around 50% of adult asthmatics in the developed world probably are or have been smokers.Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role, sometimes associated with airway structural changes, often described as airway remodeling. Smoking favors the development of a complex pathophysiology of inflammatory airway disease. Tobacco smoking makes asthma more difficult to control, results in more frequent exacerbations and produces a more rapid decline in lung function and poorly responsive to glucocorticosteroids. However, the mechanisms whereby cigarette smoking influence the development and expression of asthma are complex and interactive, so still poorly understood today.Epidemiological studies have linked cigarette smoking to community-acquired pneumonia, higher incidences of lung cancer, respiratory infections. It is well accepted that many of these health consequences may be attributable to cigarette smoke-induced changes in host immunity. Cigarette smoke contains nicotine, an immunomodulatory component that is thought to affect immune surveillance and increase the progression of diseases.Imbalance between Th1 and Th2, higher levels of Th2 cytokine IL-4, 1L-5 and IL-13 and lower levels of Thl cytokine IFN-γis prominent in the pathogenesis of asthma. More recent studies clearly demonstrated that Th2 cells and their typical cytokines play a critical role not only in airway inflammation but also in the development of airway remodeling. T helper cell (CD4+T cell, Th) differentiate into two subsets, T helper cell type 1(Thl) and T helper cell type 2 (Th2), each with distinct functions and cytokine profiles. Interferon (IFN)-γis the signature cytokine of Thl cells. Interleukin (IL)-4 is the corresponding signature cytokine of Th2 cells, which also secrete IL-5 and IL-13, these two subgroups of T helper arise in response to different immunogenic stimuli and cytokines. T box expressed in T cells (T-bet) and GATA binding protein-3(GATA-3), as the specific transcription factors of Thl and Th2 respectively and the upstream factors of Thl/Th2 balance are an active area of research. Recently significant progress has been made in identifying the transcription factors that control the transcription of a T helper cell to a Th2 or Thl cell, the results show that there is significantly a positive correlation between the level of T-bet/GATA-3 expression and IFN-γ/IL-4. A subset of CD4 cells, known collectively as CD4+CD25+regulatory T cells (Treg), are a central component of active immune suppression. Forkhead transcription facter3 (Foxp3) is a critical transcription factor in regulatory T cells development. The pathogenesis of asthma has been regarded as between Thl and Th2 cells, which is now considered as superficies. In fact, reason for the activation and proliferation of CD4+T cells and differentiation towards Th2 cells is the dysfunction of CD4+CD25+Treg in patients with asthma.So the objective of this study is to investigate the impact of cigarette smoking on Th1/Th2 banlance and its mechanism of transcription regulatory in the pathogenesis of asthma.Contents 1. Effect of cigarette smoke exposure on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in asthmatic rat.2. Effect of cigarette smoke extract on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured splenocytes of rat sensitized by ovalbumin.3. Effect of nicotine on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured CD4+ T of rat sensitized by ovalbumin.4. Effect of cigarette smoke exposure on the percentage of CD4+CD25+T cell and the expression of transcription factor Foxp3 in asthmatic rat.MethodsPart one: Effect of cigarette smoke exposure on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in asthmatic rat.1. Forty male Wistar rats were randomly divided into four groups: control group, aerosolized ovalbumin exposure group, cigarette smoke exposure group and OVA combined cigarette, smoke exposure group. Rats were exposed to either aerosolized OVA, tobacco smoke, or both tobacco smoke and OVA. 2. The change of airway histology was observed by HE and Masson.3. The concentration of interleukin-4 (IL-4) and interferon-γ(INF-γ) in peripheral blood and lung hemogenates were measured by enzyme-linked immunosorbent assay (ELESA).4. The nucleus protein expression of T-bet and GATA-3 in the lungs were detected by Western blot.5. The mRNA expression of T-bet and GATA-3 in the lungs were detected by quantitative real time polymerase chain reaction (real time PCR) using ready-made fluorogenic probes and primers.Part two: Effect of cigarette smoke extract on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured splenocytes of rat sensitized by ovalbumin.1. Two weeks after immunization by ovalbumin, spleens were excised aseptically from rats and after lyses of RBCs Splenocytes were obtained from singled-cell suspension of spleen.2. Splenocytes cultured in 0.5, 1, 2.5 percent cigarette smoke extract (CSE) solution prepared with RPMI, with which splenocytes were treated simultaneously with ovalbumin, supernatants and cell pellets were harvested after cultured for 24 hours.3. The concentration of INF-γ/IL-4 in Supernatants was measured by enzyme-linked immunosorbent assay.4. The mRNA expression of T-bet/GATA-3 in Splenocytes were detected by quantitative real time polymerase chain reaction using ready-made fluorogenic probes and primers.Part three: Effect of nicotine on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured CD4+ T of rat sensitized by ovalbumin.1. Two weeks after immunization by ovalbumin, spleens were excised aseptically from rats and after lyses of RBCs splenocytes were obtained from singled-cell suspension of spleen.2. Splenic CD4+ T cells were purified using CD4+T cell enrichment kit.3. Purified CD4+ T cells were stimulated in ovalbumin with or without nicotine. Supernatants and cell pellets were harvested after cultured for 24 hours.4. The concentration of INF-γ/IL-4 in supernatants was measured by enzyme-linked immunosorbent assay.5. The mRNA expression of T-bet/GATA-3 in CD4+T cells was detected by quantitative real time polymerase chain reaction using ready-made fluorogenic probes and primers.Part four: Effect of cigarette smoke exposure on the percentage of CD4+CD25+T cell and the expression of transcription factor Foxp3 in asthmatic rat.1. Forty male Wistar rats were randomly divided into four groups: a control group, aerosolized ovalbumin exposure group, cigarette smoke exposure group and ovalbumin combined cigarette, smoke exposure group. Rats were exposed to either aerosolized ovalbumin, tobacco smoke, or both tobacco smoke and ovalbumin.2. Percentage of CD4+CD25+T cell was determined by flow cytometry analysis.3. The protein expression of Foxp3 in the lungs was detected by Western blot.Results1. Pathological change of the rat groupsThe thickness of airway wall and expression of collagen in OVA combined cigarette smoke exposure group was higher than that in aerosolized OVA exposure and the control group(P<0.01).2. Protein levels of INF-γ/IL-4 in plasma and lungThe plasma and lung levels of IL-4 in aerosolized OVA exposure were significantly higher than those in control group (P<0.05), respectively. The plasma and lung level of IFN-γwas significantly lower than that in control group (P<0.05), The plasma and lung levels of IL-4 in Combining OVA with cigarette smoke exposure group were significantly higher than those in aerosolized OVA exposure group (P<0.05),respectively, The plasma and lung levels of IFN-γin asthma group was significantly lower than that in aerosolized OVA exposure group(P<0.05),and there was no statistical difference between the control group and the cigarette exposure group (P >0.05).3. Protein expression of T-bet/GATA-3 in lungNucleus protein expression of T-bet in OVA combined cigarette smoke exposure group were lower than that in the control group and aerosolized OVA exposure group(all P<0.01), meanwhile nucleus protein expression of GATA-3 in OVA combined cigarette smoke exposure group were higher than that in the control group and aerosolized OVA exposure group(all P<0.01).4. mRNA expression of T-bet/GATA-3 in lungThe T-bet mRNA expression in lung in OVA combined cigarette smoke exposure group was significantly attenuated than those in aerosolized OVA exposure group and control group(P<0.05),meanwhile the GATA-3 mRNA expression was significantly enhanced(P<0.05) respectively.5. The effect of cigarette smoke extract (CSE) on the production of INF-γ/IL-4 in cultured splenocytes The production of INF-γin CSE treatment groups were significantly lower than that in the control group, P<0.05, and there were significant difference in 0.5, 1, 2.5 percent CSE treatment group in term of INF-γproduction, P<0.01. CSE treatment cause dose-dependent repression of INF-γproduction. The production of IL-4 in CSE treatment groups were significantly higher than that in the control group, P<0.01, and there were significant difference in 0.5, 1, 2.5 percent CSE treatment groups in term of IL-4 production, P<0.05. CSE treatment cause dose-dependent stimulated of IL-4 production.6. The effect of cigarette smoke extract (CSE) on mRNA expression of T-bet/GATA-3 in cultured splenocytesThe mRNA expression of T-bet in CSE treatment groups were significantly lower than that in the control group, P<0.05, and there were significant difference in 0.5, 1, 2.5 percent CSE treated group in term of T-bet The mRNA expression, all P<0.01. CSE treatment cause dose-dependent repression of T-bet the mRNA expression. The mRNA expression of GATA-3 in CSE treatment groups were significantly higher than that in the control group, P<0.01, and there were significant difference in 0.5, 1, 2.5 percent CSE treatment groups in term of GATA-3 mRNA expression, all P<0.05. CSE treatment cause dose-dependent stimulated of IL-4 production.7. The effect of nicotine on the production of INF-γ/IL-4 in cultured CD4+TWhen challenged simultaneously with OVA, exposure of nicotine not only induced IL-4 but also inhibits INF-γproduction in a dose dependent manner.8. The effect of nicotine on mRNA expression of T-bet/GATA-3 in cultured CD4+T cellsWhen challenged simultaneously with OVA, exposure of nicotine not only induced mRNA expression of GATA-3 but also inhibits mRNA expression of T-bet in a dose dependent manner.9. Percentage of CD4+CD25+T cell in peripheral bloodPercentage of CD4+CD25+T cell in aerosolized OVA group was significantly lower than that in the control group (P<0.01), meanwhile percentage of CD4+CD25+T cell in OVA combined cigarette smoke exposure group was lower than that in aerosolized OVA exposure group(P<0.01). Percentage of CD4+CD25+T cell in cigarette exposure group was significantly higher than that in the control group and the (P<0.01).10. Protein expression of Foxp3 in lungProtein expression of Foxp3 in aerosolized OVA group were lower than that in the control group(P<0.01), meanwhile protein expression of Foxp3 in OVA combined cigarette smoke exposure group were lower than that in the control group and aerosolized OVA exposure group(all P<0.01). There was no statistical difference between the control group and the cigarette exposure group.Conclusions1. In this study cigarette smoke exposure play a key role in the development of Th2-type allergic inflammation in asthma by promoting over-expression of transcription factor GATA-3 and repressing the expression of T-bet.2. Nicotine play a key role in the development of Th2-type allergic inflammation in asthma.3. The ratio of CD4+CD25+Treg and the expressions of Foxp3 decreased in athmatic rats which may play an important role in the pathogenesis of asthma.4. Cigarette smoke exposure can downregulate the ratio of CD4+CD25+Treg and the expression of Foxps which was the upstream factors in cigarette smoking-induced Th1/Th2 imbalance.