Potential Contribution Of T Helper Type17Survival To Neutrophilic Airway Inflammation In Asthma

Asthma is a chronic inflammatory disorder of the airway which involves many cells. For a long time, the eosinophils (EOS) were regarded as the most important inflammatory effector cells which cause asthma. However, with on-going research into the causes for airway inflammations of asthma, two different types of asthma are identified, namely eosinophilic inflammation (EA) and non-eosinophilic inflammation (NEA), whereas for the NEA, more than half are characterized by neutrophilic asthma (NA). In our previous study, we had demonstrated delay of human neutrophil apoptosis is a potential mechanism contributing to airways neutrophilia in asthma, but the exact mechanism that underlie this phenomenon remains unknown.Past animal experiments on asthma are completed on the traditional EA model, which failed to reflect the airway inflammations and hyperresponsiveness of NA. To avoid such shortcomings, in our study we established a NA model by sensitization through the airway. Briefly, mice were sensitized by airway delivery of100ug of the ovalbumin (OVA) solution complexed in O.lug lipopolysaccharide (LPS) on Days0,6and13. For challenges, mice were exposed to an aerosol of1%OVA in saline. It turned out that the mice showed symptoms and pathologic changes similar to asthma, neutrophilic airway inflammation, airway mucus hypersecretion which is similar to eosinophilic asthma model, and the more severe airway hyperresponsiveness (AHR). These demonstrated that we have successfully established the NA mice model and laid the foundation for further studies.TH1/TH2paradigm is regarded as the most important immunological mechanism in asthma. T helper type2(TH2) cells play a key role in asthma pathogenesis by producing a variety of cytokines such as IL-4, IL-13, IL-5, which mediate airway eosinophilic inflammation and AHR. Recent studies showed that T helper type17(TH17) cells are a new subset of T helper cells which may be involved in the pathogenesis of asthma. TH17cells can secrete a variety of cytokines, the most important of which is the IL-17A (IL-17). IL-17send out signals to target cells, and induce target cells to produce a variety of cytokines such as IL-8, IL-6, granulocyte macrophage colony factor (GM-CSF), and CXC chemokine Ligand (CXCL), leading to chemotaxis and activation for neutrophils to infiltrate to inflammatory sites. Whether the two types of immunological mechanisms are involved respectively in EA and NA or both?Our research showed that the mice experimented with the above-mentioned two asthmatic models have higher percentage of TH17and TH2cells in their spleens than mice in the control group. It suggested that the naive T cell differentiation towards TH17and TH2cells are intensified both in NA and EA mice, and that TH17and TH2cells are both involved in the mechanism of EA and NA. Based on further analysis on the proportion of TH2/TH17, we found that EA group was higher than the NA group, accompanied by significantly increased IL-17and slightly increased IL-5, suggesting that TH17cells play a more important role in the pathogenesis of NA mice. Differentiation of TH17cells advantage is achieved by increasing the positive regulation of the differentiation-specific transcription factor orphan nuclear receptor yt (RORyt) expression, elevated cytokines such as IL-6and transforming growth factor-β (TGF-β) formed in vivo microenvironment conducive to the differentiation of TH17cells. Moreover, the TH17cells differentiation advantages are relevant to down-regulated expression of suppressors of cytokine signaling3(SOCS3) in the NA mice, and the TH2cells differentiation advantages are relevant to up-regulated expression of SOCS3in the EA mice.The modern immunology suggests that when activated T cells is again encountered with stimulus, they could cause activation induced cell deaths (AICD), so as to limit excessive immune response. Any exceptional condition in the AICD process may have resulted in the body abnormal performance and even disease. Therefore, the differentiation of TH17cells and the survival of the differentiated TH17cells is likely to affect the occurrence and development of airway neutrophilic inflammation. Our research found that in the spleens of NA and EA asthmatic mice, the Ki-67expression of the TH17cells was significantly higher than that in the control group, indicating that for asthmatic mice the TH17cells apoptosis is constrained, and that the deaths of TH17cells are delayed. Interleukin-7(IL-7) is a precious pleiotropic cytokine, whose main function is to promote the growth of B and T cells, T cell survival and proliferation. The question is, whether or not the TH17cell survival mechanism of asthmatic mice is dependent on the IL7-IL7receptor signaling pathways?We detected the IL-7expression in the spleen and bronchoalveolar lavage fluid (BALF), and found that the IL-7expression was up-regulated in both EA and NA asthma mice which in turn provides a favorable microenvironment to extend the survival of TH17cells. Compared with the control group, NA mice showed increased expression of transducer and activator of transcription-5(STAT5), B-cell lymphoma-2(BCL-2) and cysteine-containing aspartate-specific proteases3(Caspase-3) expression in TH17cells of the spleen, suggesting that prolonged survival of TH17cells in NA mice by the way of IL7-IL7receptors depends on Janus kinase-signal transducer and activator of transcription-5(JAK-STAT5) signal pathway activation, and by up-regulating anti-apoptotic protein BCL-2, inhibition of apoptosis of TH17cells; at the same time activate apoptosis terminal shear enzyme Caspase-3, suggesting that Th17cells apoptosis depends on caspase pathway. But the increasing range of BCL-2was greater than that of Caspase-3, suggesting that the anti-apoptotic action of BCL-2predominated in the interaction of anti-apoptotic effect and pro-apoptotic effect. Moreover, IL-7signaling pathway can be interrupted by some negative regulation mechanisms. For example, suppressors of cytokine signaling1(SOCS1) as inhibitors of cytokine signaling, could suppress STAT5signal transmission while maintaining a moderate inflammatory reaction. In our study we found that reduced SOCS1expression in NA and EA asthmatic mice may cause IL-7signal hypersensitivity reaction in the body, resulting in the long-term survival of TH17cells.How are the TH17cells with enhanced expression engaged in the formation of the airway neutrophilic inflammation? Similar to previous studies, we found higher average IL-17levels in BALF of EA and NA asthmatic mice. We also found that the level of chemokines CXCL1, CXCL5, CXCL8/IL-8was also significantly increased, and was positively correlated with the IL-17level, suggesting that the TH17cells, at least partially by the way of inducing these chemokine expression, cause the gathering of neutrophils where the airway inflammation occurs. Consistent with our previous study on asthma patients, through NA asthmatic mice study we also found a positive correlation between the enhanced airway neutrophils expression and delayed airway neutrophil apoptosis. Moreover for asthmatic mice with nebulization for14days, we also found positive correlation between IL-8levels in BALF and AHR-suggesting a possibility that the TH17cells, by stimulating target cells to secrete a variety of cytokines through IL-17, is involved in airway neutrophils inflammation and AHR formation.Glucocorticoid is currently the most effective first-line drugs to control airway inflammations in asthma. Systemic dexamethasone application on asthmetic mice mitigates pathological changes of lung tissue, significantly reduces the number of BALF cells and neutrophils, but the level of such cells is still higher than that of the control group. TH17cell numbers and related cytokines IL-17levels are decreased, demonstrating that dexamethasone may have alleviated the airway neutrophilic inflammation symptom by reducing the TH17cell numbers and cytokine levels. However, dexamethasone does not affect the airway neutrophil apoptosis rate of NA asthmatic mice, nor could it inhibit the TH17cell survival in both EA and NA asthmatic mice.In summary, our research successfully established a neutrophilic asthma mice model, confirmed on the basis of this model that we elucidated that the NA mice were primed only modest TH2responses, but strong Th17responses. The TH17cell differentiation advantages is regulated by positive adjustment (RORyt) and a negative adjustment (SOCS3), and the elevated IL-6and TGF-β formed in vivo microenvironment conducive to the differentiation of TH17cells. Differentiated TH17cells depend on the IL7-IL7receptor signaling pathway to maintain their survival status. Increased IL-17participates in airway neutrophilic inflammation and AHR formation through a variety of ways. Dexamethasone can reduce airway neutrophilic inflammation by down-regulating the number of TH17cells and cytokine levels; but has no significant effect on airway neutrophil apoptosis rate and prolonged survival status of TH17cells; it may be one of the molecular mechanisms of steroid-resistance (SR) of asthma. This study elucidates the potential contribution of TH17cell survival to neutrophil airway inflammation in neutrophilic asthma and the related molecular mechanisms, it will provide a novel scientific basis for the asthma therapy.