Mechanisms Of Rapamycin And Autophagy In Airway Inflammation And Eosinophil Differentiation In Asthma

Asthma is a chronic airway inflammatory disease, which is characterized by reversible airway obstruction, bronchial hyperresponsiveness and airway inflammation. Among these, allergic airway inflammation in asthma is characterized by eosinophil infiltration. Studies have established that a causative relationship exists between eosinophils and the development of allergic asthma, and eosinophils participate in a variety of functions, including antigen presentation, cytokine production, chemokine production, secretion of granule mediators, and leukotriene secretion. Eosinophil differentiate from a common myeloid progenitor (CMP) in mice through an intermediate granulocyte/macrophage progenitor (GMP) and then via an eosinophil lineage-committed progenitor (EoP). The development of eosinophils is orchestrated by several transcription factors such as GATA-1, in the presence of certain cytokines, in particular Interleukin-3(IL-3), IL-5, and Granulocyte-macrophage colony stimulating factor (GM-CSF). Among these, IL-5seems to be more specific and efficient in eosinophil lineage development as it promotes the selective differentiation of eosinophils. However, little is known about the molecular mechanisms of eosinophil differentiation during asthma development.Autophagy is a regulated pathway for internal organelle or protein degradation. In this dynamic process, double-membraned autophagic vacuoles (AVs) or autophagosomes surround cytosolic organelles (e.g. endoplasmic reticulum, mitochondria) or protein, and subsequently fuse with lysosomes, where the engulfed components are degraded by lysosomal hydrolases. Autophagy provides essential functions in the maintenance of cellular homeostasis and adaptation to adverse environments. However, excessive autophagy may be associated with the activation of programmed cell death (i.e. apoptosis) through a cell-autodigestive process. The role of autophagy, whether protective or deleterious, in human diseases, or specifically in chronic lung disease remains obscure. It has been recently reported that autophagy plays an important role in smoking-induced epithelial cell injury and emphysema. However, the potential functions of autophagy in asthma, to our knowledge, have not been investigated so far.Rapamycin is a macrolide product of Streptomyces hygroscopius that was initially in a soil sample from Easter Island (Rapa Nui) in the early1970s. Its major cellular target, mammalian target of rapamycin (mTOR), is a central regulator of cell growth/differentiation and survival in many cell types. Previous works using rapamycin in animal models of allergic asthma showed controversial results. In some studies rapamycin have been suggested to inhibit cardinal features of allergic asthma, including airway hyperresponsiveness (AHR), eosinophilic airway inflammation, goblet cell hyperplasia, and IgE production; however, there are other works have also shown that it has little effects on the allergic airway inflammation and AHR. Most importantly, the underlying mechanisms that mediated the effects of rapamycin in the process of allergic asthma remain largely unknown.Recently, lots evidences showing that autophagy and mTOR play an important role in hematopoietic stem cell maintenance and differentiation. It has been shown that conditional deletion of ATG7or ATG5throughout the hematopoietic system in mice results in severe anemia and inhibition of lymphocyte survival and function. Also, mTOR provides a critical link between T cell differentiation, function and metabolism. We hypothesized that autophagy and mTOR would involve in the regulation of myeloid proliferation, especially in the eosinophil differentiation, and examined their contribution to its overall effect in allergic airway inflammation. Moreover, it will bring the new insight into the prevention of asthma and other allergic disorders for the public health.In this study, we have explored that:(1) Role of rapamycin in allergic airway inflammation and in eosinophil differentiation;(2) the different roles of autophagy in regulation of airway epithelial cell injury and eosinophil differentiation in bone marrow during asthma development.Part I Mechanisms of rapamycin in airway inflammation and eosinophil differentiation in asthmaObjective:To investigate the effect of rapamycin in eosinophil differentiation and asthma airway inflammation, and to explore the underlying molecular mechanisms.Methods:Female C57BL/6mice were randomly divided into four groups:SHAM group, OVA group, SHAM+RAPA group and OVA+RAPA group. In the OVA and OVA+RAPA group, mice sensitized and challenged with ovabumin (OVA).In SHAM and SHAM+RAPA group, mice were sensitized and challenged with normal saline by the same methods. In the RAPA and OVA+RAPA group, mice were treated with an intraperitoneal injection of rapamycin1hour before each aerosol OVA challenge.24hours after the final OVA challenge, mice were sacrificed and the number of eosinophils in bronchoalveolar fluid (BALF), blood and bone marrow were accessed. Lungs were processed for histologic staining (HE), and airway inflammation was analyzed. Levels of selected cytokines (IL-5, IL-13) in serum were assayed using ELISA kits. Lung lymphocytes were isolated and labeled with T cells surface Abs (CD4, CD8and CD25) and intracellular Abs (IL-4, IL-17A, Foxp3). Bone marrow Eosinophil progenitors (Eops) were determined as Lin-Sca-1-CD34+IL-5Ra+c-Kitlo cells. Data were collected and analyzed using a FACScan for seven-color flow cytometry. Western blot of phosphorylated S6(P-S6) ribosomal protein was analyzed to explore whether mTOR is involved in eosinophil differentiation. In vitro methylcellulose colony forming assays and liquid culture of mouse bone marrow-derived eosinophils (BM-Eos) were also performed to assess the ability of eosinophil differentiation and function. Finally, we used IL-5transgenic NJ.1638mice with treatment of rapamycin, to investigate the levels of eosinophils and apoptosis of Eos in blood and bone marrow, the levels of IL-5in serum, and Eops in BM were also analyzed.Results:Rapamycin significantly attenuated OVA-induced allergic airway inflammation, without influence on the various subtypes of T helper cells. It markedly decreased the amount of eosinophils in local airways, peripheral blood, and bone marrow, independent on levels of IL-5. In vitro colony forming unit assay and liquid culture demonstrated that rapamycin directly inhibited IL-5-induced eosinophil differentiation. In addition, rapamycin reduced the production of IL-6and IL-13by eosinophils. Rapamycin was also capable of significantly reducing the eosinophil levels in IL-5transgenic NJ.1638mice, again regardless of the constitutive high levels of IL-5, and without influence on the eosinophil apoptosis. Interestingly, rapamycin inhibition of eosinophil differentiation in turn resulted in an accumulation of eosinophil progenitors in bone marrow.Conclusion:Altogether these results clearly demonstrate a direct inhibitory role of rapamycin in eosinophil differentiation and function, and reemphasize the importance of rapamycin and possibly, mTOR in allergic airway disease. Part II Mechanisms of autophagy in airway inflammation and eosinophil dfifferentiation in asthmaObjective:To investigate the effect of autophagy in eosinophil differentiation and asthma airway inflammation, and to explore the underlying molecular mechanisms.Methods:Western blot of LC3B was analyzed to explore whether autophagy is involved in eosinophil differentiation. Eosibophil, Eosinophil progenitor level and the ability of eosinophil differentiation were assessed in Beclin-l+/-mice (autophagy related gene Beclin-1impaired). Beclin-1+/-and wild type (WT) mice were randomly divided into four group:WT/NS group, WT/OVA group, BECN/NS group and BECN/OVA group.24hours after the final OVA challenge airway hyperresponsiveness to inhaled methacholine, airway inflammation and airway mucus expression were measured. Lung tissue sections were also processed for transmission electron microscope (EM) and analyzed for formation of autophagic vacuoles (AVs). MUC5AC was induced by IL-13in BEAS-2B cells with or without starvation, and the mRNA expression was detected by Q-PCR. Finally, we transplanted the bone marrow of Beclin-1+/-mice to WT, and subjected these mice for the OVA asthma model to explore the contribution of local autophagy defect of bone marrow to its overall effect in asthma.Results:The number of eosinophil in blood and bone marrow in vivo, and Eo-CFU in vitro colony forming assays were significantly increased in Beclin-1+/-mice. However, compared with WT/OVA group, BECN/OVA group showed significantly decreased AHR, airway inflammation and mucus overproduction. We found that OVA induced the accumulation of AVs in the airway epithelia cells as determined by EM. In vitro culture, starvation increased IL-13induced MUC5AC mRNA expression. In bone marrow transplantation test, AHR and airway inflammation were increased in BECN-WT-OVA group, compared with WT-WT-OVA group.Conclusion:Our data suggested that autophagy mediated OVA-induced airway epithelial injury and Beclin-1defect protected the asthma features, including airway hyperresponsiveness, airway inflammation and airway mucus expression. On the other side, autophagy suppressed the eosinophil differentiation during asthma development. Targeting mTOR and autophagy may lead to effective therapeutics for asthma.