The Functions Of Exosome From Lung On Maintenance Of Immune Balance Of Respiratory Tract And The Underlying Mechanisms

A vast quantity of environmental antigens settle upon the respiratory mucosal surface and challenge the immune system, but respiratory immune system always maintain a dynamic balance, which suggests that there are fine regulations in respiratory immune system. Along with the evolution of human beings, our immune system gradually formed a several effective ways to maintain immune tolerance to harmless foreign antigens, including clonal anergy, clonal ignorance and by regulatory T cells secrete IL-10 and TGF-β1 inhibitory cytokines to prevent activation of the immune effector cells. Once lung immune tolerance balance is disturbed which will result in respiratory- related autoimmune diseases, such as asthma. Asthma is one of the most common respiratory diseases related immune imbalances. Currently, it is considered that the disorder of Thl/Th2 cell is the main mechanism of asthma development. Immune imbalance is an important pathogenesis basis of asthma. Therefore to further elucidate the immune tolerance mechanism of respiratory tract may also provide new ideas for the treatment of asthma.Exosomes are secreted by various live cells with lipid bilayer structure of small vesicles and its diameter is mainly between 30-100 nm, with density range from 1.09 g/ml to 1.17 g/ml. It has been demonstrated that exosome plays an important role in not only promoting but inhibiting immune responses. The source of exosome in this paper is from the lung tissues of mice. We investigate whether lung tissue-derived exosome participates in the maintenance of lung immune tolerance. In this study, through murine models of asthma, we verify our hypothesis and want to provide new ideas for rebuilding the imbalance of immune tolerance in the respiratory tract.Firstly, we identified the exosome of the lung (L-EXO). Electron microscopy results showed that diameter of the L-EXO was generally ranged from 30 to 100 nm, and the density of L-EXO fell 1.09 g/ml to 1.17 g/ml. L-EXO contained exosomal marker protein such as CD63, TSG101 and HSP70 but not plasma membrane-associated protein GRP94. In addition, L-EXO also contained TGF-β1 and IL-10.We found that L-EXO significantly inhibited the proliferation of CD4+ T cells in vitro. And then we found that the ability of L-EXO to inhibit the proliferation of CD4+ T cells was almost abolished after TGF-β1 was blocked by neutralized antibodies, which indicated that the immunosuppressive function of L-EXO depends on TGF-β1. In addition, we examined origin of L-EXO. Whereas CD8, CDllc and SPB (marker of alveolar epithelial cells) could be found in L-EXO, we isolated CD8 or CD11c positive exosome. The CD11c positive L-EXO was also positive for CD8, and vice versa. And only this L-EXO subset had the ability to inhibit the proliferation of CD4+ T cells, indicating immunosuppressive L-EXO were derived from CD8-positive dendritic cells (dendritic cells, DCs).Then, we used OVA induced murine acute asthma model to investigate whether L-EXO is involved in the maintenance of immune tolerance of lung in vivo. Through analyzing the cell compositions, various cytokines in bronchoalveolar lavage fluid (BLAF) and pathology of lung by tail vein transfer of L-EXO, we found that L-EXO could relieve the symptoms of asthma. We also found that although still has protective effect on asthma symptoms, this effect of L-EXO is not as good as wild type (WT) mice when transferred L-EXO into Smad3+/- mice. IL-10 was also found in the L-EXO. Therefore, we assessed whether IL-10 was involved in the protective of L-EXO on asthma. Similarly, transfer of L-EXO from IL-10-/- mice into C57 mice showed impaired protective effect on asthma. These data suggest that L-EXO plays an important role in maintaining immune tolerance depending on TGF-β1 and IL-10.To further confirm the role of L-EXO in maintaining immune tolerance of the respiratory tract, we used a neutral sphingomyelinase small interfering RNA to knock down Smpd3 in 3LL lung cancer cells, and found along with the knockdown of Smpd3, 3LL cells produced less exosome in vitro. The L-EXO could be reduced in mice when mice were intranasally administrated with Smpd3 interference lentivirus. Under this condition, the enhanced susceptibility of mice to OVA induced acute asthma was detected and when supplying exogenous L-EXO, the enhanced susceptibity was abrogated.In summary, we find that L-EXO carrying TGF-β1 and IL-10 possesses immunosuppressive effects and could alleviate OVA induced acute asthma in mice. These results imply that L-EXO is involved in the maintenance of immune tolerance in the lung. Our results provide a new mechanism to explain the immune tolerance of lungs. It also suggests that we can treat asthma and other respiratory diseases related to immune imbalance by increasing the secretion of exosome.