Anti-viral Innate Immunity Mediates Airway Remodeling And Alveolar Rarefication In COPD Through Regulation Of Lung Epithelial Stem/Progenitor Cells And Injury/Repair Responses

ObjectiveTo isolate and identify lung epithelial stem/progenitor cells and to define their role in repair response of lung epithelium.MethodSingle pneumocyte suspension was obtained from normal C57BL/6 mice. After stained with CD45-PE, CD31-PE and Sca-1 (Stem Cell Antigen-1)-FITC, cell suspension was analyzed with flow cytometry to assess expression and cell cycle of CD45-/CD31-/Sca-1+ cells. CD45-/CD31-/Sca-1+ cells were also isolated by FACS (Fluorescence Activated Cell Sorting) from lung tissue. They were identified by IF (ImmunoFluorescence) staining on cytospin slides and cultured in vitro to detect their capacity of proliferation. The response of CD45-/CD31-/Sca-1+ cell population to injury and its role in epithelial repair was defined with a viral PAMP (Pathogen-Associated Molecular Pattern) induced lung injury model in vivo.ResultCD45-/CD31-/Sca-1+ cells were expressed at a low level in normal murine lung. Compared with CD45-/CD31-/Sca-1+ cells, CD45-/CD31-/Sca-1+ cells were not only enriched for cell cycle entry but also grew into clonal cluster in vitro. IF staining revealed CD45-/CD31-/Sca-1+ population mainly consisted of BASCs (BronchioAlveolar Stem Cells), ATⅡ (Alveolar TypeⅡEpithelial Cells) and Clara cells. In vivo, we found the expression of CD45-/CD31-/Sca-1+population increased dramatically in response to viral double-stranded RNA (dsRNA) anolog Poly(I:C). CD45-/CD31-/Sca-1+population showed a significant increase over baseline immediately following the second dose of Poly(I:C) and peaked 72 hours following the last dose after which they declined nearly to baseline. Furthermore, it displayed similar kinetics to expression of Ki67 (a proliferating marker) in lung.ConclusionCD45-/CD31-/Sca-1+cells comprise of a heterogeneous population exhibiting some progenitor properties. As they showed similar kinetics to proliferation in Poly(I:C) induced lung injury, it is reasonable to conclude that this cell population represents a repair response in the Poly(I:C) exposed lung. Part Two The roles of lung epithelial stem/progenitor cells and injury/repair imbalances in cigarette smoke and viral PAMP-induced COPDObjectiveTo define the biology of lung epithelial stem/progenitor population and the role of injury/repair imbalances in COPD.MethodA lung injury model was established by dual exposure of cigarette smoke (CS) and Poly(I:C). We assessed physiologic alterations in airway and alveolus, expressions of lung CD45-/CD31-/Sca-1+epithelial stem/progenitor cells and levels of apoptosis and proliferation in airway and alveolus respectively through TUNEL and Ki67 staining.ResultSignificant airway fibrotic remodeling and alveolus septal destruction were observed in lung tissue exposed to CS and Poly(I:C). It manifests that this lung injury model induced by dual exposure of CS and Poly(I:C) is an effective COPD animal model mimicked airway remodeling and alveolar rarefication which are important characteristics of COPD. In this model, although a great increase was shown in contrast with control group, the expression of CD45-/CD31-/Sca-1+epithelial stem/progenitor cells declined remarkably compared with the group exposed to Poly(I:C) only. It suggests that CS inhibits the expansion of lung epithelial stem/progenitor cells induced by Poly(I:C). Data from TUNEL and IF staining showed that airway epithelium appeared higher proliferation and alveolar epithelium displayed higher apoptosis in lungs exposed to both CS and Poly(I:C).ConclusionCS and Poly(I:C) induced lung injury is an effective COPD model productively imitated airway fibrotic remodeling and alveolar septal destruction which are essential characteristics of COPD; CS inhibits Poly(I:C) induced expansion of lung CD45-/CD31-/Sca-1+epithelial stem/progenitor cells suggesting that dual exposure of CS and Poly(I:C) results in dysfunction of repair responses in lung epithelium by its effect on lung epithelial stem/progenitor population; In this COPD model, different imbalances between injury and repair responses are presented in airway and alveolus respectively:airway epithelium appears more proliferation and alveolar epithelium displays more apoptosis. It is consist with lung remodeling we observed manifesting that injury/repair imbalances are involved in tissue remodeling of COPD. Part Three The effects of antiviral innate immunity on lung epithelial stem/progenitor population and injury/repair responses in COPDObjectiveTo define the effects of TLR3 (Toll-Like Receptor 3) and RLH-MAVS (RIG-Like Helicases-Mitochondrial Antiviral Signaling Protein) antiviral signaling pathways on expression of lung epithelial stem/progenitor population and injury/repair responses in COPD.MethodWild type (WT), TLR3 null (TLR3-/-) and MAVS null (MAVS-/-) mice were insulted by CS and Poly(I:C) as previous. We assessed physiologic alterations in airway and alveolus, expressions of lung CD45-/CD31-/Sca-1+ epithelial stem/progenitor cells and levels of apoptosis and proliferation in airway and alveolus respectively through TUNEL and Ki67 staining in each type of mice.ResultCompared with WT mice, inflammatory infiltration and expansion of lung CD45-/CD31-/Sca-1+population were dramatically decreased in lungs of CS+P exposed TLR3-/- and MAVS-/- mice. While deletion of MAVS ameliorated both airway fibrotic remodeling and alveolar destruction, absence of TLR3 only improved airway hyperplasia and fibrosis. TUNEL and IF staining showed that removals of TLR3 and MAVS both inhibited epithelial apoptosis and proliferation. In CS+P exposed TLR3-/- mice, apoptosis and proliferation in airway appeared similar while proliferation was much lower than apoptosis in alveolus. However, CS+P challenged MAVS-/- mice presented nearly same levels of apoptosis and proliferation in both airway and alveolus. These data demonstrates that deletion of MAVS can prevent both CS+P induced over-repair in airway and under-repair in alveolus, but absence of TLR3 only inhibits over-repair in airway.ConclusionIn CS and Poly(I:C) induced COPD murine model, both TLR3 and RLH-MAVS antiviral signaling pathways are implicated in inflammation response in lung. These pathways also mediate lung remodeling through regulating the expression of CD45-/CD31-/Sca-1+ epithelial stem/progenitor cells and the balance between cell apoptosis and proliferation. Airway remodeling dominated by too much repair is mediated by TLR3, while alveolar destruction dominated by too little repair is mediated by RLH-MAVS.