Tsc1 Controls The Development And Function Of Alveolar Macrophages

Objective:Because hematological malignancies are often accompanied by immune deficiency and bone marrow suppression and neutrophil deficiency,hematologic patients are prone to lung infections,including bacterial pneumonia and fungal pneumonia and viral pneumonia and multiple infections.Alveolar macrophages(AMs)are lung-resident macrophages important for the maintenance of surfactant homeostasis in the alveolar space.AMs are the first responders of the pulmonary innate immune system.AMs absence display increased susceptibility to a range of bacterial and fungal infectons,which is associated with impaired innate functions AMs.Tuberous sclerosis complexl(Tsc1)and Tuberous sclerosis complex2(Tsc2)are combined to form a complex that it can inhibit mammalian rapamycin target protein l(mTORC1),which lead to cell metabolism and energy stability.Tsc1 has been proved to regulate M1/M2 polarization of peritoneal macrophage,which is preventing inflammatory diseases.Tsc1 is also an important factor in maintaining the growth and function of peritoneal macrophages.AMs pivotal for maintaining the lung homeostasis,but how the development and function of AMs regulated remains largely unknown.In the present study,we demonstrated that the number of AMs was controlled by the Tsc1 protein.Cdllc-specific deletion of Tsc1 caused inefficient transition from pre-AMs to AMs in lung,which led to a great reduction of AM population.Ablation of Tsc1 downregulated the expression of surface marker CD64 and SiglecF on AMs.We further showed that conditional knockout of Tsc1 led to enhanced proliferation and increased reactive oxygen species(ROS)production and phagocytosis in AMs.These results indicated that Tsc1 was a critical regulator of development,proliferation and function in AMs.Methods:Fist,to prove AMs highly expressed in CDllc,we analyze the ratio of CDllc+ cells to F4/80+CD68+ cells by flow cytometry.Mice with Tsc1 homozygous deletion in CDllc(Cdllccre Tsc1f/f)were obtained by crossing Tsc1f/f mice with mice expressing Cre recombinase under the control of CDllc promoter(CDllccre).The Tsc1f/fand Cdllccre Tsc1f/f mice were determined using PCR.Deletion of Tsc1 by Cdllccre-mediated recombination in macrophages was confirmed by western blot assays.For analysis of AMs surface markers,single cell suspensions were by flow cytometry.At the same time,we analysis the development of lung monocyty and pre-AMs and AMs in Tsc1f/f and CdllccreTsc1f/f naive and postnatal 7days.To determine whether proliferation and apoptosis are involved in changes AMs number.Intracellular staining with anti-Ki67 were performed with Foxp3 staining kits.Indicated AM populations were sorted from lung with FACS Aria ?.The apoptosis and growth of macrophages were determined by flow cytometry.The phagocytosis of macrophages was determined using a Vybrant TM phagocytosis assay kit.Results:In the present study,we demonstrated that the number of AMs was controlled by the Tsc1 protein.CDllc-specific deletion of Tsc1 caused inefficient transition from pre-AMs to AMs in lung,which led to a great reduction of AM population.Ablation of Tsc1 downregulated the expression of surface marker CD64 and SiglecF on AMs.We further showed that conditional knockout of Tsc1 caused enhanced proliferation and increased reactive oxygen species(ROS)production and phagocytosis in AMs.Conclusion(s):Tsc1f/f mice were crossed with Cdllccre to generate Cdllccre Tsc1f/f CDllc is generally expressed on mature alveolar macrophages.Alveolar macrophages is deleted of Tsc1 protein.These results indicated that Tsc1 is a critical regulator of development,proliferation and function in AMs.