The Effects And Mechanisms Of Alveolar Macrophages-derived Microvesicles On Acute Lung Injury

BackgroundAcute lung injury/acute respiratory distress syndrome(ALI/ARDS)has a high incidence and mortality rate.According to reports,the mortality rate of patients with severe ARDS exceeded 40%,and the complications caused by ARDS will also have a serious impact on the life quality of survived patients.ALI/ARDS is often caused by bacterial or viral infection,sepsis,and trauma.The 2019 novel coronavirus disease(COVID-19)outbreak has a major impact on the world.The cumulative incidence data showed that more than 20 million patients had been infected with COVID-19.ALI/ARDS is the main complication of COVID-19 and an important factor leading patients to death.The ALI/ARDS patients have severe inflammation.A large number of immune cells such as macrophages,neutrophils,T cells are activated and recruited,and a large number of inflammatory mediators are rapidly produced,such as TNF-α,IL-1,IL-6,etc.They have a fierce attack on the body.These inflammatory mediators destroy alveolar epithelial cells and pulmonary microvascular barriers,leading to increased lung inflammation,alveolar edema,vascular leakage and diffuse damage.Eventually,severe hypoxia,progressive dyspnea,and even respiratory failure occurred.Alveolar macrophages(AMs)are one of the earliest cells in lung tissue that are stimulated by injury factors.Alveolar macrophages are located in the alveolar cavity and play an important role in the pathological development of lung injury.Injury factors can induce alveolar macrophages to release inflammatory mediators,which causes inflammatory cell infiltration.After inflammatory mediators entering the blood circulation,they can lead to a more extensive inflammatory response.Lung epithelial cells cover 95%of alveolar tissue and are a key part of the lung barrier structure.Severe lung injury is accompanied by lung epithelial cell damage.Damage to lung epithelial cells can lead to accumulation of fluid in the alveolar cavity.Sodium channel proteins and sodium pump are the key factors for lung epithelial cells to regulate the fluid balance in the alveolar cavity.In addition,tight junction proteins that connect lung epithelial cells in lung tissue also play an important role in the barrier of lung tissue,and their damage can also lead to an imbalance of fluid exchange inside and outside the alveoli.Previous studies have shown that alveolar macrophages can affect the activity and function of lung epithelial cells,thereby realizing the regulation of lung tissue physiology and disease state microenvironment.Recent studies have shown that extracellular vesicles from alveolar macrophages are an important way of paracrine action for cells.They secrete EVs to make them act on other target cells and affect the function or structure of these target cells.EVs include apoptosis body,microvesicles and exosomes.At present,most researches are focused on microvesicles and exosomes,which often transfer intracellular biologically active molecules to act on other target cells or tissues.The latest study found that in the early stage of lung injury,a large number of EVs can be detected in bronchoalveolar lavage fluid(BALF),and MVs are the most produced type in BALF,with a particle size of about 100-1000nm.MVs in BALF can be produced by a variety of lung tissue cells or immune cells such as alveolar macrophages,lung epithelial cells,neutrophils,pulmonary vascular endothelial cells,etc.However,studies have shown that microvesicles in early lung injury are mainly derived from alveolar macrophages.However,the effect and mechanism of microvesicles produced by alveolar macrophages on lung injury remain to be studied.Therefore,this study intends to design the following contents to explore the effects and potential mechanisms of microvesicles derived from alveolar macrophages on lung injury in vitro cell experiment and in vivo experiment:(1)The BALF was obtained from early lung injury and the ratio of macrophages in BALF was tested.Then microvesicles in the BALF of early lung injury are obtained and their morphology,particle size and surface markers are identified.And the high expression of inflammatory factors in MVs are determined;(2)The effect of microvesicles derived from BALF on lung epithelial cell function was determined through in vitro experiments,mainly to determine its effect on sodium channel-related proteins,and then to explore the effect of microvesicles from alveolar macrophages that is most productive in bronchoalveolar lavage fluid on sodium channel-related proteins of lung epithelial cells in vitro and its potential mechanism;(3)The extracted microvesicles derived from alveolar macrophages were studied in vivo to observe their effects on lung injury.Part 1 The Extraction and identification of bronchoalveolar lavage fluid-and alveolar macrophage-derived microvesiclesObjective To extract and identify microvesicles(MVs)derived from bronchoalveolar lavage fluid(BALF)and microvesicles derived from alveolar macrophage(AM),and explore inflammatory factors in the obtained microvesicles.Methods Lipopolysaccharide(LPS)stimulated mice to establish an acute lung injury model,and microvesicles can be obtained by differential centrifugation.The identification of microvesicles included three methods:Transmission electron microscope(TEM),Nanometer particle size tracking technology(NTA)and Western blot(WB),which tested the morphology,particle size range and three surface markers(CD63,CD68 and ALIX molecules)respectively.(1)Extracting bronchoalveolar lavage fluid:C57 BL/6 mice were cultivated and BALF derived from PBS-stimulated mice and BALF derived from LPS-stimulated mice at an early stage were obtained.Flow cytometry was used to detect the proportion of macrophages in the two groups of BALF.(2)Extracting microvesicles of BALF:Differential centrifugation was used to extract microvesicles from BALF of mice stimulated by PBS(Con-BALF-MVs)and microvesiclesfrom BALF of lung injury model mice(LPS-BALF-MVs).The morphology,size range and surface markers of BALF-derived microvesicles in the two groups were identified by TEM,NTA,and WB methods.(3)In addition,a large number of mouse alveolar macrophages were cultured and the cells were stimulated with PBS or LPS.Microvesicles(Con-AM-MVs)derived from alveolar macrophages after PBS treatment and microvesicles(LPS-AM-MVs)obtained after LPS treatment were also extracted by differential centrifugation.TEM,NTA and WB were used to identify the morphology,size range and surface markers of microvesicles derived from alveolar macrophages.(4)Enzyme linked immunosorbent assay(ELISA)is used to determine the expression levels of inflammatory mediators TNF-α,IL-1β and IL-6 in the microvesicles extracted from BALF and levels of TNF-α protein in alveolar macrophages-derived microvesicles.Results(1)TEM clearly showed the morphology of microvesicles derived from BALF and alveolar macrophages.NTA found that the size of microvesicles was mainly concentrated in 100-200 nm.WB results showed that the microvesicles derived from BALF and the microvesicles derived from alveolar macrophages all expressed CD63,CD68 and ALIX surface markers.(2)The results of flow cytometry showed that the proportion of pro-inflammatory macrophages in the BALF from lung injury reached 44.2%,which was significantly higher than that in the control group;(3)ELISA results showed that microvesicles in BALF in the early stage of lung injury mainly express TNF-α inflammatory mediators(120.91 ± 22.32 pg).But the expression in Con-BALF-MVs was less.In addition,the expression levels of IL-1βand IL-6 were lower in the two groups.The microvesicles obtained from LPS-stimulated alveolar macrophages also expressed more TNF-α protein(196.43±68.08 pg)in the early stage,while the control group Con-AM-MVs expressed less.Conclusions(1),Stable microvesicles can be extracted from bronchoalveolar lavage fluid and alveolar macrophages by differential centrifugation.(2)Microvesicles derived from lung injury BALF and inflammatory alveolar macrophages contained high levels of TNF-α inflammatory mediators.Part 2 Effect of bronchoalveolar lavage fluid-and alveolar macrophage-derived microvesicles on lung epithelial cells in vitroObjective To observe the uptake of microvesicles by lung epithelial cells in vitro,and explore the effects of microvesicles derived from bronchoalveolar lavage fluid and microvesicles derived from alveolar macrophages on ENaC and Na+,K+-ATPase of lung epithelial cells.Methods After co-cultivating lung epithelial cells and microvesicles with high-content imaging equipment,the uptake of microvesicles by lung epithelial cells was observed.The effect of microvesicles on the protein expression levels of three ENaC subunits and Na+-K+-ATPase subunits of lung epithelial cells were detected by WB.(1)High-content screening:Two groups of microvesicles derived from bronchoalveolar lavage fluid(LPS-BALF-MVs and Con-BALF-MVs)obtained in the first part and two groups of microvesicles derived from alveolar macrophages(LPS-AM-MVs and Con-AM-MVs)are labeled with PKH-26 and added to lung epithelial cells.They were added to lung epithelial cells and co-cultured in high-content imaging equipment,and the uptake of PKH-26-labeled microvesicles by lung epithelial cells was observed in real time.(2)Effect of microvesicles derived from bronchoalveolar lavage fluid on lung epithelial cells:Western blotting was used to detect the effect of microvesicles derived from bronchoalveolar lavage fluid on the expression levels of α-,β-,y-ENaC and Na+,K+-ATPase α1,β1 in lung epithelial cells.(3)Effect of microvesicles derived from alveolar macrophages on lung epithelial cells:Western blotting was used to detect the effect of microvesicles derived from alveolar macrophages on the expression levels of α-,γ-ENaC and Na+,K+-ATPase α1,β1 in lung epithelial cells.After using TNF-α antibody to inhibit inflammatory microvesicles derived from alveolar macrophages,it was observed that whether its effects were changed.Results(1)High-content imaging showed that lung epithelial cells effectively ingested microvesicles in bronchoalveolar lavage fluid and microvesicles derived from alveolar macrophages.(2)Compared to the control group(Con-BALF-MVs group),LPS-BALF-MVs derived from bronchoalveolar lavage fluid in the early stage of lung injury reduced the expression of sodium channel protein α-and y-ENaC,as well as the expression level of Na+-K+-ATPase α1and β1 protein,but there was no effect for β-ENaC.Con-BALF-MVs have no effect on the above proteins.(3)LPS-AM-MVs derived from pro-inflammatory alveolar macrophages also reduced the expression levels of α-,γ-ENaC and Na+-K+-ATPase subunit proteins,but after inhibiting TNF-α inflammatory mediators,they partially reversed effects that the inflammatory alveolar macrophages-derived microvesicles damaged the sodium channels of lung epithelial cells.Conclusions(1)Lung epithelial cells can ingest microvesicles derived from bronchoalveolar lavage fluid and alveolar macrophages.(2)Bronchoalveolar lavage fluid-derived microvesicles after lung injury can reduce the expression of sodium channel-associated protein in lung epithelial cells.(3)Microvesicles derived from inflammatory alveolar macrophages can reduce the expression of some sodium channel-associated proteins in lung epithelial cells.Inhibiting the TNF-α mediator of microvesicles derived from inflammatory alveolar macrophages can reduce its damaged effect on lung epithelial cells sodium channel-related proteins.Part 3 The effects of alveolar macrophage-derived microvesicles on lung injury in vivoObjective To explore the effects of inflammatory microvesicles derived from alveolar macrophages on lung injury in vivo.Methods After intratracheal administration of mice with PBS,microvesicles derived from alveolar macrophages(Con-AM-MVs)and microvesicles derived from inflammatory alveolar macrophages(LPS-AM-MVs),the lung injury was observed.(1)Microvesicles derived from inflammatory alveolar macrophages were used to stimulate mice intratracheally,and the effects of different doses(20 μg and 50μg)and different times(2 h and 4 h)on the lung tissue of mice were observed to determine stimulation dose and time of inflammatory microvesicles.(2)Evans-blue solution was used to determine the effects of stimulating factors on the clearance rate of alveolar edema fluid in mice and calculate the changes in the ratio of dry and wet lung tissues in each group.The cells in BALF were smeared and staining to observe cell infiltration in lung tissue.(3)The partial pressure of oxygen in the arterial blood of mice was detected by arterial blood gas analysis and then the oxygenation index of the mice was calculated.(4)The degree of lung injury was measured by lung injury score in each group and the number of cells in the bronchoalveolar lavage fluid was counted.(5)PKH-26-labeled microvesicles were stimulated with mice,then the lung tissues were obtained to make frozen sections and perform tissue staining.Laser confocal microscope was used to observe the uptake of microvesicles by lung tissue.(6)In addition,WB and immunohistochemistry(Immunohistochemistry,IHC)were used to detect the effects of microvesicles on sodium channel-associated proteins(α-,y-ENaC and Na+-K+-ATPase subunits)and tight junction proteins(ZO-1,Occludin,Claudin-5)expressions.Results(1)50 μg microvesicles derived from inflammatory macrophages cause more obvious damage to the lung tissue after acting for 4 hours.(2)Compared with the Con-AM-MVs group,LPS-AM-MVs reduced the alveolar fluid clearance rate of mice,and increased the wet/dry weight ratio of lung tissue,and caused the mouse’s oxygenation index to decrease.(3)The lung injury scores of each group showed that LPS-AM-MVS damaged the lung tissue more seriously,which was significantly higher than control group.(4)At the same time,the results of WB and IHC showed that compared with the Con-AM-MVs group,LPS-AM-MVs reduced α-,γ-ENaC and Na+-K+-ATPase α1,β1 in lung tissue.(5)WB results showed that the expression level of tight junction protein in the LPS-AM-MVs group was significantly decreased compared to the Con-AM-MVs group.Conclusions(1)Microvesicles derived from inflammatory alveolar macrophages cause obvious damage and edema to the lungs.(2)Microvesicles derived from inflammatory alveolar macrophages reduce the expression levels of ENaC,Na+-K+-ATPase and tight junction proteins in epithelial cells of lung tissue.