Role Of Lysosome-Associated Membrane Protein1in CXCL10-CXCR3 Axis Regulation Of Macrophage Polarization And Lung Injury

Objective: acute lung injury(ALI)caused by external infection is a respiratory disease that can develop into acute respiratory distress syndrome(ARDS)in severe cases.The disease has a high morbidity(7%)and mortality(30%-40%),and there is no effective drug treatment.Macrophages are involved in the whole pathogenesis of ALI/ARDS,including the regulation of inflammatory response and tissue damage repair.CXCL10 and CXCR3 are highly expressed in the lung tissues of ALI.Both CXCL10-CXCR3 axis and autophagy regulate the polarization and function of macrophages.This study explores the role of lysosome-associated membrane protein 1(LAMP1)in CXCL10-CXCR3 axis regulated macrophage polarization and the role of lysosome-associated membrane protein1(LAMP1)in inflammatory conditions.CXCL10-CXCR3 axis regulates macrophage polarization and changes in LAMP1.To investigate the effect of CXCR3 antagonist AMG487 on poly(I:C)induced acute lung injury.Methods: 1.After LAMP1 knockdown was detected by q PCR,m RNA expressions of M1-type polarization markers i NOS,IL-1β and TNF-α treated by CXCL10 and AMG487,M2-type polarization markers Arg1,Mrc1 and Mmp9,Akt1 and Akt2 were respectively detected.Western Blot was used to detect the expressions of autophagy related proteins LC3 Ⅰ,LC3 Ⅱ and Atg5-Atg12 complex after LAMP1 and CXCL10 knockdowns.2.After the addition of CXCL10 and AMG487,the m RNA expressions of M1-type polarization markers i NOS,IL-1β and TNF-α,M2-type polarization markers Mrc1,Mmp9 and Arg1,as well as Akt1 and Akt2 m RNA were detected by q PCR.Western Blot assay was performed to detect the protein expressions of LAMP1 and UBL4 A in inflammatory macrophages after the addition of CXCL10 and AMG487.co-IP was used to detect the protein interaction between LAMP1 and UBL4 A.Western Blot analysis of LAMP1 protein expression in inflammatory macrophages after UBL4 A knockdown.3.Mice were given poly(I:C)by airway drip and AMG487 by intraperitoneal injection 6h later.The number of neutrophils in alveolar lavage fluid was detected by flow cytometry.The lung ventilation function of mice was detected by pulmonary function respiratory apparatus.The number of M2-type macrophages in lung tissue was determined by immunohistochemistry.The protein expressions of LAMP1 and UBL4 A in lung tissue were detected by Western Blot.Results: 1.The regulation of CXCL10-CXCR3 axis on the direction of macrophage polarization can be changed by LAMP1.CXCL10 induced M2-type polarization of macrophages,and after knocking down LAMP1,the macrophages shifted to M1-type polarization.AMG487 induced M1-type polarization of macrophages,and after knocking down LAMP1,the macrophages shifted to M2-type polarization.CXCL10 induced macrophage autophagy,and autophagy was inhibited after LAMP1 knockdown.2.In inflammatory macrophages,UBL4 A targets LAMP1 and participates in CXCL10-CXCR3 axis induced macrophage polarization.The inflammatory macrophages were mainly M1-type.After addition of AMG487,M1-type polarization was inhibited and changed to M2-type polarization.In inflammatory macrophages,the expression of LAMP1 protein was decreased and UBL4 A protein was increased.After addition of AMG487,the expression of LAMP1 protein was increased and UBL4 A protein was decreased.Co-immunoprecipitation showed that there was protein interaction between LAMP1 and UBL4 A.In inflammatory macrophages,after UBL4 A is knocked down,LAMP1 protein levels are reduced and inhibited.CXCL10 could up-regulate the expression of Akt1 m RNA and down-regulate the expression of Akt2 m RNA,while the knockdown effect of LAMP1 was opposite.The effect of CXCL10-CXCR3 axis induced macrophage polarization is related to Akt1/Akt2 equilibrium.3.CXCR3 antagonist AMG487 has a protective effect on acute lung injury in mice.After the intervention of AMG487,the inflammatory cell infiltration degree of ALI was significantly reduced,the pathological injury was significantly improved,and the lung tissue structure tended to be normal.After AMG487 intervention,the number of neutrophils decreased and the number of M2-type macrophages increased in ALI lung tissue.AMG487 can relieve lung function impairment in ALI.LAMP1 protein levels were decreased and UBL4 A protein levels were increased in ALI lung tissue,while LAMP1 protein levels were increased and UBL4 A protein levels were decreased after AMG487 intervention,and the results were consistent in vitro and in vivo.Conclusion: CXCL10 induced M2-type polarization of macrophages,and after knocking down LAMP1,the macrophages shifted to M1-type polarization.In inflammatory macrophages,UBL4 A targets LAMP1 and CXCL10 induces M1-type polarization in macrophages.Akt1/Akt2 balance is involved in macrophage polarization regulated by the CXCL10-CXCR3 axis.AMG487 has a protective effect against ALI and can be used as a potential drug therapeutic target.