The Role Of Macrophage MIC26 In Atherosclerosis

Objectives:Atherosclerotic cardiovascular disease is the leading cause of morbidity and mortality worldwide and is essentially a chronic inflammatory disease.Macrophages play critical role in atherosclerosis.APOO gene encodes an evolutionarily conserved mitochondrial protein MIC26,but the molecular biological function of MIC26 is still not fully elucidated.Our group has been following MIC26 for a long time,and we have constructed a conditional knockout mouse model of Apoo^flox/flox.Although several recent studies indicated that MIC26 might affect the innate immune response,there has been no study on the function of MIC26 on macrophages.This study aimed to investigate the role and mechanism of macrophage MIC26 in acute inflammatory response and atherosclerosis.Approaches:1.By crossing Apoo^flox/floxmice and Lys Mcre^+/-mice,macrophage-specific MIC26 knockout mice with genotype Apoo^flox/flox;Lys Mcre^+/-(MIC26^{Lys M})were obtained.A mouse model of endotoxemia was established by intraperitoneal injection of lipopolysaccharide.The effect of macrophage MIC26 deficiency on acute inflammation was explored by analyzing the median survival time of mice and measuring the levels of serum IL-1βafter administration.A mouse model of atherosclerosis was constructed by crossing MIC26^{Lys M} mice with Apoe^-/-mice and feeding them a high-fat diet.The effects of macrophage MIC26deficiency on atherosclerosis（chronic inflammation）were explored by Oil Red O staining and aortic root section analysis.2.We used primary macrophages derived from MIC26^{Lys M} mice and its littermate(MIC26^flox,genotype Apoo^flox/flox)to study the effect of MIC26 deficiency on macrophage functions in vitro.Macrophage polarization and NLRP3 inflammasome activation were analyzed by transcriptome analysis,qRT-PCR,Western Blot,and ELISA.Apoptosis was analyzed by Annexin V/PI staining and flow cytometry.The cholesterol efflux capacity and lipid processing capacity were detected by tritium-labeled cholesterol,liquid scintillation counter,and qRT-PCR.By co-incubation with apoptotic Jurkat cells,macrophage efferocytosis was detected by fluorescence microscopy or flow cytometry.In addition,to verify the effect of MIC26 deficiency on efferocytosis,immunofluorescence staining was used to analyze in situ efferocytosis in atherosclerotic plaques and an in vivo thymus efferocytosis assay was carried out.3.The mitochondria of MIC26-deficient macrophages were analyzed by transmission electron microscopy and fluorescence confocal microscopy.The ultrastructure of mitochondria and the mitochondrial morphology were analyzed in detail.Mitochondrial membrane potential was detected with JC-1 dye and fluorescence microscopy.The m RNA and protein levels of mitochondrial fusion and fission factors were detected by qRT-PCR and Western Blot.Results:1.The macrophage-specific deletion of MIC26 mouse model was successfully constructed,and the knockout efficiency was about 70%.Loss of MIC26 did not affect the median survival time and post-injection serum IL-1βlevels in mice with endotoxemia.However,it significantly reduced plaque burden（The percentage of plaque area in the aorta:9.78±2.99%vs.13.75±5.01%,p=0.0068）and necrosis（necrotic area:13.14（2.89-18.58）×10³μm² vs.24.83（9.93-41.28）×10³μm²,p=0.0463）in mice with atherosclerosis.2.Loss of MIC26 did not affect macrophage polarization,NLRP3inflammasome activation,apoptosis,or lipid processing capacity.However,MIC26 deletion promoted efferocytosis,and the effect was more evident in the case of heavy apoptotic burden.In atherosclerotic plaques,the ratio of macrophage-associated apoptotic cells to free apoptotic cells was significantly increased in the MIC26 deletion group,approximately 2-fold higher than in controls（p=0.0019）.Moreover,after induction of thymocyte apoptosis,there were less apoptotic cells remained in the MIC26-deficient thymus（4.00±1.51×10⁶ cells vs.6.78±2.51×10⁶cells,p=0.0167）,and thymus sections showed an increased ratio of macrophage-associated apoptotic cells to free apoptotic cells（p=0.0299）,also supporting increased efferocytosis.3.We found altered mitochondrial inner membrane structure in MIC26-deficient mitochondria.The intracristal space expanded;the direction of the cristae changed,and abnormal vesicular cristae appeared.MIC26-deficient mitochondria have altered morphology,with smaller and rounded mitochondria and fragmented mitochondrial networks.Mitochondrial membrane potential and the level of mitochondrial fusion protein OPA1 decreased in MIC26-deficient mitochondria.Conclusions:Macrophage MIC26 deletion attenuates the progression of the chronic inflammatory disease atherosclerosis.The mechanism may be the increased efferocytosis by mitochondrial fission or decreased mitochondrial membrane potential.