The Effect And Mechanism Of Macrophage-specific Junctional Adhesion Molecule Like Protein In Promoting Atherosclerosis

BackgroundAround the world,atherosclerotic cardiovascular disease(ASCVD)is the leading cause of death,which is one of the major public health emergency in China.The major manifestations of ASCVD include coronary,cerebrovascular,and peripheral arterial diseases.However,atherosclerosis(AS)is the main pathological basis of ASCVD,and its pathogenesis has not been fully elucidated to date.Thus,it is of great significance to continue in-depth study on the pathogenesis of AS and develop new therapeutic targets for reducing the ASCVD risk in our country.The growing number of evidence has suggested that AS is a chronic inflammatory disease,which is characterized by a gradual accumulation of inflammatory cells and lipids.Macrophage are the major inflammatory cell type in the atherosclerotic plaques,and the accumulation of macrophages in the intima of the arteries is a key player in the formation and development of AS.Macrophages are innate immune cells that triggers interleukin-1β(IL-1β)release through NOD-like receptor NLR family,pyrin domain containing 3(NLRP3)-inflammasome activation.Since IL-1β plays a profound role in inflammatory responses,the treatment of antiinflammatory is of particular importance.In recent years,junctional adhesion molecule like protein(JAML)have been reported to play a significant role in the regulation of the immune system and inflammation.JAML is a secretory type I transmembrane protein that initiates proinflammatory effects by binding to the coxsackievirus and adenovirus receptor(CXADR).Our previous study verified that JAML gene silencing significantly decreased the phosphorylation of extracellular signal-regulated kinase(ERK1/2)and p65,thereby reducing inflammation.Nevertheless,the mechanisms of macrophage-derived JAML inducing AS remain largely unclear.Therefore,we crossed macrophage-specific JAML knockout(JAMLM-KO)with mice carrying a knockout mutation of the ApoE gene(ApoE-/-)to generate JAMLM-KO/ApoE-/-Then,we intensively investigated the potential mechanisms of macrophage-derived JAML in AS.Objective(1)To investigate the changes of JAML in atherosclerosis progression;(2)To investigate the effect of macrophage-derived JAML on atherosclerosis;(3)To investigate the role and the potential mechanism of macrophage-derived JAML on NLRP3 inflammasome activation.Methods1,Animal models of atherosclerosisApoE-/-mice were fed a high-fat diet(HFD)for 12 weeks to induce atherosclerosis models.2.Peripheral blood samplesPeripheral blood was extracted from healthy and CHD(coronary heart disease)populations,then Peripheral blood mononuclear cells(PBMCs)were separated.3.Macrophage-specific JAML knockout miceMacrophage-specific JAML knockout mice were constructed via the CRISPR/Cas9 gene editing technique to evaluate the effect of JAML gene on atherosclerosis.4.Commonly biochemical measuresBody weight,heart rate,blood pressure and lipids were measured.5.Histopathological assay(1)Hematoxylin-eosin(H&E)staining:the morphology and area of atherosclerotic plaques were observed via HE staining.(2)Oil red O staining:the plaque area and lipid accumulation of ApoE-/-mice were observed via Oil red O staining.(3)Immunohistochemical staining:immunohistochemistry was used to measure expression levels of JAML in plaques.(4)Immunofluorescent staining:immunofluorescent was used to measure expression levels of MOMA2,CD68,NLRP3,IL-1β and JAML in plaques.6.Primary peritoneal macrophages cultureMice primary peritoneal macrophages were extracted and stimulated with LPS+ATP(NLRP3 inflammasome-specific stimulation)to observe the levels of inflammatory factors.7.ELISAELISA kits were used to detect the expression levels of IL-1β and IL-18 in the cell supernatant and serum.8.Plasmid transfectionFlag-CXADR and Myc-TLR4 plasmids were co-transfected in HEK293T cells.9.Co-immunoprecipitation(Co-IP)Specific antibodies were used to detect binding of CXADR to TLR4 proteins.10.RT-PCRThe mRNA expressions of IL-1β,IL-18 and NLRP3 were evaluated by RT-PCR.11.Western blotThe protein levels of JAML,NLRP3,Pro-caspase 1,Pro-IL-1β,ASC,Caspase 1(P10),pIKKα/β,NOX2,NOX4,IKKβ,p-p65,p-IKBα,IKBa and β-actin were detected by Western blot.12.Statistical analysesAll statistical analyses were performed using GraphPad Prism 8 software(GraphPad Software)and expressed as mean±SEM.Each experiment was repeated three independent times at least.When the data conforms to normal distribution,t-test was used for comparison between two groups and one-way analysis of variance(ANOVA)was used for comparison among multiple groups,and rank sum test was used for the data did not conform to normal distribution.P<0.05 was considered as statistically significant(*p<0.05).Results1.JAML expression was significantly up-regulated in the atherosclerosis model.(1)The levels of JAML were significantly increased in plague of ApoE-/-mice which were fed a HFD for 12 weeks compared with control which were fed a normal diet for 12 weeks.And JAML was highly colocalized with macrophages.(2)Western blot indicated that the expression levels of JAML significantly upregulated in PBMC from CAD patients.2.Generation of macrophage-specific JAML knockout miceTo investigate the role of macrophage-specific JAML in atherosclerosis,we constructed macrophage-specific JAML knockout mice.The efficiency of JAML knockout was determined by Western blot analysis.3.JAML knockout of macrophages inhibited the progression of atherosclerosisDeposition of lipid and infiltration of macrophages in the plaques were significantly decreased in JAMLM-KO/ApoE-/-mice compared with JAMLWT/ApoE-/-mice after a HFD for12 weeks.Results indicated that deficient JAML of macrophages significantly slowed the progression of plaque.4.JAML deficiency prevented NLRP3 inflammasome activationThe expression of NLRP3,IL-1β and IL-18 in JAML deletion macrophages with LPS+ATP treatment were strongly upregulated compared to controls,IL-1β and IL-18 levels in supernatant were also significantly reduced.Pro-caspase 1 and ASC were not changed significantly.Immunofluorescence and Western blot showed that the amount of NLRP3 inflammasome-related proteins were also reduced in plaques from JAMLM-KO/ApoE-/-mice.5.JAML deletion reduced oxidative stressAfter LPS+ATP stimulation,we examined the expression of NADPH oxidase 4(NOX4)and NADPH oxidase 2(NOX2),oxidative stress markers,were significantly decreased with JAML knockout macrophages compared to controls.DHE staining revealed that the expression level of ROS was significantly inhibited in JAML knockout group.6.JAML knockout inhibited NF-κB pathway activationToll-like receptors(TLRs)induced the NLRP3 inflammasome formation and activation via the activation of the NF-κB signaling pathway.Thirty minutes after LPS administration,the phosphorylation of IKKα/β and NF-κB significantly reduced in macrophages of JAMLM-KO mice as compared to controls.Conclusion(1)Expression of JAML in plaque increased with the progression of atherosclerosis.(2)Macrophages were the major cellular source expressing JAML in the plaque.(3)Macrophage-specific JAML promoted atherosclerosis progression.(4)Macrophage-specific JAML exacerbated inflammation in atherosclerotic plaque via promoting NLRP3 inflammasome activation,a critical mechanism accelerating atherosclerosis.