Inhibiting CD36 Decrease Macrophages Phagocytosis Via Lyn/JNK Pathway

Background and Objective : The engulfing and usually the destruction of particulate matter by macrophages that serves as an important bodily defense mechanism.Actin cytoskeleton,a major actor of cellular morphology,is the primary force driving phagocytosis.Actin cytoskeleton remodeling produces distinct Fibrous-actin(F-actin)-rich membrane structures called phagocytic cups.Cup shaped invaginations of the cell membrane that subsequently close at their distal margins to form phagosomes during phagocytosis.By progression of its rim along the particle surface,this phagocytic cup envelops and eventually encloses the particle by separation of the phagosome membrane from the cell membrane.Cluster differentiated 36(CD36),a member of the class B scavenger receptor family,is widely expressed on a variety of cells.Current works have showed that CD36 modulates spreading,migration and adhesion of cells in phagocytosis.However,it remains unclear whether CD36 is involved in the regulation of phagocytic cup formation.Therefore,this study intends to explore the potential mechanism of CD36 involved in phagocytosis.Methods: The first part: In vivo phagocytosis assay,CD36 Wild type(CD36WT),CD36 Knockdown(CD36KD)and CD36 Knockout(CD36KO)mice(6–8 weeks old)were injected intraperitoneally with 1.0 ml of Liquid paraffin.After 6 days,GFP-expressing Escherichia coli(E.coli)or CFSE-labelled Red Blood Cell(RBC)were injected into the peritoneum of mice.Primary peritoneal macrophages were harvested after 2 h and were stained with PE-F4/80 antibody and phagocytosis was measured by flow cytometry.Detection of phagocytic cups,macrophages was seeded onto confocal dish for overnight,to allow adhesion and purification.Macrophages and CFSE-labelled RBCs were incubated at 37 °C for 2 h.The cells were stained with Phalloidin-iFluor 647 for 60 minutes.Phagocytic cup was observed with a confocal microscope.The second part: small interfering RNAs targeting CD36(CD36i)or negative control siRNA(NCi)were transfected with into THP-1 cells to establish low-CD36 expression cell model.THP-1 derived macrophages were obtained by treating THP-1 cells with phorbol 12-myristate 13-acetate(PMA)(100 nM).The expression of CD36 was detected by Western blot.When stimulated with E.coli or RBC for 2 h,the phosphorylated c-Jun N-terminal kinase(JNK)and extracellular regulated protein kinases(ERK)were determined by Western blot.Macrophages were pretreated with or without anisomycin(JNK agonist)for 24 h.Next CFSE-labeled RBC was added to a confocal dishes and incubated with macrophages for 2 h.Then macrophages were stained with Phalloidin-iFluor 647 for 60 minutes.Phagocytic cup was observed by a confocal microscope.Phagocytosis was measured by flow cytometry.Pretreated with anisomycin for 24 h,Stimulated with E.coli or RBC for 2 h,the expression of Lyn kinase(Lyn)in macrophages was determined by Western blot.Macrophages were pretreated with tolimidone(Lyn agonist)for 12 h,then incubated with E.coli or RBC for 2 h,the phosphorylated JNK was determined by Western blot.Macrophages were pretreated with or without tolimidone for 12 h.Next CFSE-labeled RBC was added to a confocal dishes and incubated with macrophages for 2 h.Then THP-1 cells were stained with Phalloidin-iFluor 647 for 60 minutes.Phagocytic cup was observed by a confocal microscope.Phagocytosis was measured by flow cytometry.Results: In the first part: Compared with that in the CD36 WT group,the percentage of macrophages that had taken up E.coli or RBC in the CD36 KD and CD36 KO group was decreased(P < 0.05).The mean fluorescence intensity of GFP or CFSE,representing numbers of E.coli or RBC taken up by macrophages,was decreased in CD36 KD and CD36 KO macrophages,compared with CD36 WT macrophages(P<0.05).There were very few phagocytic cups formed in RBC-treated CD36 KO and CD36 KD macrophages compared with CD36 WT macrophages.Moreover,the Pearson’s correlation was higher in CD36 WT macrophages than in CD36 KD and CD36 KO macrophages(P<0.05).The Pearson’s correlation between CD36 KD and CD36 KO macrophages was significantly reduced(P <0.05).In the second part: Western blot results show that expression of CD36 was suppressed by transfection of its siRNAs in macrophages,the inhibition efficiency reached 60%(P <0.05).When exposure to E.coli or RBC,JNK phosphorylation were significantly reduced in CD36 i macrophages compared with NCi macrophages(P<0.05).But there were no significant differences were observed in phosphorylated ERK between NCi and CD36 i macrophages.Furthermore,in the absence of anisomycin,CD36 interference significantly reduced phagocytic cup formation and macrophage phagocytosis;after pretreatment with anisomycin,inhibition of CD36 could not induce phagocytic cup formation and macrophages phagocytosis(P<0.05).Western blot results show that the CD36 i macrophages exhibited decreased expression of Lyn compared to the NCi macrophages(P<0.05).Furthermore,tolimidone abrogated CD36 downregulation-induced decrease of JNK phosphorylation(P<0.05).Furthermore,in the absence of tolimidone,CD36 interference significantly reduced phagocytic cup formation and macrophage phagocytosis;after pretreatment with tolimidone,inhibition of CD36 could not induce phagocytic cup formation and macrophages phagocytosis(P<0.05).Conclusion: 1.CD36 knockdown and CD36 knockout reduce the formation of phagocytic cup and macrophages phagocytosis.2.Inhibition of CD36 reduces the formation of phagocytic cup via inhibiting the Lyn/JNK pathway,thereby reducing macrophage phagocytosis.Anisomycin and tolimidone can significantly improve the CD36 low expression induced reduction of macrophage phagocytosis.