The Effects Of Different Subtypes Of Polarized Macrophages And The Macrophages Efferocytosis Mediated By Mer Receptor Tyrosine Kinase On Degenerative Chondrocytes Induced By Interleukin-1β

Part 1: The Effects of Different Subtypes of Polarized Macrophages on Degenerative Chondrocytes Induced by Interleukin-1βObjective: Osteoarthritis(OA)is the most common degenerative joint disease.Synovial macrophages are thought to be involved in the progression of OA.Macrophages are often classified into different polarization types based on the different stimulation as well as the different characteristics of transcription and secretion.However,there is no study directly comparing the paracrine effects of three polarized macrophages which were stimulated respectively by lipopolysaccharide(Lipopolysaccharide,LPS)combined with interferon-γ(Interferon-γ,IFN-γ),interleukin(Interleukin,IL)-4,and glucocorticoids(glucocorticoid,GC)to degenerative articular chondrocytes.Therefore,in this part,the effects of three kinds of polarized macrophages which were stimulated respectively by IFNγ+ LPS,IL-4 and Fluticasone propionate(FP,a kind of GC)on degenerative articular chondrocytes were compared in vitro.Methods:(1)Articular chondrocytes of mouse were extracted by the digestion useing trypsin and type Ⅱ collagenase;and then the extracted primary chondrocytes were identified by toluidine blue staining and collagen type Ⅱ immunofluorescence staining.(2)The polarization of J774 A.1 macrophages were induced by IFNγ+ LPS,IL-4 and FP respectively.The three types of polarized macrophages were denoted as M(IFNγ+ LPS),M(IL-4)and M(FP),respectively.The Macrophage Conditioned Medium(MCM)of this three macrophage types were collected,and the concentrations of IL-6,CCL17 and CXCL13 in MCM were detected by ELISA kit to verify the successful induction of polarization.(3)To simulate the chondrocytes state of OA patients,degenerative chondrocytes were obtained by pretreating chondrocytes with IL-1β(10 ng/ml)for 24 hours.(4)In order to compare the paracrine effect of this three kinds of polarized macrophages on the viability of degenerative chondrocytes,degenerative chondrocytes were cultured with each MCM,and the effect of MCM on the viability of degenerative chondrocytes was detected by CCK8 kit.(5)To compare the paracrine effect of this three kinds of polarized macrophages on the proliferation,apoptosis,anabolism and inflammatory catabolism of degenerative chondrocytes,the Transwell co-culture model was used to make degenerative chondrocytes co-culture with this three polarized macrophages respectively.The proliferation and apoptosis of chondrocytes in each co-culture group were respectively measured by Ed U staining and Annexin V-APC/7-AAD apoptosis kit(flow cytometry).Western Blot was used to measure the protein expression of Aggrecan,Collagen Ⅱ,SOX9,MMP13,MMP9,and ADAMTs5 in chondrocytes form each co-culture group.Results:(1)A large number of chondrocytes with high purity were obtained by the digestion useing trypsin and type Ⅱ collagenase.(2)J774A.1 macrophages were successfully polarizad to M(IFNγ+ LPS),M(IL-4)and M(FP)after respectively treating with IFNγ+ LPS,IL-4 and FP.(3)Compared with normal chondrocytes,IL-1β(10 ng/ml)reduced the viability,proliferation and the protein expression of Aggrecan,Collagen Ⅱ and SOX9,and promoted apoptosis and protein expression of MMP13,MMP9 and ADAMTs5 in chondrocytes.(4)Compared with the IL-1β treatment group,co-culture with M(IFNγ+ LPS)(or MCM)reduced the viability,proliferation and protein expression levels of Aggrecan,Collagen Ⅱ and SOX9,and also promoted cell apoptosis and protein expression of MMP13,MMP9 and ADAMTs5 in Degenerative chondrocytes.However,co-culture with M(IL-4)and M(FP)(or MCM)increased the viability,proliferation and protein expression of Aggrecan,Collagen Ⅱ and SOX9,and reduced the apoptosis and protein expression of MMP13,MMP9 and ADAMTs5 in degenerative chondrocytes.However,there was no significant difference between M(IL-4)co-culture(or MCM)group and M(FP)co-culture(or MCM)group.Conclusions: In vitro,the paracrine effect of pro-inflammatory M(IFNγ+ LPS)can significantly aggravate the apoptosis and inflammation of chondrocytes pretreated with IL-1β,and inhibit the cell viability,cell proliferation and anabolism level.On the contrary,the paracrine effects of anti-inflammatory M(IL-4)and M(FP)could significantly alleviate the apoptosis and inflammatory state of chondrocytes pretreated with IL-1β,and enhance their cell viability,cell proliferation and anabolism.Forthemore,there was no significantly different in the paracrine protective effect on degenerative chondrocytes between M(IL-4)and M(FP).These results provide new directions and ideas for OA treatment targeting synovial macrophage polarization.Part 2: The Macrophages Efferocytosis Mediated by Mer Receptor Tyrosine Kinase on Degenerative Chondrocytes Induced by Interleukin-1βObjective: The process by which apoptotic cells are eliminated by phagocytic cells(mainly macrophages)is called Efferocytosis.The macrophages polarized by glucocorticoid is characterized by high expression of Mer Receptor Tyrosine Kinase(MerTK),and MerTK mediates the efferocytosis.Efferocytosis has been shown to enhance the anti-inflammatory state of macrophages while eliminating the apoptotic cells.However,the role of MerTK-mediated macrophage efferocytosis to influence degenerative chondrocytes remains unclear.Therefore,the aim of this study is to investigate the effect of MerTK-mediated macrophage efferocytosis on degenerative articular chondrocytes in vitro.Methods:(1)The MerTK protein expression levels of M(IFN γ + LPS),M(IL-4),and M(FP)were determined by Western Blot.(2)The apoptosis of EL-4-B5 cells was induced by ultraviolet(UV)-ray and the apoptosis rate was determined by Annexin-APC / 7-AAD cell apoptosis detection kit.(3)Immunofluorescence was used to reveal the efferocytosis of M(FP)and the inhibited efferocytosis by MerTK inhibitor UNC2250: Macrophages in the MerTK inhibition group were pretreated with UNC2250 to inhibit MerTK,while macrophages in the efferocytosis group were not treated accordingly.Apoptotic EL-4-B5 cells were labeled with CFDA,and they were then co-cultured with M(FP)with or without UNC2250 treatment for 2 hours.The M(FP)was then labeled with CY3-F4/80.Ultimately,The double positive situation in which both CFDA and CY3 positivity(macrophage phagocytosis of at least one apoptotic cell was defined as positive)is measured by immunofluorescence microscopy.(4)The M(FP)group(control),efferocytosis group and MerTK inhibition group were established,and MCM of each group were collected to culture degenerative chondrocytes.The effect of MCM on the viability of degenerative chondrocytes was then detected by CCK8 kit.(5)To explore the paracrine effect of M(FP),which has gone through efferocytosis,on the degenerative chondrocytes proliferation,apoptosis,anabolism and inflammatory catabolism,the Transwell co-culture model was used to make degenerative chondrocytes co-culture with macrophages that from each group respectively.The proliferation and apoptosis of chondrocytes in each co-culture group were respectively measured by Ed U staining and Annexin V-APC/7-AAD apoptosis kit(flow cytometry).Western Blot was used to measure the protein expression of Aggrecan,Collagen Ⅱ,SOX9,MMP13,MMP9,and ADAMTs5 in chondrocytes form each co-culture group.Results:(1)The expression of MerTK protein was significantly higher in M(FP),but significantly lower in M(IFNγ+ LPS)and M(IL-4).(2)UV-ray successfully induced the apoptosis of EL-4-B5 cells,and the proportion of apoptotic cells was 74.36±3.09%,which could be used for subsequent experiments.(3)The efferocytosis of M(FP)was successfully induced,and this process could be significantly inhibited by MerTK inhibitor UNC2250.(4)Compared with the M(FP)from control group,the viability,proliferation and the protein expression of Aggrecan,Collagen Ⅱ and SOX9 in degenerative chondrocytes can be further improved by M(FP)which has gone through efferocytosis.It also reduced the apoptosis of degenerative chondrocytes and the protein expression of MMP13,MMP9 and ADAMTs5.These results suggest that the paracrine protective effect of M(FP)on degenerative chondrocytes is enhanced after efferocytosis.Moreover,this enhanced paracrine protection was partially(but not completely)reversed by the MerTK inhibitor UNC2250,suggesting that MerTK-mediated efferocytosis is responsible,at least in part,for this enhanced paracrine protection.Conclusion: We compared the paracrine effects of macrophages,which were from M(FP)group(control),efferocytosis group and MerTK inhibition group,on degenerative chondrocytes using an in vitro co-culture model(or MCM).The results demonstrate that M(FP)significantly enhanced its paracrine protective effect on degenerative chondrocytes after efferocytosis.And this enhanced paracrine protection is mediated,at least in part,by MerTK.Therefore,targeting MerTK-mediated efferocytosis of M(FP)may be a potential therapeutic approach for OA.