The Mechanism Of Immunomodulation Role Of CXCL12-CXCR4/CXCR7 Axis In Implants Induced Foreign Body Response

Part Ⅰ: The study of the role of CXCL12-CXCR4/CXCR7 axis in regulating the function of macrophagesAim: To explore the polarization and function of macrophage under the stimulation of the inflammatory materials and the relationship between macrophage function and CXCL12-CXCR4/CXCR7 signalings.Materials and methods: PMMA was used as the model material to induce macrophage inflammatory response in this project.After sterilization,PMMA plates were put in the 6-well plates and co-cultured with macrophages in vitro.Cells cultured under normal 6-well culture plates were considered as control group.The macrophage cell lines used in this project were human macrophage cell line THP-1 cells,mouse macrophage cell line RAW 264.7 cells and rat macrophage cell line NR8383 cells.THP-1 cells’ differentiation into mature macrophages and attachment to the culture plates require 24-h PMA exposure.Immunofluorescence staining method was used to explore the adherence and growth of macrophages cultured on PMMA plates or normal culture plates.The expression of functional markers and cytokines in the macrophages was tested by immunofluorescence staining and RT-PCR assay.Moreover,the activation of CXCL12-CXCR4/CXCR7 axis in the macrophages was also detected by the RT-PCR assay and western blot assay.Furthermore,AMD3100,simvastatin and CXCR7 si RNA were used to regulate the activation of CXCL12-CXCR4/CXCR7 axis.And then macrophage function and polarization were detected by immunofluorescence staining,RT-PCR assay,and flow cytometry.The downstream signals that involved in this modulation were also explored.Results: Immunofluorescence staining results showed that THP-1 cells and RAW 264.7 cells attached to the PMMA plates or normal culture plates and grew well.And both the THP-1 cells and RAW 264.7 cells in PMMA groups showed significantly higher level of CCR7 positive staining compared to that in the control group.Besides,RT-PCR results suggested that the stimulation of PMMA induced significantly increased m RNA expression level of M1-polarization related markers and inflammatory cytokines(CCR7,INOS,IL-1β,IL-6,IFN-γ,TNF-α,IL-12 m RNA).Furthermore,CXCR7 expression is highly upregulated and the CXCR4 expression is decreased by PMMA stimulation.AMD3100 treatment induced more serious inflammatory response in macrophages.While Simvastatin treatment decreased the expression level of CXCR7 and inhibited the expression of inflammatory cytokines in macrophages and promoted macrophage polarization to M2 phenotype.The treatment of CXCR7 si RNA showed the similar results with simvastatin group.Furthermore,western blot analysis of the downstream regulation mechanism showed that STAT signals and NF-κB was involved in the role of CXCL12-CXCR4/CXCR7 axis on regulating macrophage function and polarization.Conclusion: CXCL12-CXCR4/CXCR7 axis has an important role in regulating the macrophage function.Through inhibition of CXCL12-CXCR7 signals and activation of CXCL12-CXCR4 signals,macrophage polarization could be significantly switched from pro-inflammatory phenotype to anti-inflammatory phenotype.Part Ⅱ: The study of the role of CXCL12-CXCR4/CXCR7 axis in regulating the foreign body responseAim: The goal of this part is to explore the biological impact of CXCL2-CXCR4/CXCR7 axis on macrophage function and biomaterial implantation induced foreign body response in vivo.Material and methods: Rat femur defect model was constructed first.PMMA materials was used as inflammatory model material in this study and was made into implants with diameter of 2.2 um and length of 7 um.PMMA implants was implanted into the defect area of the femur.The experimental animal were divided into 4 groups: PMMA group;CXCL12 group(10 ul CXCL12 solution(50 ng/ul)was injected into the bone marrow cavity,PMMA implants immersed with 50 ng/ul CXCL12 solution before implantation);AMD3100 group(CXCL12 treatment was same with CXCL12 group,daily-treatment of AMD31000 was given after surgery)and Simvastatin group(CXCL12 treatment was same with CXCL12 group,daily-treatment of Simvastatin was given after surgery).1 week,2 weeks and 4 weeks after surgery,experimental animal were executed and femur with PMMA implants were dissected.The inflammatory reaction induced by PMMA implantation was tested by HE staining and RT-PCR assay.Immunofluorescence staining was used to detect the macrophage polarization profile in the implantation area.Immunohistochemistry staining was performed to detect the expression level of CXCR4 and CXCR7 around the implantsResults: In PMMA group,HE staining results showed that 1 week after surgery,inflammatory cells were accumulated in the tissue around PMMA implants in the PMMA group.2 weeks after surgery,the inflammatory response around the PMMA implants was aggravated with the time.4 weeks after surgery,a dense collagenase layer was formed around the PMMA implants with few vessels and a number ofinflammatory cells in it.Moreover,the bone tissue around the PMMA implants was seriously damaged.Besides,immunohistochemistry staining showed that CXCR7 is highly expressed in the inflammatory tissue around the PMMA implants in the PMMA group.There is no significant difference between PMMA group and CXCL12 group.The amplification of CXCL12-CXCR7 signals was induced by daily-treatment of AMD3100 and even aggravated the inflammatory response compared to PMMA group and CXCL12 group.In the contrast,the down-regulation of CXCL12-CXCR7 signal and up-regulation of CXCL12-CXCR4 signal was induced by daily-treatment of Simvastatin.In the meanwhile,the accumulation of M2 macrophage was significantly enhanced while the number of M1 macrophage was decreased sharply compared to that of the other three groups.4 weeks after surgery,the fibrous tissue formed around the PMMA implants was significantly prohibited in the Simvastatin group compared to that of the other three groups.Conclusion: CXCL12-CXCR4/CXCR7 axis has an important regulating role on foreign body response after biomaterials implantation via regulating the function of macrophages.The inhibition of CXCL12-CXCR7 signal and activation of CXCL12-CXCR4 signal could promote macrophage polarization toward M2 phenotype and then promote the good integration between host tissue and implants.Thus,CXCL12-CXCR4/CXCR7 axis could be used as an effective target to regulate foreign body response.