IL-10 Ameliorates Particle-induced Inflammation And Osteolysis Through Macrophage Polarization

First PartIL-10 Polarize Macrophages in Vitro StudyBackground:In recent decades, the functions of macrophages have been generally recognized. Macrophages, derived from monocyte precursors, are a versatile cell population which commits to a variety of phenotypes in response to different local tissue environments. Monocytes from bone marrow are distributed into the blood circulation system and migrate to most tissues within the body where they can differentiate to mature macrophages. A subset of macrophages reside in special organs, such as liver Kupffer cells, brain microglia, lung alveolar macrophages, spleen macrophages, intestinal macrophages, peritoneal macrophages, adipose tissue macrophages, atherosclerotic plaque foam cells and bone osteoclasts. Macrophages stimulated by IL-10 or TGF-β can differentiate into M2c subtype which functions as a controller in the process of inflammation or wound healing. M2 macrophages play critical roles in inhibiting inflammation and facilitating wound healing. M2 macrophages take participate in the treatment of various autoimmune diseases (AD), such as rheumatoid arthritis (RA), experimental autoimmune encephalomyelitis (EAE), idiopathic thrombocytopenic purpura (ITP) and multiple sclerosis (MS). Aseptic loosening (AL) has become the most common long-term complication. In the process of AL. wear particles and other byproducts are phagocytized by macrophages and other local cells, which results in the release of pro-inflammatory substances, such as cytokines and chemokines. These substances provoke the active biological cascade, which is an important reason of the periprosthetic osteolysis. Wear particles constantly existing and stimulating the interface tissue between periprosthesis and bone is similar like the pathological process of autoimmune diseases.Objective:To investigate the function of IL-10 in the bone marrow derived macrophage polarization in vitro.Firstly, to confirm the perfect dose of IL-10 in the process of macrophage polarization. Secondly, to investigate the gene expression level of bone marrow derived macrophages challenged by PMMA particles or PMMA particles plus IL-10.Thirdly, to investigate the protein expression level of bone marrow derived macrophages challenged by PMMA particles or PMMA particles plus IL-10.Materials and methods1. RAW 264.7 cells (2×105cells/well) were seeded into a 6-well plate. After 4-6 hours, IL-10 were added to wells and cells were divided randomly into three groups:Ong IL-10; 10ng IL-10; 20ng IL-10. RAW 264.7 cells challenged by IL-10 for 48 hours were lysed and proteins were isolated and applied for western blot to detect the expression level of the total STAT3 and phosphor-STAT3.2. Primary bone marrow derived macrophages isolation and culture Primary bone marrow derived macrophages (BMDMs) were obtained from the femurs and tibiae of Sprague Dawley rat donors sacrificed by CO2 gas. The bone marrow was flushed into a 50 ml centrifuge tube with sterile DMEM medium containing 10% fetal bovine serum,100 U/ml penicillin and 100 μg/ml streptomycin. The cells were filtered with a 100 μm cell strainer and spun down, followed by addition of the1 cold red blood cell lysis buffer. After centrifugation, the cells were cultured with 30% L929 cultural supernatant and 70% DMEM containing 10% FBS and antibiotics in a flask.3. BMDMs were seeded into a 12-well plate at the concentration of 5 x 105 cells/well. After 24 hours, cells were divided into three groups and respectively challenged with DMEM (negative control group),2 mg PMMA particles in 0.2 ml PBS (positive group), or PMMA particles plus 20ng IL-10 (treatment group). After 5 days of treatment, total RNA was extracted and real-time RT-PCR was employed to detect the gene expression level of IL-1β、CD80、 MHCⅡ CD163、IL-10、TGF-β1 and CCR2. 4. BMDMs were seeded into a 8-well chamber slide at the concentration of 5 ×103cells/well. After 24 hours, cells were divided into three groups and respectively challenged with DMEM (negative control group),2 mg PMMA particles in 0.2 ml PBS (positive group), or PMMA particles plus 20ng IL-10 (treatment group). After one week, immunocytochemical staining was applied for detecting the expression of iNOS and CD163. Results1. Western blot was used to detect the protein expression of total STAT3 andphosphor-STAT3 of RAW 264.7 challenged by different dose of IL-10. The data indicate that RAW 264.7 cells challenged by IL-10 express more phosphor-STAT3 with the increase of IL-10 dose, however, there is no difference in the total STAT3 expression among three groups.2. Real time RT-PCR was applied for detecting the gene expression of IL-1β、CD80、MHC Ⅱ、CD163、 IL-10、 TGF-β1 and CCR2 in the bone marrow derived macrophages challenged by PMMA particles or PMMA plus IL-10. The data show that PMMA particles can induce BMDMs to produce more gene expression of IL-1β,CD80 and MHC Ⅱ. IL-10 can inhibit the function of PMMA particles in facilitating the gene expression of IL-1β (p<0.01), however, there is no difference between PMMA particles group and IL-10 treatment group in the gene expression of CD80 and MHC Ⅱ. Meanwhile, IL-10 can significantly facilitate the gene expression of CD163, IL-10, TGF-β1 and CCR2 (p<0.01).3. Immunocytochemical (ICC) staining was used to check the protein expression of Ml mark (iNOS) and M2 marker (CD 163). Immunocytochemical staining revealed that the PMMA particles provoked the BMDMs to differentiate to an M1 phenotype, resulting in an increased expression of iNOS (p<0.001). Meanwhile, IL-10 treatment weakened the polarization effect of PMMA particles, confirmed by a dramatic decrease in iNOS expression (p<0.01). Also, IL-10 treatment facilitated the over-expression of CD163, which implied that BMDMs differentiated into M2 following the IL-10 treatment (p<0.001).Conclusions1. The perfect dose of IL-10 is 20ng.2. IL-10 can facilitate the gene expression of M2 macrophages.3. IL-10 can not only facilitate BMDMs challenged by PMMA particles differentiation into M2 macrophages but also inhibit the BMDMs challenged by PMMA particles differentiation into M1 macrophages.Second PartIL-10 ameliorate particle-induced osteolysis through macrophage polarizationBackground:All of the over one million total joint replacements implanted in China each year are estimated to perform revision after 15-25 years of use, because of slow subtle inflammation at the bone implant interface. Total hip and knee replacements are paradigm of successful surgical interventions with overall success rates of>90%. Aseptic loosening is the long-term complication of total joint replacement because of the high clinic risks of revision surgery and the associated high health-care cost. With the improvements of surgical techniques, materials, and implant designs, particle production has been reduced, however, debris-induced osteolysis has not been solved. Therefore, we need to improve the long-term performance of joint replacement implants. In recent decades, many researchers focus on the mechanism of aseptic loosening. Macrophages phagocytose wear particles and other byproducts and produce pro-inflammatory cytokines and chemokines. Macrophage phagocytosis provoke a biological cascade, which is the most important reason of particle-induced osteolysis. Recent studies demonstrate that M1 macrophages are predominant in the process of aseptic loosening. Given that wear particles active the innate immune systom, the local cells around implants response to wear particles and produce cytokines and chemokines, such as IL-1β、IL-8、IL-12、TNF-α IFN-γ M-CSF、MCP-1 and MIP-1α. Osteoblasts express more RANKL after wear particles activate the biological cascade. RANK/RANKL signaling pathway plays a pivotal role in the activation of osteoclasts and downstream molecule in normal and pathological conditions. Wear particles can activate osteoclasts while osteoclasts in turn are capable of phagocytosing wear particles and remain fully functional bone resorbing cells. Macrophages activated by wear particles produce pro-inflammatory factor and activate osteoclast resulting in osteolysis which exacerbates the production of wear particles. This cascade leads to a vicious circle.IL-10 can polarize macrophages to differentiate into M2 macrophages which inhibit inflammation. Because IL-10 can ameliorate the symptoms of autoimmune diseases, we hypothesize that IL-10 can inhibit particle-induced osteolysis through macrophage polarization.Objective:1. To establish the imitative animal model of aseptic loosening.2. To investigate whether IL-10 can ameliorate particle-induced osteolysis or not.3. To investigate the mechanism of IL-10 ameliorate particle-induced osteolysis.Materials and methods1. Establish air pouch animal modelThe dorsal area of BALB/c mouse was cleaned with povidone-iodine/alcohol and shaved to prepare the pouch site, followed by 3 ml of filtered air injection into subcutaneous tissue with a 5 ml syringe. Filtered air was given every other day to maintain the air pouch. At the sixth day, a femur section (0.5 cm), or a fragment of calvarium (0.6 cm × 0.3 cm), from genetically identical mouse donors, was surgically placed into the pouch. After 24 hours, mice were divided into three groups (8 mice per group) to respectively receive injections into the pouches of 0.2 ml PBS (negative control group),2 mg UHMWPE in 0.2 ml PBS (positive control group), or UHMWPE suspension (2 mg/0.2 ml) plus 20 ng IL-10 (treatment group). IL-10 or PBS vehicle was repeatedly injected to individual pouch every other day.2. Micro CT scanningMicroCT scanning was conducted at 24 hours after surgery and at sacrifice using a SCANCO microCT system. The parameters were 30 μm isotropic voxel size,400 projections,200 ms exposure time,70 kW voltage and 114 μA current. The image data were analyzed with microCT Evaluation program V6.5-1 software to generate three-dimensional images and focal bone mineral density (BMD). The bone volume (BV) to total volume (TV) ratio was also calculated to assess total bone lose.3. Histopathological assayAll mice were euthanized seven days after the first IL-10 injection, and the bone-implanted pouch membranes were harvested. A portion of the air pouch membrane was stored for molecular analyses at-80℃. Real time RT-PCR or western blot was used to detected STAT1、 NF-κBp65、JNK1、STAT3、SHIP、STAT6 and PPAR-y at the gene level or the protein level. A portion of air pouch membrane with the intact bone tissue was either embedded in tissue freezing medium (calvarium-implanted) for crystallized sectioning or fixed in 10% buffered formalin (femur-implanted) for paraffin embedding. Frozen sections were stored at -8OoC and used to detect the number of TRAP+ cells. Implanted femora were decalcified with 10% EDTA solution for 7 days before paraffin embedding. H&E staining was used to observe the difference of thickness and cellular infiltration of inflammatory membrane among groups. Immunohistochemical staining was used to assess the protein expression of iNOS and CD163.Results1. The appearance of air pouch membranesUHMWPE particles significantly provoked local inflammation, resulting in dramatic increases in membrane thickness and cellular infiltration compared with control pouch membranes. The mean thickness of control pouch membranes was 125.8±5.58 μm, while UHMWPE particle-challenged air pouch membranes increased to 449.81 ± 10.59 μm (p<0.001). Also, the cell counts in the pouch membranes were elevated from 4749±144 cells/mm2 (controls) to 6564±144 cells/mm2 (UHMWPE particle-challenged, p<0.001). Although the proportion of macrophages was higher in particle-challenged pouch membranes than in control samples (p<0.001), it appears that there was a significant decrease of the total cell infiltration in IL-10 treatment group (5674±139 cells/mm2, p<0.001) compared to the UHMWPE group.2. The function of IL-10 in M2 polarization and particle-induced osteolysisHistological assessment of the femur-bearing pouch membranes among the groups indicated that both iNOS and CD 163 positive macrophages in the membranes challenged with UHMWPE particles were significantly more than naive pouch membranes (p<0.01). However, it appeared that treatment with IL-10 resulted in less iNOS-expressing cells when compared to UHMWPE challenged samples (p<0.05). On the other hand, there was a significantly higher CD 163 expression in the IL-10 treated air pouch membranes in comparison with the UHMWPE-particle controls (p<0.05).TRAP staining shows that significantly decreased TRAP±cells appeared in the sections from the IL-10-treated group, while samples from UHMWPE particle controls exhibited very strong TRAP activities (p＜0.001). MicroCT scanning demonstrated that osteolysis was ameliorated by IL-10 treatment and BMD in treatment group significantly preserved compared to that in positive group (p<0.05). Meanwhile, significantly higher bone volume/total tissue volume was observed after IL-10 treatment (p<0.05).3. Real time RT-PCR detection of STAT1、 NF-κBp65、JNK1、STAT3、SHIP、STAT6 and PPAR-yThe data demonstrated that IL-10 not only diminished the gene expression of STAT1 (p<0.05), NF-κB p65 (p<0.05), and JNK1 (p<0.01), but also increased the STAT3 gene expression (p<0.05). There was no statistical significant difference in gene expression levels of STAT6, SHIP and PPAR-γ between the two experimental groups.4. Western blot assaySpecimens from IL-10 treatment group exhibited decreased productions of phosphor-STATl and phosphor-NF-κB p65 compared to those from non-treated UHMWPE-challenged group. Interestingly, besides elevated phosphor-STAT3 expression was observed in IL-10 treated group, the total STAT3 protein expression appeared also higher in this group compared to negative control group.Conclusions1. IL-10 can ameliorate particle-induced osteolysis.2. IL-10 can polarize macrophages to differentiate into M2 macrophages.3. IL-10 polarizes macrophages through inhibiting the activation of JAK/STAT1 signaling pathway and NF-κB p65 signaling pathway and enhancing the activation of JAK/STAT3 signaling pathway.