Mouse Models Of Spondyloarthritis Induced By Human Cartilage Proteoglycan

BackgroundSpondyloarthritis（Sp A）is a common autoimmune arthritis,which is widespread in all kinds of people all over the world.Sp A is characterized by enthesitis,bone destruction and new bone formation,and often involves axial bone joints,peripheral joints,and enthesis.Patients often complain of inflammatory low back pain.In advanced patients,there may be systemic bone destruction and new bone formation,which can lead to joint function loss and even disability,which will cause a great burden to the patient’s life and the society.The pathogenesis of Sp A is complex,human leukocyte antigen-B27（HLA-B27）and other genes,interleukin-23/interleukin-17（IL-23/IL-17）,mechanical stress and microbial flora may be involved in the pathogenesis of Sp A.Therefore,the construction of an animal model that simulates the characteristics of human Sp A and the establishment of a series of research methods are still problems that need to be solved urgently in this profession.In this study,we established two Sp A mouse models,namely,human cartilage proteoglycan-induced BALB/c mouse spondyloarthritis（PG-induced Sp A,PGISp A）model and human cartilage proteoglycan-induced HLA-B27/hβ₂m transgenic mouse spondyloarthritis（proteoglycan induced human leukocyte antigen-B27/humanβ₂ macroglobulin-spondyloarthritis,PGI-B27-Sp A）model by using human cartilage proteoglycan（PG）to immunize two different strains of mice,wild-type BALB/c mice and HLA-B27/humanβ₂ microglobulin（human leukocyte antigen-B27/humanβ₂ microglobulin,HLA-B27/hβ₂m）double transgenic C57BL/6 mice.The clinical phenotype,radiological and histopathological characteristics,the expression level of inflammatory cytokines of the models are described in detail to evaluate their similarity with human Sp A,and provide experimental basis and conditions for further research on the pathogenesis and intervention treatment of human Sp A.MethodsPart I:We collected articular cartilage from patients with osteoarthritis after knee joint replacement.After grinding,extraction,ultra-high-speed centrifuge（purification）,deglycosylated side chains,dialysis,freeze-drying,we prepared human cartilage proteoglycan（PG）.20 female BALB/c mice aged 14 weeks were immunized by intraperitoneal injection of a mixed emulsion of PG and complete Freud’s adjuvant（CFA）in 0 weeks and a mixed emulsion of PG and incomplete Freund’s adjuvant（IFA）in 3,6weeks;10 control mice were intraperitoneally injected with the same amount of PBS.The joint and extra-articular manifestations were observed 3 times a week,and the arthritis was scored.At 45 weeks after immunization,the spine and sacroiliac joints were examined by micro-CT,and the bone density（BMD）and bone volume fraction（BV/TV）of the spine were analyzed.The histopathological changes of peripheral joints,spine and sacroiliac joints of mice were evaluated by HE and Safranin O-fast green staining.Tumor necrosis factor alpha（TNF-α）and interleukin 17A（IL-17A）in serum were detected by ELISA at 0,20,and 45 weeks after immunization.Part II:6 female HLA-B27/hβ₂m transgenic C57BL/6 mice aged 22 weeks were immunized by intraperitoneal injection of a mixture of PG and DDA adjuvant at 0,3,6weeks.6 female HLA-B27/hβ₂m transgenic C57BL/6 mice were used as controls,given the same amount of PBS.The joint and extra-articular manifestations were observed 3 times a week.At 18 weeks after immunization,mice were anesthetized by intraperitoneal injection of pentobarbital and underwent micro-CT examination,and BMD,BV/TV,trabecular thickness（Tb.Th）and trabecular number（Tb.N）of the spine were analyzed.The pathological changes of mouse spinal inflammation,cartilage hyperplasia and new bone formation were evaluated by HE,toluidine blue and safranin-fast green staining.The expression of hypertrophic chondrocytes was analyzed by immunohistochemical staining（IHC）of type X collagen.ResultsPart I1.Compared with wild-type BALB/c mice,45 weeks after immunization,6 out of 20mice induced by PG（PGISp A）showed peripheral joint swelling and joint stiffness,accompanied by weight loss.HE staining of the ankle joint showed a little inflammatory cell infiltration,as well as proliferation of synovial cells and chondrocytes.2.Micro-CT results showed that the spine and sacroiliac joints of PGISp A mice with peripheral arthritis showed new bone formation and joint fusion,and BMD and BV/TV of the vertebral body were reduced,compared with control mice.3.Histological staining showed the proliferation of chondrocytes in the sacroiliac joints of PGISp A mice with peripheral arthritis,the proliferation of chondrocytes and osteoblasts in the spinal joints,and the proliferation of fibrous connective tissue and abnormal new bone formation at the enthesis of the spine.4.TNF-αand IL-17A increased at 20 weeks after immunization and TNF-αdecreased at 45 weeks in the serum of PGISp A mice,while IL-17A continued to increase.Part II1.Compared with the HLA-B27/hβ₂m transgenic C57BL/6 mice in the control group,4 out of 6 PGI-B27-Sp A showed significant bone loss of the axial skeleton at 18 weeks after immunization,accompanied by significant weight loss.HE staining of the small intestine of PGI-B27-Sp A mice showed that the villi were sparse and shorted,and no inflammatory cell infiltration was seen.2.Radiologically,the bone-related parameters of the thoracic and lumbar vertebrae showed that BMD,BV/TV and Tb.Th of PGI-B27-Sp A mice were significantly reduced,and Tb.N was significantly increased compared with the HLA-B27/hβ₂m mice in the control group.Tartrate resistant acid phosphatase（TRAP）staining showed that there were many TRAP-positive multinucleated cells,namely osteoclasts,on the bone surface in the vertebral bone marrow cavity of PGI-HLA-B27 mice,while no TRAP-positive cells were seen in the control mice.3.The three-dimensional reconstruction image of Micro-CT showed that the intervertebral space of some thoracic vertebrae of PGI-HLA-B27 mice had stenosis and articular surface fusion.The histological staining of the corresponding thoracic vertebrae showed that the nucleus pulposus,annulus fibrosus and part of the cartilage endplate were replaced by abnormally proliferated chondrocytes and osteoblasts,and a large number of abnormal bone hyperplasia was seen in the subchondral bone of the vertebral endplate.At the same time,abnormal proliferation of chondrocytes could be seen on the outside of the annulus fibrosus in some of the proximal caudal vertebrae.IHC staining showed that the type X collagen-positive hypertrophic chondrocytes of the cartilage end plate of PGI-HLA-B27 mice were significantly increased compared with the control mice.4.HE staining of the spine showed that a large number of inflammatory cell infiltrations could be seen in the posterior longitudinal ligament and the enthesis of the lumbar spine and lower thoracic spine of PGI-HLA-B27 mice,including a large number of multinucleated cells and a small amount of mononuclear cells.The inflammation eroded the ligament and adjacent bone cortex.Conclusion1.We successfully established a PGISp A model immunized BALB/c mice by human cartilage PG,which was similar with human Sp A in imaging,histology,and inflammatory factor expression.The establishment of this model and research platform provided a basis for the research of Sp A and other model animals.2.We established a PGI-B27-Sp A model immunized HLA-B27/hβ2m transgenic C57BL/6 mice by human cartilage PG for the first time,which showed bone destruction and new bone formation on imaging and enthesitis,bone destruction and bone hyperplasia on histology had strong similar with human Sp A.Moreover,the PGI-B27-Sp A model had an earlier onset time than the PGISp A model established by PG immunized BALB/c mice,and the incidence rate was also significantly increased This model had laid a solid foundation for carring out Sp A cellular and molecular research and new drug interventions.