CCR2⁺ Macropahge Promotes Development Of Rheumatoid Arthritis Via CD4⁺ T Cell,Chondrocyte And Fibroblast-like Synoviocyte

Rheumatoid arthritis（RA）is an autoimmune disease characterized by chronic progressive inflammation of the synovium and joint destruction.In the synovial tissue of RA patients,the massive infiltration of immune cells dominated by macrophages and T cells and the dysfunction of fibroblasts lead to joint,bone and cartilage damage.Macrophages are one of the key pathogenic cells in RA and participate in the development of RA.Numerous studies have shown that macrophages in the synovium interact with a variety of immune cells and non-immune cells,leading to the development of RA.The current research shows that the pathogenesis of RA is complex and closely related to the interaction of cells.Genetic factors and environmental factors are indispensable links in the pathogenesis of RA.At the same time,the existing treatment methods for RA patients are not effective,and intolerance and serious adverse reactions limit the treatment of RA patients.Therefore,further research is needed on the pathogenesis and treatment of RA.Given the critical role of macrophages in RA,restoring the homeostasis of macrophages and other cells in the synovium of joints is one of the core issues in the discovery of new strategies for the treatment of RA.Macrophages interact with a variety of cells to further affect the occurrence and development of RA.In the inflammatory synovium of RA,macrophages recruit a large number of CD4+T cells to infiltrate the inflammatory sites and play the pro-inflammatory role of Th1/Th17 cells or anti-inflammatory effects of Th2/Treg cells by affecting the ability of CD4+T cells to differentiate into Th1/Th2/Th17/Treg.Macrophages play an important role in the activation,expansion,migration and differentiation of CD4+T cells.In RA patients,bone and cartilage destruction is the result of disease progression,so the protection of chondrocytes has become a priority in RA treatment.Different subtypes of macrophages have different effects on chondrocytes.M1 macrophages inhibit the function of chondrocytes and do not utilize cartilage synthesis,while M2 macrophages can enhance the function of chondrocytes.In addition,fibroblast-like synoviocyte（FLS）is resident cell in synovial tissue.And with the onset of inflammatory diseases,FLS begins to exhibit abnormal function and thus aggravates the development of RA.Macrophages affect FLS functional changes,including the ability to secrete matrix metalloproteinases and inflammatory factors and invasive capacity.And even Macrophages affect FLS-mediated cartilage degradation.It can be seen that macrophages have regulatory effects on CD4+T cells,chondrocytes and FLS.CCR2 is a G protein-coupled transmembrane receptor and a specific receptor for the chemokine CCL2.It is mainly expressed on monocytes/macrophages and has a pro-inflammatory effect.Monocytes specifically recognize and bind CCL2 through CCR2,which induces monocytes to aggregate to the site of inflammation.Targeted inhibition of CCR2 has been shown to improve disease progression in a variety of disease models,including lung cancer,breast cancer,kidney damage,inflammation and fibrosis,and allergic rhinitis.However,the current animal experiments and clinical experiments show that CCR2 neutralizing antibodies or inhibitors do not exhibit good effect as expected.Further research is needed on the role of targeting CCR2 in the treatment of RA.In this study,macrophages were used as target cells to study the effect of CCR2 on the polarization of macrophages and the regulation of CCR2-mediated macrophages on CD4+T cells,chondrocytes and FLS.At the same time,CIA model was constructed to study the therapeutic effect of CCR2 inhibitor INCB3344 on CIA mice by intragastric administration,which provided an experimental basis for the study of the pathogenesis of RA and new therapeutic strategies.Objective:peritoneal macrophages from CCR2-/-mice were used to study the effect of CCR2 on macrophage polarization.At the same time,the effect of peritoneal macrophages from CCR2-/-mice or siRNA-mediated CCR2 knockdown of macrophage THP-1-M on the functions of CD4+T cells,chondrocytes and FLS were observed,which preliminarily explained the cellular mechanism of CCR2 macrophage polarization and targeted inhibition of CCR2+macrophages to regulate CD4+T cells,chondrocytes and FLS to improve RA.Mouse CIA model was constructed in vivo to clarify the therapeutic effect of CCR2 inhibitor INCB3344 on arthritis mice.Methods:1.In order to study the expression of CCR2 in synovial tissue and synovial macrophages of RA patients,western blot was used to detect the expression of CCR2 in synovial tissue of RA patients and immunofluorescence was used to detect the localization of CCR2 in synovial macrophages of RA patients.2.To investigate the effect of CCR2 on the polarization function of mouse peritoneal macrophages,peritoneal macrophages from WT/CCR2-/-mice were isolated.Flow cytometry was used to detect the changes in the proportion of LPS/IFNγ M1 type macrophages and IL4 M2 type macrophages as well as the change of M1/M2 type ratio in peritoneal macrophages from WT/CCR2-/-mice.At the same time,qRT-PCR was used to detect the expression of iNOS/IL1β/IL6 in peritoneal macrophages from WT and CCR2-/-mice stimulated with LPS/IFNγ.3.To investigate the effect of CCR2+macrophages on the behavior of CD4+T cells,chondrocytes and FLS cells,CD4+T cells were sorted by flow cytometry,and FLS of RA patients and mouse knee chondrocytes were isolated and routinely cultured.Using transwell co-culture chambers（0.4 μm）,peritoneal macrophages from WT/CCR2-/-mice were co-cultured with CD4+T cells and chondrocytes,respectively.CCR2 knockdown macrophage THP-1-M was constructed by siRNA and co-cultured with RA-FLS.Flow cytometry was used to detect the differentiation of CD4+T cells into Th1/Th2/Th17/Treg cells and transwell chamber（8 μm）was used to detect the chemotactic migration ability of CD4+T cells after co-culture of peritoneal macrophages from WT/CCR2-/-mice and CD4+T cells.After peritoneal macrophages from WT/CCR2-/-mice were co-cultured with chondrocytes,the cell viability of chondrocytes was detected by CCK 8,and the expression of damage factors（matrix metalloproteinases MMP3/MMP9/MMP13 and admast4）and protective factors（Acan and Col2a）was detected by qRT-PCR.After THP-1-M was transfected with NC/CCR2 siRNA,it was co-cultured with RA-FLS.CCK8 and transwell（8 μm）were used to detect the cell proliferation and migration ability of RA-FLS,respectively.qRT-PCR was used to detect the expression of inflammatory factor IL1β/IL6 and matrix metalloproteinases MMP1/MMP3/MMP9.4.In order to study the therapeutic effect of the CCR2 inhibitor INCB3344 on the mouse CIA model,the mouse CIA model was constructed using chicken type II collagen.And the normal group,CIA model group,CIA+CCR2 inhibitor INCB3344 group and CIA+MTX group were included.INCB3344 and MTX were administered by gavage.Changes in the overall indicators of each group of mice were observed,including body weight,arthritis index and number of joint swelling.X-ray was used to observe joint deformation and cartilage damage in mice.Ultrasound was used to observe the changes of blood flow signal in mouse joint synovium.H＆E staining was used to observe joint pathological changes.The ratio of M1/M2 macrophages in the peritoneal cavity of mice in each group was detected by flow cytometry.Results1.The expression of CCR2 in synovial tissue of RA patientsWestern blot results showed that CCR2 was highly expressed in the synovial tissue of RA patients compared with OA patients.Immunofluorescence results showed that CCR2 expression was significantly increased on synovial macrophages from RA patients compared with synovial macrophages from OA patients.2.The effect of CCR2 on the polarization of mouse peritoneal macrophagesM0 macrophages were induced to polarize into M1 and M2 macrophages by LPS/IFNγ or IL4,respectively.Flow cytometry results showed that compared with WT mouse macrophages,the proportion of CCR2 knockout M1 type macrophages were significantly reduced,and the M1/M2 type ratio was also significantly reduced.qRT-PCR results showed that CCR2 knockout M1 macrophages had reduced ability to express iNOS,IL1β and IL6 compared with WT mice.3.The effect of peritoneal macrophages of CCR2-/-mice on the function of CD4+T cellsPeritoneal macrophages of CCR2-/-mice were indirectly co-cultured with CD4+T cells.Transwell（8 μm）migration assay showed that peritoneal macrophages of CCR2-/mice inhibited the migration ability of CD4+T cells.Flow cytometry results showed that peritoneal macrophages from CCR2-/-mice increased the levels of IL4 and Foxp3 and decreased the levels of IFNγ and IL17 compared with peritoneal macrophages from WT mice,suggesting that peritoneal macrophages from CCR2-/-mice could promote the development of CD4+T cells to an anti-inflammatory phenotype.4.The effect of peritoneal macrophages of CCR2-/-mice on the function of chondrocytesPeritoneal macrophages of CCR2-/-mice were indirectly co-cultured with chondrocytes.The CCK8 results showed that peritoneal macrophages of CCR2-/-mice promoted the cell viability of chondrocytes.The qRT-PCR results also showed that peritoneal macrophages of CCR2-/-mice inhibited the expression of damage factors MMP3/MMP9/MMP13/admast4 and promoted the expression levels of protective factors Acan and Col2a in chondrocytes,indicating that peritoneal macrophages of CCR2-/-mice could protect chondrocytes and reduce chondrocyte damage.5.The effect of CCR2 siRNA-mediated THP-1-M on the function of RA-FLSIndirect co-culture of THP-1-M transfected with CCR2 siRNA and RA-FLS.Transwell（8 μm pore size）migration assay results and CCK8 results showed that macrophages transfected with CCR2 siRNA attenuated RA-FLS migration ability and cell viability compared with NC.Macrophages transfected with CCR2 siRNA also reduced the expression of inflammatory cytokines IL6 and matrix metalloproteinases MMP3/MMP9 in RA-FLS.6.The therapeutic effect of CCR2 inhibitor INCB3344 on mice CIA modelCompared with the CIA model group,after administration of INCB3344,mice tend to regain weight,and the arthritis index and the number of joint swelling were reduced in the CIA mice,indicating that INCB3344 had a significant therapeutic effect on the overall indicators of the mice.INCB3344 attenuated knee joint injury and cartilage destruction in CIA mice,and inhibited the formation of new blood vessels in the synovial tissue of CIA mice knee joints.The expression of M1/M2 macrophages in the peritoneal macrophages of mice in each group was detected by flow cytometry.The results showed that the ratio of CD11b+CD86+M1 macrophages and CD11b+CD206+M2 macrophages was significantly decreased,suggesting that INCB3344 could promote the polarization of macrophages towards an anti-inflammatory phenotype.Conclusion:1.CCR2 is highly expressed in the synovial tissue and synovial macrophages of RA patients.Knockout of CCR2 can reduce the polarization of peritoneal macrophages to M1 type,resulting in a lower M1/M2 type ratio.It is suggested that CCR2 is involved in macrophage polarization and changes the homeostasis of M1/M2 type.2.Peritoneal macrophages of CCR2-/-mice attenuate the chemotactic ability of CD4+T cells,reduce the differentiation of CD4+T cells to Th1/Th17 cells,and promoted the differentiation of Th2/Treg cells,suggesting that peritoneal macrophages of CCR2-/mice can reduce the infiltration of CD4+T cells and affect the change of the proportion of CD4+T cells.Peritoneal macrophages of CCR2-/-mice enhance the cell viability of chondrocytes,promote the expression of protective factors,and reduce the levels of damaging factors,suggesting that peritoneal macrophages of CCR2-/-mice can protect chondrocytes.Macrophages transfected with CCR2 siRNA could attenuate the proliferation,migration ability,inflammatory factor and matrix metalloproteinase expression of RA-FLS,suggesting that macrophages transfected with CCR2 siRNA could alleviate the abnormal function of RA-FLS.3.The CCR2 inhibitor INCB3344 reduce the M1/M2 ratio of peritoneal macrophages,suggesting that INCB3344 can mediate macrophage polarization and alleviate arthritis inflammation and pathological changes in CIA mice.