| Rheumatoid arthritis（RA）is an inflammatory autoimmune disease,which characterized by persistent synovitis,systemic inflammation and autoantibodies production.It can cause pain and swelling of multiple joints in the body and even organic impairment.Immune dysfunction and related antigen-presenting cells,lymphocytes,and inflammatory cytokines play a crucial part in the development of RA.With the rapid development of"immunometabolism",abnormal metabolism of immune cells led to the imbalance of immune homeostasis,which has gradually become a hot spot in RA research.In RA,metabolism and immunity were regulated by each other,and immune homeostasis was unbalanced in the pathogenesis of RA.And immune function disorders caused by immune cell metabolism disorders may mediate the pathological process of RA.Tryptophan（Trp）,an essential aromatic amino acid,participates in the synthesis of proteins and nucleic acids and plays a key role in maintaining physiological activities.More than 95%of Trp is metabolized through the Kynurenine pathway（KP）.The decrease of Trp level and the increase of Kyn and related metabolites in RA patients indicate that KP metabolism is abnormal.Therefore,targeted regulation of the KP restored abnormal metabolism to normal and restored the balance of immune homeostasis,which could provide a new strategy for the clinical treatment of RA.Tryptophan-2,3-dioxygenase 2（TDO2）is the rate-limiting enzymes in the first step of KP.A series of downstream active metabolites were produced of KP.Recently,with the increasing research on TDO2,it has been found that TDO2 has an immunomodulatory effect in tumors,neurological diseases,and inflammatory immune-related diseases.However,the role of TDO2 in RA has not been reported.Macrophages are of critical importance in rheumatoid arthritis（RA）.The activation of macrophages can be used as an early indicator of RA,and the increase of macrophages is a significant feature of inflammatory lesions.Moreover,macrophages can enhance the inflammatory response by secreting cytokines and interacting with other immune cells（such as T cells and B cells）and non immune cells（such as osteoclasts）,promote the destruction of cartilage and bone,and then affect the process of RA disease.Macrophages are mainly divided into pro-inflammatory macrophages（M1）and anti-inflammatory macrophages（M2）.Under physiological conditions,the two kinds of macrophages are in balance.Nevertheless,the proportion of M1/M2 macrophages was unbalanced of RA patients.The effect of abnormal KP mediated by TDO2 on macrophage function in CIA mice has not been reported.Therefore,this project takes macrophages as the target cells to investigate the regulation of TDO2 on its function by knocking out and overexpressing the TDO2 gene.In vivo,we constructed a CIA model to study the role of the TDO2-mediated KP and explore new drug targets of RA.OBJECTIVEThe effects of TDO2 on the function of RAW264.7 cells were investigated by combining gene knockout and gene overexpression.Through the establishment of CIA model,the expression and activity of TDO2 in CIA mice were further explored,the effect of TDO2 on peritoneal macrophages（PMs）polarization in CIA mice and the therapeutic effect of LM10 on CIA mice were clarified.We provide an experimental basis for revealing that TDO2 participates in the macrophages polarization of RA and that TDO2 is a potential target for RA treatment.METHODSCRISPR/Cas9 technology was used to construct stable RAW264.7-TDO2-/-cell line.The overexpression of TDO2 gene in RAW264.7 cells was constructed by plasmid transfection.CIA mice model was established by chicken type II collagen.LM10 and MTX were given by gavage respectively.LM10 and MTX were given by gavage respectively.To evaluate the arthritis index and swollen joint count.The pathological changes of the ankle joint were observed by HE staining.Immunohistochemistry was used to test the expression of TDO2 in spleen,ankle joint and liver of CIA mice.The expression of CD86 and CD206,and the phagocytosis of macrophages was detected by flow cytometry.The expressions of TDO2,iNOS,and Arg1 in RAW264.7 cells and PMs were detected by western blot.Flow cytometry was employed for detecting the proportion of T cell subtypes in spleen of CIA mice.The levels of IL-10,TGF-β,IL-1β,and TNF-αin serum and PMs supernatant were detected by ELISA.RESULTS1、The effect of TDO2 knockout on the function of RAW264.7 cells.RAW264.7-TDO2-/-stable cell line was successfully constructed by CRISPR/Cas9method.Western blot results showed that compared with the control group,the expression of iNOS in RAW264.7 cells was decreased and the expression of Arg1 was increased.Flow cytometry showed that the ratio of M1/M2 and phagocytic capacity of RAW264.7-TDO2-/-cells were significantly lower than control group.Compared with the control group,the results of ELISA displayed that the levels of IL-10 and TGF-βwere increased and the levels of IL-1βand TNF-ɑwere decreased in the cell supernatant of the RAW264.7-TDO2-/-cells.2、The effect of TDO2 overexpression on the function of RAW264.7 cells.The overexpression of TDO2 in RAW264.7 cells was constructed by plasmid transfection.Western blot results showed that compared with the control group,the expression of iNOS increased and Arg1 decreased in RAW264.7 cells after overexpression of TDO2.The results of flow cytometry showed that the ratio of M1/M2and phagocytic capacity of RAW264.7 cells overexpressed with TDO2 were increased compared with the control group.ELISA results showed that compared with the control group,the levels of IL-10 and TGF-βin the supernatant of RAW264.7 cells overexpressed with TDO2 were decreased,while the levels of TNF-ɑwere increased.3、The effect of 680C91 on RAW264.7 cell function.680C91（5 n M,10 n M,20 n M）was administered to RAW264.7 cells for 24 hours in vitro.The results of western blot showed that the 680C91 group can reduce the expression of iNOS in RAW264.7 cells and increase the expression of Arg1.Flow cytometry presented that 680C91 could downregulate the ratio of M1/M2 and decrease the phagocytic capacity of RAW264.7 cells.ELISA analysis displayed that the levels of IL-10 and TGF-βwere increased and the levels of IL-1βand TNF-ɑwere decreased in the cell supernatant of the 680C91 group than the control group.4、Therapeutic effect of TDO2 inhibitor LM10 on CIA mice.LM10,an inhibitor of TDO2 in vivo,has a therapeutic effect on CIA mice,which could significantly reduce the arthritis index,swollen joint count,spleen index,ankle pathological score,and spleen cell proliferation in CIA mice.The results of immunohistochemistry showed that LM10 can reduce the expression of TDO2 in the ankle joint,spleen and liver of CIA mice.Western blot results showed that LM10 can reduce the expression of TDO2 in PMs of CIA mice.Flow cytometry results presented LM10 can downregulate the ratio of CD3+CD4+T cells and CD4+IL-17+Th17 cells in the spleen of CIA mice,upregulate the ratio of Th17/Treg,and reduce the ratio of M1/M2 in PMs.ELISA analysis displayed that LM10 could increase the levels of IL-10and TGF-βand reduce the levels of IL-1βand TNF-ɑin the serum and PMs supernatants of CIA mice.CONCLUSIONS1.The inhibition of TDO2 could decrease the level of M1 and increase the level of M2in RAW264.7 cells,decrease the phagocytic capacity of RAW264.7 cells,and affect the secretion of RAW264.7 cells.After overexpression of TDO2 gene,the polarization and phagocytosis of RAW264.7 cells were enhanced,and the levels of cytokines in the cell supernatant were changed.These results suggest that the KP metabolic pathway mediated by tdo2 is involved in the regulation of macrophage function（polarization,phagocytosis and secretion）.2.LM10,an inhibitor of TDO2,can reduce the polarization ability of PMs in CIA mice.LM10 may restore the abnormal metabolism of KP by inhibiting the expression and enzyme activity of TDO2,thus alleviating the arthritis symptoms of CIA mice. |
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