Expression Of TDO2 In Immune Cells Of Collagen-Induced Arthritis Mice And The Regulation Of Phagocytosis In Macrophages

INTRODUCTIONRheumatoid Arthritis(RA)is a chronic systemic autoimmune disease,characterized as joint synovial inflammation.The pathogenesis of RA is not clear yet.Numerous studies have described that the pathogenesis and disease process of RA are closely related to the combined action of innate immune cells,adaptive immune cells and fibroblast-like(FLS)and inflammatory mediators such as tumor necrosis factor and prostaglandin E2(PGE2).Tryptophan(Trp)is an essential amino acid,involved in protein production and nucleic acid synthesis to maintain cells activity,and play an important role in immune regulation by produce the active metabolites.More than 95%of the free Trp is metabolized by the kynurenine pathway(KP).Studies have shown that the abnormalities in Trp metabolism play an important role in the pathogenesis of RA.tryptophan-2,3-dioxygenase2(TDO2),indoleamine-2,3-dioxygenase1(IDO1)and indoleamine-2,3-dioxygenase2(IDO2)is the first-rate limiting enzyme of KP,Trp is metabolized to kynurenine(Kyn)through an enzymatic reaction and produce a series of downstream active metabolites.The studies have shown that IDO1 is widely expressed in tissues and immune cells of mammalian and plays an immunomodulatory role by regulating KP metabolic pathways and participating in signal pathways.However,there are few studies on the role of TDO2 in the immune system.Previous research by the research group have showed that TDO2 expression was increased in synovial tissue of RA patients,and TDO2 expression was increased in adjuvant-induced arthritis(AA)rats'synovial tissue and liver.In AA rats,the fuctions like proliferation,migration and invasion of FLS were inhibited by use the TDO2 inhibitor 680C91.It is suggested that TDO2 may play a role in the process of RA.Some literatures have shown that the expression of cyclooxygenase-2(COX2)and PGE2 receptor EP4 at the level of transcription and protein in glioma models are correlated with TDO2 expression.It is suggested that TDO2 may be directly involved in the immune regulation through non-enzymatic pathways,and affecting TDO2 expression and enzyme activity may be a new strategy for treating RA.However,there are no reports on the changes of TDO2expression in tissues and immune cells in RA and the immune regulation effect of TDO2 on cells.OBJECTIVE1. Identify the distribution and expression of TDO2 in tissues and immune cells,and to analyze the changes of TDO2 expression in CIA mouse tissues and immune cells.2.Clarify the regulation of PGE2-EP4-TDO2 signaling pathway on phagocytosis of macrophages.METHODS7-8 weeks of male DBA/1 mice were used to detect TDO2 expression in tissues by immunohistochemistry;TDO2 m RNA expression in tissues were detected by real-time fluorescent quantitation PCR(RT-q PCR);TDO2 expression in spleen immune cells were dectected by flow cytometry(FCM)and Imaging flow cytometry;The mouse peritoneal macrophages(PM)were identified by FCM;The expression and distribution of TDO2 in PM were detected by confocal laser scanning microscope analysis;CIA model was established and analysis the weight,arthritis index,thymus and spleen index,and the lesions were observed using X-rays;The ankle joints,spleen and liver pathological changes of CIA mice were observed by HE staining;The TDO2 expression in CIA and normal mouse tissues were dectected by immunohistochemical analysis;The expression of TDO2 in the B cells,T cells,dendritic cell(DC)and natural killer cells(NK)of CIA and normal mice by FCM;The expression of TDO2 in the PM of CIA and normal mice were dectected by RT-q PCR and confocal laser scanning microscope analysis.In vivro study,the Raw264.7 cell was separated to five group:control group,TDO2 small interfering RNA(si RNA)group,680C91 group,CAY10598 group and PGE2 group.The phagocytosis of cells was detected by FCM;The m RNA expression of EP4 and TDO2 were detected by RT-q PCR;Western blot and laser confocal detection were used to detect TDO2 protein expression;The Kyn content in the cell supernatant was detected by the method to reflect the enzyme activity.RESULTS1. The gene and protein expression of TDO2 in DBA/1 mouse tissuesTDO2 m RNA was expressed in the lymph nodes,thymus,brain,kidney,lung,spleen,liver and heart of DBA/1 mice.With the heart as the control group,the expression was highest in the liver and lowest in the kidney.The result of immunohistochemistry showed that TDO2 protein was positively expressed in liver,brain,kidney,spleen,lung,heart and ankle of mice.In the brain,TDO2-positive cells are mainly scattered around the hippocampus.In the kidney,TDO2 is mainly expressed in renal tubular epithelial cells.In cardiac tissue,TDO2 is uniformly expressed throughout the myocardial cells.TDO2 was also positively expressed in alveolar stromal cells.In the ankle joint,TDO2 is mainly distributed in the synovial tissue of the ankle joint.The spleen contains a variety of immune cells in the sheath of lymphocytes around small arteries,and this area also contains a large number of cells that express TDO2 positively.2. The expression of TDO2 in immune cells of spleen and lymph node in DBA/1miceFlow cytometry results showed that compared with the negative control group,TDO2 was significantly expressed in T,B lymphocytes,DC(CD11c~+)and NK(CD3~-/CD49b~+)in spleen of mice.The results of imaging flow cytometry further verified and showed that TDO2 was mainly expressed in the cytoplasm of immune cells.3. The expression of TDO2 in peritoneal macrophages of DBA/1 mouseThe percentage of CD11b~+/F4/80~+positive cells which isolated and cultured was reached 99%.Compared with the negative control group,TDO2 was significantly expressed in PM and was mainly localized in cytoplasm.4. The arthritis global assessment of CIA miceThe arthritis index of CIA mice began to increase on d26 and peaked on d35-d38.X-ray results showed that compared with the normal group,the ankle joints of the CIA mice were significantly swollened and the joint worm morphology was changed.The weight began to decrease on the d22 and significant decrease on the d30.Compared with the normal group,the thymus and spleen of CIA mice were significantly enlarged,and the thymus index and spleen index were significantly increased.5. The pathological changes of ankle joint,spleen and liver in CIA miceCompared with the normal group,synovial tissue hyperplasia,inflammatory cell infiltration,bone and cartilage destruction were observed in the joint of CIA mice.CIA mice spleen showed white pulp hyperplasia,germinal center emergence,red pulp hyperplasia,lymphoid follicular hyperplasia and inflammatory cell infiltration.Compared with the normal group,inflammatory cells in the liver of CIA mice were significantly increased.6. The expression of TDO2 in ankle joint,spleen and liver of CIA miceCompared with the normal group,the positive expression of TDO2 was significantly increased in the synovial tissue of the ankle joints,periarterial lymph sheath of the spleen and the hepatocytes of liver in CIA mice.7. The expression of TDO2 in spleen and lymph node immune cells of CIA miceCompared with the normal group,the expression of TDO2 in B cells(CD19~+),plasma cells(CD19~-/CD138~+),memory B cells(CD19~+/CD27~+),NK and DC of spleen in CIA mice were significantly increased,in the Th cells(CD3~+/CD4~+),cytotoxic T cells(CD3~+/CD4~-/CD8~+),Th17 cells(CD4~+/IL-17~+)and Treg cells(CD4~+/CD25~+/Foxp3~+)of spleen,the expression of TDO2 have showed no significant differences.Compared with the normal group,the expression of TDO2 in B cells of lymph node in CIA mice was significantly increased and have no significant difference in Th cells and cytotoxic T cells.8. The expression of TDO2 in peritoneal macrophages of CIA mouseCompared with the normal group,the m RNA and protein expression of TDO2 in PM of CIA mice were significantly increased.9. Effect of TDO2 on phagocytosis and EP4 m RNA expression in Raw264.7 cellsCompared with the control group,the phagocytic of the TDO2 si RNA-Raw264.7cell group and the 680C91-Raw264.7 cell group was significantly reduced,and the m RNA expression level of EP4 was significantly increased.1 0. Effect of EP4 receptor activation on phagocytosis and TDO2 m RNA expression and activity of Raw264.7 cellsCompared with the control group,the phagocytosis of CAY10598 group was reduced,and the relatively m RNA expression and enzyme activity of TDO2 were significantly reduced.11.Effects of PGE2 on phagocytosis and TDO2 expression and enzyme activity of Raw264.7 cellsAfter stimulating with different concentrations of PGE2 for 24 hours,compared with the control group,the phagocytosis of Raw264.7 cells which stimulated by PGE2(100n M,1?M,10?M)was significantly reduced and TDO2 expression and enzyme activity were also reduced.CONCLUSIONS1.TDO2 is widely expressed in the heart,brain,kidney,lung,spleen,liver and ankle joint of DBA/1 mice.TDO2 is expressed in immune cells of DBA/1 mice,including:T,B lymphocytes,PM,DC,and NK,mainly distributed in the cytoplasm.2.Compared with the normal group,the expression of TDO2 in the ankle joint,spleen and liver of CIA mice was significantly increased,and the expression of TDO2 was increased in spleen B cells,plasma cells,memory B cells,NK and DC;There was no significant differences of TDO2 expression in total Th cells,cytotoxic T cells,Th17cells and Treg cells.3.TDO2 expression and activity were positively correlated with the phagocytosis of macrophages and negatively correlated with EP4 expression.The TDO2 expression and enzyme activity,EP4 expression and phagocytosis function of macrophage were reduced by stimulated with PGE2.