TDO2-overexpressed Dendritic Cells Possess Tolerogenicity and Ameliorate Collagen-induced Arthritis by Modulating the Th17/Regulatory T Cell Balance.

Tolerogenic dendritic cells are promising for restoring immune homeostasis and may be an alternative therapy for autoimmune diseases such as rheumatoid arthritis. The kynurenine pathway is a vital mechanism that induces tolerance in dendritic cells (DCs). Tryptophan 2,3-dioxygenase (TDO2) is an important rate-limiting enzyme in the kynurenine pathway and participates in immune regulation. However, the role of TDO2 in shaping the tolerogenic phenotypes of DCs remains unclear. In this study, we investigated the effects and mechanisms of TDO2-overexpressed DCs in regulating the T cell balance both in vivo and in vitro. TDO2-overexpressed DC2.4 and TDO2-/- mouse bone marrow-derived DCs (BMDCs) were generated to verify the role of TDO2 in DC maturation and functionality. TDO2 overexpression in BMDCs via PGE2 treatment exhibited an immature phenotype and tolerogenic state, whereas TDO2-/- BMDCs exhibited a mature phenotype and a proinflammatory state. Furthermore, transplant of TDO2-overexpressed BMDCs alleviated collagen-induced arthritis severity in mice, which was correlated with a reduction in Th17 populations and an increase in regulatory T cells. Collectively, these results indicate that TDO2 plays an important role in the tolerogenic phenotype and may be a promising target for the generation tolerogenic DCs for rheumatoid arthritis treatment.

Activation of the kynurenine-aryl hydrocarbon receptor axis impairs the chondrogenic and chondroprotective effects of human umbilical cord-derived mesenchymal stromal cells in osteoarthritis rats.

It has been proven that intra-articular injection of mesenchymal stromal cells (MSCs) can alleviate cartilage damage in osteoarthritis (OA) by differentiating into chondrocytes and protecting inherent cartilage. However, the mechanism by which the OA articular microenvironment affects MSCs' therapeutic efficiency is yet to be fully elucidated. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in various cellular processes, such as osteogenesis and immune regulation. Tryptophan (Trp) metabolites, most of which are endogenous ligand for AHR, are abnormally increased in synovial fluid (SF) of OA and rheumatoid arthritis (RA) patients. In this study, the effects of kynurenine (KYN), one of the most important metabolites of Trp, were evaluated on the chondrogenic and chondroprotective effects of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). hUC-MSCs were cultured in conditioned medium containing different proportions of OA/RA SF, or stimulated with KYN directly, and then, AHR activation, proliferation, and chondrogenesis of hUC-MSCs were measured. Moreover, the chondroprotective efficiency of short hairpin-AHR-UC-MSC (shAHR-UC-MSC) was determined in a rat surgical OA model (right hind joint). OA SF could activate AHR signaling in hUC-MSCs in a concentration-dependent manner and inhibit the chondrogenic differentiation and proliferation ability of hUC-MSCs. Similar results were observed in hUC-MSCs stimulated with KYN in vitro. Notably, shAHR-UC-MSC exhibited superior therapeutic efficiency in OA rat upon intra-articular injection. Taken together, this study indicates that OA articular microenvironment is not conducive to the therapeutic effect of hUC-MSCs, which is related to the activation of the AHR pathway by tryptophan metabolites, and thus impairs the chondrogenic and chondroprotective effects of hUC-MSCs. AHR might be a promising modification target for further improving the therapeutic efficacy of hUC-MSCs on treatment of cartilage-related diseases such as OA.

Paeoniflorin-6'-o-benzene sulfonate ameliorates the progression of adjuvant-induced arthritis by inhibiting the interaction between Ahr and GRK2 of fibroblast-like synoviocytes.

Aryl hydrocarbon receptor (Ahr) is thought to be a crucial factor that regulates immune responses, which may be involved in the pathogenesis of autoimmune inflammation including rheumatoid arthritis (RA). The results of our group in recent years have shown that Paeoniflorin-6'-O-benzene sulfonate (code: CP-25), a novel ester derivative of paeoniflorin, has a good effect on improving RA animal models. However, whether the anti-arthritis effect of CP-25 is related to Ahr remains unclear. Here, we showed that CP-25 treatment ameliorated adjuvant-induced arthritis (AA), a rat model of RA, by inhibiting Ahr-related activities in fibroblasts like synoviocytes (FLS). AA rats were treated with CP-25 or paroxetine from days 17 to 33 after immunization. We showed that CP-25 alleviated arthritis symptoms and the pathological changes. Treatment with CP-25 decreased the expression of Ahr in the synovium of AA rats. CP-25 inhibited the expression of Ahr and the G protein-coupled receptor kinase 2 (GRK2) as well as the co-expression of GRK2 with Ahr in FLS of AA rats. Furthermore, CP-25 down-regulated the production of Kyn in FLS of AA rats. These results suggested that CP-25 may inhibit the expression and activation of Ahr. Besides, treatment with CP-25 reduced the proliferation and migration of MH7A caused by Ahr activation. In addition, we also demonstrated that CP-25 down-regulated the total and nuclear expression of Ahr and the expression of GRK2 in Kyn-treated MH7A. Moreover, the co-expression and co-localization of Ahr and GRK2in Kyn-treated MH7A were also repressed by CP-25. The data presented here demonstrated that CP-25 suppressed FLS dysfunction in rats with AA, which were associated with reduced Ahr activation and the interaction between Ahr and GRK2.

Tryptophan 2,3-dioxygenase 2 plays a key role in regulating the activation of fibroblast-like synoviocytes in autoimmune arthritis.

BACKGROUND AND PURPOSE: Abnormal kynurenine (Kyn) metabolism has been closely linked to the pathogenesis of rheumatoid arthritis (RA). The aims of this study were to investigate the role of tryptophan 2,3-dioxygenase 2 (TDO2), a rate-limiting enzyme that converts tryptophan (Trp) to Kyn, in regulating fibroblast-like synoviocyte (FLS)-mediated synovial inflammation in autoimmune arthritis. EXPERIMENTAL APPROACH: The expression of TDO2 was determined by immunohistochemistry, confocal laser scanning fluorescence microscopy, imaging flow cytometry and Western blot. TDO2 activity was tested by HPLC and colorimetric assay. TDO2 siRNA and TDO2 inhibitor 680C91 were used to inhibit TDO2 in AA-FLS function in vitro. A rat model of adjuvant-induced arthritis (AA) was used to evaluate the in vivo effect of allopurinol (Allo), a TDO2 inhibitor. KEY RESULTS: TDO2 expression was strongly increased in synovial tissue and FLS of RA and AA. Immune cells were found to express high amount of TDO2 proteins at the peak stage of AA. Pharmacological inhibition or knockdown of TDO2 in AA-FLS resulted in a reduced proliferation, secretion, migration and invasion. Kyn restored the inhibitory effect of TDO2 inhibition on activation of AA-FLS. Allo treatment ameliorated the arthritis severity and decreased the activity of TDO2. CONCLUSION AND IMPLICATIONS: Our results suggest that elevated TDO2 expression may contribute to synovial inflammation and joint destruction during arthritis. Therefore, targeting TDO2 activity and the Kyn pathway of Trp degradation may represent a potential therapeutic strategy in RA.

sTNFRII-Fc modification protects human UC-MSCs against apoptosis/autophagy induced by TNF-α and enhances their efficacy in alleviating inflammatory arthritis.

BACKGROUND: Tumor necrosis factor (TNF)-α inhibitors represented by Etanercept (a fusion protein containing soluble TNF receptor II (sTNFRII) and the Fc segment of human IgG1) play a pivotal role in Rheumatoid arthritis (RA) treatment. However, long-term use increases the risk of infection and tumors for their systemic inhibition of TNF-α, which disrupts the regular physiological function of this molecular. Mesenchymal stem cells (MSCs)-based delivery system provides new options for RA treatment with their "homing" and immune-regulation capacities, whereas inflammatory environment (especially TNF-α) is not conducive to MSCs' therapeutic effects by inducing apoptosis/autophagy. Here, we constructed a strain of sTNFRII-Fc-expressing MSCs (sTNFRII-MSC), aiming to offset the deficiency of those two interventions. METHODS: Constructed sTNFRII-Fc lentiviral vector was used to infect human umbilical cord-derived MSCs, and sTNFRII-MSC stable cell line was generated by monoclonal cultivation. In vitro and vivo characteristics of sTNFRII-MSC were assessed by coculture assay and an acute inflammatory model in NOD/SCID mice. The sTNFRII-MSC were transplanted into CIA model, pathological and immunological indicators were detected to evaluate the therapeutic effects of sTNFRII-MSC. The distribution of sTNFRII-MSC was determined by immunofluorescence assay. Apoptosis and autophagy were analyzed by flow cytometry, western blot and immunofluorescence. RESULTS: sTNFRII-Fc secreted by sTNFRII-MSC present biological activity both in vitro and vivo. sTNFRII-MSC transplantation effectively alleviates mice collagen-induced arthritis (CIA) via migrating to affected area, protecting articular cartilage destruction, modulating immune balance and sTNFRII-MSC showed prolonged internal retention via resisting apoptosis/autophagy induced by TNF-α. CONCLUSION: sTNFRII-Fc modification protects MSCs against apoptosis/autophagy induced by TNF-α, in addition to releasing sTNFRII-Fc neutralizing TNF-α to block relevant immune-inflammation cascade, and thus exert better therapeutic effects in alleviating inflammatory arthritis.

A comprehensive analysis of TDO2 expression in immune cells and characterization of immune cell phenotype in TDO2 knockout mice.

Tryptophan 2,3-dioxygenase (TDO2) was an initial rate-limiting enzyme of the kynurenine (Kyn) pathway in tryptophan (Trp) metabolism. We undertook this study to determine a comprehensive analysis of TDO2 expression in immune cells and assess the characterization of immune cell phenotype in TDO2 knockout mice. The expression of TDO2 in various tissues of DBA/1 mice was detected by quantitative real-time PCR (qPCR) and immunohistochemistry. Both flow cytometry and immunofluorescence were used to analyze the expression of TDO2 in immune cells. Furthermore, TDO2 knockout (KO) mice were generated by CRISPR/Cas9 technology to detect immune cell phenotype. TDO2 protein level in liver was tested by western blot. High-performance liquid chromatography was used to detect the level of Trp and Kyn. Flow cytometry was used to test the proportions of splenic lymphocyte subsets in wild-type (WT) and TDO2 KO mice. We found that TDO2 was expressed in various tissues and immune cells, and TDO2 staining was mainly observed in the cytoplasm of cells. There was no difference in the development of immune cells between TDO2 KO mice and WT mice, including T cells, B cells, memory B cells, plasma cells, dendritic cells, and natural killer cells. Interestingly, the reduced M1/M2 ratio was observed in the peritoneal macrophages of TDO2 KO mice. Taken together, these findings enriched the known expression profile of TDO2, especially its expression in immune cells. Our study suggested that TDO2-mediated Trp-Kyn metabolism pathway might be involved in the immune response.

A High Precision Terahertz Wave Image Reconstruction Algorithm.

With the development of terahertz (THz) technology, the applications of this spectrum have become increasingly wide-ranging, in areas such as non-destructive testing, security applications and medical scanning, in which one of the most important methods is imaging. Unlike remote sensing applications, THz imaging features sources of array elements that are almost always supposed to be spherical wave radiators, including single antennae. As such, well-developed methodologies such as Range-Doppler Algorithm (RDA) are not directly applicable in such near-range situations. The Back Projection Algorithm (BPA) can provide products of high precision at the the cost of a high computational burden, while the Range Migration Algorithm (RMA) sacrifices the quality of images for efficiency. The Phase-shift Migration Algorithm (PMA) is a good alternative, the features of which combine both of the classical algorithms mentioned above. In this research, it is used for mechanical scanning, and is extended to array imaging for the first time. In addition, the performances of PMA are studied in detail in contrast to BPA and RMA. It is demonstrated in our simulations and experiments described herein that the algorithm can reconstruct images with high precision.