Yujing Wu

Therapeutic effects of TACI-Ig on rats with adjuvant-induced arthritis via attenuating inflammatory responses

OBJECTIVE To investigate the effects of TACI-Ig, a recombinant fusion protein that modulates B- and T-cell activation by binding and neutralizing B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), in an established adjuvant-induced arthritis (AA) rat model. METHODS Rats with experimental arthritis were randomly separated into different groups and then treated with TACI-Ig (0.7, 2.1, 6.3 mg/kg), rhTNFR-Fc (2.8 mg/kg), MTX (0.5 mg/kg) or IgG-Fc (6.3 mg/kg), from Day 16 to Day 34 after immunization. Arthritis was evaluated by hind paw swelling, polyarthritis index and histopathological examination. Activities of BLyS, APRIL, IL-1β, IL-2, IL-10, TGF-β1, PGE(2), TNF-α, IFN-γ, immunoglobulin (Ig)G1, IgG2a, IgM and IgA were assessed by ELISA. Cluster of differentiation (CD)20 expression was detected by immunohistochemical analysis. RESULTS TACI-Ig (2.1, 6.3 mg/kg) treatment significantly reduced the severity of established arthritis using the methods of clinical observation and histopathological examination. TACI-Ig treatment inhibited expression of IgM, decreased the expression of BLyS and APRIL and regulated the balance of pro-inflammatory and anti-inflammatory cytokines in serum of AA rats. Immunohistochemical analysis demonstrated that CD20 production was reduced in spleen. CONCLUSIONS Data presented here demonstrate that administration of TACI-Ig significantly attenuates progression of experimental arthritis, with reductions in inflammatory response and bone and joint destruction.

Therapeutic effects of TACI-Ig on rat with adjuvant arthritis

Transmembrane activator and calcium modulator and cyclophilin ligand interactor‐immunoglobulin (TACI‐Ig) is a human fusion protein that binds and neutralizes both B lymphocyte stimulator (BLyS), a cytokine shown to be a key regulator of B cell maturation, proliferation and survival, and a proliferation‐inducing ligand (APRIL). Rat adjuvant arthritis (AA) is an experimental animal model of rheumatoid arthritis (RA), which is mainly dependent on T cells and neutrophil‐mediated cytokine production. The purpose of the present study was to investigate the effects of TACI‐Ig on rat AA. Rat AA was induced by intradermal injection of 0·1 ml complete Freunds adjuvant (CFA). TACI‐Ig (0·7, 2·1 and 6·3 mg/kg), recombinant human tumour necrosis factor‐α receptor (rhTNFR) : Fc (2·8 mg/kg) and IgG‐Fc (6·3 mg/kg) were administered subcutaneously every other day from days 16 to 34 after immunization. Arthritis was evaluated by arthritis global assessment and swollen joint count (SJC). The ankle joint and spleen were harvested for histopathological examination. Spleen index and thymus index were calculated. The levels of BLyS, interleukin (IL)‐17, interferon (IFN)‐γ, IgG1, IgG2a and IgM in AA rat spleen were measured by enzyme‐linked immunosorbent assay. Administration of TACI‐Ig significantly reduced the arthritis global assessment and SJC, decreased spleen index and ameliorated histopathological manifestations of rat AA. Suppressing the levels of BLyS, IL‐17, IFN‐γ and Ig in AA rat spleen were observed after administration of TACI‐Ig. These results showed that TACI‐Ig significantly inhibited the degree of rat AA, and the inhibitory effects might be associated with its ability to reduce BLyS, proinflammatory cytokines and Ig levels in spleen.

BAFF/BAFF-R involved in antibodies production of rats with collagen-induced arthritis via PI3K-Akt-mTOR signaling and the regulation of paeoniflorin

ETHNOPHARMACOLOGICAL RELEVANCE Paeoniflorin (Pae) is extracted from the root of paeonia lactiflora which have attracted attention for anti-rheumatic and immune modulating properties. AIM OF THE STUDY To investigate the role of PI3K/Akt/mTOR signaling mediated by BAFF/BAFF-R in antibodies production and the regulation of Pae on the signaling pathway in rats with collagen-induced arthritis (CIA). MATERIALS AND METHODS CIA rats were randomly separated into different groups and treated with Pae (25, 100mg/kg) from day 18 to day 38 after immunization. The effects of Pae on B lymphocytes of CIA rats were evaluated by the levels of BAFF, anti-CII antibody, IgA, IgG and IgM, and the expressions of BAFF-R, PI3K, p-Akt and mTOR. RESULTS In CIA rats, the levels of anti-CII antibody, IgA, IgG and IgM in serum enhanced, BAFF, BAFF-R, PI3K, p-Akt and mTOR were highly expressed. Pae (100mg/kg) obviously decreased arthritis score, relieved ankle and paw swelling, improved spleen histopathology in CIA rats, decreased the levels of IgA, IgM, IgG and anti-CII antibody, and significantly decreased the expressions of BAFF, BAFF-R, PI3K, p-Akt and mTOR. CONCLUSION PI3K/Akt/mTOR signaling mediated by BAFF/BAFF-R participates in antibodies production by B lymphocytes of CIA rats. Pae had therapeutic effects on rats with CIA. These effects might be relative to regulating PI3K/Akt/mTOR signal mediated by BAFF/BAFF-R, and down regulate the antibodies production further.

Pro-apoptotic effect of epigallo-catechin-3-gallate on B lymphocytes through regulating BAFF/PI3K/Akt/mTOR signaling in rats with collagen-induced arthritis.

To investigate the role of PI3K/Akt/mTOR signaling mediated by B cell-activating factor belonging to the TNF family (BAFF) involved in anti-apoptosis of B lymphocytes in rats with collagen-induced arthritis (CIA) and the regulation of epigallo-catechin-3-gallate (EGCG). Sprague-Dawley rats were immunized to induce CIA. CIA rats were randomly separated into different groups and treated with EGCG (40, 80 mg/kg), Paeoniflorin (100mg/kg) from day 18 to day 38 after immunization. The effects of EGCG on B lymphocytes were evaluated by the levels of BAFF, anti-CII antibody, IgA, IgG and IgM, and the expressions of BAFF receptor, P110δ, p-Akt, mTORC1, Bcl-xL and Bim. B lymphocyte proliferations were analyzed by MTT assay. Apoptosis of B lymphocyte were assayed by flow cytometry. Results showed that, in CIA rats, the levels of BAFF, anti-CII antibody, IgA, IgG and IgM enhanced. BAFF receptor, P110δ, p-AKT, mTORC1 and Bcl-xL were expressed highly, while Bim expression decreased. EGCG (40, 80 mg/kg) and Paeoniflorin decreased the levels of BAFF, anti-CII antibody, IgA, IgG, IgM and the expressions of BAFF receptor, P110δ, p-AKT, mTORC1, Bcl-xL in CIA rats, and increased Bim expression. Further studies showed that EGCG could reduce the expression of P110δ and mTORC1 in vitro. EGCG inhibited B lymphocyte proliferation and induced B lymphocyte apoptosis. In conclusion, BAFF/BAFF receptor might regulate B cell anti-apoptosis through PI3K/Akt/mTOR pathway. EGCG had therapeutic effects on CIA rats, which might be relative to the inhibition effects of EGCG on BAFF and PI3K/Akt/mTOR signaling, and then the apoptosis of B lymphocytes was promoted further.

Total glucosides of paeony inhibit the inflammatory responses of mice with allergic contact dermatitis by restoring the balanced secretion of pro-/anti-inflammatory cytokines

The present study aimed to investigate the regulation exerted by the total glucosides of paeony (TGP) on the production of interleukin-2 (IL-2), IL-4, IL-10 and IL-17 in the serum and lymphocytes of mice with allergic contact dermatitis (ACD). ACD in mice was induced by the repeated application of 2,4-dinitrochlorobenzene (DNCB) to their skins. The mice were orally administered TGP (35, 70, and 140mg/kg/d) and prednisone (Pre, 5mg/kg/d) from day 1 to day 7 after immunization. The inflammatory responses were evaluated by ear swelling and histological examination. Thymocyte proliferation was assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide assay. The cytokine production in the serum and lymphocytes supernatant was measured by enzyme-linked immunosorbent assay. The results indicated that the topical application of DNCB to the skin provoked obvious inflammatory responses. The oral administration of TGP (70 and 140mg/kg/d) and Pre (5mg/kg/d) significantly inhibited skin inflammation, decreased the thymus and spleen indices, and inhibited thymocyte proliferation in mice treated with DNCB. Further study indicated that TGP increased IL-4 and IL-10 production but decreased the production of IL-2 and IL-17 in the serum and lymphocyte supernatant. The correlation analysis suggested significantly positive correlations between IL-2 and IL-17 production and the severity of skin inflammation, whereas negative correlations were obtained for IL-4 and IL-10 production and skin inflammation. In summary, these results suggest that the therapeutic effects of TGP on ACD may result from its regulation of the imbalanced secretion of IL-2/IL-4 and IL-10/IL-17.

BF02, a recombinant TNFR2 fusion protein, alleviates adjuvant arthritis by regulating T lymphocytes in rats

Aim:To investigate the therapeutic effects of BF02 on adjuvant arthritis (AA) in rats and the regulatory effects of BF02 on T lymphocyte function.Methods:SD rats received a single intradermal injection of Freunds complete adjuvant emulsion into the right hind metatarsal footpad. After the onset of AA, the rats were injected BF02 (1, 3, or 9 mg/kg, sc) every 3 d for a total of 15 d. Intragastric administration of methotrexate (MTX, 0.5 mg/kg, every 3 d for a total of 15 d) was taken as the positive control drug. Arthritis index, swollen joint count, ankle joint histopathology, spleen histopathology and the paw radiography were used for evaluating the drug effects on AA rats. T lymphocyte function was assessed by measuring T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression levels, and percentage of T lymphocyte subsets.Results:In the AA rats, remarkable secondary inflammatory responses exhibited, accompanied by significantly higher levels of IL-1, IL-6, TNF-α, IL-17, LTα, RANKL, and MMP-13. The expression of IL17 and TNF-α mRNAs was also substantially higher than in normal rats. The percentages of CD3+CD4+ and CD4+CD25+ T lymphocytes were increased, whereas the percentages of CD4+CD62L+ and CD4+CD25+FoxP3+ T lymphocytes were decreased. Treatment of the AA rats with BF02 (9 mg/kg) or MTX significantly decreased the arthritis index, swollen joint count and arthritis global assessment. Moreover, both BF02 (9 mg/kg) and MTX significantly inhibited T lymphocyte proliferation, and blocked the above mentioned aberrance in T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression, and percentages of T lymphocyte subsets.Conclusion:BF02 exerts therapeutic effects on AA rats via the regulation of T lymphocytes.

Angiotensin II type 2 receptor correlates with therapeutic effects of losartan in rats with adjuvant‐induced arthritis

The angiotensin II type 1 receptor (AT1R) blocker losartan ameliorates rheumatoid arthritis (RA) in an experimental model. In RA, AT2R mainly opposes AT1R, but the mechanism by which this occurs still remains obscure. In the present study, we investigated the role of AT2R in the treatment of rats with adjuvant‐induced arthritis (AIA) by losartan. Adjuvant‐induced arthritis rats were treated with losartan (5, 10 and 15 mg/kg) and methotrexate (MTX; 0.5 mg/kg) in vivo from day 14 to day 28. Arthritis was evaluated by the arthritis index and histological examination. Angiotensin II, tumour necrosis factor‐α, and VEGF levels were examined by ELISA. The expression of AT1R and AT2R was detected by western blot and immunohistochemistry analysis. After stimulation with interleukin‐1β in vitro, the effects of the AT2R agonist CGP42112 (10−8–10−5 M) on the chemotaxis of monocytes induced by 10% foetal calf serum (FCS) were analysed by using Transwell assay. Subsequently, the therapeutic effects of CGP42112 (5, 10 and 20 μg/kg) were evaluated in vivo by intra‐articular injection in AIA rats. After treatment with losartan, the down‐regulation of AT1R expression and up‐regulation of AT2R expression in the spleen and synovium of AIA rats correlated positively with reduction in the polyarthritis index. Treatment with CGP42112 inhibited the chemotaxis of AIA monocytes in vitro, possibly because of the up‐regulation of AT2R expression. Intra‐articular injection with CGP42112 (10 and 20 μg/kg) ameliorated the arthritis index and histological signs of arthritis. In summary, the present study strongly suggests that the up‐regulation of AT2R might be an additional mechanism by which losartan exerts its therapeutic effects in AIA rats.

Etanercept attenuates collagen-induced arthritis by modulating the association between BAFFR expression and the production of splenic memory B cells

Anti-tumour necrosis factor-α (TNF-α) drugs are approved for the treatment of rheumatoid arthritis (RA). Many studies have investigated the effect of these drugs on the T cell response; however, some clues have indicated that it may also target B cells. This study was carried out to explore the potential effects and mechanisms of etanercept, a soluble TNF-α receptor, on the function of B cells and their development into memory B cells in type II collagen (CII)-induced arthritis (CIA). Beginning on day 24 after CII immunisation, the mice were evaluated every 2-3 days to determine two clinical parameters: their arthritis global assessment and swollen joint count (SJC). The serum concentrations of IgG1, IgG2a and anti-CII antibodies and the splenic pathology and proliferation of B cells were measured. The percentage of total memory B cells in the spleen was analysed with flow cytometry. BAFFR was detected by immunohistochemistry. In CIA mice, etanercept markedly suppressed the arthritis global assessment and the SJC, reduced the production of anti-CII, IgG1 and IgG2a antibodies, and prevented spleen histopathology to varying degrees; however, it had no obvious effect on splenic B cell proliferation. Etanercept also decreased the percentage of total CD27(+) memory B cells in the spleen. Treatment with etanercept was associated with a further increase in BAFFR expression, a significant reduction in CD27 expression, and a negative correlation between the levels of BAFFR and the percentage of memory B cells. Our findings showed that increased BAFFR expression has a regulatory effect on the activation of B cells and the generation of memory B cells, which may be one of the mechanisms of the therapeutic effects of etanercept.

Regulation of PGE2 signaling pathways and TNF-alpha signaling pathways on the function of bone marrow-derived dendritic cells and the effects of CP-25.

This study was to investigate PGE2 and TNF-alpha signaling pathway involving in the maturation and activation of bone marrow dendritic cells (DCs) and the effect of CP-25. Bone marrow DCs were isolated and stimulated by PGE2 and TNF-alpha respectively. The markers of maturation and activation expressed on DCs, such as CD40, CD80, CD83, CD86, MHC-II, and the ability of antigen uptake of DCs were analyzed by flow cytometry. The proliferation of T cells co-cultured with DCs, the signaling pathways of PGE2-EP4-cAMP and TNF-alpha-TRADD-TRAF2-NF-κB in DCs were analyzed. The results showed that both PGE2 and TNF-alpha up-regulated the expressions of CD40, CD80, CD83, CD86, and MHC-II, decreased the antigen uptake of DCs, and DCs stimulated by PGE2 or TNF-alpha could increase T cell proliferation. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) decreased significantly the expressions of CD40, CD80, CD83, CD86 and MHC-II, increased the antigen uptake of DCs, and suppressed T cell proliferation induced by DCs. PGE2 increased the expressions of EP4, NF-κB and down-regulated cAMP level of DCs. TNF-alpha could also up-regulate TNFR1, TRADD, TRAF2, and NF-κB expression of DCs. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) decreased the expressions of EP4 and NF-κB, increased cAMP level in DCs stimulated by PGE2. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) also could down-regulate significantly TNFR1, TRADD, TRAF2, and NF-κB expression in DCs stimulated by TNF-alpha. These results demonstrate that PGE2 and TNF-alpha could enhance DCs functions by mediating PGE2-EP4-cAMP pathway, TNF-alpha-TNFR1-TRADD-TRAF2-NF-κB pathway respectively. CP-25 might inhibit the function of DCs through regulating PGE2-EP4-cAMP and TNF-alpha-TNFR1-TRADD-TRAF2-NF-κB pathways.

Comparative efficacy of TACI-Ig with TNF-alpha inhibitor and methotrexate in DBA/1 mice with collagen-induced arthritis.

The efficacies of TACI-Ig, rhTNFR:Fc and Methotrexate were compared in collagen-induced arthritis (CIA) mice. Sixty animals were divided into six groups: TACI-Ig (9 mg/kg), rhTNFR:Fc (4 mg/kg), Methotrexate (2mg/kg) and IgG-Fc (9 mg/kg) groups and were given medication for six weeks. Meanwhile, normal and CIA mice were given as control. The different efficacies of drugs were evaluated by the analyses of ankle joints and spleens pathology, cytokines, T and B lymphocytes subsets. TACI-Ig and rhTNFR:Fc reduced arthritis scores seven days later than that of Methotrexate. TACI-Ig and Methotrexate were superior to rhTNFR:Fc in reduction synovial hyperplasia and cell infiltration scores. The same result was observed for scores of spleens histopathology. TACI-Ig and Methotrexate presented higher efficacy than rhTNFR:Fc on B lymphocyte stimulator, but TACI-Ig was inferior to rhTNFR:Fc on TNF-alpha. TACI-Ig and Methotrexate could reduce significantly IgA and IgM, but rhTNFR:Fc had no effects on these immunoglobulins. TACI-Ig had more efficacy than rhTNFR:Fc in the decrease of CD4(+)CD154(+) T cell. TACI-Ig and Methotrexate also reduced CD4(+)CD69(+) T cell, rhTNFR:Fc had no effects on the T cell subset. TACI-Ig and Methotrexate were superior to rhTNFR:Fc on CD4(+)CD62L(+)T cells. TACI-Ig and Methotrexate could reduce CD19(+)IgD(+) and CD19(+)CD21(+) B cells, but rhTNFR:Fc had no obvious effect on above B cells subsets. TACI-Ig is as effective as rhTNFR:Fc and Methotrexate on CIA mice by ameliorating joint and spleen pathology, regulating T and B lymphocytes function, although different mechanisms among them. This study would be useful for treatment selection of rheumatoid arthritis in different pathological conditions.