Effect Of RhTNFR: Fc And MTX Combinaiton Treatment On Collagen-induced Arthritis And The Mechanisms Of Tumor Necrosis Factor-a In Regulating Ep4Signaling In Human Fibroblast-like Synoviocytes

Rheumatoid arthritis （RA） is a systemic autoimmune disease that primarily targets thesynovial membrane, cartilage, and bone. Tumor necrosis factor-α （TNF-α） is a keymediator in the inflammatory response which is implicated in the onset of a number ofdiseases, such as RA. TNF-α exerts its biological effects by interacting with twodifferent receptors: TNF receptor1（TNFR1） and TNFR2. TNFR1presents a deathdomain and triggers cell death processes. In contrast to TNFR1, TNFR2does not havea death domain and prevents the triggering of cell death processes. The developmentof TNF inhibitor therapy has revolutionized the treatment of RA. The recombinanthuman TNFR2:IgG Fc （rhTNFR:Fc）, a recombinant fusion protein that binds andneutralizes TNF-α, has been shown therapeutic effects on RA. The superiority of thecombination therapy of MTX and anti-TNF biological agents over anti-TNFmonotherapy in MTX-na ve patients with RA has been demonstrated, however, themolecular mechanism is unclear. TNFR2expresses on T cells and other immune cellswhich regulate T-lymphocytes survival, activation and proliferation. CD4⁺T cellsubsets play a different role in the pathogenesis of RA. The imbalance of T cellsubsets and pro-inflammatory cytokines are the key factors leading to the chronic inflammation of RA. Therefore, effect of rhTNFR: Fc and MTX on T cellsdifferentiation and function need to be deeply discussed.In our prior work, we found that after TNF-α treatment, the prostaglandin E₂（PGE₂）-PGE₂receptor4（EP4） signal transduction pathway of synoviocytes incollagen-induced arthritis （CIA） rats was unbalanced, TNF-α could downregulate EP4expression and cyclic adenosine monophosphate （cAMP） level, resulting in cellsproliferation. EP4receptor is a typical G protein coupled receptor （GPCR）, linked tostimulatory G protein （Gαs）, then increased intracellular cAMP level.G-protein-coupled receptor kinases （GRKs） and β-arrestin2are the importantregulators of GPCR. But on earth how TNF-α regulated the signaling of EP4receptorthrough its downstream molecules in human fibroblast-like synoviocytes （hFLS）remains unknown.Therefore, the present study was designed to screen rhTNFR:Fc and MTX for CIAtargetting on T lymphocytes and to explore how TNF-α regulated the signaling of EP4receptor through its downstream molecules in hFLS and, while providing theory basisto explore the role of TNF-α in RA.Objective These works were carried out to investigate the effect of rhTNFR:Fc andMTX for CIA targetting on T lymphocytes and the potential molecular mechanism ofTNF-α in regulating EP4signaling pathway.Methods A preparation of0.1ml of heterologous CCII was injected intradermallyinto the back and the base of the tail of40DBA/1mice to induced CIA on day0,followed by abooster injection on day21. After the onset of arthritis, animals weredivided into five groups randomly. CIA mice were treated with rhTNFR:Fc （4.5mg/kg） subcutaneously, MTX （2mg/kg） intragastrically or rhTNFR:Fc （4.5mg/kg）⁺MTX （2mg/kg） once every3days from day26to day41. The effect of rhTNFR:Fcon CIA mice was valuated by arthritis scores, joints and spleens histopathology, aswell as indices of thymus and spleen. T lymphocytes proliferation was determined by [3H]-TdR incorporation. Levels of TNF-α, LT-α, IL-1β, RANKL, IL-10, IL-17, IFN-γand IL-6were detected by ELISA. The subsets of T lymphocytes including CD4⁺,CD8⁺, CD3⁺CD4⁺, CD4⁺CD25⁺, CD4⁺CD62L⁺and CD4⁺CD25⁺Foxp3cells werequantified using flow cytometry.Normal FLS were isolated from primary synovial tissue obtained from patients.TNF-α-induced PGE₂production was inhibited by Indometacin （INN）, a COXinhibitor. Proliferation of hFLS was detected by MTT assay. The levels of TNFR1,TNFR2and total TRAF2were quantified by flow cytometry and western blotting.Cellular cAMP level was measured using125I-cAMP radioimmuno assay （RIA） kit.The expressions of total Gαs, activated Gαs, EP4, as well as the levels of EP4andGRK2in plasma membrane and cytoplasm were analysed by werstern blotting. Thegene expression of TRAF2was silenced by small interfering RNA （siRNA）transfection. The ability of TRAF2interact biochemically with GRK2/β-arrestin2andactivated Gαs were tested by co-immunoprecipitation. The concentration of PGE₂inculture supernatants was determined with a human-specific enzyme linkedimmunosorbent assay （ELISA） kit.Results1Effect and mechanism of rhTNFR:Fc and MTX on CIA mice.1.1Effect of rhTNFR:Fc and MTX on arthritis scores of CIA mice.From day35to day40, MTX （2mg/kg） and rhTNFR:Fc （4.5mg/kg）⁺MTX （2mg/kg）reduced the arthritis scores significantly. From day38, rhTNFR:Fc （4.5mg/kg）treatment obviously reduced the arthritis scores. Combination therapy showed asuperior efficacy compared with rhTNFR:Fc monotherapy in decreasing arthritisscores.1.2Effects of rhTNFR:Fc and MTX on histopathology of joints and spleens inCIA miceIn CIA mice, synoviocytes proliferated to multilayers, and articular cartilages were destructed and infiltrated with inflammatory cells. The hyperplastic synovium inmodel mice formed a large number of fibroblasts and new blood vessels. In micegiven rhTNFR:Fc, MTX or combined treatment, the synovial hyperplasia and thepannus were significantly inhibited and the destruction of articular cartilages wasalleviated. Inflammatory scores of joints in model mice were increased in comparisonwith normal mice. The combined treatment group performed significantly betterinhibition in synovial inflammation than rhTNFR:Fc group, and in synovialhyperplasia than MTX group.In model mice, white pulp proliferated, germinal centers emerged, and spleens wereinfiltrated with inflammatory cells. rhTNFR:Fc, MTX or the combined treatmentgroup alleviated these abnormalities on varying degrees. It is mainly showed that redpulp congestion mitigated, germinal center disappeared, white pulp reduced andnearly closed to normal. The combined therapy was consistently superior torhTNFR:Fc group in all pathological changes of spleen, and was superior to MTXgroup monotherapy in inhibition of lymphoid follicles hyperplasia.1.3Effects of rhTNFR:Fc and MTX on indices of thymus and spleen in CIA miceResults showed that thymus index and spleen index of model mice were increasedcompared with normal mice. rhTNFR:Fc and the combined treatment groupmoderated the thymus and spleen indices of CIA mice significantly. There are nosignificant differences among three groups. MTX had no obvious effect on the indexof spleen.1.4Effects of rhTNFR: Fc and MTX on T lymphocytes proliferation in CIA micerhTNFR:Fc, MTX or rhTNFR:Fc⁺MTX could reduce ConA-induced T lymphocytesproliferation obviously.There are no significant differences among three groups.1.5Therapeutic effects of rhTNFR:Fc and MTX on CIA mice by regulatingserum and macrophage supernatant cytokines balance. Results showed that the concentrations of serum IL-1β, TNF-α, IL-6and RANKLwere significantly elevated, while IFN-γ was significantly decreased in model mice.MTX or rhTNFR:Fc⁺MTX administration could reduce serum IL-1β and IL-6expression significantly; rhTNFR:Fc or rhTNFR:Fc⁺MTX administration couldreduce serum TNF-α and RANKL expression significantly. No group had obviouseffect on the level of serum IFN-γ in CIA mice. The combined treatment groupperformed significantly better inhibition on IL-6than rhTNFR:Fc group and MTXgroup.The concentrations of macrophage supernatant LT-α and IL-17were significantlyelevated, while IL-10was dramatically decreased in model mice. The concentration ofmacrophage supernatant IL-10in MTX or the combined group was notably increased.rhTNFR:Fc or rhTNFR:Fc⁺MTX could reduce the concentrations of macrophagesupernatant LT-α and IL-17. There are no significant differences among three groups.1.6Therapeutic effects of rhTNFR:Fc and MTX on CIA mice by regulating Tlymphocytes.In both CIA thymus and spleen, the ratio of CD4⁺/CD8⁺and the proportion ofCD3⁺CD4⁺（analogous to helper T, Th）, Teff cells were obviously higher comparedwith normal mice, while CD4⁺CD62L⁺（analogous to na ve） and Treg cells weresignificantly lower.In CIA thymus, rhTNFR:Fc and the combined treatment group could decrease theproportion of Th, Teff cells and increase the proportion of na ve and Treg cellssignificantly. And the combined treatment group performed significantly betterpromotion in Treg cells than MTX group.In CIA spleen, rhTNFR:Fc, MTX and the combined treatment group could decreasethe proportion of Th, Teff cells and increase the proportion of na ve cells significantly.rhTNFR:Fc and the combined treatment group could increase the number of Tregcells obviously. And the combined treatment group performed significantly betterpromotion in Treg cells than MTX group. Each group could obviously decrease the ratio of CD4⁺/CD8⁺in both thymus andspleen, and the combined treatment group performed significantly better inhibition onspleen CD4⁺/CD8⁺than rhTNFR:Fc group and MTX group.2The molecular mechanism of TNF-α in regulating EP4signaling pathway.2.1Effect of TNF-α on hFLS proliferation in vitro.The result of MTT suggested that the suboptimal concentration of TNF-α was20ng/ml and optimum time point was48h for FLS proliferation. The concentration ofcAMP was measured and the result showed that under TNF-α （20ng/ml） or TNF-αplus INN （20ng/ml⁺1×10-6mol/L） stimulation, intracellular cAMP level wassignificantly decreased. Both TNF-α and TNF-α plus INN treatment could increasecells proliferation obviously, and TNF-α group performed significantly betterpromotion in cells proliferation than TNF-α plus INN group.2.2Effect of TNF-α on TNF-α signaling pathway.Results showed that the expression of TNFR2was obviously increased after TNF-αstimulated. But there was no significant change on TNFR1expression. The proteinexpression level of TRAF2was significantly higher in hFLS after stimulated byTNF-α plus INN than TNF-α group.2.3Effect of TNF-α on total Gαs, activated Gαs, total EP4, membrane andcytoplasmic EP4expression.The results demonstrated that groups of TNF-α or TNF-α plus INN greatly increasedthe expression of total and cytoplasmic EP4, meanwhile, significantly decreased theexpression of membrane EP4and EP4gene. Further evidences found that total Gαsexpression had no notably change. However, TNF-α or TNF-α plus INN decreasedactivated Gαs expression obviously.2.4TNF-α decreased the level of cAMP by promoting the desensitization of EP4 by reducing TRAF2-GRK2complex.We found that an interaction occurs between TRAF2and GRK2/β-arrestin2, and theTRAF2-GRK2interaction was strong in normal hFLS. After stimulated by TNF-α orTNF-α plus INN, the interaction of TRAF2and GRK2was significantly decreased.Next, we noticed that the cell was expressed at a high level of membrane GRK2. Butthe interaction of TRAF2and β-arrestin2did not change significantly.2.5Effect of TNF-α on hFLS cellular PGE₂level.Because TNF-α is known to induce PGE₂production in hFLS, we tested theconcentration of cells supernatant PGE₂after stimulated by TNF-α or TNF-α plusINN. Results showed that the concentration of PGE₂was significantly increased byTNF-α treatment. And TNF-α plus INN group could significantly decrease theconcentration of PGE₂.2.6Effect of TNF-α on hFLS proliferation after TRAF2deficiency.In TRAF2defective hFLS, TNF-α or TNF-α plus INN group significantly increasedthe level of cAMP. However, the stimulation of TNF-α plus INN failed to promotehFLS proliferation.2.7Effect of TNF-α on total EP4, membrane and cytoplasmic EP4, GRK2expression after TRAF2deficiency.The total protein level, gene expression and membrane EP4level were prominentlyincreased by TRAF2siRNA. Furthermore, the protein level of cytoplasmic EP4wasobviously lower. After TRAF2deficiency, the TRAF2-GRK2interaction becameweaker. Next, we found that membrane GRK2was prominently decreased andcytoplasmic GRK2was increased absolutely.2.8Effect of TNF-α on hFLS cellular PGE₂level after TRAF2deficiency.Results showed that TRAF2silencing had no effect on the TNF-α induced PGE₂ production after each group stimulated.Conclusion1. In CIA mice, T lymphocytes proliferated obviously; the concentrations of serumIL-1β, TNF-α, IL-6and RANKL were significantly elevated, while IFN-γ wassignificantly decreased; the concentrations of LT-α and IL-17in macrophagesupernatant were significantly elevated, while IL-10was dramatically decreased;pathological changes were observed in spleen and joints; in both CIA thymus andspleen, the ratio of CD4⁺/CD8⁺and the proportion of Th, Teff cells wereobviously higher compared with normal mice, while na ve and Treg cells weresignificantly lower. rhTNFR:Fc, MTX and rhTNFR:Fc⁺MTX treated groupsreduced the arthritis scores significantly, reduced ConA-induced T lymphocytesproliferation obviously, regulated serum and macrophage supernatant cytokinesand T lymphocyte subsets balance. Combination therapy showed a superiorefficacy compared with rhTNFR:Fc or MTX monotherapy in arthritis scores,inflammatory scores of joints, synovial hyperplasia, all pathological changes ofspleen, serum IL-6, both thymus and spleen Treg cells and the ratio ofCD4⁺/CD8⁺.2. TNF-α group obviously promoted the proliferation of hFLS not only depend onTNF-α signaling pathway but also regulating EP4signaling pathway by bindingGRK2to control the dissociation of membrane GRK2. In normal hFLS, theTRAF2-GRK2interaction was strong, after TNF-α stimulated, the expression ofTRAF2was increased, and then TRAF2could control the dissociation ofmembrane GRK2, regulate the internalization and desensitization of EP4receptorand decrease the level cAMP.