Bioactive Analysis Of Tumor Necrosis Factor Receptor â…¡-Adiponectin Globular Domain Fused Protein

BackgroundTNF was first described by Lloyd Old et al. in1975as an endotoxin-induced glycoprotein, which caused hemorrhagic necrosis of solid tumors. TNF is released as a soluble cytokine (sTNF, a homotrimer of17kDa monomers) after being enzymatically cleaved from its cell-surface-bound precursor (transmembrane TNF [tmTNF], a homotrimer of26kDa monomers) by TNF-converting enzyme. Both of them are biologically active. TNF is a potent inducer of inflammatory response, which monitors the Thl response to defend the infection of intracellular bacteria and virus invading. However, when the expression of TNF is dis-regulated some pathological situations will ensue, such as immune-mediated inflammatory diseases (IMIDs), e.g GaIN/LPS-induced acute liver failure, LPS-induced endotoxic shock and rheumatoid arthritis (RA). Therefore, TNF has been collectedly considered as a target for the strategies against these diseases.The engaged receptors for TNF are divided into TNFRI (p55) and TNFRII (p75). Naturally, TNF binds sTNF or tmTNF in form of homo-trimer, which activate the down-stream signal transduction leading to responding cellular biology or pathology. Thus, it will contribute to the engagement between TNF and TNFR by mimicking the homologous TNF trimer. Till now, the TNF antagonists used in clinic are mainly bivalent which comprise TNFR fusion protein such as sTNFRII-Fc and monoclonal antibody (Infliximab, Adalimumab). Because of the enormous application market investigation for novel efficacious TNF antagonist has becoming the hotspot in this field.Adiponectin is a cytokine mainly secreted by adipocytes, which promotes intake of glucose and oxidation of by skeletal muscle cell and hepatocyte in addition to enhancing the sensitization of insulin. Utilizing the inclination of gAD to form homologous trimer naturally we produced a novel trimerized sTNFRII fusion protein, namely, sTNFRII-gAD.It is indicated that exposure to LPS give rises to excessive TNF production leading to liver injury, which has been testified in studies in which LPS-induced acute liver failure was ameliorated via utilizing TNF specific antibody or anti serum. It has been demonstrated in a few investigations that activation of pro-inflammatory cytokines including TNF is considered to play a important role in the pathology of clinical hepatic injury. Therefore it will bee helpful to cope with septic shock induced by G" bacteria infection through antagonizing the excessively produced TNF.On the other side, RA is collectively seemed as a systemic, progressive inflammatory autoimmune disfunction despite the specific pathogenesis of it remains to investigate till now. Mainly it manifestates synovium injury which leads to cartilage erosion and even articular injury. Although the traditional disease symptom modifying anti-rheumatoid drugs (DMARDs) could improve the symptoms in addition to enhancement of the activity to work, the progressive joint injury can’t be hampered. Studies imply that TNF expression increases significantly in the inflammatory synovium particularly joint-pannus. In another research, human TNF transgenic mice display excessively produced TNF followed by polyarthritis with the incidence being100%. Based on these evidences it is proposed that TNF antagonism would be an enterprising orientation for therapy of RA in clinic.ObjectiveThis study testified TNF antagonizing activity of sTNFRII-gAD produced via eukaryotic expression in coping with GaIN/LPS-induced acute liver injury and CII-induced rat arthritis. It would lay a solid foundation for the final application of this novel recombinant fusion protein in clinic.Methods1. In vitro TNF antagonism analysis of sTNFⅡ-gAD1.1Comparison of TNF antagonism between two antagonist①L929cells in exponential phase was digested by0.25%trypsin followed by adjustment of cell density appropriately.②100μl cell suspension was seeded in96-well microplate,incubated at37℃,5%CO2atmosphere.③Dilution of sTNFRII-gAD, sTNFRII-Fc, riding off supernatants.④Adding prepared samplel, going on incubating for18-20h.⑤Adding MTT,incubation for4h at37℃.⑥Meltting the sediment with DMSO, measuring OD57onm on ELISA reader.1.2TNF-sTNFRII-Fc/sTNFRII-gAD complex measurement①96-well plate was coated with rmTNF.②Wells were blocked with4%BSA.③Experimented samples were added and incubated.④Wells were then washed and incubated with mouse anti-human sTNFRII monoclonal antibody.⑤HRP-conjugated secondary antibody was added after washing.⑥Wells were washed once more and incubated with TMB.⑦Optical intensity was determined spectrophotometrically at450nm.2. The therapeutical efficacy of GaIN/LPS-induced ALI2.1The establishment of GaIN/LPS-induced ALIFast mice were subjected to intraperitoneal injection with GaIN/LPS mixture. Negative control was given sterile saline. Afterward, all experimented mice were monitored continuously for12hours.2.2The ameliorative effects of sTNFRII-gAD on GaIN/LPS-induced ALI①The mice survival after therapy:mice were divided into4groups randomly. Part of animals (N=6)were given administration of GaIN/LPS followed by treatments while others(N=6) were treated by experimented drugs1h later past ALI construction. After treatments, mice were monitored continuously for12.②Detection of serum aminotransferase activity:Post construction of GaIN/LPS-induced ALI with sublethal GaIN/LPS injection mice were subjected to serum aminotransferase (including ALT, AST) measurements at2and5h, respectively.③Pathological analysis:5h post therapy some mice were sacrificed and10mm3liver tissue was fixed in10%neutral formalin for H-E staining.2.3The mechanisms for the therapeutical effect of sTNFRII-gAD①Serum TNF quantification and bioactive measurement:at different points serum TNF quantification and bioactive analysis were conducted.②Serum sTNFRII-gAD-TNF complex detection:At5h after therapy, blood was collected and the isolated serum was used for measurement of sTNFRII-gAD-TNF by sandwich ELISA.③Western bloting:obtained liver tissues were determined for apoptotic protein Casepase-3by Western bloting.④TUNEL assay for in situ apoptosis: obtained liver tissues were subjected to apoptosis detection via TUNEL3. Therapeutical efficacy of sTNFRII-gAD on collagen-induced arthritis3.1Establishment of collagen-induced arthritis①Equal quantity of bovine collagen solution in acetic acid was mixed with complete Freuid adjuvant(CFA), emulsification by stiring at4℃.②Female Wistar rats were given intradermal injection of emulsified collagen(200g).③Rats were immunized again at7th with the same antigen(100μg).3.2sTNFRII-gAD allievates the severity of CIA①After collagen immunization, rats were divided into negative control, positive control (sTNFRII-Fc), sTNFRII-gAD treated group randomly. Normal Wistar rats were used as control.From10day on all immunized rats were treated every two days until two days later post the third therapy(day16). During that interval CIA incidence, ankle swelling and clinic evaluation were recorded every two days, respectively.②Serum anti-CII determination:blood sample was collected and anti-coagulated by heparin before or post therapy. The separated serum was detected for anti-CII by specific ELISA kit.③After CIA rats been sacrificed, obtained ankle tissues were fixed in10%neutral formalin solution followed by decalsification in0.5%EDTA for a week. H-E staining was carried out after embedding, slicing.3.3Mechanistic analysis of the protective effects for sTNFRII-gAD on CIA①On day10,16in this experiment TNF and IL-17A were examined in collected CIA serum in favour of evaluating the dynamic variarion of these cytokines.②Analysis of peripheral Treg:Peripheral lymphocytes obtained at day10and16was surface-labled by CD4-FITC, CD25-PE Ab followed by intracellular marking by FOXP3-APC Ab. Flow cytometry was employed to analysize the CD4+CD25+FoxP3+Treg variations.Results1. sTNFRII-gAD neutralized TNFa effectively①Both sTNFRII-gAD and sTNFRII-Fc neutralized TNFa effectively in a dose-dependent manner. Compared with sTNFRII-Fc, neutralizing activity of sTNFRII-gAD was significantly higher (P<0.001) at the dose of0.22-1.1nM.②ELISA assay showed that sTNFRII-gAD or sTNFRII-Fc was in complex with TNFa in vitro. Moreover, sTNFRII-gAD had higher (F=596.284, P<0.001) affinity with TNFa than sTNFRII-Fc under600nM.2. sTNFRII-gAD attenuated GalN/LPS-induced ALI①sTNFRII-gAD treatment improved the mice survival:Following immediate therapy after ALI construction, the mortality in negative group was100%in4.5-7h post therapy, while the death in positive group was observed until6h later after therapy and the survival at12h was64%compared to100%in sTNFRII-gAD group which both showed statistical difference relative to negative control (P<0.001). When therapy was administered1h post ALI construction, the survival at12h after sTNFRII-Fc treatment was33.3%while at the same time sTNFRII-gAD mounted to66.7%. Furthermore, the latter postponed death relative to positive group, but without statistical significance (P=0.127). ②sTNFRII-gAD alleviated liver injury efficaciously:For evaluation of the efficacy of these two antagonists, colorimetric method was carried out to analysize serum ALT, AST in addition to H-E staining for observing the pathological variations. The obtained results demonstrated that when therapy followed ALI construction instantly, the obtained ALT determined at2h,5h was indicated significant among these groups (F=100.367, P<0001); Similarly the differences compared in these groups were significant when therapy was initiated after ALI construction immediately (F=39.168, P<0001). As the same of ALT, AST of negative group increased significantly compared to other three groups(P<0.001); On the other hand, when therapy was given1h post ALI construction, ALT determined at5h was23.7±10.2、395.4±18.7,406.7±26.1,311.8±19.7U/L for them, respectively, which were higher apparently than them examed at2h. The difference among these groups was significant (F=70.780, P<0.001). AST was39.2±10.7、179.1±15.4,208.2±9.0,96.3±23.2U/L for these groups at5h post therapy, the difference for the variations of them was statistical(F=38.089, P<0.001). Compared to PBS (P=0.011) and positive group (P=0.002), sTNFRII-gAD significantly inhibited AST rise. Macroscopically, negative control displayed more obvious liver swelling, hyperemia in comparison with groups treated with TNF antagonists. The liver/body weight index was0.049±0.001、0.061±0.002,0.052±0.002,0.050±0.001(F=104.711, P<0.001) when therapy initiated instantly after ALI construction and0.065±0.001,0.054±0.002,0.053±0.003(F=32.160, P<0.001) when therapy was administered1h post ALI construction. It indicated significant differences between negative control and TNF antagonist treatments (P<0.001). On the other hand, when treatments were administered instantly post ALI establishment, sTNFRII-gAD allievated ALI more efficaciously in comparison with sTNFRII-Fc (P=0.041) while no statistical differentiation when treatments were implemented1h after ALI coPBStruction (P=1.000).In histopathology, negative control manifestated obvious liver injury including loss of nucleus, rare intact liver construction, pyknosis, destroyed membrane and invasive leukocyte. In contrast to that, liver injurys were improved apparently after therapy by sTNFRII-Fc, sTNFRII-gAD, particularly the latter. In addition, liver injury was more severe when ALI construction followed by concomitantly therapy compared to it when therapy was postponed after GaIN/LPS injection.③sTNFRII-gAD treatment led to increasing TNF but without less activity:2h post therapy serum TNF was89.6±21.3、441.2±176.8,1114.4±276.6,1359.3±183.5pg/ml for these three groups, respectively. Among them two groups treated with TNF antagonists showed apparently higher TNF than negative control and normal rats (F=62.059, P<0.001);5h past treatment following ALI construction instantly, serum TNF for these groups was870.8±192.1,1830.1±78.6,1884.2±259.2pg/ml, respectively, the differences among them was significant (F=193.902, P<0.001), which obviously exceeded nomal control (89.6±21.3pg/ml). When treatment was administered1h post ALI construction, it turned out that at2,5h, groups treated with TNF antagonists displayed significantly increasing TNF compared to negative control(F2h=193.902, P2h<0.001; F5h=27.436, P5h<0.001). Moreover, there’s no statistical significance(P2h=1.000;P5h=0.531) among these two antagonist-treated groups with respect to TNF when treatments were conducted immediately following ALI construction while sTNFRII-gAD demonstrated higher efficacy compared to sTNFRII-Fc(P2h=0.028; P5h=0.029) after therapy1h post ALI construction. L929cytotoxicity assay demonstrated that at2,5h later after instant therapy following ALI construction, TNF activity in these groups treated with TNF antagonists decreased significantly in comparison with negative control(F=130.374, P<0.001); Similarly, when ALI mice were treated1h later, no matter of2h(97%,80.54%,80.62%) or5h(80.13%,74.35%,74.86%) TNF activities in other two groups were lower than it in negative control, the differences being significant (F=168.219, P<0.001).④sTNFRII-gAD hampered the apoptosis in GaIN/LPS-induced ALI:Aiming to observe if sTNFRII-gAD could antagonize apoptosis taken place during ALI, obtained liver tissues were subjected to TUNEL assay and Western bloting for detecting Caspase-3. Results turned out that in negative control wide-spread apoptosis was performed apparently while it was less in other two groups handled with sTNFRII-Fc or sTNFRII-gAD, particularly the latter. In expression of Caspase-3, negative control manifestated more active Caspase-3compared to other two groups because the cleaved Caspase-3fragments (19kD,17kD) in negative control were more. Additionally, it demonstrated that the difference between two TNF antagonist-treated groups wasn’t significant in terms of Caspase-3activation when treatment was administered concurrently following ALI construction, but when treatment was carried out1h post ALI construction, sTNFRII-Fc treated-group showed enhanced active Caspase-3activity relative to sTNFRII-gAD which showed no cleaved fragments of Caspase-3.3. The therapeutical effects of sTNFRⅡ-gAD on CⅡ-induced arthritis①sTNFRII-gAD reduced the incidence and severity of CII-induced arthritis:In all treated groups CIA began to emerge from day12. However, at the end of this experiment negative control group showed higher incidence than other two groups (X2=7.156, P=0.028) in which CIA model group showed highest mean rank and sTNFRII-gAD-treated group performed lowest mean rank.In order to assess the swelling of CIA ankle, drainage method was used to measure the ankle swelling of CIA rats. Ankle swelling in negative control was1.00±0.12,1.16±0.15,1.32±0.08(ml) at day12,14,16, respectively. The ankle swelling in other two groups was0.91±0.06,0.99±0.13,1.08±0.21(sTNFRII-Fc) and0.90±0.09,0.97±0.17,1.03±0.19(sTNFRII-gAD). The differences of ankle swelling compared among these groups turned out to be statistical (F=4.725, P=0.020). However, the difference between these two antagonist-treated groups wasn’t indicated significant (P=1.000). Additionally, sTNFRII-Fc treatment showed no statistical difference with respect of ankle swelling compared to negative control (P=0.062).With respect of clinical score of CIA, the observations turned out that the clinical scores correlated with CIA progression closely. In comparison with nagetive and positive groups sTNFRII-gAD diminished the CIA clinical performance significantly(X2=7.962,P=0.047).②Influence of sTNFRII-gAD on serum TNF:An ELISA was employed to determine serum TNF level. It demonstrated that except normal rats (120.2±19.7 pg/ml), TNF level was251.9±14.7,255.5±25.9,257.4±13.6(pg/ml) for other three groups, respectively, which displayed statistical difference among these four groups (F=62.047, P<0.001); Contrastly, TNF level in negative group decreased to some degree, but it increased significantly after these two TNF antagonist treatments.TNF levels in these three groups after therapy were228.9±33.3、319.7±28.4、358.1±18.3(pg/ml), respectively. Compared to normal rats it showed significant variations in terms of TNF level among these four groups (F=84.948, P<0.001). Among them TNF antagonist treatments significantly retained serum TNF relative to negative control (P<0.001), however it demonstrated no statistical difference between these two antagonists (P=0.187).③The rise of serum anti-CII was inhibited by sTNFRII-gAD: Before treatments three groups displayed comparable anti-CII in serum, which turned out no statistica difference (F=0.417, p=0.668); Post therapy, rats of other two groups treated by TNF antagonists displayed much lower serum anti-CII than negative group, which had statistical difference (F=39.235, P<0.001). Intreastingly, sTNFRII-gAD treatment significantly surpressed the rise of serum anti-CII in comparison with negative, positive group (P=0.001).④sTNFRII-gAD suppress IL-17A in serum: Prior to treatment, all three groups showed comparable IL-17A, which was distinguished from normal rats (12.3±1.10pg/ml) statistically (F=19.260, P<0.00l). But, after therapy serum IL-17A in negative group increased significantly in comparison with other three groups (F=75.865, P<0.001). IL-17A in groups treated with TNF antagonists were comparable (P=0.700).⑤sTNFRII-gAD contributed to recovery of peripheral Treg: To ascertain whether sTNFRII-gAD could contribute the Thl differentiation towards Treg, flow cytometry was employed to measure the dynamics of Treg in circulation. The results turned out that rats CIA severity was related with the variation of peripheral Treg. Furthermore, prior to therapy Treg in all four groups demonstrated statistical difference (F=22.253, P<0.001), the number of Treg in these three groups was3.0±0.145(negative control),2.9±0.160(sTNFRII-Fc), and3.1±0.249 (sTNFRII-gAD). After therapy, Treg number in three groups was2.9±0.24,5.3±0.26,5.4±0.19, respectively, which indicated statistical difference (F=131.886, P<0.001). Treg of CIA model group hesitated at the level of it determined before therapy, which showed significant difference (t=0.756, P=0.492) while groups treated with antagonists demonstrated Treg numbers near normal group, which turned out statistical variations relative to them at the start of therapies(P<0.001).⑥Histopathological and photographical variations after TNF antagonism:After sacrificing through dislocation, collected ankles of CIA rats were fixed in10%neutral formalin solution prepared for histopathological analysis; parts of ankles were subjected to photographical exam. In negative control, synovial lumen narrowing, disseminated leucocyte invasion, hyperplasia of cartilage and grave synovium destruction appeared obviously. These observations were rare in rats treated with sTNFRII-gAD and were moderate in sTNFRII-Fc administered rats. In X-ray examination, soft tissue swelling adding joint erosion was obvious in negative control but not other two groups.ConclusionTaken together, data obtained in this study confirmed that sTNFRII-gAD capture and binds TNF efficaciously. In some aspects, it showed more efficacy in TNF antagonism than sTNFRII-Fc which its application in RA therapy has been permited. Probably through hampering apoptosis elicited by excessively produced TNF, sTNFRII-gAD attenuated GaIN/LPS-induced acute liver injury effectively. Additionally, in this study, the evidences imply that through neutralizing excessively produced TNF, which probaly leads to inhibiting Thl lymphocyte differentiation toward Th17cell and rendering recovery of Treg, sTNFRII-gAD could prevent or diminish collagen-induced arthritis and influence its clinical progression effectively. Therefore, sTNFRⅡ-gAD as a novel TNF antagonistic biologics would be much potential in the strategy(s) against diseases associated with excessive TNF. Furthermore, this study testifies the feasibility that cytokine receptor-gAD fused protein produced via genetic recombinant technology would play a important role in anti-cytokine strategy involved in some inflammatory diseases.