| Objective:Explore the signaling molecules during the differentiation of Th17 cells co-stimulated by mGITRL protein in vitro. Established the collagen-induced arthritis (CIA) models and administrated with mGITRL for study the downstream molecules of GITR/GITRL pathway which take part in the pathogenesis of CIA.Methods:1. Naive T cells were purified by MACS from the normal murine spleens. The recombinant mGITRL protein or the control protein was added into the cells culture, and incubated under a Th17 incomplete differentiation condition for 72 hours. The frequency of Th17 cells was analyzed by FCM.2. Purified naive T cells were treated with anti-CD3 mAb and mGITRL for 72 hours for activation and restimulated by mGITRL protein. The phosporylation level of p38 MAPK was detected by Western-Blot; the recombinant mGITRL protein, mGITRL protein+SB203580 (inhibitor of p38 MAPK) and mGITRL protein+DMSO (carrier of SB203580) was added into the cells culture, and incubated under a Th17 incomplete differentiation condition for 72 hours. The frequency of Th17 cells was analyzed by FCM, relative expression of related factors was detected by qRT-PCR (IL-17, RORγt mRNA), and the concentration of IL-17A and IL-21 was detected by ELISA, also the relative expression of IL-21 mRNA was detected by qRT-PCR.3. Purified naive T cells were activated and stimulated with mGITRL protein and their phosporylation level of STAT3Tyr705 and Ser727 was detected by Western-Blot; pre-treated the activated naive T cells with SB203580 before re-stimulated with mGITRL and their phosporylation level of STAT3 Tyr705 and Ser727 was detected by Western-Blot. The recombinant mGITRL protein, mGITRL protein+ Stattic (inhibitor of STAT3) and mGITRL protein+DMSO was added into the cells culture, and incubated under a Th17 incomplete differentiation condition for 72 hours. The concentration of IL-17A and IL-21 was detected by ELISA, and relative expression of IL-21 mRNA was detected by qRT-PCR.4. The collagen-induced arthritis (CIA) model in DBA/1 mice was established. The mGITRL protein (CIA-GITRL group) or the mGITRL protein combine with SB203580 (CIA-GITRL-SB203580 group) or its solution (CIA-GITRL-Carrier group) was injected into the CIA mice, and its role on the development of the disease (clinical scores, CIA incidence, histology) was observed. The frequency of Th17 cells from spleens and draining lymph nodes were analyzed by FCM, the expression of IL-17 and RORyt were detected by qRT-PCR, the phosphorylation of STAT3 Tyr705 and STAT3 Ser727 was detected by Western-Blot.Results:1. The percentages of Thl7 cells in CD4+cells cultured with control protein was 5.03% and the percentages of Th17 cells in CD4+cells cultured with mGITRL protein was 27.22%.2. The activated naive T cells have a higher phosphorylation level of p38 when stimulated with 1.0ug/ml mGITRL protein; and stimulated with mGITRL (1.Oug/ml) for 10 or 20 mins the activated naive T cells have a higher phosphorylation of p38. There was a clear inhibition of Th17 cells proportion, IL-17 production, IL-17 and ROR y t mRNA expression by SB203580. Also, there was a dose-dependent of these inhibitions by SB203580. The incensement of IL-21 production, IL-21 mRNA expression by GITRL protein could be inhibited with SB203580.3. When stimulated with 1.0ug/ml GITRL protein there was a higher phosphorylation of STAT3 Tyr705 and Ser727 sites in the activated naive T cells. The phosphorylation level of Tyr705 and Ser727 sites of STAT3 were accelerated at 20 or 40 mins and the phosphorylation level of Ser727 sites of STAT3 were accelerated at 10 or 20 mins after stimulated by mGITRL. There was a restraint activity of STAT3 at Tyr705 and Ser727 sites under the pretreated of p38 inhibitor. The production of IL-17 was inhibited by stattic in the condition of Th17 cells differentiation co-stimulated with mGITRL protein. The incensement of IL-21 production, IL-21 mRNA expression by GITRL protein could be inhibited with stattic.4. The model of CIA was established successfully. With the CIA model, we observed that compared with the occurrence time (day 24) and the peak time (day 36) of CIA-GITRL group, CIA-GITRL-SB203580 group had a later onset of symptoms (day 30). The pathology of mouse joints showed that there were significant cell infiltration and synovium hyperplasy within the articular space in the CIA-GITRL group, but the inflammatory changes in the CIA-GITRL-SB203580 group was relieved. Compared with the CIA-GITRL group (2.97×105±9.10×105) and CIA-GITRL-Carrier group (3.75×105±11.5×105), the number of Thl7 cells in spleen of the CIA-GITRL-SB203580 group (2.14×105±3.61×105) decreased significantly. Compared with the CIA-GITRL group (8.7×104±6.9×104) and CIA-GITRL-Carrier group (3.60×104±1.04×104), the number of Th17 cells in drain lymph nodes of the CIA-GITRL-SB203580 group (1.79×104±0.69×104) decreased significantly. The percentage of Th17 cells in CD4+splenocytes was decreased significant in CIA-GITRL-SB203580 group (2.21±0.39%) compared with CIA-GITRL group (4.16±1.90%) and CIA-GITRL-Carrier group (4.40± 1.29%). The percentage of Thl7 cells in CD4+lymphocytes was decreased significant in CIA-GITRL-SB203580 group compared with CIA-GITRL group (1.81±1.18%) and CIA-GITRL-Carrier group (1.99±0.42%). IL-21 production and IL-21 mRNA relative expression was inhibited by SB203580 in CD4+splenocytes from GITRL-CIA mice.Conclusions:1. Murine GITRL protein could co-stimulates the differentiation of Th17 cells, promotes the phosphorylation of p38 MAPK in activated naive T cells; the activity of p38 MAPK was play an important role in the Th17 cells differentiation so-stimulated by mGITRL protein.2. GITRL protein could promote the phosphorylation of STAT3 at Tyr705 and Ser727 sites via p38 MAPK; p38 MAPK-STAT3 pathway could influence the production of IL-21 during the co-stimulation of Thl7 cells differentiation by mGITRL.3. Our results suggested that mGITRL can relief the development of collagen-induced arthritis in mice, which may correlate with the decreasing of Th17 cells and the expression of IL-21mRNA. |
PDF Full Text Request