The Influence Of MGITRL On The Production Of Murine Th17 Cells And Its Role In The Development Of CIA

Objective:Glucocorticoid-induced tumor necrosis factor receptor(GITR) and its ligand(GITRL) play significant roles in regulating immune responses. To explore the role of mGITRL on murine Th17 cells in vitro and in vivo; and to observe its role on the development of the collagen-induced arthritis(CIA) in mice and research the mechanism.Methods:(1) The murine GITRL fusion protein was expressed in E.coli BL-21 with Isopropylβ-D-1- thiogalacto- pyranoside(IPTG),and identified by SDS-PAGE.The target protein was purified by IMAC column,and then identified by SDS-PAGE.The endotoxin was removed by Endotoxin Removal Kit.The rabbit was immunized by the purified recombinant mGITRL protein as antigen combined with the Freud's complete or incomplete adjuvant for three times.The rabbit anti-mouse GITRL serum was collected,the titer and specificity of the antibody was detected by ELISA or Western blot respectively.(2) CD4~+T cells were purified by MACS from the normal murine spleens,and the purity of the cells was analyzed by flow cytometry (FCM).The recombinant mGITRL protein or the control protein was added into the CD4~+T cells culture,and incubated under a Th17-differentiation condition for 96 hours.The frequency of Th17 cells was analyzed by FCM,and the supernatant was harvested to dectect IL-17 by ELISA method.(3) The mGITRL protein or the control protein was injected into normal mice,respectively.After 10 days,the frequency of Th17 cells and the expression of transcription factor RORγt from spleens were measured by FCM,the IL-17 in serum were examined by ELISA method.(4) The collagen-induced arthritis(CIA) model in DBA/1 mice was established.The mGITRL protein or the control protein was injected into the CIA mice,and its role on the development of the disease(clinical scores,CIA incidence,histology) was observed.The frequency and number of Th17 cells from spleens and draining lymph nodes,and the expression of transcription factor RORγt were analyzed by FCM;the levels of IL-17 in serum were examined by ELISA method.Results:(1) The mGITRL fusion protein was expressed in E.coli BL-21 at 30℃after 1mmol/L IPTG induction for 6 hours.The molecular weight of the fusion protein was about 40KD and the purity was larger than 90%. Rabbit anti-mouse GITRL polyclonal antibody was prepared.It showed that the titer of the antibody was 1:20000 by ELISA and the mGITRL protein could be specifically recognized by the antibody by Western blot.(2) The purity of murine CD4~+T cells purified by MACS is larger than 95%.Compared with the control protein(14.1%),we observed an increase frequency of Th17 cells(16.1%) in the culture supplemented with mGITRL protein.An increase level of IL-17 in the supematant was systematically along with increasing concentration of mGITRL protein (0.5,1,5,10μg/ml) from 387.51 pg/ml to 512.70pg/ml and the frequency of Th17 cells was from 15.81%to 17.90%.(3) Compared with the control group(CTL group),the frequency of Th17 cells in the murine spleens of GITRL group increased significantly (0.86±0.40%vs 1.77±0.49%,p＜0.01) and the percentage of CD4~+RORγt~+T cell also increased significantly(1.21±0.55%vs 1.91±0.60%,p＜0.05),and the level of IL-17 in murine serum of GITRL group (3.68±1.37 pg/ml vs 6.54±1.32 pg/ml,p＜0.05) increased significantly.(4) The model of CIA was established successfully.With the CIA model,we observed that compared with the the occurrence time(day 32) and the peak time(day 46) of CTL-CIA group,the group dealed with mGITRL protein(GITRL-CIA group) had an earlier onset of symptoms (day 28) and reached the peak score rapidly(day 38).The peak score of CTL-CIA group is 9.1,and the GITRL-CIA group reached 10.1.The pathology of mouse joints showed that there were significant cell infiltration and synovium hyperplasy within the articular space in the CTL-CIA group,but there were more serious inflammatory changes in the GITRL-CIA group,including pannus formation and artilage damage, besides significant cell infiltration and synovium hyperplasy.While CTL-CIA and CIA groups showed no difference in their clinical scores and pathology change.Compared with the CIA group(2.49×10~5±9.07×10~4) and CTL-CIA group(2.07×10~5±7.01×10~4),the number of Th17 cells in draining lymph nodes of the GITRL-group increased significantly(2.30×10~6±9.11×10~5)(p＜0.05).Compared with the CIA group(8.93±3.20 pg/ml) and CTL-CIA group(8.15±2.56 pg/ml),the level of IL-17 in serum of the GITRL-CIA group increased significantly (17.59±3.46pg/ml)(p＜0.05).Conclusions:(1) The mGITRL fusion protein was expressed and purified successfully.Rabbit anti-mouse GITRL polyclonal antibody was prepared.(2) Mouse GITRL can increase the percentage of Th17 cells and the production of IL-17 in vitro,and it showed a dose-dependent relationship.(3) Mouse GITRL can raise the frequency of Th17 cells in spleens and the level of IL-17 in serum in vivo,with an increase level of RORγt in spleens.(4) Our results suggested that mouse GITRL can exaberbate the development of collagen-induced arthritis in mice,which may correlate with the increasing number of Th17 cells in draining lymph nodes and the level of IL- 17 in serum.