Rapamycin reverses the senescent phenotype and improves immunoregulation of mesenchymal stem cells from MRL/lpr mice and systemic lupus erythematosus patients through inhibition of the mTOR signaling pathway

Abstract

We have shown that bone marrow (BM)-derived mesenchymal stem cells (BM-MSCs) from SLE patients exhibit senescent behavior and are involved in the pathogenesis of SLE. The aim of this study was to investigate the effects of rapamycin (RAPA) on the senescences and immunoregulatory ability of MSCs of MRL/lpr mice and SLE patients and the underlying mechanisms. Cell morphology, senescence associated β-galactosidase (SA-β-gal) staining, F-actin staining were used to detect the senescence of cells. BM-MSCs and purified CD4+ T cells were co-cultured indirectly. Flow cytometry was used to inspect the proportion of regulatory T (Treg) /T helper type 17 (Th17). We used small interfering RNA (siRNA) to interfere the expression of mTOR, and detect the effects by RT-PCR, WB and immunofluorescence. Finally, 1x106 of SLE BM-MSCs treated with RAPA were transplanted to cure the 8 MRL/lpr mice aged 16 weeks for 12 weeks. We demonstrated that RAPA alleviated the clinical symptoms of lupus nephritis and prolonged survival in MRL/lpr mice. RAPA reversed the senescent phenotype and improved immunoregulation of MSCs from MRL/lpr mice and SLE patients through inhibition of the mTOR signaling pathway. Marked therapeutic effects were observed in MRL/lpr mice following transplantation of BM-MSCs from SLE patients pretreated with RAPA.

Keywords: immunoregulation; mesenchymal stem cells (MSCs); rapamycin (RAPA); senescence; systemic lupus erythematosus (SLE).

Figure 1. RAPA improves lupus nephritis by influencing the cellular senescence of BM-MSCs from MRL/lpr mice

(A) The treatment group had intragastric administration of RAPA 3mg/kg/day between 16 and 28 weeks of ages. Survival curves observed that The survival rate of the RAPA-treated MRL/lpr mice was higher than that of control group. (B-E) We recorded the survival rate and the weight of mice. 24-hours urinary protein was measured by coomassie brilliant blue method. Mice were killed and were taken peripheral blood in orbit. Elisa showed that anti-ds-DNA antibody titer in serum in RAPA -treated group is lower than control group. HE-staining showed that renal pathological changes of control group is significant,including glomerular sclerosis, mesangial cell proliferation,matrix widened, and formation of crescent, a number of lymphocytes infiltrating the interstitium (HE×300). However, histopathological changes of RAPA -treated group were remarkable alleviated. (F) Cellular morphology observed that the RAPA-treated MSCs from MRL/lpr mice were less hypertrophic than untreated group (magnification; ×200). (G) MSCs were fixed and stained for β-gal. The number of SA-β-gal- positive cells among treated groups decreased in comparison with untreated group. (H) Immunofluorescence showed that the abnormal F-actin distribution in MSCs from MRL/lpr mice was reversed by RAPA treatment. (I) Cell Counting Kit (CCK)-8 method was used to detect the cell proliferation rate. (J) Flow cytometry was used to detect the distribution of cell cycle. (K) Western Blotting was used to detect the changes of cell cycle-related proteins. However, no remarkably differences were found between treated and untreated MSCs. (L) P4 MSCs transwell cultured with CD4+ T cells for 72 h. The count of Treg cells decreased and Th17 cells increased in MSCs from MRL/ lpr mice compared to the normal group by flow cytometry analysis. MSCs from RAPA-treated group could reverse the abnormal changes. The statistical results revealed that the treatment of RAPA could up-regulated the ratio of Treg/Th17 from MRL/lpr mice MSCs. (M) The supernatants of MSCs were collected. RAPA-treated group induced the secretion of IL-10 and TGF-β but reduced IL-17 and IL-6 by ELISA. All data were expressed as the mean±SEM (n = 3, *P<0.05 compared with normal group, #P<0.05 compared with the untreated group).

Figure 2. RAPA inhibited the over-activation mTOR pathway to revers the senescence of MSCs from MRL/lpr mice

(A) The over-expression of p-mTOR, p-S6K and p-S6 in p4 MSCs from MRL/lpr mice compared with normal group were determined by western blot analysis. The treatment of RAPA could obviously inhibit the expression of those proteins. GAPDH was used as the internal control. (B) P4 MSCs cultured into 24-well plates. The expression of p-mTOR, p-S6K and p-S6 analyzed by immunofluorescence staining showed that their over-activation in MSCs from MRL/lpr mice could be inhibited by RAPA treatment. Counterstaining with DAPI displays the localization of the nucleus (Scale bar = 50 μm). All data were expressed as the mean±SEM (n = 3,*P<0.05 compared with normal group, #P<0.05 compared with the untreated group).

Figure 3. Over-activation of mTOR pathway was involved in the senescence of MSCs from SLE patients

(A) The over-expression of p-mTOR, p-S6K and p-S6 in MSCs from SLE compared with normal group were determined by western blot analysis. GAPDH was used as the internal control. (B) P4 MSCs from SLE patients and normal group were cultured in 24-well plates. Immunofluorescence staining of p-mTOR, p-S6K and p-S6 verified their over-activation in SLE MSCs. Counterstaining with DAPI displays the localization of the nucleus (Scale bar = 50 μm). (C-D) P4 MSCs from SLE patients cultured at the different concentration of RAPA for 72 h. RAPA achieved maximal effects at about 500 nM by assaying the inhibition of p-70S6. (E-F) BMMSCs were depleted of mTOR by RNAi. The third one was chosen as the best siRNA by western blotting. (G) P4 MSCs from SLE patients were treated with 500 nM RAPA and si-mTOR or not for 72 h. Cellular morphology showed that the RAPA and si-mTOR treated SLE MSCs were less hypertrophic than untreated group (magnification; ×200). (H) MSCs were fixed and stained for β-gal. The number of SA-β-gal- positive cells obviously decreased among treated SLE MSCs in comparison with untreated group. (I) Immunofluorescence showed that RAPA and si-mTOR reversed abnormal F-actin distribution in MSCs from SLE patients. (J) P4 SLE MSCs were treated with 500 nM RAPA and the third si-mTOR or not, then transwell cultured with CD4+T cells for 72 h. The count of Treg cells decreased and Th17 cells increased in SLE MSCs compared to the normal group by flow cytometry analysis. But si-mTOR and RAPA-treated MSCs could reverse the abnormal changes. The statistical results revealed that RAPA could up-regulated the ratio of Treg/Th17 from SLE MSCs, which was down-regulated compared with normal group. (K) The supernatants of MSCs were collected. si-mTOR RAPA-treated SLE MSCs induced the secretion of IL-10 and TGF-β but reduced IL-17 and IL-6 by ELISA. All data were expressed as the mean±SEM (n = 3, *P<0.05 compared with normal group, #P<0.05 compared with the untreated group).

Figure 4. MSCs from SLE patients pretreated with RAPA have a significant effect on LN of MRL/lpr mice

(A) 24 MRL / lpr mice were divided into three groups: SLE BM-MSCs transplantation group (G1), RAPA-pretreated SLE BM-MSCs transplantation group (G2), normal BM-MSCs transplantation group (G3). Three groups were injected with BM-MSC in tail vein. (B) Survival curves observed that The survival rate of G2 and G3 MRL/lpr mice was higher than that of control group. (C) Three groups MRL/lpr mice were weighed one time two weeks. Weight between G2, G3 and G1 had obvious difference from 24 weeks. (D) 24-hours urinary protein was measured by coomassie brilliant blue method. (E) mice were killed and were taken peripheral blood in orbit. Elisa showed that anti-ds-DNA antibody titer in serum in G2 and G3 is lower than G1. (F) HE-staining showed that renal pathological changes of G1 is significant, including glomerular sclerosis, mesangial cell proliferation, matrix widened, and formation of crescent, a number of lymphocytes infiltrating the interstitium (HE×300). However, histopathological changes of other groups were remarkable alleviated. (G) HE-staining showed that pulmonary pathological changes of G1 is significant, including Pulmonary vascular congestion and edema, lymphocyte and mononuclear cell infiltration, and consolidation of lung (HE×300). However, histopathological changes of other groups were remarkable alleviated. There is not statistical difference between G2 and G3. All data were expressed as the mean±SEM (n = 3, *P<0.05 compared with normal group, #P<0.05 compared with the untreated group).