Targeting HMGB1 by ethyl pyruvate ameliorates systemic lupus erythematosus and reverses the senescent phenotype of bone marrow-mesenchymal stem cells

Abstract

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease involving multiple organs and systems. Mesenchymal stem cells (MSCs) from SLE patients have demonstrated defects such as impaired growth, senescence phenotype and immunomodulatory functions. Some studies have suggested the close connection between inflammation microenvironment and cellular senescence. In the current study, we detected cytokines levels in bone marrow supernatant by the quantitative proteomics analysis, and found the expression of HMGB1 was remarkably increased in bone marrow from SLE patients. Senescence associated-β-galactosidase (SA-β-gal) staining, F-actin staining and flow cytometry were used to detect the senescence of cells. After stimulation of HMGB1 in normal MSCs, the ratio of SA-β-gal positive in BM-MSCs was increased, the organization of cytoskeleton was disordered, and TLR4-NF-κB signaling was activated. Finally, Ethyl pyruvate (EP) (40 mg/kg and 100 mg/kg, three times a week), a high security HMGB1 inhibitor, was injected intraperitoneally to treat MRL/lpr mice for 8 weeks. We demonstrated that EP alleviated the clinical aspects of lupus nephritis and prolonged survival of MRL/lpr mice. In the meantime, EP reversed the senescent phenotype of BM-MSCs from MRL/lpr mice. HMGB1 could be a promising target in SLE patients, and might be one of the reasons of recurrence after MSCs transplantation.

Keywords: HMGB1; inflammation; mesenchymal stem cells; senescence; systemic lupus erythematosus.

Figure 1

(A) We collected bone marrow supernatant from normal people and SLE patients. Bone marrow supernatant samples were analyzed using a proteomics approach. (B) Elisa showed that HMGB1 expression levels were high in bone marrow supernatant and serum of SLE patients. (Bar represents mean ± SD,*P < 0.05 compared with the normal group) (NOR=normal group, SLE=systemic lupus erythematosus patients group).

Figure 2

Normal BM-MSCs were treated with bone marrow supernatant from normal persons or SLE patients, or MSCs were cocultured with SLE bone marrow supernatant treated with anti-HMGB1 mAb. Five groups were analyzed. (A, B) BM-MSCs were fixed and stained with SA-β-gal. (C) MSCs were stained by fluorescein isothiocyanate-conjugated phalloidin. The distribution of F-actin was disordered after treatment with bone marrow supernatant from SLE patients by Immunofluorescence. (D, E) Cell viability was assessed by flow cytometry analysis. (F, G) Normal BM-MSCs were treated with SLE bone marrow supernatant or 100 ng/ml HMGB1, or MSCs were cocultured with SLE bone marrow supernatant treated with anti-HMGB1 mAb. Five groups were analyzed. TLR4, p-IRAK1, p-p65, p53 and p27 expressions were analyzed by western blot. GAPDH was used as the internal control. (Bar represents mean ± SD,*P < 0.05 compared with the normal group, #P < 0.05 compared with the normal group, &P < 0.05 compared with the normal group) (NOR=normal group, SLE=systemic lupus erythematosus patients group).

Figure 3

NOR MSCs were cultured at the different concentration (1,10,100,500ng/ml) of HMGB1 for 48 h. (A, B) The expressions of TLR4, p-IRAK1, p-p65 and IκBa in MSCs were determined by western blot analysis. GAPDH was used as the internal control. (C, D) BM-MSCs were fixed and stained with SA-β-gal. (E)The distribution of F-actin was disordered after treatment with exogenous HMGB1 by Immunofluorescence. (F, G) Cell viability was assessed by flow cytometry analysis. (Bar represents mean ± SD,*P < 0.05 compared with the normal group, #P < 0.05 compared with the normal group, &P < 0.05 compared with the normal group) (NOR=normal group, SLE=systemic lupus erythematosus patients group).

Figure 4

(A, B) The expression of TLR4, p-IRAK1, p-p65 and IκBa from SLE compared with normal group were determined by western blot analysis. GAPDH was used as the internal control. (C, D) MSCs were depleted of TLR4 by RNAi. The second one was chosen as the best siRNA by western blotting. (E, F) MSCs were fixed and stained for SA-β-gal. The number of SA-β-gal-positive cells obviously decreased among treated SLE MSCs in comparison with untreated group. (G) MSCs were stained by fluorescein isothiocyanate-conjugated phalloidin. The disordered distribution of F-actin was reversed in siTLR4-treated MSCs. (Bar represents mean ± SD,*P < 0.05 compared with the normal group, #P < 0.05 compared with the SLE group,) (NOR=normal group, SLE=systemic lupus erythematosus patients group).

Figure 5

(A) 30 MRL/lpr mice were divided into three groups: MRL/lpr mice treated with normal saline, Ep-40mg/kg-treated MRL/lpr mice and Ep-100mg/kg -treated MRL/lpr mice. Ep was injected intraperitoneally to treat MRL/lpr mice aged 14 weeks for 8 weeks. (B) The expressions of HMGB1 in serum of MRL/lpr mice were examined by Elisa. (C) Survival curves observed that the survival rate of Ep-40mg/kg group and Ep-100mg/kg group was higher than that of control group. (D) Three groups MRL/lpr mice were weighed one time two weeks. (E) 24-hours urinary protein was measured by coomassie brilliant blue method. (F) HE-staining showed that renal pathological changes of MRL/lpr mice were significant, including glomerular sclerosis, mesangial cell proliferation, matrix widened, and lymphocytes infiltrating the interstitium. However, histopathological changes of other groups were alleviated. (Bar represents mean ± SD,*P < 0.05 compared with the control group, #P < 0.05 compared with the control group) (Con=Control group).

Figure 6

(A, B) the size comparison of spleen in three groups: MRL/lpr mice treated with normal saline, Ep-40mg/kg-treated MRL/lpr mice and Ep-100mg/kg -treated MRL/lpr mice. (C, D) Treg cell analysis in cells obtained from lymph nodes, spleen and peripheral blood. EP treatment upregulated the number of Treg cells. (E, F) BM-MSCs from MRL/lpr mice were isolated, then were fixed and stained for SA-β-gal. (G, H) Cell viability was assessed by flow cytometry analysis. (I, J) Expressions of TLR4, p-p65, and IκBa in MSCs from MRL/lpr mice, normal group and EP-treated group by Western blot analysis. (Bar represents mean ± SD,*P < 0.05 compared with the control group) (Con=Control group).

Figure 7

BM-MSCs and bone marrow supernatant were isolated from bone marrow of healthy donors and SLE patients. SLE bone marrow supernatant led MSCs to senescence via HMGB1/TLR4/NF-κB signaling pathway, HMGB1 had great significance. Ethyl pyruvate (EP), a high security HMGB1 inhibitor, was injected intraperitoneally to treat MRL/lpr mice aged 14 weeks for 8 weeks. EP alleviated the clinical aspects of lupus nephritis and prolonged survival of MRL/lpr mice, by reversing the senescent phenotype of BM-MSCs from MRL/lpr mice.