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

HMGB1 was elevated in bone marrow supernatant from SLE patients

To discover the crucial factor in bone marrow microenvironment which was involved in MSCs senescence, we collected bone marrow supernatant from SLE patients and healthy controls. Our proteomics results showed that expressions of multiple inflammatory cytokines were abnormal in bone marrow supernatant of SLE patients, including HMGB1, IFN-γ, and so on (Figure 1A). To verify the data from proteomics results, bone marrow supernatant level of HMGB1 was measured by ELISA, and the elevated expression of HMGB1 was also detected in SLE patients, compared to the healthy control (Figure 1B).

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).

Bone marrow supernatant from SLE patients led MSCs to senescence via HMGB1/TLR4/NF-κB signaling pathway

To further determine the effects of SLE bone marrow microenvironment on senescence of BM-MSCs, bone marrow supernatant was collected and used to stimulate BM-MSCs. Our results showed there were more SA-β-gal-positive cells in BM-MSCs when treated with bone marrow supernatant from SLE patients, interestingly, which could be reversed by a neutralizing anti-HMGB1 monoclonal antibody (Figure 2A, 2B). Furthermore, Immunofluorescence analysis showed that the distribution of F-actin in BM-MSCs was disordered after treatment with bone marrow supernatant from SLE patients (Figure 2C). The G1 phase was induced when normal BM-MSCs were treated with bone marrow supernatant from SLE patients by cell cycle analysis (Figure 2D, 2E). Western blotting analysis showed that expressions of cell cycle relation proteins, p53 and p27 were high in BM-MSCs treated by SLE bone marrow supernatant (Figure 2F). Anti-HMGB1 mAb treatment in SLE bone marrow supernatant did not lead to any significant changes in F-actin distribution and G1 phase. TLR4, one of HMGB1 receptors, can modulate MSCs function, trigger intracellular signaling pathways, and lead to the induction of inflammatory cytokines [12]. To discover the mechanism of bone marrow supernatant in the senescence of MSCs from SLE patients, we examined the expression of components of TLR4/NF-κB pathway. As shown in Figure 2F, bone marrow supernatant from SLE patients promoted the expression of TLR4 and its proteins expressed by its downstream regulated genes, p-IRAK1 and p-p65 (Figure 2F, 2G). These results confirmed that inflammatory microenvironment played facilitation to senescence of BM-MSCs, and HMGB1 had great significance.

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).

HMGB1 accelerated the senescence of normal BMMSCs by activating TLR4/ NF-κB signaling pathway

To further confirm the role of HMGB1 in the senescence of MSCs from SLE patients, different concentrations of HMGB1(1,10,100,500ng/ml) were used to stimulate BM-MSCs, which resulted in the activation of TLR4/NF-κB signaling pathway and the senescence of BM-MSCs. We discovered that HMGB1 promoted TLR4 expression at a concentration of 10-100 ng/ml and achieved maximal effects at doses of 100-500 ng/ml (Figure 3A, 3B). Thus, 48 h of treatment with 100 ng/ml of HMGB1 was used in the subsequent experiments. Then, we explored the effects of HMGB1 on the senescence of BM-MSCs from SLE patients and found that there were more SA-β-gal-positive cells in the BM-MSCs from normal persons when treated with HMGB1 (Figure 3C, 3D). Furthermore, after treatment with HMGB1, the distribution of Factin in BM-MSCs was disordered (Figure 3E). In addition, G1 phase was induced via cell cycle analysis (Figure 3F, 3G). Our results showed that HMGB1 could activate the expression of TLR4/NF-κB signaling and promoted cellular senescence in normal BM-MSCs in vitro, which further implied that the TLR4/NF-κB signaling pathway may play an essential role in the senescence of SLE BM-MSCs.

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).

TLR4 signaling pathway participated in the senescence of BM-MSCs from SLE patients

Previous studies and our experiment results have shown that SLE MSCs exhibited signs of senescence [7]. To further determine the effects of the TLR4/NF-κB signaling pathway on MSCs senescence. We next studied the expression of components of the TLR4 signaling pathway in BM-MSCs by western blotting. The results showed that expressions of TLR4, p-IRAK1 and p-p65 were clearly increased in SLE BM-MSCs compared to that in normal persons, and degradation of IκBα was induced in SLE BM-MSCs (Figure 4A, 4B). Next, TLR4-siRNA was used to knockdown the expression of TLR4 in SLE BM-MSCs to clarify the correlation between TLR4 signaling and cellular senescence. SLE BM-MSCs were transfected with TLR4-siRNA#1–4 or control siRNA. After 48 h, the levels of TLR4 were evaluated by western blot analysis. It was found that TLR4-siRNA#2 significantly knocked down the expression of TLR4 compared with the control siRNA (Figure 4C, 4D). Furthermore, the ratio of SA-β-gal-positive cells (Figure 4E, 4F) and the distribution of F-actin (Figure 4G) in BMMSCs from SLE patients was effectively reversed after treatment with TLR4-siRNA. These results further implied that. the TLR4 signaling pathway might play an important role in the senescence of SLE BM-MSCs

EP improved the survival rate and lupus nephritis by inhibiting the expression of HMGB1 in MRL/lpr mice

Previous study revealed that HMGB1 inhibition by a specific antibody could ameliorate albuminuria in MRL/lpr lupus-prone mice [13]. EP treatment significantly reduced circulating levels of HMGB1 in animals models and had protective effect [14]. Therefore, to further determine the effect of EP on MRL/lpr mice, mice were treated with EP according to the experimental design (Figure 5A). As expected, the expression of HMGB1 declined in serum of Ep-100mg/kg group, and the expression of HMGB1 in serum of Ep-40mg/kg group decreased moderately (Figure 5B). The survival rates in Ep-100mg/kg group were higher than that in saline solution-treated group (Figure 5C). The weight of the mice in Ep-40mg/kg group and Ep-100mg/kg group gradually increased (Figure 5D). The 24-hours urinary protein in these two groups were lower than those in control group (Figure 5E). In terms of pathology, glomerular sclerosis and interstitial fibrosis were ameliorated in Ep-100mg/kg group (Figure 5F). These results demonstrated EP had a significant therapeutic effect on LN by inhibiting HMGB1 in MRL/lpr mice.

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).

EP improved Treg immune regulation and reversed the senescence of BM-MSCs via inhibiting TLR4/ NF-κB signaling pathway

In the present study, we examined the influence of EP intraperitoneal injection on immune response of MRL/lpr mice, especially Treg immune. As shown in Figure 6A–6B, the size of spleen was significantly reduced in Ep-100mg/kg group. At the same time, EP treatment upregulated the number of Treg cells in peripheral blood, spleen, lymph nodes (Figure 6C, 6D). BM-MSCs from MRL/lpr mice showed senescent behavior, characterized by flattened and enlarged cell morphology, increased SA-β-gal activity, and cell cycle arrest. Interestingly, we observed decelerated cell senescence signs in BM-MSCs in Ep-100mg/kg group (Figure 6E, 6F). Cell-cycle data showed that BM-MSCs from MRL/lpr mice treated by EP displayed a significantly lower number of cells arrested in the G0/G1 phase compared with the control group (Figure 6G, 6H). Next, we investigated the expressions of TLR4, p-p65, and IκBa in MSCs from MRL/lpr mice, normal group and EP-treated group by Western blot analysis. We found higher levels of TLR4 and p-p65 in MSCs from MRL/lpr mice compared to the EP-treated group (Figure 6I, 6J). These results revealed that EP might be an effective therapeutic approach to SLE.

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).

Patients

Twelve SLE patients between 13 and 43 years of age (mean 28.25±9.69 years) were enrolled in this study. The SLE diagnosis was made based on the criteria proposed by the American College of Rheumatology (2009). The Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) was used to measure the disease activity. All patients were categorized as active using a cut off SLEDAI score of eight. Twelve healthy subjects were used as the normal group. All research subjects were females with a similar age distribution (mean 26.58±7.97 years). All participators gave consent to the study, which was approved by the Ethics Committee of the Affiliated Hospital of Nantong University.

Proteomics

Bone marrow supernatant samples were analyzed using the PEA and the Olink Immuno-oncology panel (Olink Bioscience AB). This panel includes 92 proteins. The protein analysis is reported as normalized protein expression levels (NPX), which are Ct values normalized by the subtraction of values for extension control, as well as interplate control; the scale is shifted using a correction factor (normal background noise) and reported in log2 scale.

Isolation, cell culture and identification of BM-MSCs from SLE and normal subjects

MSCs were isolated and expanded from iliac crest BM of all the SLE patients and normal subjects. Five milliliters of heparinized BM were mixed with an equal volume of phosphate-buffered saline (PBS) (Gibco, USA). Then, the resuspended cells were layered over Ficoll solution (1.077 g/mL) and centrifuged at 1,000×g for 30 min at 24 °C. The mononuclear cells were collected at the interface and resuspended in low-glucose Dulbecco Modified Eagle Medium (L-DMEM) (Gibco, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco, USA). Then, the cells were plated at a density of 2×10⁷ cells per 25 cm² dish and cultured at 37 °C in a 5% CO₂ incubator, and the medium was replaced and non-adherent cells were removed after 5 days and every three days thereafter. When the MSCs became nearly confluent, the adherent cells were released from the dishes with 0.25% trypsin (Gibco, USA), and then replanted at a density of 1×10⁶ cells per 25 cm² dish.

Western blotting

Cells were washed twice with cold-buffered PBS and were then lysed in RIPA buffer. Equal amounts of protein were separated by 10% SDS polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred to polyvinylidene difluoride (PDVF) membranes. Membranes were incubated with primary antibodies (1:500) at 4 °C overnight and incubated with horseradish peroxidase conjugated mouse anti-rabbit secondary antibody for 2 h at room temperature. The blots were developed using an enhanced chemiluminescence kit. The following primary antibodies were used: TLR4 (anti-rabbit, Santa Cruz), GAPDH (anti-rabbit, Santa Cruz), β-actin(anti-rabbit, Sangon Biotech), p-IRAK1 (anti- rabbit, Santa Cruz), p-p65 (anti-mouse, Santa Cruz), p65 (anti-rabbit, Santa Cruz), IκBa (anti-rabbit, Santa Cruz), p53 (anti-mouse, Santa Cruz), p27 (anti-mouse, Sangon Biotech).

SA-β-gal assay

The SA-β-gal activity was determined using a kit from the Chemical Company following the manufacturer’s instructions(Beyotime, China). MSCs were passaged into the 6-well culture plates at a density of 5×10⁴ cells per well for 72 h. Then cells were washed twice with PBS and fixed with fixing solution for 15 min. After incubation with staining SA-β-gal detection solution at 37°C without CO₂ overnight, the slips were washed and analyzed under the microscope.

Immunofluorescence assay of the skeleton of MSCs

MSCs were washed twice with PBS and fixed in 4% PFA for 1 h. After permeabilization and bloking, they were incubated with fluorescein isothiocyanate-conjugated phalloidin(Thermo Fisher, Waltham, USA). The stained cells were then examined by a Zeiss Confocal Laser Scanning Microscope.

Flow cytometry

For cell cycle analysis, MSCs collected and fixed with 70% ethanol at -20°C for 24 h. After being washed with PBS and then treated with 100 μg/ml RNase (Sigma, USA) for 20 min, cells stained with 50 μg/ml propidium iodide (PI) solution (Sigma, USA) for 20 minutes and analyzed by the flow cytometry machine (FACS Calibur, BD Biosciences, USA). The fraction of cells in the G0/G1, S, and G2/M phases were quantified with the ModFit LT system.

For Treg cell analysis, Cells obtained from lymph nodes, spleen and peripheral blood were blocked with anti-CD16/32 and stained with monoclonal anti-mouse CD4 antibody (eBiosciences; San Diego, CA, USA) to assess T cell percentages. Treg cells were characterized by staining for CD4, CD25 and Foxp3, according to the manufacturer’s instructions (Mouse Treg Staining Kit, eBiosciences). All data were acquired in a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA) and analysed using FlowJo X 10.0.7 software.

Elisa

Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000g. Aspirate serum and assay immediately or aliquot and store samples at ≤ −20 °C. Bone marrow supernatant and serum HMGB1 levels were measured using a commercially available ELISA kit according to the manufacturer’s instructions (Arigo Biolaboratories, USA)

Cell transfection

BM-MSCs were seeded 2.5 x 10⁵ cells in 6-cm dish with MEM-α medium containing 10% FBS (Gibco) for one day. Transient transfection was performed using the transfection reagent Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The MSCs were then transfected with TLR4 small interfering RNA (siRNA) (GenePharma Co. Ltd.) and a non-specific control siRNA. SiRNA was mixed with Lipofectamine transfection reagent in serum-free medium according to the manufacturer’s instructions.

Mice and treatments

8-week-old female MRL/lpr mice were obtained from Shanghai SLAC Laboratory Animal Institute Co. Ltd. MRL/lpr mice (n=30) and BALB/c mice (n=10) were maintained in a specific pathogen-free animal facility of the Experimental Animal Center in Nantong University. The animal experimentation was approved by Ethics Committee of Nantong University. The mice were intraperitoneally injected with EP (40 mg/kg or 100 mg/kg) or vehicle control (saline) three times per week. MRL/lpr mice were divided into three groups. All animal procedures were approved by the institute’s Institutional Animal Care and Use Committee.

Isolation, culture and identification of BM-MSCs from MRL/lpr mice

The BM was flushed out of the femurs and tibias removed from MRL/lpr and BALB/c mice using 10 ml PBS with 100 U/ml heparin in a syringe. The cells were centrifuged at 1000 ×g for 10 min. The cell pellet was resuspended in 5 ml L-DMEM supplemented with 10% FBS (Gibco, USA) and plated in a 25 cm2 dish to allow the MSCs to adhere. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37°C.

Albuminuria

24h urine samples were collected from each mouse by metabolic cages method once every two weeks. Urinary albumin levels were measured using a commercially available ELISA kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instructions.

Renal histology studies

At the time of euthanization, kidney specimens were obtained, fixed in 10% formaldehyde and embedded in paraffin. Sections (4 μm thickness) were prepared and then stained with haematoxylin and eosin (H&E). The kidney sections were coded and examined by two independent observers who were blinded to the treatment groups.

Statistical analysis

All data are presented as the mean±standard deviation (SD) from at least three independent experiments. All statistical analyses were performed using the SPSS 21.0 software, and the data were analyzed using Student’s t test with P<0.05 considered statistically significant.