Myeloid-Derived Suppressor Cells Induce Podocyte Injury Through Increasing Reactive Oxygen Species in Lupus Nephritis

The expansion of myeloid-derived suppressor cells (MDSCs) has been documented in murine models and patients with lupus nephritis (LN), but the exact role of MDSCs in this process remains largely unknown. In this study, we investigated whether MDSCs are involved in the process of podocyte injury in the development of LN. In toll-like receptor-7 (TLR-7) agonist imiquimod-induced lupus mice, we found the severe podocyte injury in glomeruli of lupus mice and significant expansion of MDSCs in spleens and kidneys of lupus mice. The function of TLR-7 activated MDSCs was enhanced including the increased generation of reactive oxygen species (ROS) in vivo and in vitro. Moreover, the ROS production of MDSCs induced podocyte injury through activating the p-38MAPK and NF-kB signaling. Furthermore, we verified that podocyte injury was indeed correlated with expansion of MDSCs and their ROS secretion in LN of pristane-induced lupus mice. These findings first indicate that the podocyte injury in LN was associated with the increased MDSCs in kidney and MDSCs may be a promising therapeutic target of LN in the future.

The expansion of myeloid-derived suppressor cells (MDSCs) has been documented in murine models and patients with lupus nephritis (LN), but the exact role of MDSCs in this process remains largely unknown. In this study, we investigated whether MDSCs are involved in the process of podocyte injury in the development of LN. In toll-like receptor-7 (TLR-7) agonist imiquimod-induced lupus mice, we found the severe podocyte injury in glomeruli of lupus mice and significant expansion of MDSCs in spleens and kidneys of lupus mice. The function of TLR-7 activated MDSCs was enhanced including the increased generation of reactive oxygen species (ROS) in vivo and in vitro. Moreover, the ROS production of MDSCs induced podocyte injury through activating the p-38MAPK and NF-kB signaling. Furthermore, we verified that podocyte injury was indeed correlated with expansion of MDSCs and their ROS secretion in LN of pristane-induced lupus mice. These findings first indicate that the podocyte injury in LN was associated with the increased MDSCs in kidney and MDSCs may be a promising therapeutic target of LN in the future.
Keywords: lupus nephritis, myeloid-derived suppressor cells, podocytes, toll-like receptor-7, reactive oxygen species inTrODUcTiOn Systemic lupus erythematosus (SLE) is a typical autoimmune disease and mostly occurs in females (9:1 prevalence) around child-bearing age (1). Lupus nephritis (LN), one of the most severe manifestations of SLE, is characterized by proteinuria, hematuria, and renal failure caused by deposition of immune complexes and tightly associated with high morbidity and mortality (2)(3)(4). Although there have been great advances in treatment and improved prognosis for patients with LN for over the past 30 years, LN treatment remains challenging. Indeed, only 50-70% of patients with LN achieved remission and 10-20% of patients will progress to end-stage renal disease within 5 years of diagnosis (5). In order to find an effective treatment, the detailed pathogenic mechanism of LN is still needed to be clarified.
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous collection of myeloid cells comprised myeloid precursors, immature granulocytes, mononuclear macrophages, and dendritic cells (6,7). MDSCs are broadly characterized by CD11b + Gr-1 + cells in mice and HLA-DR − CD11b + CD33 + cells in humans, which further divided into granulocytic polymorphonuclear MDSCs (G-MDSCs) and monocytic MDSCs (M-MDSCs). G-MDSCs are phenotypically and morphologically similar to neutrophils, whereas M-MDSCs are similar to monocytes (8). Increasing evidence suggests that MDSCs exert a powerful influence on the regulation of autoimmunity in autoimmune diseases. It had been reported that MDSCs induced the expansion of regulatory B cells by inducible nitric oxide synthase (iNOS) to ameliorate autoimmunity in murine model of SLE (9). MDSCs suppressed the progression of collagen-induced arthritis by inhibiting the proinflammatory immune response of CD4 + T cells in autoimmune arthritis (10). The frequency of MDSCs was positively correlated with the levels of serum arginase-1 (Arg-1) activity, T helper 17 (TH17) responses, and disease severity in SLE patients. In addition, MDSCs were essential for the associated renal injuries in a humanized SLE model (11). Recently, new evidence revealed that MDSCs play a crucial role in the regulation of LN. The granulocyte colony stimulating factor treatment ameliorated LN through reducing the number of G-MDSCs and M-MDSCs and promoting the preferential expansion of CD4 + CD25 + Foxp3 + Tregs in the spleen and kidney of NZB/W F1 female mice (12). Consistent with these results, in our previous study, the proportion of MDSCs in the kidney gradually increased along with the progression of SLE in MRL/lpr lupus-prone mice (13). Nevertheless, laquinimod suppressed LN in (NZB × NZW) F1 prone mice partly by inducing expansion of MDSCs and promoted a shift from proinflammatory type I monocyte/macrophages to anti-inflammatory type II monocyte/macrophages (14). These studies suggest that MDSCs may play a dual role in the development of LN. Although current evidence manifests that MDSCs are involved in LN, it is still unclear on their mechanisms in LN development.
Podocytes exert crucial influence on maintaining the normal renal function as the major component of the glomerular filtration barrier (15,16). Podocyte injury is correlated with the generation of proteinuria, one of the major features of renal dysfunction in LN (17). Indeed, podocytes injury is common in patients with LN, as shown in a large cohort study of patients with renal-biopsy-proven LN (18). It had been reported the diverse mechanisms were involved in podocyte injury, including genetic factors, inflammation, toxic injury, and metabolic disturbances (19). Previous study revealed that the activated NLRP3 inflammasomes in podocytes from lupus-prone mice and LN patients was involved in the pathogenesis of podocyte injuries and the development of proteinuria in LN (20). Furthermore, oxidant stress-mediated aldosterone/MR-induced podocyte injury by triggering both Chop-dependent apoptosis and autophagy via activating ER stress (21). However, it has been unclear about the effect of MDSCs on podocyte injury in LN so far.
In the present study, we found that the significant expansion of MDSCs and severe podocyte injury in glomeruli of kidneys in toll-like receptor-7 (TLR-7) agonist imiquimod or pristaneinduced lupus mice. Moreover, MDSCs generated the enhanced reactive oxygen species (ROS) via TLR-7 activation in vivo or in vitro, since TLR-7 is thought to trigger glomerulonephritis in experimental lupus erythematosus. Furthermore, the ROS production of MDSCs could induce podocyte injury through activating the p-38MAPK and NF-kB signaling. These results first indicate that the podocyte injury in LN was associated with the increased MDSCs in kidney and MDSCs may be a promising therapeutic target of LN in the future.

Mice
Female BALB/c mice (6-8 weeks old) and female C57BL/6 mice (6-8 weeks old) were obtained from Model Animal Research Center of Nanjing University (Nanjing, China) and kept under pathogen-free and housing conditions in a 12-h light and dark cycle. All experiments were conducted in accordance with institutional guidelines for animal care and used based on the Guide for the Animal Care Committee at Nanjing University. 10-weekold BALB/c mice received a single intraperitoneal injection of 0.5 ml pristane or PBS and monitored for the following 7 months. C57BL/6 mice were kept for 1 week. The skin on the right ears of the mice was treated topically, every other day, with 1.25 mg of 5% imiquimod cream for 10 weeks.

isolation of MDscs From Kidney and spleen
Kidneys were cut into small fragments and digested to singlecell suspensions with 1 mg/ml collagenase type D (Roche) and 0.1 mg/ml DNase I (Roche) in HBSS (Hanks balanced saline solution) at 37°C for 30 min, the isolated cells were layered on a 30/40/75% Percoll gradient, followed by centrifugation for 20 min at 600 g, and collected the cells on the 40/75% interface, containing mostly leukocytes. After washing in FACS buffer, the cells were immediately processed for FACS.
Spleens were digested to single-cell suspensions with 1 mg/ml collagenase type D (Roche) and 0.1 mg/ml DNase I (Roche) in HBSS (Hanks balanced saline solution) at 37°C for 30 min. The cells were collected and suspended in ACK for the lysis of red blood cells and then centrifuge at 1,500 rpm for 5 min. After washing, collecting cells were purified from spleen using magnetic-activated cell sorting beads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacture's protocol. The purity of cells after separation was >90%. Cells were then analyzed using flow cytometer (22).

generation of BM-Derived MDscs
BM cells were isolated as described previously from mice by flushing femurs and tibiae, and then BM cells were centrifuged and resuspended in culture medium supplemented with murine IL-6 and GM-CSF (both 40 ng/ml; Miltenyi Biotec, Auburn, CA, USA) and were cultured for 4 days (23,24). Negative immune-selection of lineage-negative bone-marrow progenitors was performed using the Myeloid-Derived Suppressor Cell Isolation Kit.

Flow cytometry analysis
To detect mouse MDSC subsets, cell suspensions isolated from the spleens and kidneys were first incubated with Fc-blockeranti-CD16/32 antibody (dilution 1:20, Miltenyi Biotech) for 15 min, stained and pre-incubated with anti-CD11b-APC (1 μl/test) and anti-GR-1-PE (0.3 μl/test) for 30 min at 4°C in the dark. Cells were then washed with buffer to remove the excess stains and analyzed in a FACS (Becton Dickinson, San Diego, CA, USA). To detect TLR7 of MDSCs in pristane-treated mice, after stained with anti-CD11b-APC and anti-GR-1-PE, fixed and permeabilized by BD Fixation/Permeabilization Solution Kit (BD Pharmingen, USA), stained with anti-TLR7-FITC (Invitrogen, USA) for 30 min at 4°C.

apoptosis assay
Podocyte apoptosis was measured by flow cytometry using an annexin V-FITC apoptosis detection kit (Becton Dickinson, San Diego, CA, USA). Briefly, podocytes were collected and suspended in 500 µl binding buffer, followed by staining with annexin V-FITC and propidium iodide (PI) at room temperature for 5 min in the dark. After removing the unbound annexin V-FITC and PI by centrifugation, the cells were resuspended in excess binding buffer. For each measurement, at least 10,000 cells were analyzed by FACS flow cytometer (Becton Dickinson, San Diego, CA, USA).

rOs Detection
The oxidation-sensitive dye DCFDA (Beyotime, Shanghai, China) was used to measure ROS production. Cells were incubated at 37°C in RPMI in the presence of 2.5 µM DCFDA for 30 min.

reverse Transcription and real-Time Quantitative Pcr analysis
Total RNA was isolated from cells or tissues using TRizol reagent (Invitrogen) according to the manufacturer's instructions. The condition of reversed transcription for cDNA with reverse transcription system was 42°C for 5 min, 99°C for 20 min, and 4°C for 5 min. The SYBR Green PCR Master Mix (Bio RAD) according to the manufacturer's instructions was performed for quantitative real-time RT-PCR (qRT-PCR). The qPCR assays were carried out on a Step One Plus, and data collected with this instrument. Relative gene expression was calculated with the 2 −ΔΔCT formula, and results were normalized to the expression of GAPDH. All the premier sequences are shown in Table S1 in Supplementary Material.

statistical analysis
All statistical calculations were performed using commercially available statistical software GraphPad Prism (GraphPad, San Diego, CA, USA). Results were expressed as mean ± SEM of three independent experiments and each experiment included triplicate sets. Data were statistically evaluated by one-way ANOVA followed by Dunnett's test between control group and multiple dose groups. Statistical significance was defined at equal to or more than 95% confidence interval or P ≤ 0.05.

Podocyte injury in Tlr-7 agonist imiquimod-induced lupus Mice
It has been report that TLR-7 is involved in the development of SLE (26). The up-regulation of TLR-7 in patient with SLE and TLR-7 activation in wild-type mice leads to lupus-like systemic autoimmune disease with elevated levels of autoantibodies and multiple organ involvement (27,28). According to previous study (28), we initially applied imiquimod (IMQ), a TLR-7 agonist, to induce lupus mice. Consistent with previous report, the spleens of IMQ-treated mice became swollen ( Figure 1A) and significant changes have been found in renal morphology ( Figure 1B). Moreover, the level of urinary protein was significantly higher in urine of IMQ-treated mice than in normal controls ( Figure 1C). Furthermore, WT-1 and nephrin are required during kidney development for the maturation of podocytes and their expressions are down-regulated in acquired glomerular diseases. The deficiency of WT-1 and nephrin is considered a pathologic feature of glomerular injury. The analysis of immunofluorescence showed that the expression of WT-1 and nephrin was obviously decreased in kidney of IMQ-treated mice compared with wild mice (Figures 1D,E). The down-regulated expression of WT-1 and nephrin at mRNA level was confirmed by qRT-PCR (Figures 1G,H). Meanwhile, electron microscopy revealed that the foot process effacement of podocyte was exacerbated in the IMQ-treated mice compared with wild mice. These results indicated that podocyte injury of the glomerulus was obviously more serious in TLR-7 agonist imiquimod-induced lupus mice than normal mice.

expansion of MDscs correlates Positively With Podocyte injury in imiquimodinduced lupus Mice
Previously, we found significant expansion of MDSCs in lupus mice, and expanded MDSCs correlated positively with disease severity in diseased lupus-prone mice (13). Here, we verified the percentage of MDSCs in kidneys and spleens by flow cytometry again, and the results showed the frequency of MDSCs markedly elevated in both kidneys and spleens of IMQ-treated mice compared with wild-type mice (Figures 2A,B). We further examined whether the expansion of MDSCs was associated with the decreased expression of WT-1 and nephrin. The results showed the expanded MDSCs were negatively correlated with the reduced expression of WT-1 and nephrin at mRNA level (Figures 2C,D). Expectedly, the function of MDSCs was also changed, i.e., the expression levels of their functional genes, including NOX component P47 phox , iNOS, and Arg-1 were evidently increased in MDSCs purified from kidneys and spleens of IMQ-treated mice (Figures 1E,F and 2E,F). These results indicate the expanded MDSCs correlated positively with renal

Tlr-7-activated MDscs induce Podocyte injury In Vitro
To explore the specific role of MDSCs in podocyte injury, we respectively incubated podocytes alone as control group, treated podocytes with R848 as positive group, co-cultured podocytes with BM-induced MDSCs by transwells in vitro, stimulated with or without resiquimod (R848), a TLR-7 agonist. The results showed that most significant change was observed in the live podocytes co-cultured with R848-stimulated MDSCs, while morphologic change was also observed in co-cultured group of MDSCs. But no change was observed in control group and positive control group ( Figure 3A). Moreover, the significant reduced expression of WT-1 and nephrin, two molecular markers of podocytes, was determined by immunofluorescence in the co-cultivation group of podocytes and R848-stimulated MDSCs (Figures 3B,C). In consistence with these results, the expression of WT-1 and nephrin was also manifestly decreased at both mRNA level and protein levels in the co-cultured group of podocytes and R848-treated MDSCs (Figures 3D,E). In addition, the expression of desmin, which is a sensitive marker of podocyte injury (29), was up-regulated at mRNA level in the co-cultured podocytes and R848-treated MDSCs, compared to control groups ( Figure 3F). As previous study shown, podocytes can also secrete vascular endothelial growth factor (VEGF) to maintain the integrity of glomerular endothelial cells and promote the formation of new capillaries (17). The expression of VEGF was measured by qRT-PCR and the results showed that the down-regulated expression of VEGF in podocytes co-cultured with R848-treated MDSCs ( Figure 3F). These data reveal that TLR-7-activated MDSCs indeed induce podocyte injury in vitro.
To further directly investigate the association of TLR-7activated MDSCs with podocyte injury. We measured the cell apoptosis of podocytes alone, R848-treated, co-cultured with MDSCs stimulated with or without R848 using transwell by flow cytometer, respectively. The results showed that MDSCs treated with R848 promoted obviously podocytes apoptosis, compared with the treatment of podocytes alone ( Figure 3H). Moreover, the expression of inflammatory factors, such as IL-1β, IL-6, and TNF-α were up-regulated markedly in podocytes co-cultured with MDSCs treated with R848 ( Figure 3G). These results indicate TLR-7-activated MDSCs can cause podocyte apoptosis and induce podocytes to secrete inflammatory factors.

Podocyte injury is attributed to rOs From Tlr-7-activated MDscs
To elucidate the mechanism of TLR-7-activated MDSCs impair podocytes, we focused on functional molecules of MDSCs. The results showed that the functional molecules of MDSCs were significantly up-regulated in a dose-dependent effect of TLR-7 agonist R848 treatment (Figures 4A-C). The function of MDSCs was also enhanced by activating TLR-7 signaling. Previous study revealed that renal overexpression of ROS could directly lead to podocyte injury in diabetic nephritis model mice and increased production of oxidative stress markers in the renal tissue of SLE patients (30,31). To further determine the role of the functional molecule of MDSCs in podocyte injury, we co-cultured podocytes with MDSCs treated with R848, or pretreated with ROS inhibitors N-acetyl-l-cysteine (NAC) or iNOS inhibitors L-NMMA for 1 h before treated with R848. The results showed that the expression of WT-1 and nephrin was increased, while the expression of IL-1β, IL-6, and TNF-α was reduced in podocytes co-cultured with MDSCs pretreated with NAC before treatment with R848, compared with control group, and co-cultured with MDSCs treated with R848 ( Figure 4D). In neutralizing iNOS using L-NMMA, podocyte injury and the levels of inflammatory factors were not significantly alleviated (Figure 4E). The results indicated that podocyte injury may be attributed to ROS generation from TLR-7-activated MDSCs.

rOs Promotes Podocyte injury via p-38MaPK and nF-kB signaling
To verify further the effect of ROS secreted by MDSCs on podocyte injury, apoptosis of podocytes was tested. We first observed that podocytes became shrinking and round in cellular morphology when co-cultured with MDSCs treated with R848. In contrast, the change in podocytes was reversed when co-cultured by adding R848 to MDSCs after pretreated with NAC for 1 h (Figure 5A). Moreover, we detected podocyte injury marker desmin and cell apoptosis after co-incubated with R848-treated MDSCs following NAC-pretreatment. The results showed that NAC-pretreatment significantly reduced expression of desmin ( Figure 5B). In consistence with the results, NAC-pretreatment obviously decreased the cell apoptosis of podocytes (Figures 5C,D). These results indicate that inhibition of ROS by NAC markedly blocked the deteriorative effect of MDSCs on podocyte injury. In addition, previous studies have shown that ROS might promote podocyte injury via activation of p-38MAPK or NF-kB pathway (32,33). Thus, we measured the protein expression of p-p38MAPK, p-38MAPK, p-p65, and p-65 by Western blot. The results showed that the expressions of p-p38MAPK and p-p65 were significantly increased in podocytes co-cultured with R848stimulated MDSCs, compared with those in other group of the treated podocytes ( Figure 5F). These data indicate that TLR-7-activated MDSCs can induce podocyte injury through ROS-activated p-38MAPK and NF-kB pathways.

correlation of Podocyte injury With expansion of MDscs is Verified in Pristane-induced lupus Mice
Pristane, an isoprenoid alkane induces a lupus-like syndrome in several non-autoimmune prone mouse strains (34). We constructed the pristane-induced lupus-prone mice and confirmed that the model mice showed significant renal damage. Consistent with the results of IMQ-treated mice, 7 months after pristane or PBS challenge, histologic sections were stained by PAS and analyzed for pathologic features. The results showed that glomerular injury with basement membrane thickening, mesangial matrix expansion, endocapillary hypercellularity, and PAS positive deposits in pristane-induced lupus mice ( Figure 6A). Immunofluorescence staining of renal tissues showed that the number of WT-1 + and nephrin + podocytes was significantly lower in the glomerular of pristane-treated mice than in PBS-treated mice (Figures 6B,C). Moreover, the results of electron microscopy revealed the exacerbated podocyte foot process effacement in pristane-treated mice compared with wild mice (Figure 6D). We also found that the expression levels of WT-1 and nephrin were significantly down-regulated in the kidneys of pristane-treated mice by qRT-PCR ( Figure 6E). The above results suggest that pristaneinduced lupus model mice encounter obvious renal damage, especially the glomerular podocyte injury. Of note, percentage of MDSCs was also increased by immunofluorescence staining for CD11b and GR-1 in the kidneys of pristane-treated mice ( Figure 6F). Moreover, TLR-7 amplified the activation of MDSCs (Figure 6G). The overexpression of ROS was also detected by ROS fluorescent probe, DHE, in the pristanetreated mice glomerular ( Figure 6H). The mRNA level of P47phox was up-regulated in kidneys of pristane-treated mice ( Figure 6I). These results verified that podocyte injury was correlated with expansion of MDSCs and their ROS secretion in LN of pristane-induced lupus mice. model (11). According to previous results, a significant increase in MDSCs was correlated positively with renal injury in LN.

DiscUssiOn
Previously, we have already demonstrated that the expansion of splenic MDSCs and the degrees of renal injuries was increased with disease progression in SLE (13). In this study, we further confirmed significant renal injury symptoms, proteinuria, and the expansion of MDSCs in spleens and kidneys in both pristane and imiquimod-induced lupus-like model mice. These results suggested that MDSCs might be involved in the immune regulation of renal microenvironment and induce kidney lesions.
Podocytes are highly differentiated cells characterized by expressing markers, such as synaptopodin, Wilms' tumor protein (WT-1), glomerular epithelial protein 1, and nephrin. These molecules play important roles to maintain filtration barrier integrity (35). In this study, we found the decreasing expression of WT-1 and nephrin by immunofluorescence and qRT-PCR in kidneys of both pristane and imiquimodinduced lupus-like model mice. These results indicated severe podocytes injury. Furthermore, we found the frequency of MDSCs was negatively correlated with the expression of WT-1 and nephrin in pristane-or imiquimod-induced lupus-like model mice, which has been demonstrated the correlation with nephrotic-range proteinuria and extensive effacement of podocyte foot processes in LN (17,36). In vitro, the co-cultivation experiment of MDSCs and podocytes shown MDSCs could lead to the down-regulated expression of podocytes markers, WT-1 and nephrin, at mRNA level and protein level detected by immunofluorescence and qRT-PCR. Moreover, the up-regulated expression of desmin and the down-regulated expression of VEGF were significant in co-cultivation experiments. The damage effect was enhanced by R848, and the results were further demonstrated by cell apoptosis assay. In addition, R848-treated MDSCs promoted the up-regulated expression of IL-1β, IL-6, and TNF-α in podocytes. A recent report demonstrated that IL-1β, IL-6, and TNF-α can influence the permeability of the glomerular basement membrane and alter glomerular filtration in a ROSdependent mean (37). These data indicated that MDSCs could possibly affect podocytes injury with a ROS-dependent way to promote renal injuries in LN and the activation of TLR-7 in MDSCs exerted an important role.
It has been reported that endogenous agonists of TLR-4 were present in glomerular diseases that may induce podocyte dysfunction independent of TIL7/9 activation seen with endocytosed DNA and RNA immune complexes in SLE (38). Moreover, a number of studies demonstrated that LPS and oligosaccharide could cause comparable podocyte injury in vitro and in vivo (39). In the present study, TLR-7 activation of MDSCs occurred in kidneys of IMQ-treated mice and pristane-treated mice. Furthermore, ROS was increasing secreted by MDSCs stimulated with R848 and in vitro and in vivo. These results suggest that the role of TLR-7 activation is important in the generation of ROS of MDSCs. A common link of acute and chronic kidney injuries with the enhanced generation of ROS and reactive nitrogen species during injury progression was documented (40)(41)(42). Here, although the level of iNOS and NO was up-regulated in R848-treated MDSCs, the expression of WT-1 and nephrin was not significantly reversed in podocytes co-cultured with L-NMMA-pretreated MDSCs treated with R848, compared with podocytes cocultured with R848-treated MDSCs. These data suggested that In the present study, we demonstrated that MDSCs induce podocytes injury by ROS and were involved in the subsequent development of proteinuria in LN for the first time. Furthermore, TLR-7-activated MDSCs enhanced podocyte injury through activating p-38MAPK and NF-kB pathways by ROS. In summary, our study further defined the role of MDSCs in the pathogenesis of LN, and we proposed that therapeutic approaches directed against MDSCs may lead to alleviation of the disease. In a word, our findings deepen our understanding of the occurrence of LN and provide a new ideas and targets for the clinical treatment of LN.

eThics sTaTeMenT
All aspects of this study were approved by the Research Ethics Committee of Nanjing University, and all experiments were conducted in accordance with institutional guidelines for animal care and used based on the Guide for the Animal Care Committee at Nanjing University. All these specimens were handled and anonymized according to ethical and legal standards.
aUThOr cOnTriBUTiOns DZ and JX conceived, designed the experiments, drafted the article, performed experiments, data analysis, and wrote the manuscript. JR, LD, GS, and DL supported the materials. YH provided the concept and designed this study. HD and YH co-designed experiments and co-wrote the manuscript. All authors reviewed and approved the manuscript final version.
acKnOWleDgMenTs This work was supported by the National Natural Science Foundation of China (91542113 and 31570909) and supported by the Fundamental Research Funds for the Central Universities (021414380342).