Abstract
Objective
Glycogen synthase kinase 3β (GSK-3β) has been demonstrated to be involved in immune and inflammatory responses via multiple signaling pathways, leading to the production of proinflammatory cytokines. The purpose of this study was to investigate the role of GSK-3β in the pathogenesis of lupus nephritis in 2 mouse models.
Methods
Thiadiazolidinone 8 (TDZD-8), a selective inhibitor of GSK-3β, was administered intraperitoneally to 12-week-old MRL/lpr mice for 8 weeks or to 22-week-old (NZB × NZW)F1 mice for 12 weeks. The expression of GSK-3β and NLRP3 inflammasome components was analyzed. Proteinuria, biochemical parameters, proinflammatory cytokines, anti–double-stranded DNA (anti-dsDNA) antibody levels, and renal pathology were examined. In vitro, the effect of GSK-3β–directed small interfering RNA (siRNA) on NLRP3 inflammasome activation was evaluated in bone marrow–derived macrophages (BMMs) from the mice and in the J774A.1 macrophage cell line.
Results
The incidence of severe proteinuria and renal inflammation was significantly attenuated in both models, with a significant reduction in anti-dsDNA antibody production, immune complex deposition in the kidney, and circulating proinflammatory cytokine levels. TDZD-8 inhibited the activation of GSK-3β and caspase 1, with a concomitant decrease in interleukin-1β (IL-1β) synthesis. In vitro, GSK-3β siRNA transfection of mouse BMMs and the J774A.1 cell line with GSK-3β siRNA inhibited the expression of GSK-3β, the activation of caspase 1, and the production of IL-1β.
Conclusion
These results show that GSK-3β promotes lupus nephritis at least partly by activating the NLRP3/IL-1β pathway. The linking of GSK-3β to the NLRP3/IL-1β pathway is a novel observation in our study. Our results suggest that the GSK-3β/NLRP3/IL-1β pathway may be a potential therapeutic target for lupus in humans.
Lupus nephritis is a major complication associated with poor prognosis in patients with systemic lupus erythematosus (SLE) (1). The long-term outcome of lupus nephritis remains unsatisfactory, considering both the renal damage and treatment-related adverse events (2). Novel therapeutic strategies for lupus nephritis are therefore needed, especially for patients who do not respond to therapy and those who experience relapse after conventional treatment. Accumulated data have demonstrated that complex signaling pathways are aberrantly expressed and involved in SLE. Small-molecule inhibitors that target the key molecules responsible for these pathways offer perspective for more effective and less toxic therapy in SLE (3).
Glycogen synthase kinase 3β (GSK-3β) is a multifunctional serine/threonine kinase that was initially identified as a key enzyme in the regulation of glycogen metabolism. Subsequent studies demonstrated that GSK-3β is a positive regulator of NF-κB activation and plays a pivotal role in inflammation (4). GSK-3 activity is necessary for the Toll-like receptor (TLR)–mediated production of proinflammatory cytokines such as interleukin-6 (IL-6), IL-1β, and tumor necrosis factor α (TNFα) (5). GSK-3β inhibition can increase the stability and function of Treg cells (6), and GSK-3 is a critical determinant in the differentiation of pathogenic Th17 cells (7). In vivo, GSK-3 inhibition was shown to significantly alleviate experimental autoimmune encephalomyelitis (8). Of special interest is the finding that GSK-3 may be involved in anti–double-stranded DNA (anti-dsDNA) autoantibody production and glomerulonephritis in MRL/lpr mice (9).
Pattern-recognition receptors (PRRs) were initially identified as sensor proteins crucial for innate immune responses. However, some PRRs, including TLRs and nucleotide-binding oligomerization domain–like receptors (NLRs), are also expressed and are functional in cells of the adaptive immune system, bridging the innate and adaptive immunity (10). TLRs play a crucial role in autoimmunity and inflammation, and antagonists of TLRs are being tested in human SLE (11). However, the role of the NLR family in SLE is poorly understood. The NLRs represent a family of cytosolic pattern-recognition molecules that trigger multiple signaling pathways in inflammation and immunity (12). NLRP3 is the best-characterized member of the NLR family, and its role in health and disease has recently attracted increasing attention.
The NLRP3 inflammasome is a multiprotein complex that activates caspase 1, leading to the processing and secretion of the proinflammatory cytokines IL-1β and IL-18 (12). This inflammasome has been implicated in the pathogenesis of SLE. The renal NLRP3 inflammasome has been shown to be activated in (NZB × NZW)F1 lupus-prone mice (13). Our previous data indicated that the NLRP3 inflammasome is up-regulated in the kidneys of MRL/lpr mice and that blockade of the inflammasome attenuated the lupus nephritis in MRL/lpr mice (14). Purinergic receptor P2X7 has been proposed to lie upstream of NLRP3 activation, and inhibition of P2X7 was shown to suppress NLRP3/ASC/caspase 1 inflammasome assembly, the autoimmune response, and the severity of nephritis in MRL/lpr mice and NZM2328 mice with interferon-α (IFNα)–accelerated disease (15).
Although GSK-3β is involved in the inflammatory response via regulation of TLRs (5,16), it remains unclear whether GSK-3β regulates NLRs. In the present investigation using lupus-prone MRL/lpr and (NZB × NZW)F1 mice, evidence was obtained to support this hypothesis.
Materials and Methods
Mice and treatments
Female MRL/lpr mice (Shanghai SLAC Laboratory Animal Company) and female (NZB × NZW)F1 mice (The Jackson Laboratory) were maintained in the specific pathogen–free animal facility at the Experimental Animal Center at Sun Yat-sen University. All experiments were approved by the Institutional Animal Care Committee of Sun Yat-sen University. Age- and sex-matched female C57BL/6 mice (provided by the Experimental Animal Center, Sun Yat-sen University) served as normal controls.
In one experiment, 12-week-old MRL/lpr mice (n = 10 per group) were treated for 8 weeks with thiadiazolidinone 8 (TDZD-8; Sigma-Aldrich), which is the selective antagonist of GSK-3β, or with an equal volume of vehicle (DMSO). TDZD-8 was administered by intraperitoneal injection at a daily dosage of 5 mg/kg of body weight dissolved in DMSO (final concentration 1%). This dosage of TDZD-8 was based on the published literature (8). Mice were euthanized under anesthesia at the end of their twentieth week of age.
In a separate experiment, 22-week-old female (NZB × NZW)F1 mice (n = 10 per group) were treated for 12 weeks as described above. Mice were euthanized under anesthesia at the end of their thirty-fourth week of age.
Mouse bone marrow–derived macrophages (BMMs) and J774A.1 cells
Mouse BMMs were generated from (NZB × NZW)F1 mice as described by Siegfried et al (17). Femora and tibiae were flushed with cold RPMI 1640 medium, and cells were suspended in RPMI 1640 medium containing 10% fetal calf serum (HyClone) and cultured at 37°C in a humidified atmosphere containing 5% CO2 for 6 days with 20 ng/ml of macrophage colony-stimulating factor (5 × 105/ml; Life Technologies). The medium was changed every 2 days. Flow cytometric analysis using both phycoerythrin-conjugated mouse anti-CD11b and fluorescein isothiocyanate (FITC)–conjugated mouse anti-F4/80 (both from eBioscience) was performed to confirm the purity (>95%) of the BMMs. The mouse macrophage cell line J774A.1 was obtained from ATCC.
Determination of proteinuria and blood urea nitrogen (BUN) levels
Mice were placed in metabolic cages for the purpose of collecting 24-hour urine specimens every 2 weeks. Proteinuria was determined using Siemens Multistix 10 SG reagent strips and was scored on a scale of 0–4, where 0 = none, 1 = 30–100 mg/dl, 2 = 100–300 mg/dl, 3 = 300–2,000 mg/dl, and 4 = ≥2,000 mg/dl (15). Severe proteinuria was defined as ≥300 mg/dl. BUN levels were determined at the end of the study, using a commercial autoanalyzer (Beckman Coulter).
Measurement of anti-dsDNA antibody and cytokine levels
Serum anti-dsDNA antibodies were detected by enzyme-linked immunosorbent assay (ELISA) as previously described (15). Briefly, 96-well plates were precoated with methylated bovine serum albumin (BSA) at a concentration of 10 mg/ml, followed by 5 μg/ml of calf thymus dsDNA (Sigma-Aldrich). After blockade with 1% BSA, sera were added in serial dilutions and incubated for 1 hour at room temperature. After washing, horseradish peroxidase–conjugated goat anti-mouse IgG antibody (Sigma-Aldrich) was added to detect the bound anti-dsDNA, followed by the peroxidase substrate tetramethylbenzidine. The reaction was terminated with 1M H2SO4, and the absorbance at an optical density of 450 nm was determined. Normal mouse IgG was used as a negative control.
Levels of IL-1β, IL-17, and IFNγ were determined with ELISA kits (R&D Systems) according to the manufacturer's instructions.
Analysis of renal histology and immune complex deposition
Mouse kidneys were harvested, fixed in 10% buffered formalin, and embedded in paraffin. Sections (4 μm) were stained with hematoxylin and eosin (H&E). Pathologic changes in the kidneys were scored semiquantitatively on a scale of 0–3 (0 = no changes; 3 = severe cell proliferation/infiltration and crescent formation) as described previously (15).
FITC-conjugated anti-mouse IgG (Santa Cruz Biotechnology) and anti-mouse complement C3 (Cedarlane) were used to examine immune complex deposition in the kidneys. The mean intensity of the green fluorescence was scored on a scale of 0–3 and subjected to semiquantitative analysis as described previously (18).
Western blot analysis
Western blot analysis was carried out as described previously (15). Cells or kidney tissues were lysed in cell lysis buffer (Cell Signaling Technology) and homogenized, followed by centrifugation for 10 minutes at 15,000g. Protein concentrations were determined using the bicinchoninic acid protein assay. Electrophoresis was conducted with 10% or 12.5% running gels and 5% stacking gels. Proteins were transferred onto a nitrocellulose membrane. After blockade with 5% skim milk in Tris buffered saline–0.1% Tween 20, membranes were incubated overnight at 4°C with the following primary antibodies: mouse anti–GSK-3β (Cell Signaling Technology), anti–phospho–GSK-3β (phosphorylated at Ser9; Cell Signaling Technology), anti-NLRP3 (AdipoGen), anti–caspase 1-p20 (AdipoGen) and anti-GAPDH antibodies (Santa Cruz Biotechnology). After washing, blots were incubated with their corresponding secondary antibodies. The signals on the membranes were detected via enhanced chemiluminescence analysis (Cell Signaling Technology).
In vitro transfection with GSK-3β small interfering RNA (siRNA)
The predesigned GSK-3β siRNA was purchased from Santa Cruz Biotechnology. Mouse BMMs or J774A.1 cells were transfected with GSK-3β siRNA using Lipofectamine RNAiMAX (Life Technologies) in serum-free medium according to the manufacturer's instructions. Mock transfection using Lipofectamine RNAiMAX and nontargeting siRNA was performed in each experiment. Cells were incubated at 37°C for 48 hours before treatment. The silencing efficacy of GSK-3β siRNA was assessed by Western blotting using a mouse anti-GSK-3β antibody (Cell Signaling Technology).
Evaluation of NLRP3 inflammasome activation
GSK-3β–directed siRNA was used to assess how inhibition of GSK-3β in conjunction with ATP affected NLRP3 inflammasome activation. After transfection, BMMs or J774A.1 cells were primed with 1 μg/ml of lipopolysaccharide (LPS; Sigma-Aldrich) for 4 hours prior to stimulation. Media were then removed, and cells were incubated in serum-free RPMI 1640 (Sigma-Aldrich), followed by treatment for 1 hour with the NLRP3 inflammasome agonist ATP (5 mM; Santa Cruz Biotechnology).
Following different treatments, cell supernatants were collected for quantification of IL-1β using a commercially available ELISA kit (R&D Systems) according to the manufacturer's instructions. Cells were subjected to Western blot analysis of the active caspase 1-p20 to assess NLRP3 inflammasome activation.
Human peripheral blood mononuclear cells (PBMCs)
Patients with SLE met the America College of Rheumatology classification criteria for the disease (19). The participants were enrolled from the First Affiliated Hospital, Sun Yat-sen University. Patients with concurrent infection and other autoimmune diseases were excluded.
Blood was obtained from SLE patients before treatment was initiated. Samples were drawn into heparinized tubes. After the blood draw, PBMCs were immediately isolated by Ficoll-Hypaque density-gradient centrifugation. PBMCs were also separated from whole blood that had been drawn from healthy volunteer donors. Evaluation of NLRP3 inflammasome activation was conducted as described above. Written informed consent was obtained from all subjects before study entry, and the study was approved by the Ethics Committee of First Affiliated Hospital, Sun Yat-sen University.
Statistical analysis
Data are reported as the mean ± SD. Chi-square analysis was performed to compare the incidence of severe proteinuria between groups. Student's unpaired t-test was used to compare continuous variables between 2 groups. All P values were 2-tailed. P values less than 0.05 were considered significant. Analysis was performed using SPSS 16.0 software.
Results
Protective effects of TDZD-8 treatment against glomerulonephritis in MRL/lpr mice
As shown in Figure 1A, the incidence of severe proteinuria (≥300 mg/dl) was significantly decreased in TDZD-8–treated MRL/lpr mice as compared to vehicle-treated mice (P < 0.05). In addition, BUN levels were significantly lower in TDZD-8–treated MRL/lpr mice than in vehicle-treated mice at week 20, the end of this experiment (P < 0.05) (Figure 1B). H&E staining of kidney sections obtained from MRL/lpr mice at week 20 demonstrated enlarged glomeruli, with diffuse cellular infiltration and marked interstitial mononuclear cell infiltration in the vehicle-treated group (Figure 1C). In contrast, TDZD-8 treatment significantly inhibited glomerulonephritis and interstitial inflammatory infiltration (Figure 1C). Histologic scoring of the pathologic changes in the kidneys showed significantly lower scores in the TDZD-8–treated group (P < 0.05) (Figure 1D).
Figure 1.
Reduction of severe proteinuria and renal injury following 8 weeks of treatment with the glycogen synthase kinase 3β (GSK-3β) inhibitor thiadiazolidinone 8 (TDZD-8) in MRL/lpr mice. A, Incidence of severe proteinuria (≥300 mg/dl), as recorded biweekly in vehicle-treated and TDZD-8–treated mice. B, Levels of blood urea nitrogen (BUN) at the end of the study. C, Representative hematoxylin and eosin–stained sections of glomerular areas from 20-week-old MRL/lpr mice treated with vehicle or TDZD-8. Original magnification × 400. D, Semiquantitative analysis of histologic lesions in the kidneys. Values in B and D are the mean ± SD of 10 mice per group. * = P < 0.05 versus vehicle-treated controls.
Protective effects of TDZD-8 treatment against glomerulonephritis in (NZB × NZW)F1 mice
TDZD-8 treatment of (NZB × NZW)F1 mice resulted in a significant decline in the incidence of severe proteinuria (P < 0.05) (Figure 2A). TDZD-8–treated (NZB × NZW)F1 mice also showed a significant reduction in serum BUN levels (P < 0.05) (Figure 2B). Consistent with the above results, H&E staining of kidney tissues obtained at week 34 demonstrated that the renal damage was markedly attenuated by TDZD-8 treatment as compared with vehicle treatment (Figures 2C and D).
Figure 2.
Reduction of severe proteinuria and renal injury following 12 weeks of thiadiazolidinone 8 (TDZD-8) treatment in (NZB × NZW)F1 mice. A, Incidence of severe proteinuria (≥300 mg/dl), as recorded biweekly in vehicle-treated and TDZD-8–treated mice. B, Levels of blood urea nitrogen (BUN) at the end of the study. C, Representative hematoxylin and eosin–stained sections of glomerular areas from 34-week-old (NZB × NZW)F1 mice treated with vehicle or TDZD-8. Original magnification × 400. D, Semiquantitative analysis of histologic lesions in the kidneys. Values in B and D are the mean ± SD of 10 mice per group. * = P < 0.05 versus vehicle-treated controls.
Inhibitory effects of TDZD-8 treatment on serum anti-dsDNA levels and renal immune complex deposition
Serum levels of anti-dsDNA antibodies were significantly lower at week 20 in MRL/lpr mice treated with TDZD-8 (P < 0.01) (Figure 3A). Furthermore, glomerular deposits of IgG and C3 were significantly reduced in TDZD-8–treated mice as compared with controls (P < 0.01) (Figure 3B).
Figure 3.
Reduction of serum anti–double-stranded DNA (anti-dsDNA) levels and renal immune complex deposition following 8 or 12 weeks of thiadiazolidinone 8 (TDZD-8) treatment in lupus-prone mice. A, Reduced serum anti-dsDNA antibody levels in TDZD-8–treated versus vehicle-treated MRL/lpr mice. B, Reduced renal deposition of IgG and C3 in TDZD-8–treated versus vehicle-treated MRL/lpr mice. C, Reduced serum anti-dsDNA antibody levels in TDZD-8–treated versus vehicle-treated (NZB × NZW)F1 (NZB/W F1) mice. D, Reduced renal deposition of IgG and C3 in TDZD-8–treated versus vehicle-treated (NZB × NZW)F1 mice. Representative fluorescein isothiocyanate–stained sections are shown. Original magnification × 200. Values are the mean ± SD of 10 mice per group. ** = P < 0.01 versus vehicle-treated controls.
Similar results were obtained in the experiment with (NZB × NZW)F1 mice. There was a pronounced decrease in both anti-dsDNA antibody levels and renal immune complex deposition in the TDZD-8–treated (NZB × NZW)F1 mice as compared with the vehicle-treated controls (P < 0.01) (Figures 3C and D).
Inhibitory effects of TDZD-8 treatment on proinflammatory cytokine levels in lupus-prone mice
As shown in Figure 4, TDZD–8 treatment resulted in a significant reduction of serum levels of IL-1β, IL-17, and IFNγ in both MRL/lpr and (NZB × NZW)F1 mice as compared with vehicle-treated control mice (P < 0.01). These results suggest that TDZD-8 treatment inhibits systemic inflammation in lupus-prone mice.
Figure 4.
Reduced production of proinflammatory cytokines following 8 or 12 weeks of thiadiazolidinone 8 (TDZD-8) treatment in lupus-prone mice. At the end of the study, serum levels of interleukin-1β (IL-1β), IL-17, and interferon-γ (IFNγ) in MRL/lpr mice (A) and (NZB × NZW)F1 (NZB/W F1) mice (B) were determined by enzyme-linked immunosorbent assay. Values are the mean ± SD of 10 mice per group. ** = P < 0.01 versus vehicle-treated controls.
Correlation of renal IL-1β levels with enhanced activities of GSK-3β and caspase 1 in the kidneys of lupus-prone mice
Renal activity of GSK-3β was determined in 20-week-old female MRL/lpr mice (n = 5 mice per group) after establishment of severe proteinuria. Age- and sex-matched C57BL/6 mice were used as controls. As demonstrated by Western blot analysis (Figure 5A), phosphor–GSK-3β (phosphorylated at Ser9), which is the inactive form of GSK-3β (20,21), was significantly reduced in the kidneys of MRL/lpr mice. The diminished ratio of phosphor–GSK-3β to GSK-3β reflected the activation of the GSK-3β pathway as compared with the controls.
Figure 5.
Suppression of glycogen synthase kinase 3β (GSK-3β) activity and NLRP3 inflammasome activation in the kidneys of lupus-prone mice following treatment with thiadiazolidinone 8 (TDZD-8). A and B, Representative Western blots (each band represents 1 mouse kidney sample) as well as analysis of GSK-3β activity in the kidneys of 20-week-old MRL/lpr mice (A) and 34-week-old (NZB × NZW)F1 mice (B) as compared with age- and sex-matched normal C57BL/6 mice (n = 5 per group). C and D, Representative Western blots as well as analysis of GSK-3β activity and NLRP3 inflammasome activation in the kidneys of MRL/lpr mice following 8 weeks of TDZD-8 treatment (C) and in the kidneys of (NZB × NZW)F1 mice following 12 weeks of TDZD-8 treatment (D) (n = 10 per group). TDZD-8 treatment inhibited renal GSK-3β activity and NLRP3 inflammasome activation (as indicated by the presence of caspase 1-p20 [p20]) in both experiments. E and F, Reduced renal levels of interleukin-1β (IL-1β) following TDZD-8 treatment in MRL/lpr mice (E) and (NZB × NZW)F1 mice (F), as determined by enzyme-linked immunosorbent assay. Values are the mean ± SD. # = P < 0.01; * = P < 0.05; ** = P < 0.01 versus vehicle-treated controls.
As shown in Figure 5C, caspase 1-p20, the active form of caspase 1, was readily detected in the kidneys of vehicle-treated MRL/lpr mice, indicating that the NLRP3 inflammasome was activated. In these kidneys, the ratios of phospho–GSK-3β to GSK-3β were small, indicating activation of the GSK signaling pathway. TDZD-8 treatment increased the ratios of phospho– GSK-3β to GSK-3β and inhibited the activation of caspase 1 (Figure 5C). These biochemical changes resulted in diminished renal production of IL-1β (Figure 5E). Similar data were obtained in (NZB × NZW)F1 mice at age 34 weeks, when the mice developed severe proteinuria (Figures 5B, D, and F).
Inhibitory effects of GSK-3β knockdown by GSK-3β siRNA on NLRP3 inflammasome activation in mouse macrophages
BMMs were derived from the (NZB × NZW)F1 mice. Cells were transfected with GSK-3β siRNA. Silencing efficacy was shown by Western blot analysis to be as high as 80% (Figure 6A). More inhibition of GSK-3β transcription was seen in the macrophage cell line J774A.1 (Figure 6A). Caspase 1 was significantly activated with the addition of LPS and ATP, as evidenced by the increase in caspase 1-p20 detected by Western blot analysis (Figure 6B). This enhancement was accompanied by an increase in IL-1β in the supernatant (Figure 6C). Both caspase 1 activation and IL-1β secretion were significantly inhibited with GSK-3β siRNA, but not by control siRNA. Similarly, caspase 1-p20 protein expression and IL-1β production were decreased in J774A.1 by GSK-3β siRNA (Figures 6D and E).
Figure 6.
Inhibition of NLRP3 inflammasome activation in mouse macrophages following the silencing of glycogen synthase kinase 3β (GSK-3β). After GSK-3β–directed small interfering RNA (siRNA) transfection, mouse bone marrow–derived macrophages (BMMs [BMDMs]) or J774A.1 macrophages were primed with lipopolysaccharide (LPS) for 4 hours prior to stimulation, followed by 1 hour of treatment with the NLRP3 inflammasome agonist ATP. NLRP3 inflammasome activation was evaluated by measuring caspase 1-p20 (p20) protein expression and interleukin-1β (IL-1β) production. A, Silencing efficacy of GSK-3β siRNA was as much as 80%, as demonstrated by Western blotting. B, GSK-3β silencing inhibited the expression of p20 in BMMs. C, GSK-3β siRNA significantly reduced the production of IL-1β by BMMs in the cell supernatants. D, GSK-3β silencing inhibited the expression of p20 in the mouse macrophage cell line J774A.1. E, GSK-3β siRNA significantly reduced the production of IL-1β by J774A.1 macrophages in the cell supernatants. Values are the mean ± SD of 3 independent experiments. ** = P < 0.01 versus controls.
Discussion
GSK-3β has been reported to be involved in the pathogenesis of multiple diseases. In the area of autoimmune diseases, it has been shown that inhibition of GSK-3β attenuates symptoms in experimental autoimmune encephalitis (8) and collagen-induced arthritis (22). In this study, the involvement of GSK-3β in the pathogenesis of lupus nephritis was explored in two mouse models of lupus. TDZD-8, a selective inhibitor of GSK-3β, attenuated the development of lupus nephritis in MRL/lpr mice, as indicated by the lower incidence of severe proteinuria, reduced levels of BUN, and reduced levels of histopathologic damage. Additionally, inhibition of GSK-3β reduced the production of autoantibody, the deposition of immune complexes, and the production of proinflammatory cytokines. In contrast to MRL/lpr mice, in which the disease is primarily driven by inflammation resulting from Fas deficiency, (NZB × NZW)F1 mice, with intact Fas gene, spontaneously develop a lupus-like disease that closely resembles SLE in humans (23). Similar inhibitory effects of TDZD-8 on the development of autoantibodies and immune complex–mediated glomerulonephritis were reproduced in (NZB × NZW)F1 mice. These observations suggest that GSK-3β plays a major role in the development of lupus nephritis.
Since its initial characterization more than 30 years ago as a critical enzyme involved in glycogen synthesis, GSK-3β has been shown to be a point of convergence for many signaling pathways involved in a number of physiologic processes (21,24). Subsequent studies have indicated that GSK-3β is a key regulator in inflammation and immune response (24). GSK-3 regulates the inflammatory response by differentially affecting nuclear levels of the transcription factors NF-κBp65 and CREB that interact with the coactivator CREB binding protein in endotoxin shock (5). Because of its involvement in multiple important signaling pathways, GSK-3β has also been considered a point of convergence for the host inflammatory response (24). Thus, it is not surprising that GSK-3β inhibition has been shown to reduce the synthesis of multiple proinflammatory cytokines, including IL-12, TNFα, IFNγ, IL-1β, and IL-6, while increasing the production of IL-10, in monocytes (5,25,26). Our results showed that inhibition of GSK-3β resulted in significant reduction in serum levels of IL-1β, IL-17, and IFNγ in MRL/lpr and (NZB × NZW)F1 mice, which supports an essential role of GSK-3β in the production of proinflammatory cytokines.
Because of the observation that down-regulation of the NLRP3/IL-1β pathway has a significant attenuating effect on mouse models of lupus nephritis (14,15), we investigated the effect of TDZD-8 on this pathway. The results showed that GSK-3β promotes murine lupus nephritis, at least in part, by activating the NLRP3 inflammasome. GSK-3β involvement in the NLRP3/IL-1β pathway was further confirmed in macrophages generated from the bone marrow of (NZB × NZW)F1 mice and in the mouse macrophage cell line J774A.1. Thus, the linking of GSK-3β to the NLRP3/IL-1β pathway is established, and this is a novel observation.
The NLRP3 inflammasome has recently been demonstrated to play a role in the pathogenesis of SLE. U1 small nuclear RNP, which plays a pathogenic role in SLE, activates the NLRP3 inflammasome in human monocytes, leading to IL-1β production (27). Self ds-DNA induces IL-1β production in human monocytes by activating the NLRP3 inflammasome in the presence of anti-dsDNA antibodies (28). Recently, Kahlenberg et al (29) showed that mice lacking caspase 1 were significantly protected against pristane-induced vascular dysfunction and immune complex glomerulonephritis. Our previous data have shown that blockade of NLRP3 or its upstream members in the signaling pathway attenuated lupus nephritis in mice (14,15). In the present study, we found that TDZD-8 treatment suppressed GSK-3β activity and NLRP3 inflammasome activation, resulting in a decrease in IL-1β synthesis. The incidence of severe proteinuria and renal inflammation was significantly attenuated in both lupus models with a significant reduction in anti-dsDNA antibody production, renal immune complex deposition, and circulating proinflammatory cytokine levels. These observations suggest that GSK-3β is a potential therapeutic target for the treatment of lupus nephritis and warrants further investigation.
Diverse GSK-3 inhibitors have been reported and have been used in cellular and in vivo animal models of human diseases (30). In addition, it is increasingly recognized that moderate inhibition of a cellular target, particularly for long-term treatment, provides a more favorable outcome than does complete inhibition (30). In support of our results, we also tested the effects of TDZD-8 on NLRP3 inflammasome activation in PBMCs from 8 SLE patients and 8 healthy volunteer donors. We found that TDZD-8, selective inhibitor of GSK-3β, also blocked NLRP3/IL-1β activation in normal and lupus PBMCs (Supplementary Figure 1 available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.38993/abstract).
In conclusion, we identified a novel role of GSK-3β in murine lupus nephritis. Inhibition of GSK-3β effectively attenuated lupus nephritis in 2 lupus models by suppressing NLRP3 inflammasome activation. Our results support the hypothesis that GSK-3β is a target for therapeutic intervention in lupus nephritis and that small molecules that inhibit GSK-3 signaling for treatments of other human diseases, such as cancer and Alzheimer's disease, may be applicable to the treatment of lupus nephritis.
Supplementary Material
Acknowledgments
Supported by the National Natural Science Foundation of China (grants 81273278 and 81471598), the Ministry of Education of China (PhD Program Foundation grant 20120171110064), the Guangdong Natural Science Foundation (grants S2012010008780 and S2011010004578), the Guangzhou Science and Technology Planning Program (grant 2012J4100085), and the Sun Yat-sen Innovative Talents Cultivation Program for Doctoral Graduate Students. Drs. Gaskin and Fu's work was supported by grants from the NIH (R01-AR-047988 and R01-AR-049449) and the Alliance for Lupus Research.
Footnotes
Author Contributions: All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Yang had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Zhao, H. Wang, Yang, Fu.
Acquisition of data. Zhao, H. Wang, Huang, Zhang, S. Wang, Gaskin, Yang, Fu.
Analysis and interpretation of data. Zhao, H. Wang, Huang, Zhang, S. Wang, Gaskin, Yang, Fu.
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