Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with systemic organ involvements and the current therapies are limited to suboptimal treatments. Peroxisome proliferator-activated receptor γ (PPARγ) ago- nists are widely prescribed for the pharmacological treatment of type 2 diabetes by their insulin-sensitizing properties. PPARγ agonists also exert anti-inflamma- tory and immunomodulatory effects, and the beneficial impacts of rosiglitazone were reported for the amelioration of the autoantibody production, renal disease, and atherosclerosis in MRL-lpr mice depending on the induction of adiponectin  and also in NZBWF1 mice  . SLE is associated with increased circulating apoptotic autoantigens and increased type I interferon (IFN) signaling which simulate the production of autoantibody including anti-dsDNA antibodies  . Genetic depletion of PPARδ decreased the expression of opsonins such as C1qb and Mertk, which was resulted in the impairment of clearance for apoptotic cells and autoimmune kidney disease resembling lupus nephritis in human  . Engulfment of apoptotic cells by macrophages activated liver X receptor (LXR) and induced the transcriptional activation of Mertk. The lack of LXR also manifested the defects of self-tolerance and developed autoimmune glomerulonephritis  . Thus, the administration of PPARδ agonist or LXR agonist may be beneficial for the treatment of SLE by enhancing the clearance of apoptotic bodies. The efforts were made to develop the PPARγ/δ dual agonists without the deleterious side effects associated with full PPARγ agonists such as weight gain, edema and osteoporosis  and simultaneous stimulation of both PPARγ and PPARδ may be beneficial in the treatment of SLE. In the current investigation, we explored the efficacy of LXR agonist (GW3965) or dual treatment of PPARγ (pioglitazone) and PPARδ (GW0742) agonists in SLE animal model in mice, female MRL/MpJ-Fas
Female BALB/cAJcl mice (CLEA Japan) were injected with pristane at 10 weeks of age and they were divided into three groups, control (BALB/c; n = 10), 20 mg/kg/day of GW3965 treated (LXR; n = 11), and 25 mg/kg/day of pioglitazone plus 1 mg/kg/day of GW0742 treated (PPARγ/δ; n = 10) (Sigma-Aldrich) groups from 11 to 23 weeks of age. Sera and urine samples were collected at 17 weeks of age, and they were sacrificed at 23 weeks of age and used for following experiments.
The methods were carried out in accordance with the approved guidelines. All experimental protocols were approved by the Animal Care and Use Committee of the Department of Animal Resources, Advanced Science Research Center, Okayama University.
2.2. Measurement of Urinary Albumin Excretion and dsDNA Antibodies
Urinary albumin excretions were measured with mouse albumin ELISA kit (Shibayagi, Japan) and standardized by the concentrations of urine creatinine. dsDNA antibodies were measured with mouse anti-dsDNA ELISA kit (Shibayagi, Japan).
2.3. Histopathology and Scoring
Kidney specimens were fixed with 10% buffered formalin and embedded in paraffin. Serial 2-μm sections were stained with periodic acid Schiff (PAS) for histological examination by light microscopy. Glomerular lesions were graded according to reported criteria: grade 0, no recognizable lesion in glomeruli; grade 1, mild cell proliferation and/or cell infiltration; grade 2, the same as grade 1 with mesangial proliferation, lobulation and hyaline droplet, associated with macrophage infiltration; grade 3, the same as grade 2 with crescent and granuloma formation and/or hyalinosis  . Glomeruli of more than 20 renal glomeruli examined in each mouse.
2.4. Real-Time RT-PCR
Kidney tissues were homogenized by Tissuelyser Adapter Set 2X24 (QIAGEN) with 1 ml QIAzolLysis Reagent (QIAGEN) and total RNAs extracted by RNeasy Lipid Tissue Mini Kit (QIAGEN). Then, 1 μg total RNAs were used to synthesize cDNA by using High Capacity RNA-to-cDNA Kit (Applied Biosystems). Quantitative real-time PCR was performed in Step One Plus Real-Time PCR System (Applied Biosystems) with specific primers and Universal Master Mix II (Life Technologies) and TaqMan Gene Expression Assays to evaluate the gene expression of intercellular adhesion molecule 1 (Mm00516023_m1), tumor necrosis factor (Mm00443258_m1), complement component 1, q subcomponent, alpha polypeptide (Mm00432142_m1), ATP-binding cassette, sub-family A (ABC1), member 1 (Mm00442646_m1), c-mer proto-oncogene tyrosine kinase (Mm00- 434920_m1). The relative abundance of mRNAs was standardized with GAPDH mRNA as the invariant control.
2.5. Statistical Analysis
Data are expressed as the mean ± standard error. The data were also analyzed with one-way analysis of variance and Tukey’s honestly significant difference test when multiple comparisons against the control were required. P < 0.05 was regarded as statistically significant. Pearson’s χ2 test was used to compare the distribution of glomeruli with grading scores from 0 to 3. The data were analyzed with JMP 8.0.2 software package (SAS Institute Inc.).
3.1. Effects of LXR Agonist and PPARγ/δ Agonists in MRL Mice
MRL mice at 8 weeks of age were administered with LXR or dual treatment of PPARγ and PPARδ (PPARγ/δ) agonists for 12 weeks. There were no statistically significant differences in body, kidney and lymph node weights in PPARγ/δ and LXR groups compared with MRL group (Figures 1(a)-1(d)). In MRL group, urine albumin-to-creatinine ratio (UACR) progressively increased at 14 and 20 weeks of age. At 20 weeks of age, the treatments with LXR agonist or PPARγ/δ agonists demonstrated the reduction of UACR; however, it did not reach statistical differences (Figure 1(e)). The administration of PPARγ/δ agonists did not demonstrate therapeutic effects on the tiler of anti-dsDNA antibodies. The titer of anti-dsDNA antibodies in LXR group was rather elevated and deteriorated compared with MRLmice with significant difference (p = 0.002) (Figure 1(f)). We next performed the histopathological grading of glomerular lesions as previously described  . The treatments with PPARγ/δ and LXR agonists reduced the average scores of glomerular lesions but they were not statistically significant (Figure 2(a)). Furthermore, the distribution of glomeruli with grading scores from 0 to 3 was not significantly altered by the treatments with PPARγ/δ and LXR agonists (Figure 2(b) and Figure 2(c)). The genes such as Abca1, C1qa, and Mertkare under the transcriptional control of PPARγ, PPARδ, and LXR, respectively, in addition, the genes related to inflammation, Icam1 and Tnf, may be suppressed by PPARγ agonists. The mRNA expression of these genes were reduced in PPARγ/δ and LXR groups compared with MRL mice, but they were not statistically significant again (Figure 2(d)). Taken together, the administration of LXR or PPARγ/δ agonists into MRL mice demonstrated the tendency to ameliorate the albuminuria and histological scores in MRL mice; however, it did not exhibit sufficient therapeutic potential against glomerulonephritis in MRL mice.
3.2. Effects of LXR Agonist and PPARγ/δ Agonists in Pristane-Treated BALB/cAJcl Mice
Female MRL mice demonstrate the “severe” disease phenotype with an average lifespan of 17 weeks and we further tested the preventive effects of PPARγ/δ and LXR agonists in pristane-treated BALB/cAJcl (BALB/c) mice with milder phenotype of lupus and glomerulonephritis. There were no statistically significant differences in body, kidney and lymph node weights in PPARγ/δ and LXR groups compared with BALB/c mice treated with pristane (Figure 3(a) and Figure 3(b)). The administration of PPARγ/δ and LXR agonists did not alter the
Figure 1. The administration of LXR agonist (GW3965) (LXR) or dual treatment of PPARγ (pioglitazone) and PPARδ (GW0742) agonists (PPARγ/δ) in MRL/MpJ-Fas
urinary excretion of albumin (Figure 3(c)). The glomeruli in BALB/c mice treated with pristane demonstrated mild proliferation of glomerular cells but there were no differences in PPARγ/δ and LXR groups compared with BALB/c mice with pristane.
In the current investigation, LXR or dual treatment of PPARγ/δ agonists demonstrated only minimal beneficial effects in the treatment of established and severe autoimmune phenotypes in MRL mice. We also tested the efficacy of LXR
Figure 2. Histology scoring and RT-PCR in MRL/MpJ-Fas
or PPARγ/δ agonists in BALB/c mice treated with pristane with mild autoimmune phenotypes and again it demonstrated the minimal therapeutic potential. Recent work suggested that PPARγ agonist treatment started before disease onset demonstrated the marked amelioration of disease, while it was not effective when started after disease onset  . We thought that the administration of LXR or PPARγ/δ agonists at 8 weeks of age might be too late to suppress the disease and we administered them immediately after the injection of pristane in BALB/c mice; however, they did not reveal the beneficial effects in prevention. The interventions to enhance the clearance of apoptotic body may not be sufficient to suppress the autoimmune phenotypes in SLE. In addition, PPARγ agonist is known to be beneficial to ameliorate the chronic or subclinical inflammation; however, it may not be effective to suppress the acute and severe inflammatory
Figure 3. The administration of LXR agonist (GW3965) (LXR) or dual treatment of PPARγ (pioglitazone) and PPARδ (GW0742) agonists (PPARγ/δ) in BALB/cAJcl mice treated with pristane. (a) Kidney weight, (b) Kidney/body weight, (c) Urine albumin/ creatinine, (d) PAS stain of kidney tissues. ns, not significant.
response in SLE. In gld. apoE (-/-) with accelerated atherosclerosis and SLE, PPARγ agonist demonstrated the marked amelioration in atherosclerosis and minimal impacts on autoimmune phenotype such as autoantibody production  .
The ineffectiveness of LXR or PPARγ/δ agonists in lupus animal models was also explained by the unfavorable changes in global gene expression profiles or alterations in endogenous lipid ligands. The stimulation of nuclear receptors may induce both favorable and unwanted changes in mRNA expression profile, although we did not perform the profiling such as DNA microarray. In addition, PPARγ and LXR bind to various physiological lipid ligands such as unsaturated fatty acids and hydroxylcholesterol, respectively  , and the alterations of such lipid ligands also influence the activation of nuclear receptors.
The limitation of current study is that we did not investigate the dose-effect relationship and the higher doses may exert the beneficial effects on SLE, although we employed the doses which were used in previous studies. Second, we did not investigate the simultaneous use of steroids or immune suppressants with LXR or PPARγ/δ agonists, which might be beneficial in the treatment of SLE. Finally, the results in SLE animal models in mice may not be applicable to human and the clinical studies have not been performed since LXR and PPARδ agonists have not been approved so far in the market.
The use of nuclear receptor agonists such as LXR or PPARγ/δ agonists seems to be not effective in the treatment of MRL and pristane-treated BALB/c mice. Efficacy of LXR or PPARγ/δ agonists may be tested in the patients with SLE in combination with steroids and immune suppressants.
The authors declare to have no competing interests.
This work was supported by JSPS Grant-in-Aid for Scientific Research, Grant Numbers (25126716, 26293218).
Study concept: NTT, KSW, KS and JW. Animal experiments: NTT, SZ, SH, TK, and EK. RT-PCR: SH and MY. Statistical analysis: NTT and JW. Manuscript writing: NTT, KSW and JW. All authors approved the final manuscript.
ELISA: enzyme-linked immunosorbent assay. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. PPAR: peroxisome proliferator-activated receptor. SLE: systemic lupus erythematosus. LXR: liver X receptor.
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