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Blunting Autoantigen-induced FOXO3a Protein Phosphorylation and Degradation Is a Novel Pathway of Glucocorticoids for the Treatment of Systemic Lupus Erythematosus

2016; Elsevier BV; Volume: 291; Issue: 38 Linguagem: Inglês

10.1074/jbc.m116.728840

ISSN

1083-351X

Autores

Mudan Lu, Wei Xu, Bo Gao, Sidong Xiong,

Tópico(s)

Adipose Tissue and Metabolism

Resumo

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease affecting multiple organs. Glucocorticoids (GCs), the potent anti-inflammatory drugs, remain as a cornerstone in the treatment for SLE; nevertheless, their clinical efficacy is compromised by the side effects of long term treatment and resistance. To improve the therapeutic efficacy of GCs in SLE, it is important to further decipher the molecular mechanisms of how GCs exert their anti-inflammatory effects. In this investigation, FOXO3a was identified as a molecule that was down-regulated in the course of SLE. Of interest, GC treatment was found to rescue FOXO3a expression both in SLE mice and in SLE patients. Gain- and loss-of-function studies demonstrated that FOXO3a played a crucial role in GC treatment of SLE via inhibiting inflammatory responses. Further studies showed that the up-regulation of FOXO3a by GCs relied on the suppression of pI3K/AKT-mediated FOXO3a phosphorylation and the arrest of FOXO3a in the nucleus. Finally, our data revealed that FOXO3a was critical for GC-mediated inhibition of NF-κB activity, which might involve its interaction with NF-κB p65 protein. Collectively, these data indicated that FOXO3a played an important role in GC treatment of SLE by suppressing pro-inflammatory response, and targeting FOXO3a might provide a novel therapeutic strategy against SLE. Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease affecting multiple organs. Glucocorticoids (GCs), the potent anti-inflammatory drugs, remain as a cornerstone in the treatment for SLE; nevertheless, their clinical efficacy is compromised by the side effects of long term treatment and resistance. To improve the therapeutic efficacy of GCs in SLE, it is important to further decipher the molecular mechanisms of how GCs exert their anti-inflammatory effects. In this investigation, FOXO3a was identified as a molecule that was down-regulated in the course of SLE. Of interest, GC treatment was found to rescue FOXO3a expression both in SLE mice and in SLE patients. Gain- and loss-of-function studies demonstrated that FOXO3a played a crucial role in GC treatment of SLE via inhibiting inflammatory responses. Further studies showed that the up-regulation of FOXO3a by GCs relied on the suppression of pI3K/AKT-mediated FOXO3a phosphorylation and the arrest of FOXO3a in the nucleus. Finally, our data revealed that FOXO3a was critical for GC-mediated inhibition of NF-κB activity, which might involve its interaction with NF-κB p65 protein. Collectively, these data indicated that FOXO3a played an important role in GC treatment of SLE by suppressing pro-inflammatory response, and targeting FOXO3a might provide a novel therapeutic strategy against SLE. Systemic lupus erythematosus (SLE) 3The abbreviations used are: SLE, systemic lupus erythematosus; GC, glucocorticoid; Dex, dexamethasone; ALD-DNA, activated lymphocyte-derived apoptotic DNA; UnALD-DNA, unactivated lymphocyte-derived DNA; SLEDAI, SLE disease activity index; FOXO, forkhead box class O; PBMC, peripheral blood mononuclear cell; GR, GC receptor. 3The abbreviations used are: SLE, systemic lupus erythematosus; GC, glucocorticoid; Dex, dexamethasone; ALD-DNA, activated lymphocyte-derived apoptotic DNA; UnALD-DNA, unactivated lymphocyte-derived DNA; SLEDAI, SLE disease activity index; FOXO, forkhead box class O; PBMC, peripheral blood mononuclear cell; GR, GC receptor. is a prototypical autoimmune disorder, affecting virtually every organ (1Tsokos G.C. 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Systemic corticosteroid therapy–side effects and their management.Br. J. Ophthalmol. 1998; 82: 704-708Crossref PubMed Scopus (275) Google Scholar, 11Stahn C. Buttgereit F. Genomic and nongenomic effects of glucocorticoids.Nat. Clin. Pract. Rheumatol. 2008; 4: 525-533Crossref PubMed Scopus (412) Google Scholar), and not every patient responds sufficiently to GC treatment (12Barnes P.J. Adcock I.M. Glucocorticoid resistance in inflammatory diseases.Lancet. 2009; 373: 1905-1917Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar), deeper insights into the molecular mechanisms of how GCs exert their anti-inflammatory activities may also help improve the therapeutic efficacy for these patients. FOXO3a belongs to a family of forkhead box class O (FOXO) transcription factors, which are involved in diverse physiopathological processes, such as metabolism, apoptosis, oxidative stress resistance, and senescence. In mammals, there are four FOXO proteins, FOXO1, FOXO3a, FOXO4, and FOXO6. FOXO1, FOXO3, and FOXO4 are widely distributed and are expressed in most tissues, whereas FOXO6 expression is largely restricted to neural cells (13Morris B.J. Willcox D.C. Donlon T.A. Willcox B.J. FOXO3: a major gene for human longevity–a mini-review.Gerontology. 2015; 61: 515-525Crossref PubMed Scopus (177) Google Scholar14Wang Y. Zhou Y. Graves D.T. FOXO transcription factors: their clinical significance and regulation.Biomed. Res. Int. 2014; 2014: 925350PubMed Google Scholar, 15Calnan D.R. Brunet A. The FoxO code.Oncogene. 2008; 27: 2276-2788Crossref PubMed Scopus (917) Google Scholar16Eijkelenboom A. Burgering B.M. FOXOs: signalling integrators for homeostasis maintenance.Nat. Rev. Mol. Cell Biol. 2013; 14: 83-97Crossref PubMed Scopus (711) Google Scholar). 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The activation of NF-κB through Akt-induced FOXO1 phosphorylation during aging and its modulation by calorie restriction.Biogerontology. 2008; 9: 33-47Crossref PubMed Scopus (81) Google Scholar, 20Miao H. Zhang Y. Lu Z. Yu L. Gan L. FOXO1 increases CCL20 to promote NF-κB-dependent lymphocyte chemotaxis.Mol. Endocrinol. 2012; 26: 423-437Crossref PubMed Scopus (35) Google Scholar); however, evidence indicates that FOXO3a and FOXO4 may play an inhibitory role in autoimmune and inflammatory diseases by repressing NF-κB activity (21Lin L. Hron J.D. Peng S.L. Regulation of NF-κB, Th activation, and autoinflammation by the forkhead transcription factor Foxo3a.Immunity. 2004; 21: 203-213Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 22Zhou W. Cao Q. Peng Y. Zhang Q.J. Castrillon D.H. DePinho R.A. Liu Z.P. FoxO4 inhibits NF-κB and protects mice against colonic injury and inflammation.Gastroenterology. 2009; 137: 1403-1414Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Mice with a defect in FOXO3a were found to predispose to a spontaneous multisystemic inflammatory syndrome, accompanied by an up-regulated production of pro-inflammatory cytokines (21Lin L. Hron J.D. Peng S.L. Regulation of NF-κB, Th activation, and autoinflammation by the forkhead transcription factor Foxo3a.Immunity. 2004; 21: 203-213Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 23Snoeks L. Weber C.R. Wasland K. Turner J.R. Vainder C. Qi W. Savkovic S.D. Tumor suppressor FOXO3 participates in the regulation of intestinal inflammation.Lab. Invest. 2009; 89: 1053-1062Crossref PubMed Scopus (44) Google Scholar). Accumulating evidence also indicates the expression of FOXO3a may be associated with diverse inflammatory diseases, such as asthma, ulcerative colitis Crohn's disease, and rheumatoid arthritis in clinical patients (24Barkund S. Shah T. Ambatkar N. Gadgil M. Joshi K. FOXO3a gene polymorphism associated with asthma in Indian population.Mol. Biol. Int. 2015; 2015: 638515Crossref PubMed Google Scholar, 25Min M. Peng L. Yang Y. Guo M. Wang W. Sun G. MicroRNA-155 is involved in the pathogenesis of ulcerative colitis by targeting FOXO3a.Inflamm. Bowel Dis. 2014; 20: 652-659Crossref PubMed Scopus (61) Google Scholar26Lee J.C. Espéli M. Anderson C.A. Linterman M.A. Pocock J.M. Williams N.J. Roberts R. Viatte S. Fu B. Peshu N. Hien T.T. Phu N.H. Wesley E. Edwards C. Ahmad T. et al.Human SNP links differential outcomes in inflammatory and infectious disease to a FOXO3-regulated pathway.Cell. 2013; 155: 57-69Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Of interest, it is also suggested that FOXO3a may be involved in some GC-mediated biological processes (27Ma J. Xie Y. Shi Y. Qin W. Zhao B. Jin Y. Glucocorticoid-induced apoptosis requires FOXO3A activity.Biochem. Biophys. Res. Commun. 2008; 377: 894-898Crossref PubMed Scopus (19) Google Scholar, 28Consolaro F. Ghaem-Maghami S. Bortolozzi R. Zona S. Khongkow M. Basso G. Viola G. Lam E.W. FOXO3a and post-translational modifications mediate glucocorticoid sensitivity in acute B-ALL.Mol. Cancer Res. 2015; 13: 1578-1590Crossref PubMed Scopus (21) Google Scholar). Using protein antibody array and Western blotting analysis, we found that FOXO3a was down-regulated in the course of SLE, which could be reversed by treatment with GCs. Of importance, FOXO3a was revealed to play a crucial role in GC treatment of SLE via inhibiting pro-inflammatory responses. Further studies showed that the up-regulation of FOXO3a by GCs relied on the suppression of FOXO3a phosphorylation and its ensuing degradation in cytoplasm. Moreover, it was found that the nucleus-arrested FOXO3a played an important role in GC-mediated inhibition of NF-κB activity, which might involve its interaction with NF-κB p65 protein. A total of 21 patients (male/female, 2/19; median age, 42 years) with SLE and 12 age- and gender-matched healthy individuals (male/female 1/11; median age, 41 years) were recruited. All patients took prednisone (0.5–1 mg/kg/day orally) as part of their routine therapy for 4 weeks in combination with oral hydroxychloroquine or other immunosuppressants to achieve a smooth reduction in GC dosage after 4 weeks. After the 4-week GCs treatment, the peripheral blood samples and urine samples were collected from SLE patients after informed consent. The diagnosis of SLE was established according to the 1982 revised American College of Rheumatology criteria (29Tan E.M. Cohen A.S. Fries J.F. Masi A.T. McShane D.J. Rothfield N.F. Schaller J.G. Talal N. Winchester R.J. The 1982 revised criteria for the classification of systemic lupus erythematosus.Arthritis Rheum. 1982; 25: 1271-1277Crossref PubMed Scopus (12513) Google Scholar). This study was approved by the Ethics Committee of Fudan University and performed in compliance with the Helsinki Declaration. Six-week-old female BALB/c mice were purchased from the Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China). Mice were housed in a specific pathogen-free room under controlled temperature and humidity. All animal experiments were conducted according to the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, People's Republic of China, 1998) and with the approval of the Ethical Committee of Fudan University (Shanghai, People's Republic of China). RAW264.7 cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 2 mm glutamine and 10% FBS (Hyclone, Logan, UT) in a 5% CO2 incubator at 37 °C. Primary peritoneal macrophages were obtained from 6-week-old female BALB/c mice and were maintained in DMEM (Invitrogen) supplemented with 10% FBS. For the preparation of renal macrophages, murine renal tissues were dispersed in RPMI 1640 medium containing 5% FBS and 0.1% collagenase (Sigma) at 37 °C for 30 min, followed by progressive sieving to obtain single-cell suspensions. The CD11b+/F4/80 high macrophages were further sorted from single-cell suspensions using a FACSAria (BD Biosciences) with FITC-labeled anti-F4/80 and phycoerythrin-labeled anti-CD11b (BD Biosciences). The purity of cells was more than 90%, as determined by flow cytometry (FACSCalibur; BD Biosciences). Activated lymphocyte-derived apoptotic DNA (ALD-DNA) and unactivated lymphocyte-derived DNA (UnALD-DNA) extraction and purification were performed as described previously (30Qiao B. Wu J. Chu Y.W. Wang Y. Wang D.P. Wu H.S. Xiong S. Induction of systemic lupus erythematosus-like syndrome in syngeneic mice by immunization with activated lymphocyte-derived DNA.Rheumatology. 2005; 44: 1108-1114Crossref PubMed Scopus (52) Google Scholar31Wen Z.K. Xu W. Xu L. Cao Q.H. Wang Y. Chu Y.W. Xiong S. DNA hypomethylation is crucial for apoptotic DNA to induce systemic lupus erythematosus-like autoimmune disease in SLE-non-susceptible mice.Rheumatology. 2007; 46: 1796-1803Crossref PubMed Scopus (72) Google Scholar, 32Zhang W. Xu W. Xiong S. Blockade of Notch1 signaling alleviates murine lupus via blunting macrophage activation and M2b polarization.J. Immunol. 2010; 184: 6465-6478Crossref PubMed Scopus (138) Google Scholar, 33Chen M. Zhang W. Xu W. Zhang F. Xiong S. Blockade of TLR9 signaling in B cells impaired anti-dsDNA antibody production in mice induced by activated syngeneic lymphocyte-derived DNA immunization.Mol. Immunol. 2011; 48: 1532-1539Crossref PubMed Scopus (21) Google Scholar, 34Zhang W. Xu W. Xiong S. Macrophage differentiation and polarization via phosphatidylinositol 3-kinase/Akt-ERK signaling pathway conferred by serum amyloid P component.J. Immunol. 2011; 187: 1764-1777Crossref PubMed Scopus (107) Google Scholar, 35Zhang W. Zhou Q. Xu W. Cai Y. Yin Z. Gao X. Xiong S. DNA-dependent activator of interferon-regulatory factors (DAI) promotes lupus nephritis by activating the calcium pathway.J. Biol. Chem. 2013; 288: 13534-13550Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 36Lu M. Yu S. Xu W. Gao B. Xiong S. HMGB1 promotes systemic lupus erythematosus by enhancing macrophage inflammatory response.J. Immunol. Res. 2015; 2015: 946748Crossref PubMed Scopus (48) Google Scholar, 37Mao X. Wu Y. Diao H. Hao J. Tian G. Jia Z. Li Z. Xiong S. Wu Z. Wang P. Zhao L. Yin Z. Interleukin-6 promotes systemic lupus erythematosus progression with Treg suppression approach in a murine systemic lupus erythematosus model.Clin. Rheumatol. 2014; 33: 1585-1593Crossref PubMed Scopus (18) Google Scholar38Li F. Zhu X. Yang Y. Huang L. Xu J. TIPE2 alleviates systemic lupus erythematosus through regulating macrophage polarization.Cell. Physiol. Biochem. 2016; 38: 330-339Crossref PubMed Scopus (39) Google Scholar). In brief, for the generation of activated lymphocyte-derived apoptotic DNA (ALD-DNA), splenocytes from 6- to 8-week-old female BALB/c mice were seeded at 2 × 106 cells/ml in 75-cm2 cell culture flasks and cultured for 6 days in the presence of concanavalin A (5 mg/ml) to induce apoptosis. The apoptotic cells were stained with FITC-labeled annexin V (BD Biosciences) and propidium iodide (Sigma) and then sorted using a FACSAria (BD Biosciences). Genomic DNAs from syngeneic apoptotic splenocytes were treated with S1 nuclease (TaKaRa, Dalian, China) and proteinase K (Sigma) and then purified using the DNeasy Blood and Tissue kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Unactivated lymphocyte-derived DNA (UnALD-DNA) was extracted from unactivated (resting) splenocytes using the same methods. The SLE murine model was generated as described previously (30Qiao B. Wu J. Chu Y.W. Wang Y. Wang D.P. Wu H.S. Xiong S. Induction of systemic lupus erythematosus-like syndrome in syngeneic mice by immunization with activated lymphocyte-derived DNA.Rheumatology. 2005; 44: 1108-1114Crossref PubMed Scopus (52) Google Scholar31Wen Z.K. Xu W. Xu L. Cao Q.H. Wang Y. Chu Y.W. Xiong S. DNA hypomethylation is crucial for apoptotic DNA to induce systemic lupus erythematosus-like autoimmune disease in SLE-non-susceptible mice.Rheumatology. 2007; 46: 1796-1803Crossref PubMed Scopus (72) Google Scholar, 32Zhang W. Xu W. Xiong S. Blockade of Notch1 signaling alleviates murine lupus via blunting macrophage activation and M2b polarization.J. Immunol. 2010; 184: 6465-6478Crossref PubMed Scopus (138) Google Scholar, 33Chen M. Zhang W. Xu W. Zhang F. Xiong S. Blockade of TLR9 signaling in B cells impaired anti-dsDNA antibody production in mice induced by activated syngeneic lymphocyte-derived DNA immunization.Mol. Immunol. 2011; 48: 1532-1539Crossref PubMed Scopus (21) Google Scholar, 34Zhang W. Xu W. Xiong S. Macrophage differentiation and polarization via phosphatidylinositol 3-kinase/Akt-ERK signaling pathway conferred by serum amyloid P component.J. Immunol. 2011; 187: 1764-1777Crossref PubMed Scopus (107) Google Scholar, 35Zhang W. Zhou Q. Xu W. Cai Y. Yin Z. Gao X. Xiong S. DNA-dependent activator of interferon-regulatory factors (DAI) promotes lupus nephritis by activating the calcium pathway.J. Biol. Chem. 2013; 288: 13534-13550Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 36Lu M. Yu S. Xu W. Gao B. Xiong S. HMGB1 promotes systemic lupus erythematosus by enhancing macrophage inflammatory response.J. Immunol. Res. 2015; 2015: 946748Crossref PubMed Scopus (48) Google Scholar, 37Mao X. Wu Y. Diao H. Hao J. Tian G. Jia Z. Li Z. Xiong S. Wu Z. Wang P. Zhao L. Yin Z. Interleukin-6 promotes systemic lupus erythematosus progression with Treg suppression approach in a murine systemic lupus erythematosus model.Clin. Rheumatol. 2014; 33: 1585-1593Crossref PubMed Scopus (18) Google Scholar38Li F. Zhu X. Yang Y. Huang L. Xu J. TIPE2 alleviates systemic lupus erythematosus through regulating macrophage polarization.Cell. Physiol. Biochem. 2016; 38: 330-339Crossref PubMed Scopus (39) Google Scholar). Briefly, 6-week-old female BALB/c mice were immunized s.c. with ALD-DNA (50 μg/mouse) plus complete Freund's adjuvant (Sigma) on day 1, followed by s.c. injection of ALD-DNA (50 μg/mouse) emulsified with incomplete Freund's adjuvant (Sigma) on days 14 and 28 for a total of three times. Serum and urine samples were collected every 2nd week for further experiments. Eight weeks after initial immunization, mice were sacrificed, and kidneys were collected for further cellular function and tissue histology analysis. The protein expression profiles of ALD-DNA-stimulated RAW264.7 cells in the absence or presence of GCs were analyzed using an antibody array kit (Full Moon Biosystem, Inc., Sunnyvale, CA). Briefly, protein samples obtained from RAW264.7 cells were biotinylated using biotin reagent dissolved in N,N-dimethylformamide. Biotin-labeled protein samples were then conjugated to the antibody microarray slides, followed by incubation with FITC-streptavidin solution. Slides were scanned using the GenePix 4000 Array Scanner, and the images were analyzed with GenePix Pro 6.0 (Molecular Devices, Sunnyvale, CA). To down-regulate the FOXO3a expression in RAW264.7 cells, cells were transfected with FOXO3a-specific siRNA (catalogue number 6302; Cell Signaling Technology) using mouse macrophage nucleofector kit (Lonza Amaxa, Cologne, Germany) according to the manufacturer's instructions. To inhibit the FOXO3a expression in lupus mice, 6-week-old female BALB/c mice were randomized for injection with siFOXO3a or siControl using in vivo-jetPEITM (Polyplus Transfection, Strasbourg, France) every other 3 days for 6 weeks as described previously (33Chen M. Zhang W. Xu W. Zhang F. Xiong S. Blockade of TLR9 signaling in B cells impaired anti-dsDNA antibody production in mice induced by activated syngeneic lymphocyte-derived DNA immunization.Mol. Immunol. 2011; 48: 1532-1539Crossref PubMed Scopus (21) Google Scholar, 35Zhang W. Zhou Q. Xu W. Cai Y. Yin Z. Gao X. Xiong S. DNA-dependent activator of interferon-regulatory factors (DAI) promotes lupus nephritis by activating the calcium pathway.J. Biol. Chem. 2013; 288: 13534-13550Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In brief, 24 h after the initial siFOXO3a or siControl treatment, mice were immunized with ALD-DNA (50 μg/mouse), UnALD-DNA (50 μg/mouse), or PBS three times in 4 weeks. Eight weeks after initial immunization, mice were sacrificed, and surgically resected kidneys were collected for further cellular function and tissue histology analysis. Adenoviruses encoding FOXO3a (Ad-FOXO3a) and the control (Ad-Control) were obtained from Hanheng Biological Technology (Shanghai, China). The adenovirus delivery in mice was performed as described previously (39Gui J. Yue Y. Chen R. Xu W. Xiong S. A20 (TNFAIP3) alleviates CVB3-induced myocarditis via inhibiting NF-kappaB signaling.PLoS ONE. 2012; 7: e46515Crossref PubMed Scopus (51) Google Scholar). To examine the therapeutic effects of FOXO3a, each mouse received an intravenous injection of 100 μl of Ad-FOXO3a or Ad-Control (3 × 109 plaque-forming units, pfu) 2 days before ALD-DNA immunization. For the detection of anti-dsDNA levels, ELISA plates were pretreated with protamine sulfate (Sigma) and then coated with calf thymus dsDNA (Sigma). After incubation with mouse sera, levels of anti-dsDNA antibodies were examined with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Southern Biotechnology, Birmingham, AL), followed by the measurement of enzymatic color reaction at 450 nm. Proteinuria of the mice was determined by the BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions. To assess the levels of TNF-α, IL-6, and MCP-1 in sera or the cell culture supernatants, ELISAs were performed according to the manufacturer's instructions (eBioscience, San Diego). Murine renal tissues were fixed in 4% paraformaldehyde (Sigma), processed, and coated in paraffin. H&E staining of renal tissue sections was performed according to the manufacturer's instructions and assessed by a pathologist blinded to treatment group. The kidney score of glomerulonephritis was determined by using the ISN/RPS2003 classification (40Weening J.J. D'Agati V.D. Schwartz M.M. Seshan S.V. Alpers C.E. Appel G.B. Balow J.E. Bruijn J.A. Cook T. Ferrario F. Fogo A.B. Ginzler E.M. Hebert L. Hill G. Hill P. et al.The classification of glomerulonephritis in systemic lupus erythematosus revisited.Kidney Int. 2004; 65: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar). Pictures were acquired with a Nikon Eclipse TE2000-S microscope (Nikon, Tokyo, Japan) with magnification ×200. Fluorescent staining of cryosections was used for autoantibody deposition analysis in the glomeruli. Sections were fixed in acetone for 10 min and incubated with FITC-conjugated goat anti-mouse IgG antibody (catalogue number sc-2010; Santa Cruz Biotechnology, Santa Cruz, CA) or FITC-conjugated C3 antibody (catalogue number orb102204; Biorbyt Corp., Cambridgeshire, UK) for 30 min. Pictures were acquired with a Nikon Eclipse TE2000-S microscope (Nikon, Tokyo, Japan) with magnification ×200. Paraffin-embedded kidney tissue sections were cut at 4 μm, placed on 3-aminopropyltriethoxysilane-pretreated slides. Sections were then deparaffinized and rehydrated according to the standard protocol. For immunohistochemistry analysis, sections were incubated with anti-Hexon (catalogue number LS-C63694; LSBio, Seattle, WA) or anti-FOXO3a (catalogue number 2497; Cell Signaling Technology), followed by the incubation of biotin-labeled secondary antibody and avidin-biotin complex. Peroxidase stain was developed with 3,3′-diaminobenzidine solution and counterstained with hematoxylin. Pictures were acquired with a Nikon Eclipse TE2000-S microscope (Nikon, Tokyo, Japan) with magnification ×200. The nuclear factor-κB (NF-κB) p65 DNA binding assay was performed according to the manufacturer's instructions (catalogue number 10007889; Cayman Chemical Co.). Briefly, 10 μg of nuclear extract, diluted to 100 μl, was added to the wells coated with oligonucleotides containing the NF-κB consensus binding site. For the detection of activated NF-κB, antibodies against the p65 were used, followed by HRP-conjugated secondary antibody. The colorimetric readout (450 nm) was done with an ELISA plate reader (Thermo Scientific). Western blotting assay and coimmunoprecipitation assay were performed as described previously (41Gao B. Wang Y. Xu W. Duan Z. Xiong S. A 5′ extended IFN-stimulating response element is crucial for IFN-γ-induced tripartite motif 22 expression via interaction with IFN regulatory factor-1.J. Immunol. 2010; 185: 2314-2323Crossref PubMed Scopus (19) Google Scholar, 42Gao B. Wang Y. Xu W. Li S. Li Q. Xiong S. Inhibition of histone deacetylase activity suppresses IFN-γ induction of tripartite motif 22 via CHIP-mediated proteasomal degradation of IRF-1.J. Immunol. 2013; 191: 464-471Crossref PubMed Scopus (23) Google Scholar). Antibodies against β-actin (catalogue number sc-130300), tubulin (catalogue number sc-9104), and lamin B (catalogue number sc-374015) were obtained from Santa Cruz Biotechnology. Antibodies against p-FOXO3a (catalogue number 13129), FOXO3a (catalogue number 2497), p-NF-κB p65 (catalogue number 3033), NF-κB p65 (catalogue number 4765), p-IκBα (catalogue number 2859), IκBα (catalogue number 4812), p-Akt (catalogue number 9721), Akt (catalogue number 9272), p-JNK (catalogue number 9251), p-ERK1/2 (catalogue number 9101), and p-p38 (catalogue number 9211) were from Cell Signaling Technology (Beverly, MA). Experimental data were presented as mean ± S.E. of at least three independent experiments. The Student's t test was used to compare differences between two groups, whereas comparison of multiple groups was performed using analysis of variance with post hoc tests to compare differences between individual groups. Pearson correlation analysis was used to assess the association between GC therapy of SLE and the pro-inflammatory responses. A p value of <0.05 was considered to be statistically significant. Data were entered and analyzed using a statistical software package (SPSS18.0). To investigate the therapeutic effect of GCs on SLE patients, we detected the clinical indicators of SLE patients before and after GC treatment. It was found that after GC treatment, levels of serum anti-dsDNA antibody (Fig. 1A) and urine protein (Fig. 1B) were decreased; the serum C3 level (Fig. 1C) was elevated; and the SLE disease activity index (SLEDAI) (Fig. 1D) was significantly attenuated. We also tested the therapeutic effect of GCs on SLE using a mouse model, which was established by immunized female BALB/c with an autoantigen, activated lymphocyte-derived apoptotic DNA (ALD-DNA), as described previously (30Qiao B. Wu J. Chu Y.W. Wang Y. Wang D.P. Wu H.S. Xiong S. Induction of systemic lupus erythematosus-like syndrome in syngeneic mice by immunization with activated lymphocyte-der

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