A Possible Role of HMGB1 in DNA Demethylation in CD4+ T Cells from Patients with Systemic Lupus Erythematosus

The aberrant activity of CD4+ T cells in patients with systemic lupus erythematosus (SLE) is associated with DNA hypomethylation of the regulatory regions in CD11a and CD70 genes. Our previous studies demonstrated that Gadd45a contributes to the development of SLE by promoting DNA demethylation in CD4+ T cells. In this study, we identified proteins that bind to Gadd45a in CD4+ T cells during SLE flare by using the method of co-immunoprecipitation and mass spectrometry, High mobility group box protein 1 (HMGB1) is one of identified proteins. Furthermore, gene and protein expression of HMGB1 was significantly increased in SLE CD4+ T cells compared to controls, and HMGB1 mRNA was correlated with CD11a and CD70 mRNA. A significant, positive correlation was found between HMGB1 mRNA and SLEDAI for SLE patients. Our data demonstrate that HMGB1 binds to Gadd45a and may be involved in DNA demethylation in CD4+ T cells during lupus flare.


Introduction
Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that affects multiple organ systems. The etiology and pathogenesis of SLE remain to be fully defined. Recent studies have shown that T-cell DNA demethylation plays an important role in the pathogenesis of SLE [1]. We previously demonstrated that inhibition of DNA methylation in T cells resulted in the demethylation of regulatory sequences and increased gene expression of CD11a (ITGAL) and CD70 (TNFSF7) [2][3][4]. Increased expression of these genes results in T-cell autoreactivity and autoantibody production by the B lymphocyte lineage. The molecular mechanisms that mediate gene demethylation in SLE CD4 + T cell are not fully understood, but it is possible that growth arrest and DNA damage-inducible gene (Gadd45a/Gadd45) may contribute since increased expression of Gadd45a gene has been shown to repress DNA methylation, and factors that induce lupus flare such as ultraviolet have been shown to promote DNA demethylation in SLE CD4 + T cells [5][6][7].
In this study, we identified protein(s) that bind to Gadd45a, assessed their gene and protein expression and their association with CD11a, CD70 mRNA, and lupus disease activity. HMGB1 was identified as a major protein that bind to Gadd45a, which may contribute to DNA demethylation.

Patients and Control
Subjects. Ten SLE patients (28.50 ± 10.23 years of age) were recruited from the outpatient clinics and inpatient Department of Dermatology at the Second Xiangya Hospital, Central South University. All patients fulfilled at least 4 of the SLE classification criteria of the American College of Rheumatology [8]. Disease activity was quantified by the SLE Disease Activity Index (SLEDAI) [9], and the mean SLEDAI score of patients recruited into this study was 7.20 ± 1.93 (Table 1). Age-and sex-matched healthy controls (25.80 ± 6.89 years of age) were recruited from the medical staff at the Second Xiangya Hospital. This study   was approved by the human ethics committee of the Second Xiangya Hospital.

Isolation of CD4 + T Cells.
Peripheral blood mononuclear cells were isolated from heparin-anticoagulated whole blood using Ficoll-Hypaque density gradient centrifugation (Shanghai Hengxin Chemical Reagent Co., Ltd., Shanghai, China), and CD4 + T cells were isolated by positive selection using Miltenyi beads, according to the manufacturer's instructions (Miltenyi, Germany). The purity of the enriched subsets was validated by flow cytometry and was >95%.

Coimmunoprecipitation and LS-MS/MS. CD4 + T cells were solubilized with cell lysis buffer (KeyGEN, China).
Aliquots of CD4 + T-cell lysates were incubated overnight at 4 ∘ C with rabbit anti-human Gadd45a antibody (Santa Cruz Biotechnology, USA) or isotype-matched rabbit IgG (Beyotime, China) prior to the absorption with protein A/G PLUS-agarose beads (Millipore, USA) at 4 ∘ C for 1 hour.
Proteins that bound to Gadd45a were centrifuged and eluted. Immunoprecipitated samples were incubated with 2 × SDS electrophoresis sample buffer at 95 ∘ C for 5 min and subjected to SDS-PAGE followed by staining with Coomassie blue. Bands of interest were excised, and sent to BGI (Shenzhen, China) for identification using LS-MS.

Western Blot.
Aliquots of CD4 + T-cell proteins were denatured, separated by SDS-PAGE, and transferred to PVDF membranes (Amersham Bioscience, GE Healthcare Company, USA). The membranes were incubated overnight at 4 ∘ C with rabbit anti-human HMGB1 polyclonal antibody (diluted 1 : 500, Abcam, USA) followed by incubation with horseradish peroxide-conjugated mouse anti-rabbit antibody (diluted 1 : 3000, Santa Cruz, USA). Bands were detected with Pierce chemiluminescence detection system (Thermo Scientic, Rockford, IL, USA). All bands were normalized to -actin.

Identification of Proteins That Bound to Gadd45a.
To identify proteins that bind to Gadd45a, total proteins extracted from CD4 + T cell were immunoprecipitated with anti-human Gadd45a antibodies, separated by SDS-PAGE, and stained with Coomassie blue. Anti-Gadd45a antibodies but not isotype-matched control immunoprecipitated two bands with MW of ∼30 and ∼26 kDa (Figure 1). A protein band with MW ∼55 kDa was detected by both anti-Gadd45a antibodies and control IgG. These bands were excised, subjected to in-gel trypsin digestion, and subjected to Tandem MS. Data obtained were analyzed against the Gene Ontology annotations to proteins in the UniProt Knowledgebase using Mascot 2.3.02 software. (Proteins listed in Table 2 are MW of ∼30 KDa and ∼26 KDa proteins precipitated by anti-Gadd45a antibodies and MW ∼55 KDa proteins which are different proteins between the anti-Gadd45a antibodies precipitation group and control antibody precipitation group.)

Gene and Protein Expression of HMGB1 in CD4 + T Cells from SLE Patients with Active
Disease. HMGB1 was identified as the protein that bound to Gadd45a by immunoprecipitation. Elevated expression was confirmed in PBMC and serum in SLE patients in previous studies [10][11][12]. In addition, it may interact with various proteins, including methy-CpG binding protein2 (MeCP2) [13]. So we compared HMGB1 mRNA levels in CD4 + T cells from SLE patients and healthy controls. Gene and protein expression of HMGB1 was significantly increased in SLE CD4 + T cells compared to CD4 + T cells isolated from healthy subjects ( = 0.01 and = 0.018, resp.) (Figures 2, 3(a), and 3(b)).

Discussion
Over the past two decades, global DNA hypomethylation and gene-specific DNA demethylation have been observed in T cells in patients with idiopathic lupus and drug-induced lupus. Gadd45a (Gadd45a/Gadd45) is a nuclear protein that plays an important role in the maintenance of genomic stability, DNA repair, and suppression of cell growth. In our previous studies, we have demonstrated that Gadd45a contributes to autoimmune diseases by promoting DNA demethylation [5]. In this study, we identified proteins that bind to Gadd45a in SLE CD4 + T cells that may assist in DNA demethylation. Using LS-MS/MS, we identified HMGB1 as one of the proteins that bind to Gadd45a. Gene and protein expression of HMGB1 was increased in SLE CD4 + T cells compared to control CD4 + T cells. HMGB1 is a nuclear DNA binding protein secreted by monocytes and macrophages and plays a critical role in innate immunity, inflammation, and sterile injury [14]. Serum HMGB1 levels are increased in SLE patients and its expression is increased in skin lesions in lupus patients [10][11][12]15].
HMGB1 interacts with numerous proteins such as methyl-CpG binding protein-2 (MeCP2), a protein that participates in the recognition of methylated DNA in CpG islands [13]. In this study, we demonstrated that gene and protein expression of HMGB1 in CD4 + T cells is significantly increased in SLE patients compared to healthy controls thereby corroborating previous studies. Furthermore, HMGB1 mRNA in CD4 + T cells is correlated with SLEDAI for SLE patients. Given that CD4 + T cells promote inflammation and autoimmunity, it is plausible to suggest that HMGB1 contributes to the pathogenesis of SLE [16].
CD11a plays a key role in cellular adhesion and costimulatory signaling. Its expression is increased in T cells consequent to hypomethylation and correlates with the development of T-cell autoreactivity in SLE patients [3,4]. CD70 is a methylation-sensitive gene present in T cells. Costimulatory signals mediated through CD70-CD27 interactions regulate   study, we demonstrated that HMGB1 mRNA is significantly correlated with CD11a and CD70 mRNA.
In conclusion, we have demonstrated that HMGB1 binds to Gadd45a and its increased gene expression which is associated with a concomitant increase in CD11a and CD70 mRNA may be involved in DNA demethylation in CD4 + T cells, thus contributing to the pathogenesis of SLE. Further studies are currently ongoing to delineate the function of HMGB1 and its underlying mechanism contributing to DNA demethylation in CD4 + T cells during pathogenesis of SLE.