Detection and characterization of IgG-and sIgA-abzymes capable of hydrolyzing histone H1

Immunoglobulins IgG and sIgA actively hydrolyzing histone H1 have been detected on analyzing proteolytic activity of antibodies isolated by chromatography on Protein A-agarose from blood serum of patients with multiple sclerosis and from colostrum of healthy mothers. These antibodies hydrolyze other histones less actively and virtually failed to cleave lysozyme of chicken egg. By gel filtration at acidic pH and subsequent analysis of protease activity of chromatographic fractions, it was shown that IgG and sIgA molecules were responsible for hydrolysis of histone H1. Anti-histone H1 antibodies of IgG and sIgA classes were purified by affinity chromatography on histone H1-Sepharose from catalytically active antibody preparations. The protease activity of anti-histone H1 IgG antibodies was inhibited by serine proteinase inhibitors, whereas anti-histone H1 sIgA antibodies were insensitive to inhibitors of serine, asparagine, and cysteine proteases.     

Autoimmunity and immune system dysregulation in schizophrenia: IgGs from sera of patients hydrolyze myelin basic protein

Several different theories of schizophrenia (SCZ) were discussed; the causes of this disease are not yet clear. Using ELISA, it was shown that titers of autoantibodies against myelin basic protein (MBP) in SCZ patients are ~1.8‐fold higher than in healthy individuals but 5.0‐fold lower than in patients with multiple sclerosis. Several rigid criteria were checked to show that the MBP‐hydrolyzing activity is an intrinsic property of SCZ IgGs. Approximately 82% electrophoretically homogeneous SCZ IgGs purified using several affinity sorbents including Sepharose with immobilized MBP hydrolyze specifically only MBP but not many other tested proteins. The average relative activity of IgGs from patients with negative symptoms was 2.5‐fold higher than that of patients with positive symptoms of SCZ, and it increases with the duration of this pathology. It was shown that abzymes are the earliest statistically significant markers of many autoimmune pathologies. Our findings surmise that the immune systems of individual SCZ patients can generate a variety of anti‐MBP abzymes with different catalytic properties, which can attack MBP of the myelin‐proteolipid shell of axons. Therefore, autoimmune processes together with other mechanisms can play an important role in SCZ pathogenesis. MBP‐hydrolyzing antibodies were previously detected in the blood of 80% to 90% of patients with systemic lupus erythematosus (SLE) and multiple sclerosis (MS). In addition, some similar neuropsychiatric indicators of disease common to SLE, MS, and SCZ were described in the literature. Thus, the destruction of the myelin sheath and the production of MBP‐hydrolyzing antibodies can be a common phenomenon for some different diseases.     

Non‐erythropoietic erythropoietin‐derived peptide protects mice from systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease, which results in various organ pathologies. However, current treatment towards SLE is suboptimal. Erythropoietin (EPO) has been shown to promote SLE recovery, but clinical application can be limited by its haematopoiesis‐stimulating effects. EPO‐derived helix‐B peptide (ARA290) is non‐erythrogenic but has been reported to retain the anti‐inflammatory and tissue‐protective functions of EPO. Therefore, here we investigated the effects and potential mechanisms of ARA290 on SLE. The administration of ARA290 to pristane‐induced SLE and MRL/lpr mice significantly suppressed the level of serum antinuclear autoantibodies (ANAs) and anti‐dsDNA autoantibodies, reduced the deposition of IgG and C3, and ameliorated the nephritis symptoms. Moreover, the serum concentrations of inflammatory cytokine IL‐6, MCP‐1 and TNF‐α in SLE mice were reduced by ARA290. Further, ARA290 decreased the number of apoptotic cells in kidney. In vitro experiment revealed that ARA290 inhibited the inflammatory activation of macrophages and promoted the phagocytotic function of macrophages to apoptotic cells. Finally, ARA290 did not induce haematopoiesis during treatment. In conclusion, ARA290 ameliorated SLE, which at least could be partly due to its anti‐inflammatory and apoptotic cell clearance promoting effects, without stimulating haematopoiesis, suggesting that ARA290 could be a hopeful candidate for SLE treatment.     

Mechanism of action of DNA-hydrolyzing antibodies to DNA from blood of patients with systemic lupus erythematosus

Four fractions of IgG antibodies to native DNA (nDNA) were obtained from blood of patients with systemic lupus erythematosus (SLE). These antibodies displayed a thermostable DNA-hydrolyzing activity and were different in affinity for DNA-cellulose and sorption on DEAE-cellulose. DNA-hydrolyzing antibodies to nDNA are metal-dependent endonucleases, cause mainly single-strand breaks in DNA, and are active over a wide range of pH. By atomic-force microscopy, three-dimensional images of DNA complexes with DNA-hydrolyzing antibodies to nDNA were obtained with nanometer resolution, and a nonprocessive action mechanism was shown for the DNase activity of antibodies to nDNA.     

Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein

Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP‐Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26–27 kDa). Seventy‐two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty‐two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7–9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease‐like and three thiol protease‐like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca²⁺, Mg²⁺_, Mn²⁺, Ni²⁺, Zn²⁺, Cu²⁺, and Co²⁺ was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti‐MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti‐MBP abzymes, which can attack MBP of myelin‐proteolipid sheath of axons and play an important role in MS and SLE pathogenesis. Copyright © 2015 John Wiley & Sons, Ltd.     

Application of protein-A indirect ELISA (PAI-ELISA) for the detection of anti-Smith antibodies in systemic lupus erythematosus patients

An indirect form of protein-A ELISA (PAI-ELISA) was optimized and, when used to detect anti-Smith antibodies in sera of 31 systemic lupus erythematosus (SLE) patients, gave results comparable with those using a commercial immunodiffusion kit. The number of sera found to be positive for anti-Smith antibodies by ELISA was seven, four of which were also found positive by immunodiffusion.B.O. Siti-Rohana is with the Universiti Kebangsaan Malaysia (UKM), Faculty of Medicine, Kuala Lumpur, Malaysia. I.B. Ahmad is with the Department of Microbiology, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, Bangi, 43600 UKM, Malaysia; B.A. Nasaruddin is with the Institute for Medical Research, Malaysia.     

Substrate specificity of IgGs with peroxidase and oxidoreductase activities from sera of patients with systemic lupus erythematosus and multiple sclerosis

The analysis of IgGs to protect humans from oxidative stress through oxidation of harmful compounds was carried out. We have compared here for the first time peroxidase (in the presence of H₂O₂) and oxidoreductase (in the absence of H₂O₂) activities of IgGs from sera of healthy humans and patients with systemic lupus erythematosus (SLE) and multiple sclerosis (MS). In addition, substrate specificity of SLE and MS IgG preparations in the oxidation of different compounds was analyzed: 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), 3,3′‐diaminobenzidine (DAB), homovanillic acid (HVA), o‐phenylenediamine (OPD), α‐naphthol, 3‐amino‐9‐ethylcarbazole (AEC), p‐hydroquinone (pHQ), and adrenaline. IgGs of healthy humans and SLE and MS patients oxidized DAB, ABTS, and OPD due to their peroxidase and oxidoreductase activities, while other compounds were substrates of IgGs only in the presence of H₂O₂: adrenaline was not oxidized by both activities of IgGs. The average SLE IgGs peroxidase activity increased statistically significant in comparison with abzymes from healthy humans in the order (‐fold): OPD (1.2) <  DAB (1.7) < α‐naphtol (2.2) ≤ AEC (2.4) < ABTS (4.5) < 5‐ASA (10.6), while with oxidoreductase activity: OPD (1.8) ≤ DAB (2.1‐fold) < ABTS (5.0). Only HVA was oxidized by IgGs with peroxidase activity of healthy donors faster than by SLE (1.3‐fold) and MS abzymes (2.4‐fold). In the oxidation of several substrates, only three IgGs of MS patients were used. The data speak of a tendency to increase the peroxidase and oxidoreductase activities of MS IgGs in comparison with healthy donors, but to a lesser extent: OPD (1.1 to 1.2‐fold) ≤ ABTS (1.2 to 1.8‐fold). It was shown that development of SLE and MS leads to increase in peroxidase and oxidoreductase activities of IgGs toward most of classical substrates. Thus, abzymes can serve as an additional factor of reactive oxygen species detoxification protecting of patients with SLE and MS from some harmful compounds somewhat better than healthy peoples.     

Affinity and catalytic heterogeneity and metal-dependence of polyclonal myelin basic protein-hydrolyzing IgGs from sera of patients with systemic lupus erythematosus

It was shown using enzyme-linked immunosorbent assay (ELISA) that titers of antibodies against human myelin basic protein (hMBP) in systemic lupus erythematosus (SLE) patients 4.2-fold higher than in healthy individuals, but 2.1-fold lower than in patients with multiple sclerosis (MS). Approximately 86% electrophoretically and immunologically homogeneous SLE immunoglobulin Gs (IgGs) purified using several affinity resins including Sepharose with immobilized hMBP specifically hydrolyze only hMBP but not many other tested proteins. Several rigid criteria were applied to show that the hMBP-hydrolyzing activity is an intrinsic property of SLE IgGs but not from healthy donors. In contrast to MS IgGs, abzymes from SLE patients are more sensitive to ethylenediaminetetraacetic acid and less sensitive to specific inhibitors of serine-like proteases. We present the first evidence demonstrating a significant diversity of different fractions of SLE IgGs in their affinity for hMBP-Sepharose, the ability of IgGs to hydrolyze hMBP at different optimal pHs (5-10) and be activated by different metal ions: Ca(2+) > Mg(2+) ≥ Co(2+) ≥ Fe(2+) ≥ Ni(2+) ≥ Zn(2+) ≥ Cu(2+) ≥ Mn(2+) . Combinations of Ca(2+) + Mg(2+) and Ca(2+) + Co(2) lead to a significant increase in the antibody proteolytic activity as compared with Ca(2+) , Co(2+) , or Mg(2+) ions taken separately. Our findings suggest that the immune systems of individual SLE similar to MS patients can generate a variety of anti-hMBP abzymes with different catalytic properties, which can attack hMBP of myelin-proteolipid shell of axons and play an important role in pathogenesis not only MS but also SLE patients.     

Predictors of clinical outcomes in patients with neuropsychiatric systemic lupus erythematosus

IntroductionNeuropsychiatric systemic lupus erythematosus (NPSLE), a serious organ disorder with a variety of symptoms, has diverse therapeutic outcomes because of the variability of NPSLE manifestations. A comprehensive association study of NPSLE among clinical and immunopathogenic aspects and outcomes has not been conducted.MethodsWe analyzed the laboratory data, NPSLE symptoms, and clinical outcomes at 1 yr post-treatment and the profiles of 27 cytokines, chemokines and growth factors in cerebrospinal fluid (CSF) samples using the Bio-Plex Human 27-plex panel from 28 NPSLE patients. Univariate and multivariable competing risks regression analyses were used to determine the predictive factors of clinical response. We also tried to predict the outcome of NPSLE by the 27 cytokines/chemokines/growth factors using a weighted-voting (WV) algorithm.ResultsOf the two males and 26 females (92.9%), 16 were non-responders at 1 yr post-treatment; in the final model, the independent predictors of non-responders were longer disease durations of SLE (odds ratio [OR]: 1.490, 95% confidence interval [CI]: 1.143–2.461, p = 0.0003) and patients with more than one NPSLE symptom types (OR: 15.14, 95% CI: 1.227–452.1, p = 0.0334). The pretreatment CSF interleukin (IL)-6, IL-10, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) levels were significantly higher in the non-responders (p = 0.0207, p = 0.0054, p = 0.0242 and p = 0.0077, respectively). We identified six “minimum predictive markers:” IL-10, TNF-α, IL-6, IFN-γ, IL-4 and IL-13 by a WV algorithm that showed the highest accuracy (70.83%) and highest Matthews correlation coefficient (54.23%).ConclusionsWe have devised a numerical prediction scoring system that was able to separate the non-responders from responders. The patients with longer disease durations of SLE and those with more than one NPSLE symptom types had poorer outcomes. Our findings may indicate both the importance of making a diagnosis at an earlier phase for better therapeutic response and the usefulness of measuring multiple cytokines to predict NPSLE therapeutic outcomes.     

Affinity and catalytic heterogeneity of polyclonal myelin basic protein‐hydrolyzing IgGs from sera of patients with multiple sclerosis

Human myelin basic protein (hMBP)‐hydrolyzing activity was recently shown to be an intrinsic property of antibodies (Abs) from multiple sclerosis (MS) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from MS patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hBMP at different optimal pHs (3–10.5). IgGs containing λ‐ and κ‐types of light chains demonstrated comparable relative activities in the hydrolysis of hMBP. IgGs of IgG1–IgG4 sub‐classes were analyzed for catalytic activity. IgGs of all four sub‐classes were catalytically active, with their contribution to the total activity of Abzs in the hydrolysis of hMBP and its 19‐mer oligopeptide increasing in the order: IgG1 (1.5–2.1%) < IgG2 (4.9–12.8%) < IgG3 (14.7–25.0%) < IgG4 (71–78%). Our findings suggest that the immune systems of individual MS patients generate a variety of anti‐hMBP abzymes with different catalytic properties, which can attack hMBP of myelin‐proteolipid shell of axons, playing an important role in MS pathogenesis.     

系统性红斑狼疮患者血清中CD83 与自身抗体的表达及其相互关系

目的:检测系统性红斑狼疮(systemic lupus erythematosus,SLE)患者血清中 CD83(soluble CD 83,sCD 83)和多种自身抗体的表达水平,并探讨其相互关系.方法:ELISA 检测患者可溶性 CD 83 和AnuA的表达,应用间接免疫荧光的方法检测抗cmDNA 抗体,应用乳凝法检测血清中的DNP,采用胶体金标记和快速膜渗滤技术测定血清中的抗 dsDNA 抗体.结果:对照组患者血清中可溶性 CD83 的表达为(0.26±0.10)ng/ml,实验组患者血清中可溶性 CD83 的表达为(5.56±0.72)ng/mI.与对照组相比,实验组患者血清中可溶性CD 83的平均浓度明显升高.在抗dsDNA抗体阴性的 51 例系统性红斑狼疮患者中 AnuA 的阳性率明显高于抗DNP 抗体和抗 cmDNA 抗体,同样在抗 DNP 抗体阴性的 58 例系统性红斑狼疮患者中 AnuA 的阳性率明显高于 dsDNA 抗体和抗 cmDNA 抗体.系统性红斑狼疮患者中可溶性 CD83 的水平(<2.68 ng/ml)与各种自身抗体(抗 dsDNA 抗体、AnuA、抗DNP抗体和抗 cmDNA 抗体) 水平的相关系数分别为(r＝0.542,0.613,0.489和0.367).具有高水平可溶性CD83的系统性红斑狼疮患者( ≥2.68 ng/ml),与各种自身抗体(抗dsDNA抗体,AnuA,抗 DNP 抗体和抗cmDNA 抗体)水平的相关系数分别为(r＝0.711,P<0.05)、(r＝0.845,P<0.01)、(r=0.862,P<0.01)和(r=0.724,P<0.051).结论:可溶性CD83通过活化DC细胞并激活补体系统,参与系统性红斑狼疮的发生发展,联合可溶性 CD83 和多种自身抗体的检测,能更明确系统性红斑狼疮患者病情的严重程度,有利于 SLE 的诊断和治疗.     

系统性红斑狼疮患者血清中CD83与自身抗体的表达及其相互关系

目的：检测系统性红斑狼疮（systemic lupus erythematosus,SLE）患者血清中CD83（soluble CD 83,sCD 83）和多种自身抗体的表达水平,并探讨其相互关系。方法：ELISA检测患者可溶性CD 83和AnuA的表达,应用间接免疫荧光的方法检测抗cmDNA抗体,应用乳凝法检测血清中的DNP,采用胶体金标记和快速膜渗滤技术测定血清中的抗dsDNA抗体。结果：对照组患者血清中可溶性CD 83的表达为（0.26±0.10）ng/ml,实验组患者血清中可溶性CD 83的表达为（5.56±0.72）ng/ml。与对照组相比,实验组患者血清中可溶性CD 83的平均浓度明显升高。在抗dsDNA抗体阴性的51例系统性红斑狼疮患者中AnuA的阳性率明显高于抗DNP抗体和抗cmDNA抗体,同样在抗DNP抗体阴性的58例系统性红斑狼疮患者中AnuA的阳性率明显高于dsDNA抗体和抗cmDNA抗体。系统性红斑狼疮患者中可溶性CD83的水平（〈2.68 ng/ml）与各种自身抗体（抗dsDNA抗体、AnuA、抗DNP抗体和抗cmDNA抗体）水平的相关系数分别为（r=0.542,0.613,0.489和0.367）。具有高水平可溶性CD83的系统性红斑狼疮患者（≥2.68 ng/ml）,与各种自身抗体（抗dsDNA抗体,AnuA,抗DNP抗体和抗cmDNA抗体）水平的相关系数分别为（r=0.711,P〈0.05）、（r=0.845,P〈0.01）、（r=0.862,P〈0.01）和（r=0.724,P〈0.051）。结论：可溶性CD83通过活化DC细胞并激活补体系统,参与系统性红斑狼疮的发生发展,联合可溶性CD83和多种自身抗体的检测,能更明确系统性红斑狼疮患者病情的严重程度,有利于SLE的诊断和治疗。     

Elevated levels of serum antibodies against alpha-1, 6-glucan in patients with systemic lupus erythematosus or rheumatoid arthritis

This study was undertaken to investigate whether levels of anti-alpha-1, 6-glucan antibodies in human sera correlate with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Serum samples were collected from patients with SLE (n = 30), RA (n = 30) and healthy adult volunteers. IgG, IgA and IgM levels against alpha-1, 6-glucan were measured using enzyme linked immunosorbent assays. Anti-alpha-1, 6-glucan IgG prevalence was raised in patients with active SLE (73.3%) and RA (60%) compared with healthy controls (13.3%). Strong correlation between anti-alpha-1,6-glucan-IgG levels and anti-perinuclear factor (r = 0.642; p<0.05) in RA patients or anti-nuclear antibodies (r = 0.675; p<0.05) in SLE patients was observed. No significant differences in anti-alpha-1,6-glucan-IgA or-IgM levels were noted between different groups. We conclude that anti-alpha-1,6-glucan-IgG levels were significantly elevated in patients with SLE or RA and positively correlated with disease activity.     

Serum protein oxidation and apolipoprotein CIII levels in people with systemic lupus erythematosus with and without nephritis

Increased oxidative stress is a hallmark of the autoimmune disease systemic lupus erythematosus (SLE). This study compares serum protein oxidation levels in SLE patients without and with renal involvement (lupus nephritis); the latter have a significantly poorer prognosis. Similar increases in protein carbonyls and decreases in protein thiols were observed in both SLE groups compared to controls. Protein carbonyl distribution, determined by Western blotting of 2D gels, was similar in both SLE groups, suggesting factors other than oxidation also play a role in SLE complications. 2D electrophoresis examined the serum proteome further. Six proteins were significantly decreased in non-renal SLE patients compared to controls; five were identified by mass spectrometry, including one isoform of pro-atherogenic apoCIII. Total apoCIII levels (assessed by ELISA) in lupus nephritis patients were significantly elevated compared to controls or non-renal SLE patients. Thus, levels of oxidized proteins and apoCIII may be useful biomarkers in SLE studies.     

Anti-plasminogen autoantibodies from plasma of patients with systemic lupus erythematosus having anti-phospholipid antibody syndrome: isolation and some immunochemical properties

Blood plasma samples from patients with systemic lupus erythematosus having the anti-phospholipid antibody syndrome were found to contain anti-plasminogen antibodies of the IgG class. The titers of anti-plasminogen autoantibodies of the IgG class were elevated in these patients compared with normal controls. Part of the pool of IgG anti-plasminogen antibodies reacts with an epitope in the lysine-binding sites of plasminogen. Anti-plasminogen IgG isolated from patients' blood plasma is specific only for a native epitope of human plasminogen passively adsorbed on immunosorbent micro-titration plate. As shown by enzyme immunoassay, autoantibodies to plasminogen of the IgG class cross-react with human fibrinogen.     

EDTA/gelatin zymography method to identify C1s versus activated MMP‐9 in plasma and immune complexes of patients with systemic lupus erythematosus

Gelatin zymography analysis is a sensitive method and commonly used to characterize and quantify the presence of the gelatinases (MMP‐2 and MMP‐9) in biological samples. In human plasma samples from healthy controls and systemic lupus erythematosus (SLE) patients, we observed a gelatinolytic molecule at 80 kDa, suggestive for activated human MMP‐9. However, by developing and using the EDTA/gelatin zymography method and after purification of the 80 kDa entity, we proved that this molecule was the C1s subunit of the complement system. The zymolytic capacity of C1s was validated and found to be enhanced, in the absence of calcium and in the presence of EDTA. Our findings indicate that for correct identification of gelatinolytic proteins in complex biological samples the use of EDTA/gelatin zymography for enzyme development is advised. In addition, by quantification of EDTA/gelatin zymography analysis and ELISA, we observed that the levels of C1s were higher in plasma and immune complexes of SLE patients than of healthy individuals. Therefore, our data imply that C1s may become a marker for the diagnosis of SLE.     

Association of leptin and leptin receptor gene polymorphisms with systemic lupus erythematosus in a Chinese population

To explore the association of LEP and leptin receptor (LEPR) gene single‐nucleotide polymorphisms (SNPs) with susceptibility to systemic lupus erythematosus (SLE) in a Chinese population. Four LEP SNPs (rs11761556, rs12706832, rs2071045 and rs2167270) and nine LEPR SNPs (rs10749754, rs1137100, rs1137101, rs13306519, rs8179183, rs1805096, rs3790434, rs3806318 and rs7518632) were genotyped in a cohort of 633 patients with SLE and 559 healthy controls. Genotyping of SNPs was performed with improved multiple ligase detection reaction (iMLDR). No significant differences were detected for the distribution of allele and genotype frequencies of all 13 SNPs between patients with SLE and controls. The genotype effects of recessive, dominant and additive models were also analysed, but no significant evidence for association was detected. However, further analysis in patients with SLE showed that the TT genotype and T allele frequencies of the LEP rs2071045 polymorphism were nominally significantly higher in patients with pericarditis (P = 0.012, P = 0.011, respectively). In LEPR, the GA/AA genotype and A allele frequencies of the rs1137100 polymorphism were both nominally associated with photosensitivity in patients with SLE (P = 0.043, P = 0.018, respectively). Moreover, the genotype and allele distribution of rs3806318 were also nominally associated with photosensitivity in patients with SLE (P = 0.013, P = 0.008, respectively). No significant differences in serum leptin levels were observed in patients with SLE with different genotypes. In summary, LEP and LEPR SNPs are not associated with genetic susceptibility to SLE, but may contribute to some specific clinical phenotype of this disease; further studies are necessary to elucidate the exact role of LEP and LEPR genes in the pathogenesis of SLE.     

Interferon regulatory factor 1 and histone H4 acetylation in systemic lupus erythematosus

Histone acetylation modulates gene expression and has been described as increased in systemic lupus erythematosus (SLE). We investigated interferon regulatory factor 1 (IRF1) interactions that influence H4 acetylation (H4ac) in SLE. Intracellular flow cytometry for H4 acetylated lysine (K) 5, K8, K12, and K16 was performed. Histone acetylation was defined in monocytes and T cells from controls and SLE patients. RNA-Seq studies were performed on monocytes to look for an imbalance in histone acetyltransferases and histone deacetylase enzyme expression. Expression levels were validated using real-time quantitative RT-PCR. IRF1 induction of H4ac was evaluated using D54MG cells overexpressing IRF1. IRF1 protein interactions were studied using co-immunoprecipitation assays. IRF1-dependent recruitment of histone acetyltransferases to target genes was examined by ChIP assays using p300 antibody. Flow cytometry data showed significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation in SLE monocytes. HDAC3 and HDAC11 gene expression were decreased in SLE monocytes. PCAF showed significantly higher gene expression in SLE than controls. IRF1-overexpressing D54MG cells were associated with significantly increased H4K5, H4K8, and H4K12 acetylation compared to vector-control D54MG cells both globally and at specific target genes. Co-immunoprecipitation studies using D54MG cells revealed IRF1 protein-protein interactions with PCAF, P300, CBP, GCN5, ATF2, and HDAC3. ChIP experiments demonstrated increased p300 recruitment to known IRF1 targets in D54MG cells overexpressing IRF1. In contrast, p300 binding to IRF1 targets decreased in D54MG cells with IRF1 knockdown. SLE appears to be associated with an imbalance in histone acetyltransferases and histone deacetylase enzymes favoring pathologic H4 acetylation. Furthermore, IRF1 directly interacts with chromatin modifying enzymes, supporting a model where recruitment to specific target genes is mediated in part by IRF1.