In systemic lupus erythematosus, TNFα is elevated in the serum and correlates with disease activity and triglyceride levels. The stimuli that drive TNFα in this setting are incompletely understood. This study was designed to evaluate monocyte chromatin at the TNFα locus to identify semi-permanent changes that might play a role in altered expression of TNFα. SLE patients with relatively quiescent disease (mean Physician Global Assessment=0.6) and healthy controls were recruited for this study. TNFα expression was measured by intracellular cytokine staining of different monocyte subsets in patients (n=24) and controls (n=12). Histone acetylation at the TNFα locus was measured by chromatin immunoprecipitation using a normalized quantitative PCR in patients (n=46) and controls (n=24). There were no differences in the overall fractions of cells expressing CD14 in SLE patients compared to controls, however, the fraction of DR+/CD16+ cells expressing CD14 was slightly higher as was true in the monocyte subset defined by DR+/CD11b+. Within the monocyte population defined by physical characteristics and DR+/CD14+, TNFα expressing cells were more frequent in SLE patients compared to controls. Both the fraction of positive cells and the mean fluorescence intensity were higher in patients than controls. Consistent with this was the finding that monocytes from patients had increased TNFα transcripts and more highly acetylated histones at the TNFα locus compared to controls. Furthermore, patients with the highest levels of TNFα histone acetylation were more likely to have had consistently elevated erythrocyte sedimentation rates, and to have required cytotoxic use. Histone acetylation, associated with increased transcriptional competence of TNFα, may play a role in certain inflammatory aspects of the disease.
lupus; epigenetics; histone; TNFalpha
Excess type-I interferons (IFN-I) have been linked to the pathogenesis of systemic lupus erythematosus (SLE). Therapeutic use of IFN-I can trigger the onset of SLE and most lupus patients display upregulation of a group of interferon stimulated genes (ISGs). While this “interferon signature” has been linked with disease activity, kidney involvement, and autoantibody production, the source of IFN-I production in SLE remains unclear. Tetramethylpentadecane (TMPD)-induced lupus is at present the only model of SLE associated with excess IFN-I production and ISG expression. Here we demonstrate that TMPD treatment induces an accumulation of immature Ly6Chi monocytes, which are a major source of IFN-I in this lupus model. Importantly, they were distinct from interferon-producing dendritic cells. The expression of IFN-I and ISGs was rapidly abolished by monocyte depletion whereas systemic ablation of dendritic cells (DCs) had little effect. In addition, there was a striking correlation between the numbers of Ly6Chi monocytes and the production of lupus autoantibodies. Therefore, immature monocytes rather than DCs appear to be the primary source of IFN-I in this model of IFN-I dependent lupus.
autoimmunity; systemic lupus erythematosus; monocytes
To optimise a strategy for identifying gene expression signatures differentiating systemic lupus erythematosus (SLE) and antineutrophil cytoplasmic antibody-associated vasculitis that provide insight into disease pathogenesis and identify biomarkers.
44 vasculitis patients, 13 SLE patients and 25 age and sex-matched controls were enrolled. CD4 and CD8 T cells, B cells, monocytes and neutrophils were isolated from each patient and, together with unseparated peripheral blood mononuclear cells (PBMC), were hybridised to spotted oligonucleotide microarrays.
Using expression data obtained from purified cells a substantial number of differentially expressed genes were identified that were not detectable in the analysis of PBMC. Analysis of purified T cells identified a SLE-associated, CD4 T-cell signature consistent with type 1 interferon signalling driving the generation and survival of tissue homing T cells and thereby contributing to disease pathogenesis. Moreover, hierarchical clustering using expression data from purified monocytes provided significantly improved discrimination between the patient groups than that obtained using PBMC data, presumably because the differentially expressed genes reflect genuine differences in processes underlying disease pathogenesis.
Analysis of leucocyte subsets enabled the identification of gene signatures of both pathogenic relevance and with better disease discrimination than those identified in PBMC. This approach thus provides substantial advantages in the search for diagnostic and prognostic biomarkers in autoimmune disease.
Systemic lupus erythematosus (SLE) is an autoimmune disease with known genetic, epigenetic, and environmental risk factors. To assess the role of DNA methylation in SLE, we collected CD4+ T-cells, CD19+ B-cells, and CD14+ monocytes from 49 SLE patients and 58 controls, and performed genome-wide DNA methylation analysis with Illumina Methylation450 microarrays. We identified 166 CpGs in B-cells, 97 CpGs in monocytes, and 1,033 CpGs in T-cells with highly significant changes in DNA methylation levels (p<1×10−8) among SLE patients. Common to all three cell-types were widespread and severe hypomethylation events near genes involved in interferon signaling (type I). These interferon-related changes were apparent in patients collected during active and quiescent stages of the disease, suggesting that epigenetically-mediated hypersensitivity to interferon persists beyond acute stages of the disease and is independent of circulating interferon levels. This interferon hypersensitivity was apparent in memory, naïve and regulatory T-cells, suggesting that this epigenetic state in lupus patients is established in progenitor cell populations. We also identified a widespread, but lower amplitude shift in methylation in CD4+ T-cells (>16,000 CpGs at FDR<1%) near genes involved in cell division and MAPK signaling. These cell type-specific effects are consistent with disease-specific changes in the composition of the CD4+ population and suggest that shifts in the proportion of CD4+ subtypes can be monitored at CpGs with subtype-specific DNA methylation patterns.
We have analyzed DNA methylation, an epigenetic modification that influences gene expression, in lupus patients and control subjects. Our analysis was run in three different immune cell types, T-cells, B-cells, and monocytes, to discern common epigenetic effects in lupus from cell type-specific effects. We have identified a lupus-related reduction in methylation around genes that respond to interferon, a cytokine that induces inflammation in response to pathogens. This hypomethylation suggests that lupus patients are hypersensitive to interferon, as DNA methylation is typically an inhibitor of gene expression. We also find that this hypersensitivity is preserved in lupus patients beyond active stages of the disease, and this may help explain the chronic, recurrent nature of the disease. In addition, we have identified DNA methylation changes in T-cells that suggest an alteration in the proportions of these cells in lupus patients, which may help explain the disease process.
The transcription factor interferon regulatory factor 5 (IRF5) has been identified as a human systemic lupus erythematosus (SLE) susceptibility gene by numerous joint linkage and genome-wide association studies. Although IRF5 expression is significantly elevated in primary blood cells of SLE patients, it is not yet known how IRF5 contributes to SLE pathogenesis. Recent data from mouse models of lupus indicate a critical role for IRF5 in the production of pathogenic autoantibodies and the expression of Th2 cytokines and type I IFN. In the current study, we examined the mechanism(s) by which loss of Irf5 protects mice from pristane-induced lupus at early time points of disease development. We demonstrate that Irf5 is required for Ly6C(hi) monocyte trafficking to the peritoneal cavity (PC), which is believed to be one of the initial key events leading to lupus pathogenesis in this model. Chemotaxis assays using peritoneal lavage from pristane-injected Irf5+/+ and Irf5−/− littermates support an intrinsic defect in Irf5−/− monocytes. We found the expression of chemokine receptors CXCR4 and CCR2 to be dysregulated on Irf5−/− monocytes and less responsive to their respective ligands, CXCL12 and CCL2. Bone marrow reconstitution experiments further supported an intrinsic defect in Irf5−/− monocytes since Irf5+/+ monocytes were preferentially recruited to the PC in response to pristane. Together, these findings demonstrate an intrinsic role for IRF5 in the response of monocytes to pristane, and their recruitment to the primary site of inflammation that is thought to trigger lupus onset in this experimental model of SLE.
Monocytes in SLE have been described as having aberrant behavior in a number of assays. We examined gene expression and used a genome-wide approach to study the posttranslational histone mark, H4 acetylation, to examine epigenetic changes in SLE monocytes. We compared SLE monocyte gene expression and H4 acetylation with three types of cytokine-treated monocytes to understand which cytokine effects predominated in SLE monocytes. We found that γ-interferon and α-interferon both replicated a broad range of the gene expression changes seen in SLE monocytes. H4 acetylation in SLE monocytes was overall higher than in controls and there was less correlation of H4ac with cytokine-treated cells than when gene expression was compared. A set of chemokine genes had downregulated expression and H4ac. Therefore, there are significant clusters of aberrantly expressed genes in SLE which are strongly associated with altered H4ac, suggesting that these cells have experienced durable changes to their epigenome.
Genetic variants of interferon regulatory factor 5 (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). IRF5 regulates the expression of proinflammatory cytokines and type I interferons (IFN) believed to be involved in SLE pathogenesis. The aim of this study was to determine the activation status of IRF5 by assessing its nuclear localization in immune cells of SLE patients and healthy donors, and to identify SLE triggers of IRF5 activation.
IRF5 nuclear localization in subpopulations of peripheral blood mononuclear cells (PBMC) from 14 genotyped SLE patients and 11 healthy controls was assessed using imaging flow cytometry. IRF5 activation and function were examined after ex vivo stimulation of healthy donor monocytes with SLE serum or components of SLE serum. Cellular localization was determined by ImageStream and cytokine expression by Q-PCR and ELISA.
IRF5 was activated in a cell type-specific manner; monocytes of SLE patients had constitutively elevated levels of nuclear IRF5 compared to NK and T cells. SLE serum was identified as a trigger for IRF5 nuclear accumulation; however, neither IFNα nor SLE immune complexes could induce nuclear localization. Instead, autoantigens comprised of apoptotic/necrotic material triggered IRF5 nuclear accumulation in monocytes. Production of cytokines IFNα, TNFα and IL6 in monocytes stimulated with SLE serum or autoantigens was distinct yet correlated with the kinetics of IRF5 nuclear localization.
This study provides the first formal proof that IRF5 activation is altered in monocytes of SLE patients that is in part contributed by the SLE blood environment.
More than half of systemic lupus erythematosus (SLE) patients show evidence of excess type I interferon (IFN-I) production, a phenotype associated with renal disease and certain autoantibodies. However, detection of IFN-I proteins in serum is unreliable, and the measurement of interferon-stimulated gene (ISG) expression is expensive and time consuming. The aim of this study was to identify a surrogate marker for IFN-I activity in clinical samples for monitoring disease activity and response to therapy.
Monocyte surface expression of Fcγ receptors (FcγRs), chemokine receptors, and activation markers were analyzed with flow cytometry in whole blood from patients with SLE and healthy controls. FcγR expression also was measured in peripheral blood mononuclear cells (PBMCs) from healthy controls cultured with Toll-like receptor (TLR) agonists, cytokines, or serum from SLE patients. Expression of ISGs was analyzed with real-time PCR.
Circulating CD14+ monocytes from SLE patients showed increased surface expression of FcγRI (CD64). The mean fluorescent intensity of CD64 staining correlated highly with the ISG expression (MX1, IFI44, and Ly6E). In vitro, IFN-I as well as TLR7 and TLR9 agonists, induced CD64 expression on monocytes from healthy controls. Exposure of monocytes from healthy controls to SLE sera also upregulated the expression of CD64 in an IFN-I-dependent manner. Decreased CD64 expression was observed concomitant with the reduction of ISG expression after high-dose corticosteroid therapy.
Expression of CD64 on circulating monocytes is IFN-I inducible and highly correlated with ISG expression. Flow-cytometry analysis of CD64 expression on circulating monocytes is a convenient and rapid approach for estimating IFN-I levels in SLE patients.
Patients with systemic lupus erythematosus (SLE) have an increased expression of type I interferon (IFN) regulated genes because of a continuous production of IFN-α. The cellular and molecular background to this IFN-α production has started to be elucidated during the last years, as well as the consequences for the innate and adaptive immune systems. Plasmacytoid dendritic cells (pDC) activated by immune complexes containing nucleic acids secrete type I IFN in SLE. Type I IFN causes differentiation of monocytes to myeloid-derived dendritic cell (mDC) and activation of auto-reactive T and B cells. A new therapeutic option in patients with SLE is, therefore, inhibition of IFN-α, and recent data from a phase I clinical trial suggests that administration of neutralizing monoclonal antibodies against anti-IFN-α can ameliorate disease activity.
The role played by cytokines, other than interferon (IFN)-α, in the differentiation and function of dendritic cells (DCs) in systemic lupus erythematosus (SLE), remains unclear. Serum interleukin-10 (IL-10) levels are generally elevated in SLE patients, which might modulate the differentiation of DCs. In this study, DCs were induced from monocytes either by transendothelial trafficking or by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) + IL-4 + tumor necrosis factor (TNF)-α. Both systems were used to investigate the effects of elevated serum IL-10 level on DC differentiation in SLE patients. The results showed that monocyte-derived DCs induced by either SLE serum or exogenous IL-10 reduced the expression of human leukocyte antigen (HLA)-DR and CD80, decreased IL-12p40 level, and increased IL-10 level, and exhibited an impaired capacity to stimulate allogenic T-cell proliferation. These results indicate that serum IL-10 may be involved in the pathogenesis of SLE by modulating the differentiation and function of DCs.
lupus erythematosus systemic (SLE); interleukin-10 (IL-10); dendritic cells (DCs); differentiation
Both genetic and environmental interactions affect systemic lupus erythematosus (SLE) development and pathogenesis. One known genetic factor associated with lupus is a haplotype of the interferon regulatory factor 5 (IRF5) gene. Analysis of global gene expression microarray data using gene set enrichment analysis identified multiple interferon- and inflammation-related gene sets significantly overrepresented in cells with the risk haplotype. Pathway analysis using expressed genes from the significant gene sets impacted by the IRF5 risk haplotype confirmed significant correlation with the interferon pathway, Toll-like receptor pathway, and the B-cell receptor pathway. SLE patients with the IRF5 risk haplotype have a heightened interferon signature, even in an unstimulated state (P = 0.011), while patients with the IRF5 protective haplotype have a B cell interferon signature similar to that of controls. These results identify multiple genes in functionally significant pathways which are affected by IRF5 genotype. They also establish the IRF5 risk haplotype as a key determinant of not only the interferon response, but also other B-cell pathways involved in SLE.
Exacerbation of disease in systemic lupus erythematosus (SLE) is associated with bacterial infection. In conventional dendritic cells (cDCs), the Toll-like receptor 4 (TLR4) ligand bacterial lipopolysaccharide (LPS) induces interferon (IFN)-β gene expression but does not induce IFN-α. We hypothesized that when cDCs are primed by cytokines, as may frequently be the case in SLE, LPS would then induce the production of IFN-α, a cytokine believed to be important in lupus pathogenesis. Here we show that mouse cDC and human monocytes produce abundant IFN-α following TLR4 engagement if the cells have been pretreated either with IFN-β, or with supernatant from DCs activated by RNA-containing immune complexes from lupus patients. This TLR4-induced IFN-α induction is mediated by both an initial TRIF-dependent pathway and a subsequent MyD88-dependent pathway, in contrast to TLR3-induced IFN-α production which is entirely TRIF-dependent. There is also a distinct requirement for interferon regulatory factors (IRFs), with LPS-induced IFN-α induction being entirely IRF7- and partially IRF5-dependent, in contrast to LPS -induced IFN-β gene induction which is known to be IRF3-dependent but largely IRF7-independent. This data demonstrates a novel pathway for IFN-α production by cDCs and provides one possible explanation for how bacterial infection might precipitate disease flares in SLE.
Dendritic cells; Monocytes; Systemic Lupus Erythematosus; Cytokines; Lipopolysaccharide
Systemic lupus erythematosus (SLE) is a systemic inflammatory disease characterized by autoantibody production and immune complex deposition. Interleukin-10 (IL-10), predominantly an anti-inflammatory cytokine, is paradoxically elevated in SLE patients. We hypothesize that the anti-inflammatory function of IL-10 is impaired in monocytes from SLE patients who are chronically exposed to immune complexes.
CD14+ monocytes were isolated from healthy donors and SLE patients with all experiments done in pairs. Cultured CD14+ cells were treated with heat-aggregated human IgG (HIg 325 μg/ml) in the presence or absence of IL-10 (20 ng/ml). To study gene expression, RNA was extracted 3 hours after treatment. To study cytokine production, supernatants were harvested after 8 hours. To study IL-10 signaling, cell lysates were obtained from CD14+ cells treated with HIg (325 μg/ml) for 1 hour followed by IL-10 (20 ng/ml) treatment for 10 minutes. Western blot was used to assess STAT3 phosphorylation.
SLE monocytes produced more TNFα and IL-6 than control cells when stimulated with HIg. IL-10 had less suppressive effect on HIg-induced TNFα and IL-6 production in SLE monocytes, although IL-10 receptor expression was similar in SLE and control monocytes. HIg suppressed IL-10R expression and altered IL-10 signaling in control monocytes. Like SLE monocytes, IFNα-primed control monocytes stimulated with HIg were also less responsive to IL-10.
HIg and IFNα modulate IL-10 function. In SLE monocytes, which are considered IFNα-primed and chronically exposed to immune complexes, responses to IL-10 are abnormal, limiting the anti-inflammatory effect of this cytokine.
Monocytes in patients with systemic lupus erythematosus (SLE) are hyperstimulatory for T lymphocytes. We previously found that the normal program for expression of a negative costimulatory molecule programmed death ligand-1 (PD-L1) is defective in SLE patients with active disease. Here, we investigated the mechanism for PD-L1 dysregulation on lupus monocytes. We found that PD-L1 expression on cultured SLE monocytes correlated with TNF-α expression. Exogenous TNF-α restored PD-L1 expression on lupus monocytes. Conversely, TGF-β inversely correlated with PD-L1 in SLE and suppressed expression of PD-L1 on healthy monocytes. Therefore, PD-L1 expression in monocytes is regulated by opposing actions of TNF-α and TGF-β. As PD-L1 functions to fine tune lymphocyte activation, dysregulation of cytokines resulting in reduced expression could lead to loss of peripheral T cell tolerance.
There is increased expression of type I interferon (IFN)-regulated proteins in the blood and target tissues of patients with cutaneous lupus erythematosus (CLE) and systemic lupus erythematosus (SLE). Patients with SLE have increased IFN-regulated gene expression pointing towards a possible underlying genetic defect.
We measured expression levels of five type I IFN-regulated genes that are highly expressed in SLE in the peripheral blood of patients with CLE and correlated expression levels with cutaneous disease activity.
Peripheral blood was obtained from 10 healthy controls and 30 patients with CLE, including 8 with concomitant SLE. Total RNA was extracted and reverse transcribed into complimentary DNA. Gene expression levels were measured by real time PCR. Gene expression was normalized to GAPDH, standardized to healthy controls and then summed to calculate an IFN score for each patient. Disease activity was assessed with the Cutaneous Lupus Area and Severity Index (CLASI).
Patients with subacute cutaneous lupus erythematosus (SCLE) and discoid lupus erythematosus (DLE) had elevated IFN scores compared to healthy controls regardless of concomitant SLE (p< 0.01 with SLE and p<0.05 without SLE). There was no difference between patients with tumid lupus erythematosus (TLE) and healthy controls. The IFN score correlated with CLASI scores (Spearman’s Rho (r) = 0.55, p = 0.0017).
Patients with SCLE and DLE have an IFN signature, as seen in SLE. The level of gene expression correlates with cutaneous disease activity. These findings support a shared pathogenesis between SLE and some subtypes of CLE.
Atherosclerotic cardiovascular disease (ASCVD) contributes to morbidity and mortality in systemic lupus erythematosus (SLE). Immunologic derangements may disrupt cholesterol balance in vessel wall monocytes/macrophages and endothelium. We determined whether lupus plasma impacts expression of cholesterol 27-hydroxylase, an anti-atherogenic cholesterol-degrading enzyme that promotes cellular cholesterol efflux, in THP-1 human monocytes and primary human aortic endothelial cells (HAEC). THP-1 monocytes and HAEC were incubated in medium containing SLE patient plasma or apparently healthy control human plasma (CHP). SLE plasma decreased 27-hydroxylase message in THP-1 monocytes by 47 ± 8% (p < 0.008) and in HAEC by 51 ± 5.5% (n = 5, p < 0.001). THP-1 macrophages were incubated in 25% lupus plasma or CHP and cholesterol-loaded (50 µg ml−1 acetylated low density lipoprotein). Lupus plasma more than doubled macrophage foam cell transformation (74 ± 3% vs.35 § 3% for CHP, n = 3, p < 0.001). Impaired cholesterol homeostasis in SLE provides further evidence of immune involvement in atherogenesis. Strategies to inhibit or reverse arterial cholesterol accumulation may benefit SLE patients.
Lupus erythematosus; Systemic; Atherosclerosis; Cholesterol; Macrophage scavenger receptor; Foam cells
To investigate peripheral blood (PB) cell transcript profiles of systemic sclerosis (SSc) and its subtypes in direct comparison with systemic lupus erythematosus (SLE).
We investigated PB cell samples from 74 SSc patients, 21 healthy controls, and 17 SLE patients using Illumina Human Ref-8 BeadChips and quantitative polymerase chain reaction confirmation. None of the study participants were receiving immunosuppressive agents other than low-dose steroids and hydroxychloroquine. In addition to conventional statistical and modular analysis, a composite score for the interferon (IFN)–inducible genes was calculated. Within the group of patients with SSc, the correlation of the IFN score with the serologic and clinical subtypes was investigated, as were single-nucleotide polymorphisms in a selected number of IFN pathway genes.
Many of the most prominently overexpressed genes in SSc and SLE were IFN-inducible genes. Forty-three of 47 overexpressed IFN-inducible genes in SSc (91%) were similarly altered in SLE. The IFN score was highest in the SLE patients, followed by the SSc patients, and then the controls. The difference in IFN score among all 3 groups was statistically significant (P < 0.001 for all 3 comparisons). SSc and SLE PB cell samples showed striking parallels to our previously reported SSc skin transcripts in regard to the IFN-inducible gene expression pattern. In SSc, the presence of antitopoisomerase and anti–U1 RNP antibodies and lymphopenia correlated with the higher IFN scores (P = 0.005, P = 0.001, and P = 0.004, respectively); a missense mutation in IFNAR2 was significantly associated with the IFN score.
SLE and SSc fit within the same spectrum of IFN-mediated diseases. A subset of SSc patients shows a “lupus-like” high IFN-inducible gene expression pattern that correlates with the presence of antitopoisomerase and anti–U1 RNP antibodies.
Using oligonucleotide microarray, many IFN-inducible genes have been found to be highly expressed in peripheral blood mononuclear cells (PBMCs) from most patients with systemic lupus erythematosus (SLE). Among these IFN-inducible genes, IFN-induced protein with tetratricopeptide repeats 4 (IFIT4) is a novel gene whose function is unknown.
In this study we examined the role played by IFIT4 in monocyte differentiation and the correlation between IFIT4 expression and the clinical manifestation of SLE. To this end, we used plasmid transfection, flow cytometry, mixed leucocyte responses, ELISA, quantitative RT-PCR and Western blotting.
We found that both IFIT4 mRNA and protein expression levels were significantly higher in PBMCs and monocytes from SLE patients than in those from healthy control individuals. IFIT4 expression was positively correlated with antinuclear antibodies, anti-double-stranded DNA, and anti-Sm auto-immune antibodies in SLE. Patients with SLE exhibiting higher expression of IFIT4 had a higher prevalence of leucopenia, thrombocytopenia and C3/C4 decrease. IFIT4 protein was localized exclusively to the cytoplasm, and it was significantly upregulated by IFN-α in normal PBMCs. To determine the role played by IFIT4 in monocyte differentiation, the monocytic cell line THP-1 was transfected with pEGFP-IFIT4 expression plasmid and stimulated with granulocyte-macrophage colony-stimulating factor/IL-4 to generate IFIT4-primed dendritic cell-like cells (DCLCs). IFIT4-primed DCLCs acquired morphological characteristics of dendritic cells more quickly, with greater resemblance to dendritic cells, as compared with DCLCs primed with pEGFP-C1 control plasmid trasfection. Furthermore, they exhibited higher expressions of CD40, CD86, CD80, HLA-DR and CD83, along with lower expression of CD14; increased IL-12 secretion; and an increased ability to stimulate T-cell proliferation. In addition, IFIT4-primed DCLCs enhanced IFN-γ secretion (about 2.4-fold) by T cells compared with controls.
Our findings suggest that IFIT4 might play roles in promoting monocyte differentiation into DCLCs and in directing DCLCs to modulate T-helper-1 cell differentiation; these actions might contribute to the autoimmunity and pathogenesis of SLE.
Defects in phagocytosis of apoptotic cells have a role in the pathogenesis of autoimmune diseases. Decrease of phagocytosis of apoptotic cells occurs in systemic lupus erythematosus (SLE). Factors underlying this decrease are, presently, unknown.
To analyse the expression of relevant membrane receptors of monocyte derived macrophages (MDM) from patients with SLE and assess their ability to phagocytose apoptotic cells in comparison with MDM from healthy controls. Additionally, to compare phagocytosis in the presence of SLE sera with that in normal human serum (NHS).
Human peripheral blood monocytes were isolated from patients and controls, and cultured for 7 days to obtain MDM. Membrane expression of CD14, CD18, CD36, and CD51/61 was measured. MDM were incubated with apoptotic Jurkat cells in the presence of NHS or serum from patients with active or inactive disease.
No differences in phagocytosis capacity were found between MDM from patients and controls. Membrane expression of the respective receptors was comparable in patients and controls. However, when MDM from controls were incubated with apoptotic cells in patient serum, phagocytosis was significantly decreased in comparison with incubation in NHS. This effect depended on the patients' disease activity and could be reversed by addition of NHS. Reduced uptake of apoptotic cells was associated with decreased levels of complement C1q, C4, and C3, but not with levels of complement factor B.
Reduced uptake of apoptotic cells by MDM from patients with SLE is not an intrinsic defect but is serum dependent and associated with decreased levels of C1q, C4, and C3.
systemic lupus erythematosus; apoptotic cells; phagocytosis; complement
The purpose of this investigation was to assess the correlation of two biomarkers with the occurrence of renal flares in systemic lupus erythematosus (SLE). Urine levels of monocyte chemotactic protein-1 (MCP-1) and transforming growth factor beta (TGF-β) were measured at baseline, and at two and four months in five groups of patients: 25 lupus nephritis patients with active disease (active LN), 10 lupus nephritis patients with SLE in remission (remission LN), 25 patients with clinical active SLE and without nephritis (active NLN), 10 patients without nephritis with SLE in remission (remission NLN) and 10 healthy controls. We used repeated measurement and ANOVA with Duncan's post hoc to analyze the data; the urine level of the two proteins could distinguish the groups based on the existence of lupus nephritis and/or activity of SLE disease. Furthermore we performed receiver operating curve analysis to identify a cutoff point with a good sensitivity and specificity to diagnose lupus nephritis with either one of the urine proteins. Finally the samples from active LN were grouped according to whether they were Class IV or other classes. Baseline urinary MCP-1, but not TGF-β, was significantly different between the classes. Further investigation into the use of these cytokines in a prospective study is needed to determine their capacity as diagnostic tools for renal flares.
Lupus nephritis; monocyte chemotactic protein-1; systemic lupus erythematosus; transforming growth factor beta
Systemic lupus erythematosus (SLE) is a prototypic autoimmune inflammatory disease characterized by the production of autoantibodies directed against nuclear antigens such as nucleosomes, DNA and histone proteins found within the body’s cells and plasma. Autoantibodies may induce disease by forming immune complexes that lodge in target organs or by crossreacting with targeted antigens and damaging tissue. In addition to autoantibody production, apoptotic defects and impaired removal of apoptotic cells contribute to an overload of autoantigens that initiate an autoimmune response. Besides the well-recognized genetic susceptibility to SLE, environmental and epigenetic factors play a crucial role in disease pathogenesis as evidenced by monozygotic twins typically being discordant for disease. Changes in DNA methylation and histone acetylation alter gene expression and are thought to contribute to the epigenetic deregulation in disease. In SLE, global and gene-specific DNA methylation changes have been demonstrated to occur. Additionally, aberrant histone acetylation is evident in individuals with SLE. Moreover, histone deacetylase inhibitors (HDACi) have been shown to reverse the skewed expression of multiple genes involved in SLE. In this review, we discuss the implications of epigenetic alterations in the development and progression of SLE, and how therapeutics designed to alter histone acetylation status may constitute a promising avenue to target disease.
Systemic lupus erythematosus (SLE) is a complex autoimmune disease involving multiple organs. The disease is characterized by the production of pathogenic autoantibodies to DNA and certain nuclear antigens, chronic inflammation, and immune dysregulation. Genetic studies involving SLE patients and mouse models have indicated that multiple lupus susceptible genes contribute to the disease phenotype. Notably, the development of SLE in patients and in certain mouse models exhibits a strong sex bias. In addition, several lines of evidence indicates that activation of interferon-α (IFN-α) signaling in immune cells and alterations in the expression of certain immunomodulatory cytokines contribute to lupus pathogenesis. Studies have implicated factors, such as the X chromosomal gene dosage effect and the sex hormones, in gender bias in SLE. However, the molecular mechanisms remain unclear. Additionally, it remains unclear whether these factors influence the “IFN-signature,” which is associated with SLE. In this regard, a mutually positive regulatory feedback loop between IFNs and estrogen receptor-α (ERα) has been identified in immune cells. Moreover, studies indicate that the expression of certain IFN-inducible p200-family proteins that act as innate immune sensors for cytosolic DNA is differentially regulated by sex hormones. In this review, we discuss how the modulation of the expression of the p200-family proteins in immune cells by sex hormones and IFNs contributes to sex bias in SLE. An improved understanding of the regulation and roles of the p200-family proteins in immune cells is critical to understand lupus pathogenesis as well as response (or the lack of it) to various therapies.
Studies in human systemic lupus erythematosus (SLE) suggest a possible role for histone deacetylases (HDACs) in skewed gene expression and disease pathogenesis. We used the MRL-lpr/lpr murine model of lupus to demonstrate that HDACs play a key role in the heightened levels of both Th1 and Th2 cytokine expression that contribute to disease. The availability of specific HDAC inhibitors (HDIs) such as trichostatin A (TSA) and suberonylanilide hydroxamic acid (SAHA) permits the study of the role of HDACs in gene regulation. Our results indicate that HDIs downregulate IL-12, IFN-γ, IL-6, and IL-10 mRNA and protein levels in MRL-lpr/lpr splenocytes. This effect on gene transcription is associated with an increased accumulation of acetylated histones H3 and H4 in total cellular chromatin. To elucidate the in vivo effects of TSA on lupuslike disease, we treated MRL-lpr/lpr mice with TSA (0.5 mg/kg/d) for 5 weeks. Compared with vehicle-treated control mice, TSA-treated mice exhibited a significant reduction in proteinuria, glomerulonephritis, and spleen weight. Taken together, these findings suggest that increased expression of HDACs leading to an altered state of histone acetylation may be of pathologic significance in MRL-lpr/lpr mice. In addition, TSA or other HDIs may have therapeutic benefit in the treatment of SLE.
OBJECTIVE—Cells of the myeloid lineage comprise a very heterogeneous population with many phenotypes and functional activities including macrophages and dendritic cells. To investigate the status, differentiative potential and lineage commitment of monocytic cells in systemic lupus erythematosus (SLE) patients, this study isolated and cultured peripheral blood monocytes from patients and healthy donors.
METHODS—Monocytes were isolated by gradient centrifugation and adherence to plastic dishes. The cells were then cultured for three days, partially supplemented with GM-CSF and interleukin 4 (IL4) to obtain dendritic cells. The differentiation status was monitored by the expression of surface markers using flow cytometry and cytokine secretion.
RESULTS—Monocytes from SLE patients expressed significantly lower numbers of the monocytic marker CD14 and HLA-DR while secreting significantly more tumour necrosis factor α (TNFα) than monocytes from healthy donors. The addition of GM-CSF and IL4 resulted in an inhibition of TNFα secretion, but was not sufficient to generate monocytederived dendritic cells.
CONCLUSION—Monocytes from SLE patients are severely altered in phenotype and function and have a limited differentiation flexibility towards the accessory type of monocytic cells.
Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by various clinical manifestations. Several cytokines interact and play pathological roles in SLE, although the etiopathology is still obscure. In the present study we investigated the network of immune response-related molecules expressed in the peripheral blood of SLE patients, and the effects of cytokine interactions on the regulation of these molecules.
Gene expression profiles of peripheral blood from SLE patients and from healthy women were analyzed using DNA microarray analysis. Differentially expressed genes classified into the immune response category were selected and analyzed using bioinformatics tools. Since interactions among TNF, IFNγ, β-estradiol (E2), and IFNα may regulate the expression of interferon-inducible (IFI) genes, stimulating and co-stimulating experiments were carried out on peripheral blood mononuclear cells followed by analysis using quantitative RT-PCR.
Thirty-eight downregulated genes and 68 upregulated genes were identified in the functional category of immune response. Overexpressed IFI genes were confirmed in SLE patient peripheral bloods. Using network-based analysis on these genes, several networks including cytokines – such as TNF and IFNγ – and E2 were constructed. TNF-regulated genes were dominant in these networks, but in vitro TNF stimulation on peripheral blood mononuclear cells showed no differences in the above gene expressions between SLE and healthy individuals. Co-stimulating with IFNα and one of TNF, IFNγ, or E2 revealed that TNF has repressive effects while IFNγ essentially has synergistic effects on IFI gene expressions in vitro. E2 showed variable effects on IFI gene expressions among three individuals.
TNF may repress the abnormal regulation by IFNα in SLE while IFNγ may have a synergistic effect. Interactions between IFNα and one of TNF, IFNγ, or E2 appear to be involved in the pathogenesis of SLE.