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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Clin Immunol. Author manuscript; available in PMC Apr 1, 2008.
Published in final edited form as:
PMCID: PMC1905860
NIHMSID: NIHMS20625

The TNFα locus is Altered in Monocytes from Patients with Systemic Lupus Erythematosus

Abstract

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.

Keywords: lupus, epigenetics, histone, TNFalpha

Introduction

TNFα is a proinflammatory cytokine which is produced primarily by monocytes. Other immunologically important producers of TNFα are T cells and dendritic cells. Serum levels of TNFα are often elevated in systemic lupus erythematosus (SLE) patients and higher serum levels are associated with increased disease activity [1; 2; 3; 4; 5; 6]. The role of TNFα in the disease process is controversial. While elevation of serum TNFα is a consistent finding in humans, several murine models suggest that diminished TNFα is associated with an increased risk of disease and/or increased severity of disease [7; 8].

Regardless of the role of TNFα in the etiopathogenesis of SLE, increased expression of TNFα could contribute to certain inflammatory manifestations. For example, MRL/lpr mice massively overexpress TNFα. Treatment of MRL/lpr mice with a TNFα inhibitor ameliorates lung disease and skin disease [9]. Specifically in humans, TNFα is thought to contribute to the dyslipidemia of SLE and glomerular disease [10; 11]. Serum levels of TNFα correlate with serum triglycerides and TNFα is a known inhibitor of lipoprotein lipase, the major enzyme that degrades triglyceride-rich VLDLs [12]. TNFα is overexpressed in the renal tissues of many murine lupus models [13] and also in the renal tissues of human SLE patients [14].

The regulation of TNFα is complex and active disease could stimulate TNFα through many potential mechanisms. TNFα is regulated at the levels of transcription, message turnover, translation, and protein processing. Transcriptional regulation is particularly complex, with tissue-specific regulation, tissue specific repression, and stimulus-specific regulation [15; 16; 17; 18]. A core promoter region is sufficient to confer appropriate basal expression and responses to several stimuli. In addition, monocytes are “readied” for responses to stimuli by altering their chromatin conformation at the TNFα locus [19; 20]. Increased histone acetylation is associated with monocyte maturation, responses to certain priming agents, and increased production of TNFα [20]. Increased histone acetylation is a general feature of increased chromatin accessibility and increased competence for transcription [19; 21]. In general, the epigenetic changes required for competence for gene expression include DNA demethylation, localization into a euchromatin environment, and the establishment of a favorable chromatin environment. Many of these steps are often developmentally regulated such as demethylation of the perforin gene in cells capable of expressing perforin [22], localization of globin genes in euchromatin prior to expression [23], and localization of Th2 cytokine genes into a favorable spatial arrangement [24; 25]. While often related to differentiation, these epigenetic may also be induced via a specific stimulus such as is seen with IL-2 DNA demethylation in T cells after activation [26]. Nevertheless, the more common epigenetic changes that reflect an acute stimulation are histone modifications. These may include acetylation, methylation, ubiquitination and phosphorylation. Each amino acid on the histone tail undergoes characteristic modifications and both the location and the type of modification are recognized by specific binding proteins which modulate transcription. In the case of IL-4, gene expression was closely regulated by a combination of DNA methylation and histone acetylation and individual clones derived from a common progenitor exhibited significant variation in expression, which in turn correlated with levels of histone acetylation and DNA methylation [27]. There are no comparable data for TNFα, and each individual gene is regulated by its own unique combination of epigenetic variables and its own timeframe for epigenetic alterations. In spite of this limitation, there is evidence to suggest that TNFα is substantially regulated at the level of epigenetics [19; 20] and as these chromatin alterations are least partly heritable, can serve to mold the immune system and the response to subsequent stmuli.

This study examined whether patients with SLE exhibited fundamental changes at the TNFα locus which could contribute to its over-expression and perhaps lead to perpetuation of inflammation.

Methods

Patients

This study was approved by the Institutional Review Board and patients and controls consented to participation. Patients were recruited from an ongoing long term SLE cohort (n=46). Patient inclusion in this cohort is based on a clinical diagnosis of SLE [28]. A cumulative SLE database has been constructed for each patient. Patients are seen prospectively every three months after cohort entry. The specific patients in this study had quiescent disease with an average Physician Global Assessment of 0.6 and average ESRs throughout their time in the cohort of 27 mm/h. The average prednisone dose was 3.5 mg/d. Only 8% were positive for La antibodies. Controls (n=24) recruited from the community were required to have no ongoing health problems, nor acute illness, and to not be taking any medications.

Flow cytometry

Flow cytometry was performed utilizing a combination of antibodies to detect monocyte populations with intracellular cytokine staining with anti-TNFα (BD Biosciences, San Jose, CA). Monocytes were initially purified over Ficoll-Paque and subsequent adherence to plastic for 30 minutes. Purity was typically 85–90% with the major contaminant being T cells. On this fresh preparation, viability was always greater than 98%. Where indicated, cells were stimulated for 6 hours with 1μg/ml of LPS in the presence of brefeldin A. Surface staining with CD11b, CD16, CD14, and DR (BD Biosciences) was performed. Intracellular cytokine staining was performed using the reagents and methodology of the manufacturer (BD Biosciences). The FACSCalibur was set to define monocytes through the use of physical parameters and a combination of stains. The settings were conserved from sample to sample to allow comparisons of fluorescence intensity.

Transcription analyses

Chromatin immunoprecipitation (ChIP) assays were performed as per the protocol from Upstate Biotechnology (Lake Placid, NY) with some modifications [20]. Shear size was confirmed by gel analysis. Primer/probe combinations were optimized according to Applied Biosystems recommendations and competition was evaluated via serial titrations. No competition between TNFα primers and control GAPDH primers was seen at any concentration of primer. Antibodies were purchased from Upstate Biotechnology (anti-acetylated H3 and anti-acetylated H4) or Sigma-Aldrich (anti-BSA) (St. Louis, MO).

Real-time PCR was utilized to quantitate the amount of immunoprecipitated DNA from specific regions of the TNFα promoter and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. Each DNA sample was subjected to four PCR reactions for the four regions of the TNFα promoter [20]. The regions detected by each amplimer are the following: TNF1 (+99/−42), TNF2 (+32/−119), TNF3 (−100/−250), TNF4 (−195/−345) and GAPDH (−152/−211). Generally, duplicates or triplicates of samples were run and averaged separately prior to analysis. Known concentrations of genomic DNA were used as standards and positive controls with each Taqman run. Output was normalized to the GAPDH signal in each well according to the manufacturer’s instructions.

Reverse transcription PCR was performed using RNA prepared with RNeasy plus DNase (Qiagen, Valencia, CA), followed by cDNA preparation using the Advantage RT for PCR method (BD Biosciences). Real-time PCR was used to quantitate the specific TNFα cDNA. Primers and probes for the human TNFα target and the 18S internal control were from Applied Biosystems (Foster City, CA).

Statistical analysis

Comparisons of flow cytometry results utilized t-tests, as did the overall comparisons of histone acetylation between controls and patients. Error bars on all graphs indicate Standard Deviation. Oneway ANOVA was used to identify clinical characteristics associated with the patients who exhibited the highest histone acetylation. The study design incorporated these clinical features and thus, correction for multiple comparisons was not performed.

Results

Monocyte characteristics in patients with SLE

We utilized flow cytometry to characterize monocyte subsets and their competence to produce TNFα. Monocyte subsets were identified through surface stains and TNFα was characterized by intracellular staining (Figure 1). The fraction of cells within the DR+/CD16+ subset co-expressing CD14 was similar between patients and controls (p=0.26). There was a trend towards a higher number of CD14+ cells within the DR+/CD11b+ subset although not statistically significant (p=0.08). These data suggest that the monocyte compartment is not grossly altered in SLE.

Figure 1
Comparison of monocyte subsets between patients (n=24) and controls (n=12). A) The surface phenotype of monocytes is not markedly different in patients compared to controls. CD14 cells within populations defined by DR + CD11b and DR + CD16 were enumerated. ...

The monocytes were evaluated for their spontaneous and stimulated TNFα production. The unstimulated cells produced essentially no detectable TNFα in either patients or controls. The fraction of stimulated cells positive for TNFα within the DR+/CD16+ subset trended higher in patients than controls (p=0.17) but the fraction of stimulated cells positive for TNFα within the DR+/CD14+ subset was clearly higher in patients than controls (p=0.02). The mean fluorescence intensity of TNFα was also higher in patients than controls within this conventional subset of monocytes (p=0.03).

Histone acetylation at the TNFα locus

We utilized a ChIP assay to define histone acetylation at the TNFα locus in patients and controls. Purified monocytes were examined unstimulated to identify the resting characteristics of the locus. The TNFα promoter was divided into four regions for evaluation. The TNF1 region brackets the transcription initiation site and TNF4 corresponds to the distal promoter region. Increased histone acetylation is generally associated with increased competence to transcribe genes [21]. Acetylated histone H3 exhibits a characteristic pattern whereby the transcription initiation site (TNF1) is most highly acetylated. There is progressively less histone acetylation with transition to the distal upstream region (TNF4). Both patients and controls have the same pattern and have comparable levels of histone H3 acetylation (Figure 2). There is little variation between individuals with respect to H3 acetylation. Histone H4 acetylation has a different pattern with the highest levels of H4 acetylation in the distal promoter. The pattern is similar between patients and controls, however, there is far more histone H4 acetylation in patients than controls. This is particularly true for the distal TNF3 and TNF4 regions of the promoter (p=0.003 and p=0.009 respectively). There is greater variation among individual patients than in the controls. We analyzed the relationship of the TNF4 H4 acetylation with the production of TNFα in the conventional DR+/CD14+ monocyte population in 16 patients for which both data sets were available from the same specimen and 7 controls. There is a significant relationship between the expression TNFα and TNF4 H4 acetylation (Figure 3).

Figure 2
A) H3 or H4 histone acetylation was measured in resting monocytes by ChIP analysis. Real time PCR was performed using an internal standard and the results are reported as normalized units. DNA quantitation is expressed as the ratio of TNF DNA to GAPDH ...
Figure 3
Higher acetylated H4 levels at the TNF4 region are seen in the patients (n=16, solid circles) with the highest frequency of TNFα producing cells within the CD14/DR positive population (y-axis). Seven controls (grey squares) are included for comparison. ...

H3 and H4 acetylation generally is associated with enhanced competence for transcription. To determine whether the cells were producing increased transcripts, cDNA was quantitated from patient and control monocytes, before and after stimulation. Neither control nor patient cells produced substantial TNFα transcripts in the absence of stimulation. Patient cells produced substantially higher levels of TNFα upon stimulation compared to controls (Figure 4).

Figure 4
SLE patients (n=11) produce higher levels of TNFα message in their peripheral blood monocytes than controls (n=11). Approximately 10 million monocytes were purified by adherence to plastic and either mock stimulated or stimulated with 1μg/ml ...

The H4 acetylation elevated above H3 acetylation at the TNFα locus has not been seen in in vitro model systems. To determine whether a soluble mediator present in the sera of patients with lupus could induce this alteration, twelve ChIP assays were performed on control human donor-derived monocytes incubated either with control sera or sera from lupus patients at 20% for sixteen hours. There were minimal differences between cells incubated with control sera vs. lupus sera, however, the pattern did not replicate that seen in fresh patient monocytes (Figure 5).

Figure 5
Control human monocytes incubated with sera from patients with systemic lupus erythematosus do not have the same pattern of increased H4 acetylation. The primer/probe combinations define the same regions as shown in Figure 2b. There is a trend towards ...

Elevated H4 acetylation and disease features

Seventeen patients with the highest H4 acetylation at the TNF4 region were compared to the other two thirds of patients. The mean H4 acetylation at the TNF4 site in this group was 2.0 with a range of 1.25–5.5. The middle third of patients had a mean H4 acetylation at the TNF4 site of 1.0 with a range of 0.84–1.2. The bottom third of patients had a mean H4 acetylation at the TNF4 site of 0.64 with a range of 0.33–0.8. These compare with the control group which had a mean H4 acetylation at the TNF4 site of 0.75 with a range of 0.3–1.3. Thus, the top one third of patient H4 acetylation levels were higher than the controls. The presence of features commonly associated with lupus were defined (Table 1). Oneway ANOVA was performed to compare the relationship of these features with high H4 acetylation (Table 1). High H4 acetylation was associated with an increased likelihood of cytotoxic drug use, and a higher erythrocyte sedimentation rate (on the day of the sample as well as over the course of the disease). In addition, it was noted that three of the five patients with a history of cerebro-vasclar accident (CVA) had markedly elevated H4 acetylation. Due to the small number of patients, the findings were not statistically significant. The association of the elevated erythrocyte sedimentation rate and H4 acetylation is not surprising. TNFα typically drives the acute phase responses.

Table 1
Clinical Features of Patients with the Highest TNF4 H4 Acetylation

To determine whether medication use could alter the H4 acetylation, we compared current medication use in patients who were studied. There were no differences noted in the current medications (prednisone, non-steroidal anti-inflammatory agents, hydroxychloroquine, or anti-hypertensive agents) when the group with the highest TNF4 H4 acetylation was compared to the rest of the studied patients (Table 1).

Discussion

The rationale for this study was to investigate whether semipermanent changes in monocytes, as a consequence of the disease, could facilitate inflammatory gene expression and perhaps contribute to disease perpetuation. Over-expression of inflammatory genes could contribute to end organ manifestations such as the premature coronary artery disease seen in SLE [10; 29; 30; 31; 32]. This is the first study to specifically examine the chromatin conformation at the TNFα locus in patients with SLE. Epigenetic changes such as histone modifications have recently been demonstrated to predict chemotherapy responses in prostate cancer [33] and to correlate with COPD severity [34], suggesting that epigenetic changes arising during the course of the disease can alter outcome. Perhaps more significantly, interventions directed at histone acetylation have been shown to be beneficial in murine models of inflammation [35; 36] and in human disease [37; 38].

Monocytes in SLE patients have been infrequently evaluated previously. Traditional surface marker expression has not been markedly different in patients and controls in some studies, although receptors for immune complexes are down-regulated, perhaps due to internalization [39; 40; 41]. One study using single color flow cytometry, found decreased CD14 expression and decreased DR expression [41]. Increased monocyte apoptosis has been described and these results may reflect fewer monocyte cells rather than decreased expression of CD14 on an individual monocyte [42]. Interestingly, this same study identified increased spontaneous TNFα production from patients compared to controls [41], which was not seen in the current study. Intracellular flow cytometry may not have been sufficiently sensitive to detect the spontaneous secretion.

The importance of this study is in the identification of a chromatin modification which is associated with increased TNFα transcripts in SLE patients. Although only one stimulus was used, the increased H4 acetylation suggests that responses to multiple stimuli would be increased. This is unlikely to be due to simple accelerated maturation because in previous studies, both the H3 acetylation and H4 acetylation have been increased with maturation [20]. Previous studies have determined that artificial histone acetylation at the TNFα locus is sufficient to increase the cells ability to respond to stimuli but does not lead in and of itself to transcription of TNFα [20]. Thus, we would hypothesize that the high serum levels of TNFα that are seen in SLE patients are due both to an increased competence to produce TNFα and to the presence of a stimulus, either or both of which may be a consequence of the disease process [43]. The immunology of SLE is extremely complex and there is evidence that histone acetylation at certain genetic loci may correlate with biochemical or phenotypic disease features while acetylation of other loci appears to be protective [35; 44]. Thus, the effect of increased acetylation of H4 at the TNFα locus must be placed within the context of a complex network of immunologic relationships.

The finding of increased H4 acetylation specifically at the TNF4 site was unexpected. In previous studies, H3 and H4 acetylation were typically coordinately regulated [20]. The TNF4 site has recently been identified as a potential repressor binding location but has generally been felt to be transcriptionally silent based on transient transfection assays [15]. A known histone acetyltransferase, CBP/p300 has been shown to bind more proximally on the promoter, however, the TNF4 site is not known to bind any recognized histone acetyltransferases [18]. There is the potential for ATF2 to recognize two potential DNA motifs in the TNF4 region. ATF2 is a potent histone acetyltransferase, known to be activated in response to inflammatory stimuli and with a preference for H4 over H3 [45; 46]. Future studies could address the role of ATF2.

This study demonstrates that monocytes from patients with lupus are altered such that they have increased competence to produce TNFα. Increased histone acetylation has been shown to independently [20] increase the competence of the cell to produce TNFα. Patients with a TNFα promoter wrapped around nucleosomes with increased histone acetylation would therefore respond more readily to any stimulus. This quality of the monocytes could predate the disease but is more likely a consequence of the disease and may contribute to the persistence of the disease.

Acknowledgments

The authors would like to thank the patients who participated in this study. This study was supported by R01 AI051323.

Footnotes

This study was supported by R01 AI051323

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