Autoantibodies to Estrogen Receptor α in Systemic Sclerosis (SSc) as Pathogenetic Determinants and Markers of Progression

Systemic sclerosis (SSc) is a multisystem autoimmune disease of unknown etiology characterized by inflammation, autoantibody production, and fibrosis. It predominantly affects women, this suggesting that female sex hormones such as estrogens may play a role in disease pathogenesis. However, up to date, the role of estrogens in SSc has been scarcely explored. The activity of estrogens is mediated either by transcription activity of the intracellular estrogen receptors (ER), ERα and ERβ, or by membrane-associated ER. Since the presence of autoantibodies to ERα and their role as estrogen agonists interfering with T lymphocyte homeostasis were demonstrated in other autoimmune diseases, we wanted to ascertain whether anti-ERα antibodies were detectable in sera from patients with SSc. We detected anti-ERα antibody serum immunoreactivity in 42% of patients with SSc (30 out of 71 analyzed). Importantly, a significant association was found between anti-ERα antibody values and key clinical parameters of disease activity and severity. Fittingly, anti-ERα antibody levels were also significantly associated with alterations of immunological features of SSc patients, including increased T cell apoptotic susceptibility and changes in T regulatory cells (Treg) homeostasis. In particular, the percentage of activated Treg (CD4+CD45RA− FoxP3brightCD25bright) was significantly higher in anti-ERα antibody positive patients than in anti-ERα antibody negative patients. Taken together our data clearly indicate that anti-ERα antibodies, probably via the involvement of membrane-associated ER, can represent: i) promising markers for SSc progression but, also, ii) functional modulators of the SSc patients’ immune system.


Introduction
Estrogens are well-known regulators of the immune responses and several lines of evidence support a key role for them in the development or progression of numerous diseases, including autoimmune disorders [1][2][3][4][5]. Estrogens, in particular 17bestradiol, directly modulate the function of immune cells by transcriptional activity of nuclear estrogen receptors (ER), i.e., ERa and ERb [1]. Recently, the expression of functional membrane-associated ERa in different cell types including human lymphocytes has been suggested [6][7][8] and autoantibodies specific to ERa have been detected in sera from patients with systemic lupus erythematosus (SLE) [9]. These anti-ERa antibodies behave as true estrogen agonist and are able to induce cell activation and apoptotic cell death in resting lymphocytes as well as proliferation of anti-CD3 activated T cells. Interestingly, a significant association between anti-ERa antibody titer and disease activity was demonstrated [9]. Similarly to SLE, systemic sclerosis (SSc) is an autoimmune disease characterized by multiorgan involvement and circulating autoantibodies against intracellular antigens [10]. The pathogenesis of SSc is complex and incompletely understood. Immune activation, vascular damage, and connective tissue fibrosis are all known to be important in the development of this disease [11,12]. Overall, a substantial female predominance exists in SSc, with a female-to-male ratio ranging from 3:1 to 14:1 [10], suggesting that female sex hormones such as estrogen may play a role in disease pathogenesis. Nevertheless, only limited information is currently available on the role of estrogens in SSc [13][14][15] and the presence of anti-ERa antibodies has not been explored yet. Therefore, the aim of this study was to evaluate anti-ERa serum immunoreactivity in patients with SSc and to assess the possible relationship between the presence of anti-ERa antibodies and the clinical and immunological features of the disease.

Ethics Statement
This study has been conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all patients and controls, and the study was approved by the Ethical Committee of ''Policlinico Umberto I'', Rome, Italy.

Patients and Biological Samples
We analyzed sera from 71 consecutive patients with SSc ( Table 1). All patients fulfilled the preliminary criteria for SSc as defined by the American College of Rheumatology [16]. SSc was diffuse (dcSSc) in 29 patients and limited (lcSSc) in 42 patients. Disease activity was evaluated using the European Scleroderma Study Group (EScSG) activity index [17]. All 71 patients had antinuclear antibody (ANA) (indirect immunofluorescence on Hep-2000 cells), 24 out of 29 dcSSc patients had anti-topoisomerase I antibodies (anti-Scl70 antibodies) and 17 out of 41 lcSSc patients had anti-centromere (ACA) antibodies (Innogenetics, Gent, Belgium). All patients underwent nailfold capillaroscopy and were divided in three different capillaroscopic patterns: early, active and late [18]. Carbon monoxide diffusion capacity (DLCO) was measured by the single breath method, according to the American Thoracic Society standards [19]. Exclusion criteria were previous or concomitant treatments with immunosuppressive drugs. Twenty-two (31%) patients were on low dose steroids (below 10 mg of prednisone a day) at the time of inclusion in the study. The control group consisted of 90 healthy donors matched for age and sex with the SSc group. For flow cytometry analysis, 34 out of 90 healthy donors were randomly selected as representative of the whole series.

Enzyme-Linked Immunosorbent Assay (ELISA)
We analyzed serum IgG immunoreactivity to ERa by ELISA in patients with SSc and in healthy donors. ELISA was developed as previously described [9]. Briefly, polystyrene plates (Maxisorp, Nunc, Roskilde, Denmark) were coated with the antigen (2 mg/ well ERa, Sigma, St Louis, MO, USA) in 0.05 M NaHCO3 buffer, pH 9.5, and incubated overnight at 4uC. Plates were blocked with 100 ml/well of 3% milk for 1 hour at 37uC. Human sera were diluted 1:100 in phosphate buffered saline (PBS)-Tween (PBST) and 1% milk, 100 ml per well. Peroxidase-conjugated goat anti-human IgG (BioRad, Richmond, CA, USA) were diluted in PBST containing 1% milk (1:3,000) and incubated for 1 hour at room temperature. O-phenylenediamine dihydrochloride (Sigma) was used as a substrate and the optical density (OD) was measured at 490 nm (OD 490 ). Three SD above the mean OD reading in the healthy donors was considered the cutoff level for positive reactions. All assays were performed in quadruplicate. Data are presented as the mean OD corrected for background (wells without coated antigen). The results of unknown samples on the plate were accepted if internal controls (2 serum samples, 1 positive and 1 negative) had an absorbance reading within mean 610% of previous readings. To evaluate the antibody specificity, the serum pool obtained from patients with SSc who were positive for anti-ERa antibodies was diluted 1:100 in PBST and incubated overnight at room temperature in the presence of 40 mg/ml of ERa (Sigma) or, as a negative control, with 40 mg/ml of bovine serum albumin (BSA; Sigma).
Apoptosis was quantified using a FITC-conjugated annexin V and propidium iodide apoptosis detection kit (Marine Biological Laboratory, Woods Hole, MA, USA) according to the manufacturer's protocol. Reported data are referred to annexin V-positive apoptotic cells.
Acquisition was performed on a FACSCalibur flow cytometer (BD Immunocytometry Systems) and data were analyzed using the Cell Quest Pro software (BD Immunocytometry Systems).

Statistical Analyses
The results are expressed as mean 6 standard deviation (SD), unless otherwise indicated. variables, chi-square test was used. Correlations were evaluated by using Spearman's rank correlation test. Linear regression analysis was used to display a best fit line to the data. Statistical analyses were performed using GraphPad Prism, version 5.0 software (GraphPad Software, San Diego, CA, USA). All tests were 2-sided and a P value,0.05 was considered statistically significant.

Serum Anti-ERa Antibodies in Patients with SSc
We detected anti-ERa antibodies in 30/71 (42%) patients with SSc whereas no anti-ERa antibodies were found in sera from healthy donors ( Figure 1A). Preabsorption of the pooled positive sera (from 30 patients with SSc) with ERa completely inhibited the antibody immunoreactivity, thus confirming the specificity of ELISA (data not shown).

Association between Anti-ERa Antibodies and Clinical Variables
In order to evaluate whether the presence of anti-ERa antibodies plays a role in the clinical course of the disease, we divided SSc patients into subgroups according to the presence of serum anti-ERa antibodies and examined the demographic and clinical parameters in each group. Only significant results are reported in Table 2. The presence of serum anti-ERa antibodies was significantly associated with the diffuse form of the disease (P = 0.02), the presence of anti-Scl70 antibodies (P = 0.03), the EScSG activity index (P = 0.001) and the late capillaroscopic pattern (P = 0.04). Spearman's rank analysis also showed a significant positive correlation between the anti-ERa antibody level and the EScSG activity index (Spearman's rho = 0.3, P = 0.003), Figure 1B. No further significant association was found with the demographic and clinical parameters evaluated in the present study nor with the different pharmacological treatments.

Association between Anti-ERa Antibodies and Immunological Parameters
We also evaluated the association between the presence of serum anti-ERa antibodies and different immunological parameters including the peripheral distribution of T lymphocyte subsets (naive, central and effector memory lymphocytes and Treg), the expression of relevant markers associated with cell activation and cell fate (i.e., HLA-DR, CD95, Bcl-2) and the level of ex vivo apoptosis. To this aim, 34 patients, randomly selected as representative of the whole series, were studied. The demographic characteristics and the immunophenotypic profile of this patient population, divided into subgroups according to the presence or absence of serum anti-ERa antibodies (anti-ERa IgG-positive, n = 16 and anti-ERa IgG-negative, n = 18), were shown in Table 3. The proportion of CD4 + CD25 + FoxP3 + Treg cells was lower in anti-ERa antibody positive patients than in anti-ERa antibody negative patients (5.262% and 7.262%, respectively, P,0.05). A negative correlation between anti-ERa antibody levels and frequencies of CD4 + CD25 + FoxP3 + Treg was also found (Spearman's rho = 20.4, P = 0.02; Figure 1C). On the contrary, the proportion of CD4 + CD45RA 2 FoxP3bright CD25 bright aTreg was significantly higher in anti-ERa antibody positive patients than in anti-ERa antibody negative patients (0.8560.3% vs. 0.6360.2%, respectively, P,0.05). Moreover, the Spearman's rank analysis showed a significant positive correlation between the proportion of aTreg and the anti-ERa antibody level (Spearman's rho = 0.5, P = 0.004; Figure 1D). Significant differences between anti-ERa antibody positive and anti-ERa antibody negative patients were also found for CD4 + CD95 + frequencies (55613% vs. 44612%, respectively, P,0.05), ex vivo apoptosis level (1064% vs.

Discussion
In this study, we demonstrated for the first time the presence of serum anti-ERa antibodies in SSc. Anti-ERa antibodies were  n., number. For CD4 + and CD8 + lymphocyte subsets, data were expressed as the percentage of each subset within the CD4 + or CD8 + population considered as 100%.
The naive subset is defined as CD45RA + CD62L + while the remaining cells comprise the memory subsets (CD45RA 2 CD62L + , central memory subset; CD45RA 2 CD62L 2 and CD45RA + CD62L 2 , effector memory subset) [20]. T regulatory cells (Treg) are defined as CD4 + CD25 + FoxP3 + ; activated Treg (aTreg) are defined as CD4 + CD45RA 2 FoxP3 bright CD25 bright [21]. Mean fluorescence intensity (MFI) was calculated as the ratio between Bcl-2 mAb mean and negative control mean. significantly associated with disease activity and were mainly found among patients with the diffuse form of the disease, the Scl70 positivity, and the late capillaroscopy pattern. Anti-ERa antibody positivity was also associated with diverse alterations of immunological features, including increased T cell apoptotic susceptibility and a higher percentage of aTreg. Several lines of evidence suggest a hierarchical predominant role of the T cell-mediated immune response in inducing SSc [11,22,23] and an altered T cell homeostasis has been associated with a more severe disease course and phenotype [24]. Here, we found that anti-ERa antibody levels negatively correlated with the peripheral frequency of Treg. This finding is consistent with the critical role of this cell subset in maintaining self tolerance and preventing autoimmunity [25]. Interestingly, different subsets of Treg, displaying unique functional and homeostatic properties, have been recently described [21]. Notably, in this study, we found a significantly higher level of circulating aTreg in anti-ERa antibody positive SSc patients than in anti-ERa antibody negative patients. In the same vein, Mathian and coworkers [23] found higher levels of aTreg in SSc patients with the diffuse and/or active disease (here demonstrated to be also, in large part, anti-ERa positive) suggesting a compensatory, but inefficient, amplification of regulatory cells in the context of active inflammation. However, one recent report demonstrated contrary results, showing lower aTreg levels in SSc patients than in healthy donors although in this case the variable ''disease form'' (i.e., diffuse and limited) was not considered [26]. Conflicting results have been also reported for other autoimmune diseases such as SLE in which both normal [27] or decreased [21] levels of aTreg have been detected. Variables such as genetic background or different criteria for patient selection (e.g., disease stage, clinical form, duration, and treatment) possibly contribute to these contrasting results impairing a comparative analysis of aTreg in different reports, and further studies are needed to better address the distribution and the role of Treg subpopulations in autoimmune diseases.
Anti-ERa antibody positivity was also significantly associated with T cell apoptosis and apoptotic related molecules. This finding is in line with our previous in vitro data on the ability of the anti-ERa antibodies to induce apoptosis in T lymphocytes from healthy donors [9]. Apoptosis plays an important role in bypassing tolerance to intracellular autoantigens [28]. Increased apoptosis detected in autoimmune diseases, such as SSc [24], may lead to an autoantigen overload, initiating and/or perpetuating an autoimmune response and an autoantibody-mediated tissue damage [28]. Apoptotic T cells can also trigger the release of a set of cytokines, such as TGF-b [29], that play a key role in the pathogenesis of SSc [10]. Moreover, TGF-b induces differentiation and expansion of Treg [30] and therefore it could also have a role in determining the Treg levels in SSc. Finally, beside their effects on the immune system, since it was demonstrated that anti-ERa antibodies can act as estrogen agonists [9], we can not rule out the possibility that these antibodies could also work as pathogenic determinants exerting important modulatory effects, e.g., in the fibrotic progression [13,14].
Several lines of evidence indicate pathogenic homologies between SSc and SLE. We refer in particular to the so-called type 1 interferon signature and the activation of the Toll-like receptor signalling [31]. With this study we provide further evidence for a similarity between SLE and SSc. In fact, serum anti-ERa antibodies were detected in patients with SLE but not in patients with other autoimmune diseases such as rheumatoid arthritis or Behçet's disease [9]. In SSc, as well as in SLE, serum anti-ERa antibody levels were significantly related to disease activity, pointing out to the promising role of these antibodies as prognostic markers.
Overall, our study suggests that future research should be aimed at evaluating the potential therapeutic effectiveness of targeting estrogen and its receptors in SSc.