Low expression of estrogen receptor β in T lymphocytes and high serum levels of anti-estrogen receptor α antibodies impact disease activity in female patients with systemic lupus erythematosus

Abstract

Background: Current evidence indicates that estrogens, in particular 17β-estradiol (E2), play a crucial role in the gender bias of autoimmune diseases although the underlying molecular mechanisms have not yet been fully elucidated. Immune cells have estrogen receptors (ERs), i.e., ERα and ERβ, that play pro- and anti-inflammatory functions, respectively, and the presence of one estrogen receptor (ER) subtype over the other might change estrogen effects, promoting or dampening inflammation. In this study, we contributed to define the influences of E2 on T cells from female patients with systemic lupus erythematosus (SLE), a representative autoimmune disease characterized by a higher prevalence in women than in men (female/male ratio 9:1). Particularly, our aim was to evaluate whether alterations of ERα and ERβ expression in T cells from female SLE patients may impact lymphocyte sensitivity to E2 and anti-ERα antibody (anti-ERα Ab) stimulation interfering with cell signaling and display a direct clinical effect.

Methods: Sixty-one premenopausal female patients with SLE and 40 age-matched healthy donors were recruited. Patients were divided into two groups based on the SLE Disease Activity Index 2000 (SLEDAI-2K) (i.e., <6 and ≥6). ER expression was evaluated in T lymphocytes by flow cytometry, immunofluorescence, and Western blot analyses. Serum anti-ERα Ab levels were analyzed by enzyme-linked immunosorbent assay (ELISA). ER-dependent signaling pathways were measured by a phosphoprotein detection kit.

Results: Intracellular ERβ expression was significantly lower in T cells from patients with SLEDAI-2K ≥6 as compared with healthy donors and patients with SLEDAI-2K <6 and negatively correlated with disease activity. The expression of intracellular and membrane-associated-ERα was similar in SLE and control T cells. ER-dependent signaling pathways were activated in T cells from SLE patients with SLEDAI-2K ≥6, but not with SLEDAI-2K <6, when both membrane and intracellular ERs were stimulated by co-treatment with E2 and anti-ERα Abs.

Conclusions: Our results demonstrate an altered ER profile in SLE patients, possibly contributing to SLE pathogenesis and interfering with clinical activity, and highlight the potential exploitation of T cell-associated ERβ as a biomarker of disease activity.

Keywords: Anti-ERα antibodies; Estrogen; Estrogen receptor; Gender; Immunity; Systemic lupus erythematosus; T lymphocytes.

Fig. 1

Evaluation of intracellular ER expression levels in T lymphocytes from SLE patients and healthy controls. a Intracellular ERα and b intracellular ERβ expression levels were evaluated by flow cytometry. Values of ER/isotype control mean fluorescence intensity ratio (rMFI) are reported, and data are represented as box plots displaying medians, 25th and 75th percentiles as boxes, and the lowest and highest values as whiskers. Statistical differences were calculated by the Mann-Whitney U test. Correlations of intracellular ERα and ERβ expression levels with the SLEDAI-2K score are also shown. The Spearman’s rho (R) and p values were determined using the Spearman’s rank correlation analysis. Solid lines represent best fits as estimated by linear regression analysis. c Immunofluorescence analysis of intracellular ERα (left panels) and ERβ (right panels) expression (green). Representative images of T lymphocytes from the studied populations are shown. Cell nuclei were stained with Hoechst 33342 in blue. Magnification, ×2200. d ERα/ERβ ratio and its correlation with the SLEDAI-2K score are shown. Data are represented and analyzed as described above. ctrs healthy controls, iER intracellular ER, SLEDAI-2K Systemic Lupus Erythematosus Disease Activity Index 2000

Fig. 2

Flow cytometry immunophenotyping of DPN-treated T lymphocytes. Flow cytometry analysis of T cell activation markers was carried out in CD4⁺ and CD8⁺ T lymphocytes from randomly selected SLE patients with SLEDAI-2K <6 and ≥6 (n = 5 for group), arbitrarily chosen as representative of the whole series, and treated with DPN (10 nM) for 48 h. Data are represented as box plots displaying medians, 25th and 75th percentiles as boxes, and the lowest and highest values as whiskers. *p < 0.05 versus untreated cells

Fig. 3

Membrane-associated ERα expression levels and anti-ERα Ab levels. a Membrane-associated ERα expression levels were evaluated by flow cytometry. Values of mERα/isotype control mean fluorescence intensity ratio (rMFI) are reported, and data are represented as box plots displaying medians, 25th and 75th percentiles as boxes, and the lowest and highest values as whiskers. b Anti-ERα Abs (median with range) in sera from patients with SLE and healthy controls. Samples were considered positive if the OD at 490 nm was higher than the cutoff value of an OD at 490 nm of 0.2 (broken line). The cutoff value was defined as 3 SD above the mean OD at 490 nm in healthy controls. Circles represent individual samples. c Correlation of anti-ERα Ab levels with the SLEDAI-2K score. The Spearman’s rho (R) and p values were determined using the Spearman’s rank correlation analysis. Solid lines represent best fits as estimated by linear regression analysis. ctrs healthy controls, mERα membrane-associated ERα, OD optical density, SLEDAI-2K Systemic Lupus Erythematosus Disease Activity Index 2000

Fig. 4

Analysis of ER-dependent signaling pathways in T cells from SLE patients. a–d A panel of phosphoproteins was measured using the Bio-Plex Multiplex Phosphoprotein Assay in T cell lysates from randomly selected SLE patients with SLEDAI-2K <6 and ≥6 (n = 5 for group), arbitrarily chosen as representative of the whole series, after cell treatment with IVIG, E2, anti-ERα Abs, and E2 plus anti-ERα Abs (see the “Methods” section for details). The fluorescence intensity (FI) of a phospho (p)-ERK, b p-JNK, c p-Akt, and d p-NF-kB was measured and data are presented as mean ± SD. *p < 0.01 versus untreated cells. e Activation of ER with the abovementioned treatments was antagonized by addition of 10-fold molar excess of the high-affinity estrogen receptor antagonist ICI 182,780 in T cells from patients with SLEDAI-2K scores ≥6. Data are presented as mean ± SD. ^# p < 0.05 versus E2 plus anti-ERα Abs