Function of Treg Cells Decreased in Patients With Systemic Lupus Erythematosus Due To the Effect of Prolactin

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INTRODUCTION
S ystemic lupus erythematosus (SLE) is an autoimmune rheumatic disease characterized by widespread inflammation, alteration in T cell activation, and overproduction of autoantibodies. This disease is most commonly observed in women. The course of the disease is characterized by remissions and exacerbation. The exacerbation of the disease has been linked to the activity of the immune system. 1 Autoreactive T cells assist autoreactive B cells and infiltrate into the target organs to promote inflammation via cytokine secretion, which causes damage. Thus, autoreactive T cells are key players in the pathogenesis of SLE. 2 Hyperprolactinemia has been reported in several autoimmune diseases, including SLE. [3][4][5][6] Prolactin (PRL) can be synthesized in an extra-pituitary fashion by cells from the immune system, such as B and T cells, which also express the PRL receptor. 7,8 During an immune response, PRL promotes the proliferation, growth, activation, and differentiation of T cells 9,10 and intervenes in the expression of CD69 and CD154 by CD4 þ T cells. 11 In human CD4 þ T cell cultures activated with phorbol myristate acetate and subjected to PRL blockade by using an anti-PRL antibody, IL2 and IFNg secretion is decreased, indicating a role for PRL in the regulation of cytokine secretion. 12 Furthermore, PRL can decrease the function of regulatory T (T reg ) cells 13 in healthy individuals. These studies show the importance of PRL in the regulation of the immune system.
The pathogenesis of SLE involves complex interactions between genetic and environmental factors and the adaptive and innate immune systems. The breakdown of immunologic selftolerance results in the development of autoimmune diseases. 14,15 Other alterations could also be involved in regulating the immune response mediated by T reg cells. There are 2 types of T reg cells: natural T reg cells, which are generated in the thymus, and inducible T reg cells, which are generated in peripheral sites. Both cells exhibit the same CD4 þ CD25 hi C-CD127 low/À FoxP3 þ phenotype. 16,17 T reg cells exert an inhibitory effect on CD4 þ CD25 À CD127 þ conventional or effector T (T eff ) cells. 18 A numerical defect in T reg cells has been observed in autoimmune pathologies such as thyroiditis 19 and diabetes, 20 whereas in SLE, decreased [21][22][23][24][25][26] as well as normal [27][28][29][30] T reg cell numbers have been reported. Moreover, in SLE patients, conventional T cells exhibit reduced sensitivity to T reg cell inhibition. 22,31,32 The objective of our work was to determine whether PRL participates in the regulation of the immune response mediated by T reg cells in patients with SLE. We found that both percentage and function of T reg (CD4 þ CD25 hi CD127 À/low FoxP3 þ ) cells were decreased in SLE patients compared to healthy individuals. The expression of PRL receptor was found to be constitutive in both T reg and T eff cells in patients with SLE and this expression was increased compared to that in healthy individuals. PRL receptor expression varied among SLE patients; in inactive patients, the expression of the receptor was higher in T reg cells compared to T eff cells, similar to what was observed in healthy individuals. However, there was no difference in the expression of the receptor between T reg and T eff cells among active SLE patients. We also found that PRL affects the function of T reg cells. The addition of prolactin to T reg :T eff cocultures decreased the suppressor effect in T reg cells and increased IFNg secretion. These results suggest that PRL increases IFNg secretion, favoring an inflammatory environment, and decreases the suppressor function of T reg cells; this, in addition to the decrease in the number of T reg cells, contributes to the expansion of autoreactive lymphocytes, favoring disease activation.

Study Group
The Ethics Committee of Human Research of the Instituto Mexicano del Seguro Social (IMSS) and the Ethics and Research Committees of the Hospital General de México approved this study (2009-785-028). It was conducted according to the Declaration of Helsinki. Informed consent was obtained from all participants. The samples were obtained from 17 healthy women in the reproductive age (18-50 years) without menstrual disorders and with normal levels of serum prolactin (<20 ng/ml). Since T reg is a rare cell population, the cells from 1 patient are inadequate for all experiments; therefore, from a total of 68 patients with SLE (25-50 years of age), we used samples from an average of 13 patients with inactive lupus and 13 patients with active lupus for each experiment. All patients with SLE fulfilled the American College of Rheumatology (ACR) criteria for SLE. 33 Disease activity was measured by SLEDAI (systemic lupus erythematosus disease activity index). Inactive lupus was considered when the SLEDAI value was equal to 0; lupus was considered to be active when the SLEDAI value was !4. The samples were obtained between 08:00 and 11:00 AM from the cubital vein.

Prolactin
The human PRL used in this study was kindly provided by Dr. A.F. Parlow, from the National Hormone & Pituitary Program (NHPP; Harbor UCLA Medical Center, Los Angeles, CA).

T reg and T eff Cell Purification
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples by density centrifugation using Lymphoprep (Axis Shield, Oslo, Norway). T reg cells were isolated from PBMCs by using a CD4 þ CD25 þ CD127 dim/À Regulatory T cell Isolation Kit II (Miltenyi Biotec, Bergish Gladbach, Germany), according to the manufacturer's instructions. The purity of the cells ranged from 93% to 97% (Supplemental Content 1, http://links.lww.com/MD/A610).

Cell Culture and Proliferation Assays
Cells were cultured in AIM-V liquid medium (Gibco BRL, NY, New York) supplemented with 50 units/ml penicillin and 50 mg/ml streptomycin (Gibco BRL). T reg cells (CD4 þ CD25 hi CD127 low/À ) were plated at a density of 4.0 Â 10 4 cells/well in 96-well U-bottomed plates (Nunc, Roskilde, Denmark) with or without 8.0 Â 10 4 T eff cells (CD4 þ CD25 À CD127 þ ) and cultured in synthetic serum-free medium (AIM-V, Gibco BRL). We standardized the optimum ratio of T reg :T eff cells required to generate a response by using a standard curve illustrating the following ratios: 0.5:1, 1:1, 2:1, and 4:1. The suppressor effect was observed under all conditions; thus, we decided to use a 0.5:1 T reg :T eff cell ratio, on the basis of the percentage of circulating T reg cells and the feasibility of obtaining sufficient quantities for all tests.
T reg Suppression Inspector human (anti-CD2/CD3/CD28 beads; Miltenyi Biotec, Germany) was used for the functional characterization of human T reg cells by in vitro suppression assays in the presence and absence of 50 ng/ml human PRL (NHPP, Los Angeles, CA). The concentrations of Inspector and PRL were obtained using a dose-response curve. Cells were cultured for 5 days, and 1 mCi [ 3 H]-thymidine (Hartmann Analytical, Braunschweig, Germany) was added 18 hours before harvesting. Thymidine incorporation was determined using a liquid scintillation analyzer (Packard 1900 TR, Meriden, Connecticut), and the percentage of proliferation suppression was determined. All conditions were previously standardized and optimized.

Real-Time PCR Assay
Total RNA was extracted from purified T reg and T eff cells by using TRIzol Reagent (Invitrogen), according to the manufacturer's instructions. RNA concentration was determined using UV spectrophotometry, and 1 mg of total RNA was used to generate cDNA with SuperScript II reverse transcriptase (Invitrogen). The PRL receptor and b actin were then amplified by real-time PCR using a LightCycler TaqMan Master kit (Roche Diagnostic, Mannheim, Germany), hydrolysis probes, and primers designed by Roche Diagnostic; all reactions were performed according to the manufacturer's specifications. The primers and probes used are as follows: number 8 probe from the Universal Probe Library for PRL receptor determination, forward primer CTT TCC ACA TGA ACC CTG AAG and reverse primer GCA GAT GCC ACA TTT TCC TT, and number 64 probe from Universal Probe Library for b-actin determination, forward primer CCA ACC GCG AGA AGA TGA and reverse primer CCA GAG GCG TAC AGG GAT AG. Reactions were carried out in a final volume of 10 ml, and a LightCycler 1.5 instrument was used (Roche Diagnostic). The PCR conditions were as follows: 10 minutes at 958C, followed by 45 cycles of 10 seconds at 958C, 30 seconds at 598C, and 1 seconds at 728C, with a final cycle for 30 seconds at 408C. The samples were normalized to b-actin gene expression. The relative expression of PRL and its receptor was calculated using the 2 DCT formula.

Cell Surface Staining and Flow Cytometry
To determine the percentage of peripheral blood T reg cells, PBMCs were incubated with fluorescently labeled antibodies (anti-CD4, CD25, CD127, and PRL receptor or unrelated antibody) for 20 minutes at 48C in staining buffer (phosphate-buffered saline [PBS] with 0.5% bovine serum albumin [BSA] and 0.01% sodium azide). The cells were then washed and fixed in 2% PBS-paraformaldehyde (Sigma Aldrich, St. Louis, MO). Data were obtained using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec, Auburn, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR).

Intracellular Staining for FoxP3
After the superficial staining, the cells were fixed and permeabilized with the Foxp3 Staining Buffer Set (eBioscience) for 18 hours, and stained with fluorescent antibodies. After washing, the stained cells were assayed in a MACSQuant Analyzer 10 flow cytometer and the data were analyzed with FlowJo software.

Statistical Analysis
Statistical analysis was performed using the SPSS package, version 20.0 (SPSS, Inc., Chicago, IL). Normality of the data was checked using the Kolmogorov-Smirnoff test, followed by an analysis using the relevant parametric or nonparametric test. The suppressor function among the groups was assessed using the Kruskal-Wallis test. Comparisons between individual groups were tested using the unpaired Mann-Whitney U or paired Wilcoxon matched-pairs test, at a significance level of P < 0.05.

Percentage of T reg Cells
The percentage of T reg (CD4 þ CD25 hi CD127 low/À FoxP3 þ ) cells was determined based on PBMCs from healthy individuals and SLE patients (active and inactive). We found that the percentage of T reg cells decreased in a statistically significant way (P < 0.001) in patients with active and inactive SLE, compared to healthy individuals (x ¼ 2.95%), but no difference was observed between the inactive (x ¼ 1.67%) and active (x ¼ 1.19%) patients, suggesting that the number of T reg cells is less in SLE patients (active and inactive) (Figure 1).

PRL Receptor Expression in T reg and T eff Cells
Our results showed that T reg cells from SLE patients express the PRL receptor even in absence of stimuli and that both mRNA (relative expression) and protein (FMI ¼ mean fluorescence intensity) expression by T reg cells from active and inactive SLE patients was higher than that in T reg cells from healthy individuals (Table 1). This result showed a statistically significant difference (Figure 2 A and B), although no statistically significant difference were observed between the active and inactive patients. We found that the expression of PRL receptor mRNA and protein in T eff cells from active and inactive SLE patients was higher than that in cells from healthy individuals, with a statistically significant difference ( Figure 2C and D). There was no difference in the expression of PRL receptor between active and inactive patients. Moreover, the expression of PRL receptor was higher in T reg cells compared to T eff cells from patients with inactive SLE, similar to that observed in healthy individuals. However, in patients with active SLE, there was no difference in the expression of the receptor between T reg and T eff cells and the expression of the PRL receptor in T eff cells from patients with SLE was higher than in healthy controls ( Figure 2).

Percentage of Treg
Healthy Inactive SLE Active SLE

PRL Function Regarding T reg Cell-Meditated Regulation
The suppressor capacity of T reg cells stimulated with ''T reg Suppression Inspector human'' (anti-CD2/CD3/CD28 beads) in the presence and absence of PRL was evaluated through in vitro cellular proliferation studies. The proliferation of T eff cells from healthy individuals is shown in Figure 3A. We observed that the addition of PRL did not exert any effect on the proliferation of these cells when T reg cells were added (coculture T reg :T eff ), but the cells exerted suppressor activity over T eff cells by decreasing their proliferation in a significant manner (P ¼ 0.001). The addition of PRL to this coculture interfered with the activity of T reg cells, reestablishing the proliferative capacity of T eff cells to levels similar to that of T eff cells in the absence of T reg cells. PRL did not affect the proliferation of T eff cells from patients with inactive SLE. The suppressor effect exerted by T reg cells over T eff cells was observed in most patients (Supplemental Content 2, http://links.lww.com/MD/A610). However, when considering the entire group, we did not find any statistically significant difference (P ¼ 0.08) in the suppressor effect of T reg cells over T eff cells. Similar to healthy subjects, PRL does not increase the proliferation of T eff cells from inactive SLE patients. In T reg: T eff coculture, the addition of PRL decreased the regulatory effect of T reg cells, thus causing an increase in the proliferation of T eff cells, with a statistically significant difference (P ¼ 0.001; Figure 3B). In contrast, in cells from patients with active SLE, PRL activity increased the proliferation of T eff cells in a statistically significant manner (P ¼ 0.006). The T reg cells from these patients did not have the capacity to exert their suppressor activity over the T eff cells, although the addition of PRL to the T reg :T eff coculture tended to increase the proliferation of T eff cells, with no statistically significant difference (P ¼ 0.06; Figure 3C). This result suggests that the function of T reg cells is no longer adequate under this condition ( Table 2).

Cytokine Secretion by T eff Cells Cultured in the Presence and Absence of PRL
Cytokine concentrations were determined in T eff culture supernatants stimulated with ''T reg Suppression Inspector human'' (anti-CD2/CD3/CD28 beads) in the presence and absence of PRL. As shown in Figure 4A, we observed that IL10 secretion from T eff cells from patients with active or inactive SLE was decreased with respect to that from the cells from healthy individuals, with a statistically significant difference (P ¼ 0.05), but there were no differences between inactive and active patients. The addition of PRL to the T eff cell culture did not modify the secretion pattern of IL10 when using cells from any of the 3 studied groups. No difference in the secretion of IL17A, TNF, or IFNg was observed for the T eff cells from the 3 groups, and the addition of PRL did not affect IL17A and TNF secretion. However, PRL treatment increased IFNg secretion from Te ff from inactive patients, with a statistically significant difference (P ¼ 0.01) ( Figure 4B); meanwhile, in active patients, only an increase was observed, without any statistically significant difference (P ¼ 0.08; Table 3).

Cytokine Secretion by T reg and T eff Cell Coculture in the Presence and Absence of PRL
Cytokine secretion was determined in the presence and absence of PRL by using T reg: T eff cocultures from the 3 groups being studied. The addition of PRL to the T reg: T eff coculture from healthy individuals significantly increased the secretion of IL10, TNF, and IFNg, whereas IL17A secretion was unaffected. Meanwhile, PRL significantly increased IFNg secretion in T reg :T eff cocultures using cells from patients with inactive SLE (P ¼ 0.05) and IL17A secretion increased in most patients. However, we did not find any statistically significant difference (P ¼ .07) in case of the entire group; there was no difference in TNF and IL10 secretion. Cytokine secretion was not affected by the addition of PRL to the cocultures using cells from patients with active SLE ( Figure 5, Table 4).

DISCUSSION
Sex hormones such as PRL play an important role in the modulation of immune response, which depends on the type of cell expressing the PRL receptor. 7,34 Moreover, PRL has an immune-stimulating effect and promotes autoimmunity, 5 interfering with the tolerance of B cells 35 and increasing the production of antibodies. 5, 36 We previously reported that the PRL receptor is constitutively expressed in the T reg cells of healthy individuals (females), whereas the expression increases in T eff cells in response to a stimulus. 13 The results of this study showed that compared to healthy individuals, the expression of PRL receptor was higher in the T reg and T eff cells from patients with SLE (females), with the receptor being expressed even in the absence of a stimulus. This expression tended to increase in cells from active patients compared to that from inactive patients, suggesting higher activity in the disease, along with higher expression of the receptor. Which occurs in B cells from mice that developed lupus (MRL, MRL/lpr), whereby the expression of the receptor increased with the manifestation of the disease. 37,38 In addition, the expression patterns of T eff and T reg cells differed between active and inactive patients. In inactive patients, the expression of the receptor was higher in T reg cells compared to T eff cells, a behavior similar to that observed in healthy individuals. However, there was no difference in the expression of the receptor between T eff and T reg cells from active patients, most likely because the T eff cells were already active, increasing the expression of PRL receptor. This would be similar to the phenomenon in T eff cells from healthy individuals: when activated, the cells increase the expression of the receptor to a level higher than that in T reg cells. 13 T reg cells are a component of one of the peripheral tolerance mechanisms, which fail in autoimmune diseases such as SLE; therefore, these cells are important in the pathogenesis of the disease. 2 However, available data on the number and function of T reg cells in SLE are contradictory, and the definitive role of T reg cells in SLE remains unclear. 29 Therefore, we decided to explore, the percentage of T reg cells in patients with active and inactive SLE, and the role played by PRL in the regulatory function of these cells ex vivo. A statistically significant decrease was found in the percentage of T reg (CD4 þ CD25 hi CD127 À/low FOXP3 þ ) cells from patients with SLE, both active and inactive, compared to that in healthy individuals, supporting the findings of previous studies. 22,24,39,40 Additionally, the suppressor function exerted by T reg cells over T eff cells depends on the stage of the disease. In patients with inactive SLE, we observed 2 behaviors; first, T reg cells did not present any defects in their suppressor activity (majority of the patients), and second, T reg cells did not present a suppressor function in another group of patients (minority of the patients). Although the patients are clinically inactive, their immune system is probably active, and therefore, T reg cells no longer exert their suppressor effect, as observed in active patients where we did not observe T reg suppressor function, as has been reported. The decrease in the number and function of T reg cells in SLE patients favors the activation of autoreactive clones, and thus, disease manifestation. 26,40,41 Because T reg cells from SLE patients express high levels of PRL receptor, we studied whether an interaction with its PRL receptor could affect the suppressor effect of T reg cells, especially those from inactive patients, possessing suppressor function. In these patients, PRL blocked the suppressor effect of T reg cells on T eff cells, a behavior similar to healthy individuals. 13 The loss of suppressor effect cannot be attributed to the notion that PRL increases the proliferation of T eff cells, because the addition of PRL to the T eff cell culture did not increase the proliferation of these cells. It might be due to the presence of proinflammatory cytokines (IFNa, IFNg, and TNF), [42][43][44][45] as their presence in the culture reduces the suppressor effect of T reg cells. It is also known that PRL promotes the secretion of cytokines such as IFNg, IL2, IL12, and TNF. 12,46,47 Our results showed an increase in IFNg levels in the cocultures incubated with PRL (T reg :T eff of inactive patients), and although an increase in IL17 levels was observed in these cultures, it was not statistically significant. The increase in IFNg levels by the addition of PRL was also observed in T eff cell cultures (expressing PRL receptor), which makes us hypothesize that interaction of PRL with its receptor on T eff cells increases IFNg secretion, and that the presence of this cytokine in the culture decreases the suppressor function of T reg cells in patients with inactive SLE, because this cytokine is known to inhibit the generation and/or  function of T reg cells. 44,48,49 It is also possible that IFNg is secreted by T reg cells, as reported in patients with type I diabetes and rheumatoid arthritis, diseases in which T reg cells that secrete proinflammatory cytokines as IFNg and IL17. [50][51][52] Unfortunately because of the low number of T reg cells purified from patients, we could not verify whether PRL favors IFNg secretion in these cells. It will be interesting to show whether PRL favors the presence of T reg IFNg-secreting cells, especially because this has been reported for other autoimmune diseases. [50][51][52] Our results show that both T reg and T eff cells in women with inactive SLE constitutively express the PRL receptor, and therefore, an increase in serum PRL levels will favor the interaction of PRL and its receptor and, in turn, the malfunctioning of the Treg cells, probably because of presence of IFNg. This malfunction, added to the decrease in the cell  number, will contribute to the expansion of autoreactive Tlymphocytes, favoring disease activation. In patients with active SLE, different from those with inactive SLE, PRL increased the cellular proliferation of T eff cells. Thus, PRL in active patients could help in maintaining the disease active by favoring the proliferation of T eff cells among those that are autoreactive. It is worth mentioning that in our study, we did not use antigen-presenting cells (APCs); only T reg cells were coincubated with T eff cells to observe the suppressor effect of T reg cells. Other models using APCs as a suppressor of the function of T reg cells have been reported. In this sense, it has been proposed that the APCs can block T reg cell activity via overproduction of pro-inflammatory cytokines such as IFNa. 42 It would be interesting to determine whether APCs express PRL receptor, and whether PRL favors the secretion of IFNa and other inflammatory cytokines, thereby aiding the malfunction of T reg cells in SLE patients.

CONCLUSIONS
Our results showed that T reg cells from patients with SLE differed from those from healthy individuals with regard to number and function. In inactive patients, PRL acts on T eff cells, which constitutively express the receptor, increasing IFNg secretion and encouraging an inflammatory microenvironment and T reg cell malfunction. The decrease in the number of T reg cells and their malfunction can contribute to the expansion of autoreactive T-lymphocytes to favor disease activation. Additionally, in active patients, PRL increases the proliferation of inspector-stimulated T eff cells, which can further aid the T eff cells to be more resistant to regulation by T reg cells. It will be interesting to study whether PRL decreases the function of different subpopulations of T reg cells and whether this decrease occurs because PRL favors the plasticity of T reg cells toward a Th1 profile.