Heren, we analyzed Treg cells as potential biomarkers of disease activity in systemic lupus erythematosus (SLE) patients. Peripheral blood mononuclear cells from 30 SLE patients (15 active: SLEDAI > 6/15 SLE remission: SLEDAI< 6) and 15 healthy volunteers were purified. Treg immunophenotyping was performed using CD4, CD25, CD45, CD127, and FOXP3 markers. CD4+FOXP3+ Treg activation state was investigated based on CD45RA and FOXP3 expression. To increase the accuracy of our findings, a multivariate linear regression was performed. We showed a significant increase in the frequency of CD4+FOXP3+ Treg cells in SLE patients. However, unlike all other Treg cells phenotypes analyzed, only eTreg (CD4+
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the presence of antibodies against self-antigens. SLE evolves by unpredictable episodes of intense inflammatory activity and remission, with localized or systemic damage [
In clinical practice, treatment depends on the manifestations of the disease; usually corticosteroid and immunosuppressant drugs are used. However, in a long-term treatment, patients become refractory to these conventional drugs. This can reduce chances of controlling the disease activity and increases death risk [
Searching for new therapeutic strategies for autoimmune diseases, regulatory T cells (Tregs) have a prominent place [
SLE pathogenesis is related to defects in Treg cell homeostatic control [
FOXP3 is a key transcription factor for Treg pathway. Cells termed “naturally occurring Treg cells” (nTreg or tTreg) that can be identified by the phenotype CD4+FOXP3+CD25+CD127- are widely studied in SLE; however, conflicting results are reported in their definition and function [
Based on the SLE complexity, all percentage of Treg cell subpopulations and their functions vary according to the severity of the disease and the therapeutic course taken [
Thirty patients (28 women and 2 men) with an average age of 35.33 (±10.40) years were invited to participate in this study. The control group consisted of 15 healthy women with an average age of 34.19 (±11.16) without diagnosis for autoimmune disease. All patients were recruited from the Rheumatology Service of Hospital das Clinicas, at the Federal University of Pernambuco, and met the classification criteria for SLE of the American College of Rheumatology [
Clinical and demographic parameters of SLE patients.
Number of patients | N = 30 |
---|---|
Age (yrs), Mean (range) | 38.17 ± 10.43 (19-61) |
Sex, N (%) | |
Female | 28 (93.33) |
Male | 2 (6.66) |
Disease duration (Months) | |
Mean (range) | 96 ± 72.94 (2 - 300) |
Anti-dsDNA, N (%) | |
Positive | 11 (36.66) |
Negative | 19 (63.66) |
Complement, N (%) | |
Decreased | 19 (63.66) |
Normal | 11 (36.66) |
Treatment, N (%) | |
Steroids | 26 (86.66) |
Antimalarial agents | 26 (86.66) |
Azathioprine | 21 (70) |
Mycophenolate mofetil | 10 (33.33) |
Disease activity (SLEDAI), N (%) | |
Range | 0-20 |
< 6 | 15 (50) |
≥ 6 | 15 (50) |
Nephritis, N (%) | |
Active | 10(33.33) |
Inactive | 20(66.66) |
Serum samples, obtained from patients’ peripheral blood, were stored at −80°C until use. Anti-dsDNA analysis was performed by indirect immunofluorescence with
The peripheral blood collected in heparin tubes was directly added to Ficoll-Hypaque gradient (Amersham Biosciences, Uppsala, Sweden) in 50 mL falcon tube. After centrifugation at 400 x g for 40 min at 22°C, peripheral blood mononuclear cells (PBMCs) were recovered and washed twice with PBS (Phosphate-buffered saline) (pH 7.2) at 350 x g for 15 min. The PBMCs were then resuspended in RPMI 1640 medium (Roswell Park Memorial Institute) (Gibco, Thermo Fisher Scientific) supplemented with L-Glutamine, 10% Fetal Bovine Serum (Lonza), 10 mM HEPES (4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Gibco, Thermo Fisher Scientific), and 200 U/ml Penicillin/Streptomycin (Gibco, Thermo Fisher Scientific). An aliquot of these cells was removed for counting on a Neubauer chamber using Trypan blue (Sigma, St. Louis, MO) as a viability dye.
105 PBMCs were resuspended in 100uL of PBS for labeling with human cell-surface antibodies (all from eBioscience) in two different conditions: (1) antiCD25 PECy7 (BC96), antiCD127 PerCPCy5.5 (eBioRDR5), antiCD4 FITC (RPA-T4;) together, or (2) antiCD4 FITC (RPA-T4) and antiCD45RA PerCPCy5.5 (HI100). Cells were then permeabilized with the “Human FoxP3 Buffer Set” BD-Pharmingen (San Diego, CA) according to the manufacturers’ recommendation and labeled with FOXP3 PE (236A). A hundred thousand events per sample were acquired by Attune® (Thermo Fisher Scientific) flow cytometer. Analysis was done with FlowJo 7.6.5 (Tree Star® Inc.) (Figure
Data analysis that did not follow the normal distribution was performed using univariate comparisons through nonparametric tests (Mann–Whitney, Kruskal-Wallis, and Kolmogorov-Smirnov). For data that followed the normality, we apply parametric tests (
We investigated naturally occurring Treg cells in SLE patients and healthy donors according to Figure
Naturally occurring Treg lymphocytes of SLE patients and healthy individuals. (a) CD4+FOXP3+CD25+CD127- Phenotyping: CD4+FOXP3+ cells (1), CD4+FOXP3+CD25+ cells (2), CD4+FOXP3+CD25+CD127- in CD4+FOXP3+ cells (3); (b) CD4+ T Lymphocytes and CD4+FOXP3+ Treg cells; (c) CD4+FOXP3+CD25+CD127- cells in CD4+FOXP3+ Treg cells.
FOXP3+ Tregs subset phenotyping in patients with systemic lupus erythematosus (SLE) and healthy donors. (a) Gating strategy for Treg cells characterization in PBMCs: lymphocytes gate (1); TCD4+ lymphocytes (2); CD4+CD45RA+
Since SLE patients showed an increase in CD4+FOXP3+ Treg cells, we decided to investigate its subtypes. Based on the differential expression of the CD45RA marker and FOXP3 by CD4+ T lymphocytes, naïve Treg (Figure
To deepen the analysis, the influence of Treg cell subtypes on SLE activity, measured by SLEDAI through multiple linear regression analysis, was also evaluated. The eTreg, Foxp3+nonTreg, and naïveTreg together exerted an influence of 28.90% on SLEDAI score variability, with a high significance recorded by F-test (p=0.006). In this specific type of analysis, it was possible to detect that the eTreg subtype exerts an inverse influence on the severity of the disease (p=0.010), whereas Foxp3+nonTreg subtypes (p=0.003) is associated with an increased SLEDAI score. The correlation with naïve Treg and the disease activity was not significant (p=0.352). Also, the FOXP3+CD4+ phenotype (p=0.56) and FOXP3+CD4+ CD25+ CD127- (p=0.52) had low influence on disease activity (1.47%), confirming our previous remarks. Additionally, this poor correlation was indicated by F-test (p=0.771), that showed no significance between these phenotypes and the disease activity evaluated by SLEDAI score (Table
Influence of Treg cells subtypes on SLEDAI score for sample.
SLEDAI | Coef. | Std. Err. |
|
R-squared | Prob > F |
---|---|---|---|---|---|
eTreg | −1.206469 | .435602 | 0.010 | 0.2890 | 0.0065 |
Foxp3+nonTreg | .3915982 | .1208674 | 0.003 | ||
naïveTreg | .3039881 | .3204841 | 0.352 | ||
|
|||||
|
−.3861103 | .6703293 | 0.569 | 0.0147 | 0.7717 |
|
.998021 | 1.5654 | 0.529 |
Since the eTreg and Foxp3+nonTreg subtypes had the greatest influence on the SLE activity, we investigated specific SLEDAI clinical parameters that exerted influence on our sample correlated to these Treg subtypes (Table
Clinical parameters influence in eTreg cells frequencies of SLE patients.
eTreg | Coef. | Std. Err. |
|
R-squared | Prob > F |
---|---|---|---|---|---|
Proteinuria | −1.271477 | .4145096 | 0.006 | 0.3255 | 0.0163 |
Hematuria | .3083865 | .7977254 | 0.703 | ||
Pyuria | −.5732928 | 1.006636 | 0.575 | ||
Anti-dsDNA | −2.084068 | .9786697 | 0.045 | ||
serum complement | 1.581082 | 1.260822 | 0.223 | ||
Rash | −1.476643 | .5323137 | 0.011 |
SLE often evolves with hematological disorders including anemia, leukopenia, lymphopenia, and thrombocytopenia [
Aiming to understand the role of Treg cells in SLE activity, other phenotypes of these cells have been explored in the disease. However, emerging analysis of CD4+CD25+CD127- and CD4+CD25+FOXP3+ cells suggest that the latter is a promising SLE activity indicator, especially in renal involvement, and may facilitate the detection of Treg subsets with clinical relevance [
Although higher in SLE patients, CD4+FOXP3+ cells were not a good indicator of SLE disease activity. Therefore, we investigated its activation state according to differential expression of CD45RA and FOXP3, featuring the subtypes CD4+
The Miyara group [
Furthermore, these differences could depend not only on unclear definition of the Treg phenotype, as previously reported [
The analysis of naïve Treg, eTreg, and Foxp3+nonTreg together explains 28% of SLEDAI score variability in our sample. Evaluating the correlation coefficients for each subtype and their respective significance, we concluded that the eTreg subtype is inversely correlated with disease activity (p=0.010) while Foxp3+nonTreg (p=0.003) exerted a direct influence. Although naïveTreg frequencies exerts a direct influence, it was without significance for our sample (p=0.352). Additionally, we confirmed the low influence exerted by CD4+FOXP3+ and CD4+CD25+FOXP3+CD127- cells frequencies on the SLEDAI score, both related only to 1.47% of the disease activity. It was also found that proteinuria (p = 0.006), anti-dsDNA antibody (0.045), and rash (p=0.011) were associated with eTreg cell reduction. Analysis of proteinuria, hematuria, pyuria, anti-DNA, complement, and rash may explain 32.55% eTreg frequency variations. Equivalent analysis for Foxp3+nonTreg did not identify specific clinical parameters related to frequency of this subtype, probably because the Foxp3+nonTreg phenotype can be correlated to multiple variables that go beyond those described in SLEDAI score.
Unlike naïveTreg and eTreg, Foxp3+nonTreg phenotype includes cells with a Th17 potential, that have no suppressive capacity, enhanced cell proliferation response, and exhibits strong IFN-
Immunotherapies that target Treg cells and/or recovery of Treg cell homeostasis stand out in the search for more specific treatments for autoimmune diseases [
All SLE patients in this study were in treatment as shown in Table
Our data demonstrated that eTreg and Foxp3+nonTreg frequencies correlate significantly with disease activity in systemic lupus erythematosus patients. The use of CD45RA as activation marker in CD4+FOXP3+ Treg cells allowed a more accurate analysis of a potential biomarker for active SLE, unlike conventional analysis based on CD25 and CD127 expression and in FOXP3+ CD4+ Treg cells. Although heterogeneity of Brazilian population is considerable [
The data, in free formats, used to support the findings of this study are available from the corresponding author upon request.
The authors declare no conflicts of interest.
The authors would like to acknowledge the following financial support for the research, authorship, and/or publication of this article: this study was supported by the Brazilian National Research Council (CNPq) [474204/2010-3], the Research Foundation of Pernambuco State (FACEPE) [APQ-1034-4.03/10], National Institute for Science and Technology in Pharmaceutical Innovation (INCT_if) [573663/2008-4], and Coordination for the Improvement of Higher Education Personnel (CAPES).
Figure S1: additional representative analysis of the positive region delimitation. eTreg (1), Foxp3+nonTreg (2), naïveTreg (3), and CD4+FOXP3-CD45RA+ T cells (4) (A). Differential PE-conjugated anti-FOXP3 antibody fluorescence intensity in eTreg, Foxp3+nonTreg, naiveTreg, and CD4+FOXP3-CD45RA+ T cells (4) subtypes (B). FMO control for FOXP3 delimitation (all fluorochromes minus FOXP3 marker) (C). Figure S2: Th17 and Treg related cytokines in the serum of SLE patients and healthy donors (A). Foxp3+nonTreg cells related to serum concentrations of IL-17 (B), IL-23 (C), TGF-