Immune regulation in lupus nephritis : role of CD 4 + CD 25 high FOXP 3 + T regulatory cells and related cytokines ( IL-6 , IL-10 , TGF-β 1 )

Background – Aim: Loss of tolerance to self-antigens repre sents the critical factor for pathogenesis initiation in systemic lupus erythematosus (SLE) and lupus nephritis (LN). T regulatory cells (Tregs) and related cytokines (IL-6, IL-10, TGF-β1) are the main mediators of this mechanism. The aim of this study was the evaluation of Tregs and IL-6, IL-10 and TGF-β1 in the course of LN patients. Patients – Methods: Twenty LN patients (18 female, 2 male, mean age 33.8±8.8 years, mean disease duration 87.2±63.4 months) were included; twelve with a histological diagnosis (10 class IV+V, 2 class V). CD4+CD25highFOXP3+ Tregs were analyzed in 61 samples (44 active, 17 quiescent disease). IL-6, IL-10 and TGF-β1 were evaluated in 18 patients (10 active, 8 inactive LN). Disease activity was evaluated with SLE Disease Activity Index (SLEDAI). Other parameters included C3/C4d complement fragments, antidsDNA antibodies and proteinuria. Analysis was performed with Kruskal-Wallis test; p<0.05 was considered significant. Results: Tregs were significantly lower in active LN (0.71±0.29% vs. 1.14±0.19% of the CD4+ T cells, p<0.001). IL-6 and IL-10 were significantly higher (IL-6: 6.25±2.38 vs. 1.62±1.66pg/ml, p<0.001, IL-10: 5.8±3.8 vs. 1.7±2.6pg/ml, p=0.025), whereas TGFβ1 was lower (16529±7962 vs. 25957±6776pg/ml, p=0.034) in active LN. C3 and C4d were significantly lower in active disease (C3: 82.1±22.3 vs. 152±29.9mg/dl, p<0.001 and C4d: 8.7±4.3 vs. 21±7.7mg/dl, p<0.001). Conclusions: Tregs and TGF-β1 were significantly lower in active LN, while IL-6 and IL-10 were detected in higher levels. Impairment of IL-6/TGF-β1 axis seems to drive Tregs reduction in active LN; on the contrary, IL-10, despite its known regulatory profile, seems to exert mainly proinflammatory functions in LN.


Introduction
Renal involvement in systemic lupus erythematosus (SLE) Ελληνική Νεφρολογία 2014; 26 (1): 29 -36   Πρωτότυπη εργασία Immune regulation in lupus nephritis: role of CD4+CD25high FOXP3+ T regulatory cells and related cytokines (IL-6, IL-10, TGF-β1) represents a decisive prognostic factor for such patients 1 .Disease pathogenesis remains largely unknown; however, reactive lymphocytes, autoantibodies targeting nuclear antigens and immune complexes have been implicated 2 .Central immune tolerance to certain nuclear antigens is incomplete even in healthy individuals and results in reactive peripheral lymphocytes, identified in both B and T cell compartments 3,4 .Thus, a further failure in the mechanisms of peripheral tolerance is critical in initiating the pathogenetic process in lupus nephritis (LN).In this context, T regulatory cells (Tregs), which represent the main mediators for suppressing reactive lymphocytes in the periphery, are believed to play a determinant role 5 .
Following early experiments in lupus-prone mice, several investigators demonstrated quantitive and/or qualitative defects of these cells in human SLE [6][7][8] .However, significant differences between human and mice Tregs do exist; for instance, in humans, only Tregs with the highest CD25 surface expression exert suppressive capacity and this is strongly correlated with the intracellular expression of Foxp3, the master gene for Tregs' differ entia tion 9 .Consequently, this particular immunophe notype (CD4+CD25 high FOXP3+) has been succes sfully employed to quantify their frequency in peri pheral blood.
Recent studies have shown that Tregs differentiate and function in a sensitive equilibrium with their effector counterparts in LN, mainly the Th17 cells 10,11 .Soluble mediators, such as IL-6 and TGF-β, are of paramount importance; IL-6 drives the differentiation of naive CD4+ T cells towards Th17, while transforming growth factor beta 1 (TGF-β1) predominance is responsible for the expansion of Tregs 12 .Their mechanisms of action involve both cell-to-cell contact and cytokines, such as IL-10 and TGF-β1 and are able to target virtually all effector immune cells 13,14 .
The exact role of these cells and their related cytokines in LN has not been fully elucidated.Aim of the present study was the assessment of peripheral Tregs and certain cytokines (IL-6, IL-10, TGF-β1) in LN patients in regard to disease activity and response to administered therapy.

Patients -Methods
Twenty LN patients (18 females, 2 males, mean age 33.8±8.8years, mean disease duration 87.2±63.4months) were included in the study from January 2008 until December 2011.Twelve of them had a histological diagnosis; 10 with class IV+V LN and 2 with pure class V LN, according to the current pathologic classification 15 .The remaining eight patients were diagnosed with SLE, according to the American College of Rheumatology revised criteria 16,17 .LN diagnosis in these patients was based on the presence of proteinuria (>500mg/24h) and/or hematuria and/or urinary casts, according to the recent recommendations from European League Against Rheumatism (EULAR) 18 .One female patient had already developed end stage renal disease (requiring dialysis) at the time of diagnosis.Twenty age-and sex-matched healthy individuals served as controls in order to quantify the "normal" numbers of Tregs.
The Human Ethics Review Committee of the Medical School of Aristotle University of Thessaloniki approved the study protocol and a signed informed consent was obtained from each subject.
Cytokine assessment was performed in stored (-76°C) serum samples by enzyme-linked immunosorbent assay (ELISA) with the following commercially available kits: Human IL-6 BMS213HS, Human IL-10 BMS215HS and high sensitivity Human TGF-β1 BMS249/3CE, all from Bender MedSystems® GmbH, Vienna, Austria.Normal range for each cytokine was considered the one reported by the manufacturer.
Clinical assessment was made using the SLE Disease Activity Index (SLEDAI), according to its initial description 19 .Active LN was defined by the presence of significant proteinuria (>500mg/24h) and/or hematuria and/or urinary casts in the absence of urinary infection (based on negative urine culture).The SLICC/ACR (Systemic Lupus International Collaborating Clinics) index was used for assessment of cumulative damage 20 .
Patients were divided into group A (active unstable LN, n=8) and group B (inactive quiescent LN, n=12).CD4+CD25 high FOXP3+ Tregs were assessed in 61 samples (44 in group A and 17 in group B) in total.Six out of eight active patients were administered intravenous cyclophosphamide (6 monthly pulses of 500mg/m 2 /month) plus oral methylprednisolone (8-32mg/day, slow tapering) for remission induction; in these patients, Tregs were assessed monthly.In the other two active patients, mycophenolate mofetil (1080-1440mg/day) plus methylprednisolone (8-32mg/day, slow tapering) were used; Tregs were assessed quarterly for 12 months.Inactive patients (n=12) were evaluated once; five patients were re-evaluated after 12 months.Maintenance therapy in these patients included methylprednisolone (4-8mg/day) in all patients, azathioprine (100mg/day) in 6 patients and mycophenolate mofetil (720-1080mg/day) in the other 6 patients.IL-6, IL-10 and TGF-β1 were evaluated in 18 patients (10 active, 8 inactive LN) once in total; all assessments were made in parallel with Tregs' evaluation.

Statistical analysis
Analysis was performed using the non-parametric Kruskal-Wallis test for independent variables and Monte Carlo simulation.All p values were 2tailed and p<0.05 was considered to be statistically significant.Correlations were made with Pearson's correlation co-efficient.The SPSS software package (version 20.0) was used for data analysis.
Serum creatinine was not affected in neither patients' group, except for one patient in group A (where serum creatinine raised 1.5x upper limit of normal transiently) and one patient in group B (who was on dialysis since LN diagnosis).Proteinuria was more severe in active LN (1445±2348 vs 197±111mg/24h, p=0.010).All the aforementioned parameters refer to values before remission induction treatment; after remission, proteinuria in active patients was similar to inactive individuals (234±86mg/24h, p=0.34).

Discussion
The role of Tregs in human LN pathogenesis and outcome has not been extensively investigated thus far; only 5 relevant studies have been published in human subjects 10,11,[21][22][23] .In the present study, Tregs, either as a proportion of CD4+ T cells or as absolute numbers, were found in significantly lower numbers in active disease.This finding comes in agreement with most studies in the field 10,11,21,22 , although there is one study where no significant differences were demonstrated 23 .It should be mentioned that in the present study, a comparatively larger number of samples were evaluated, whereas a significant proportion of the patients were re-assessed over time in different phases of disease activity.Furthermore, Foxp3 mRNA, the critical factor for Tregs' differentiation, has been reported to be significantly up-regulated in the urine of active LN patients and predicted a poor therapeutic response 24 .The controversy between defective Tregs and overxpressed Foxp3 in active SLE may be explained by the Foxp3 expression in other T cells and/or the destruction of Tregs by their effector counterparts 24 .In terms of their functional status, experimental studies described intact suppressive activity of these cells in murine lupus, a finding that was also confirmed in human LN 23,25 .
It was recently stressed that Tregs decrement in active LN was accompanied by a compensatory expansion of the Th17 lymphocytes, along with an increment of related cytokines IL-17 and IL-23 10 .In addition, several investigators have shown that other Th17-specific cytokines, particularly IL-6, was significantly higher in active LN and may represent  a promising biomarker for assessing the activity of renal involvement in SLE [26][27][28] .Furthermore, it was recently shown that urinary levels of IL-6 are higher in active LN in comparison to patients with no renal involvement 26,29 .In a mechanistic basis, it was shown that dendritic cell derived IL-6 is able to inhibit the function of Tregs in lupus-prone mice 30 .
On the other hand, serum TGF-β1 has been repeatedly found in lower levels in such patients, while its urinary levels were reportedly higher in comparison with inactive LN 11,31,32 .Our findings are in line with these observations, further reinforcing the hypothesis that disturbances in the IL-6/TGF-β1 axis may further promote Tregs/Th17 equilibrium towards the pro-inflammatory Th17 phenotype.
Successful remission induction was accompanied by Tregs restoration in all LN patients in the present study.Administered regimens, mainly methylprednisolone and cyclophosphamide, are considered nonspecific; consequently, Tregs' increment probably represents an indirect result.Other investigators demonstrated that rituximab, a B cell targeted agent, is able to lead to a significant increment of Tregs, which is strongly related to clinical remission 21,22 .The underlying mechanism is not known, however, it can be hypothesized that B cell depletion would result in immune complexes reduction, decreased dendritic cells' stimulation and less IFN-α production.This would further lead to the over-expression of regulatory cytokines, such as TGF-β1, and Tregs expansion.
Concerning IL-10, its serum levels were found significantly higher in active LN.This finding has been reported recently, while its levels returned to normal after remission induction 33 .In addition, its levels were strongly correlated to anti-dsDNA antibodies titers 34 .Increased IL-10 levels are not paradoxically observed in SLE.This cytokine displays multiple roles in disease pathogenesis, either as a promoter of Th2 differentiation and inhibitor of Th17 expansion (by reducing IL-23) or by being the critical factor for B cell maturation [35][36][37] .In this context, IL-10 reflects disease activity rather than being a suppressive/regulatory cytokine.
Limitations to be considered in the present study include the small number of patients, which does not allow for definite conclusions, and the lack of functional assays for more precise Tregs charac-terization.Furthermore, it would be helpful to assess the urinary levels of the studied cytokines and/or Foxp3 mRNA, in parallel to serum levels, in order to clarify their importance in LN activity and response to treatment.In addition, the simultaneous study of the Th17 cell population would further elucidate disease pathogenesis.
In conclusion, Tregs were found significantly lower in active LN, while their numbers were restored after remission induction.Serum levels of IL-6 and IL-10 were higher in active disease, while TGF-β1 was decreased in the same patients.Impairment of IL-6/TGF-β1 axis seems to drive Tregs reduction in active LN.

Table 1 .
Demographic characteristics and parameters studied comparatively in active and inactive lupus nephritis (LN).SLEDAI: Systemic Lupus Erythematosus Disease Activity Index, SLICC/ACR: Systemic Lupus International Collaborating Clinics/American College of Rheumatology.