Value of Foxp3 expressing T-regulatory cells in renal tissue in lupus nephritis; an immunohistochemical study

Background Forkhead box P3 (Foxp3) functions as a master regulator in the development and function of T-regulatory (Treg) cells. Recent studies have shown that autoimmune diseases including systemic lupus erythematosus (SLE) are associated with an imbalance with the Treg cells and T helper (Th) subtypes. Objectives To evaluate immunohistochemical expression of Foxp3 positive Treg cells in lupus nephritis (LN) and analyze its association with clinicopathologic parameters. Materials and Methods Renal biopsy specimens of 50 patients with LN were studied. Specimens were divided into; group A; 25 LN cases without proliferative activity (Class II and V) and group B: 25 cases with proliferative activity (Class III and IV). Immunohistochemical staining for anti-human Foxp3 antibody and grading from grade 0 to grade 3 was done. Results Foxp3 expression in group A was (grade 0 in 14 [56.0%], grade +1 in 11 [44.0 %]) in comparison to group B (grade +1 in 6 [24.0%], grade +2 in 11 [44.0%] and grade +3 in 8 [32.0%]) (P < 0.001). Foxp3 expression was significantly correlated to National Institutes of Health (NIH) activity and chronicity indices (P < 0.05), as well as serum creatinine (P < 0.01) in both groups A and B and there was a highly significant correlation with proteinuria (P < 0.01) in group B with proliferative LN. Conclusions Immunohistochemical Foxp3 expression in renal tissue was higher in proliferative versus non-proliferative LN and is associated with activity and severity of LN. Further studies are needed to determine its prognostic value in LN.


Background
Lupus nephritis (LN) is the most important complication of systemic lupus erythematosus (SLE), which is responsible for SLE-related mortality and morbidity. LN occurs in up to 50% of patients with SLE (1). In LN, the deposition of immune complexes plays a leading role in the initiation of the disease. Subsequently, the deposited immune complexes and dendritic cells trigger the release of type I interferon and induce activation of infiltrating T cells, and this leads to intensification of T cell subgroups including the CD4+ T helper (Th) subtypes. Furthermore, CD4+ T cells are stimulated via the presentation of the putative antigens presented by dendritic cells or macrophages in the context of class II major histocompatibility complex (2,3). The Th cells have certain subtypes. Th1 and Journal of Nephropathology, Vol 5, No 3, July 2016 www.nephropathol.com 106 Th17 classically represent the inflammatory Th cells whereas the regulatory T cells (Treg) have a regulatory and suppressive role. Recent studies have detected that autoimmune diseases, including SLE, are associated with an imbalance with the Treg population and the inflammatory Th subtypes in the system. IFN-γ is the main cytokine product of the Th1 cells whereas IL-17 is the major cytokine of Th17 cells (4). Treg cells play a role in the prevention of uncontrolled immune responses against pathogens or allergens, guarantee stability of the normal microflora and prevent the escape of tumors from immune surveillance. Treg cells are also implicated in the development of cancer, autoimmunity, allergy and asthma (5-7). Forkhead box P3 (Foxp3) protein is a transcription factor of the forkhead family, which is required for the development and function of most thymus-derived, naturally occurring Treg cells (8). This molecule also plays a crucial role in the regulation of immune responses mediated by peripheral T cells, preventing autoimmunity and permitting the maintenance of Treg cells (9). Foxp3 is the most specific marker of Treg cells currently available (10)(11)(12). Few studies investigating the presence and function of Foxp3 expressing Treg cells in renal tissue in LN are done (13,14).

Objectives
The objective of this investigation was to evaluate immunohistochemical expression of Foxp3 expressing Treg cells in LN and analyze its association with various clinical and histopathological parameters.

Study population
This study included 50 renal biopsy specimens that were diagnosed as LN at the pathology laboratory, and electron microscopy (EM) laboratory of Ain-Shams University hospitals during the period of 2012 to 2014. Data extraction sheet was designed to collect data retrospectively from records as personal data, medical history and laboratory results including serum creatinine and proteinuria by protein dipstick grading with approximate daily amount classified into grade I: 1+ dipstick (0.15-1.0 g/24 h), grade II: 2+ or 3+ dipstick (>1.0-3.5 g/24 h), grade III: 4+ on dipstick (>3.5 g/24 h) (15). Cases with adequate paraffin tissue blocks for immunostaining were enrolled in the study. All renal biopsy specimens were graded according to the 2003 International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification system (16) and National Institutes of Health (NIH) activity and chronicity indices (17,18). The renal biopsy speci-mens were divided into: Group A: LN cases without proliferative activity (Classes II and V) Group B: LN cases with proliferative activity (Classes III and IV) The renal specimens were re-evaluated by light microscopy (LM) and immunohistochemical staining using anti-human Foxp3 polyclonal antibody.

Light microscopy
Formalin-fixed, paraffin-embedded tissues from all cases were retrieved from the archive of the pathology laboratory. Slides were re-evaluated by two pathologists using LM (Olympus BX31) according to the histopathological guidelines for the evaluation and scoring of LN (16)(17)(18). The pathologists were blinded to the clinical parameters of the patients.

Immunohistochemical staining method
Tissue sections were deparaffinized, rehydrated, and the endogenous peroxidase activity was quenched by 10-minute incubation in 3% hydrogen peroxide in methanol. After antigen retrieval and protein block, the primary antibody (Rabbit anti-human Foxp3 polyclonal antibody; catalog number: E18491, spring bioscience) was supplied as 7.0 mL prediluted immunogen affinity purified rabbit polyclonal antibody in TBS/1% PSA buffer pH 7.6 with less than 0.1% sodium azide. The secondary antibody (supersensitive immunodetection system (Biogenex, catalog No: AD000-SL) was then applied followed by peroxidase labeled streptavidin. Slides were incubated for 10 minutes with substrate chromogen (DAB) mixture. Finally, the slides were counterstained and mounted with Canada balsam. Tissue sections from tonsils were used as positive control. Positive control slides were included in each run.

Semi-quantitative evaluation of immunostaining
Cells revealing clear lymphocyte morphology with distinct nuclear staining for Foxp3 but unstained cytoplasm (interpreted as Treg cells) were semi-quantified according to Yazici et al, (13) as follows: Grade 0 was defined as the complete absence or weak staining in <1% of the cells; grade 1 was staining in 1%-10% of the cells; grade 2 was positive staining in 11%-50% of the cells; and grade 3 was positive staining in >50% of the cells. Glomerular and tubular staining with Foxp3 was not graded.

Ethical issues
The research followed the tenets of the Declaration of Helsinki. The work is a retrospective study which

Statistical methods
Data was revised for its completeness and consistency. Double data entry on SPSS program version 16 was done. Quantitative data were summarized by mean along with standard deviation while qualitative data were summarized by frequencies and percentages. Analysis of variance, chi-square test and Spearman's correlation coefficient test were used the analysis of significant differences or associations. A "P value" of less than 0.05 was considered statistically significant.

Results
Fifty renal biopsy specimens with LN were re-evaluated and divided into two groups. Group A included 25 cases with non-proliferative LN (19 cases of class II, and 6 cases of class V). Among these, 24 (96%) were females and one (4%) was male, with mean age of 24.6 ± 12.7 years. Group B included 25 cases with proliferative LN (class III, IV), where 18 (72%) were females and 7 (28 %) males, with mean age of 29.3 ± 13.9 years. The patients' characteristics, laboratory results and NIH activity and chronicity indices of LN of both studied groups are described in Table 1. Group B showed significantly higher mean serum creatinine and higher mean activity and chronicity indices compared to group A (P < 0.001). Immunohistochemical expression of Foxp3 among studied groups is shown in Table 2 and Figures 1, 2 and 3. Group B showed significantly higher percentage of Foxp3 immunostaining with grade +2 and grade +3 in comparison to group A (P < 0.001), while group A showed higher percentage of negative Foxp3 staining. Immuohistochemical expression of Foxp3 was significantly correlated to activity index and chronicity index in group A (P < 0.05) and in group B (P < 0.001). There was significant association between Foxp3 expression and presence of wire loops (χ 2 = 7.26, P = 0.005), fibrinoid necrosis (χ 2 = 6.21, P = 0.029), cellular crescents (χ 2 = 6.57, P = 0.023) and interstitial inflammation (χ 2 = 4.48, P = 0.032) by Fisher's exact test in group B as parameters of activity of LN. There was highly significant positive correlation between Foxp3 expression and serum creatinine in both non-proliferative (group A) and proliferative LN (group B) (P < 0.001) and proteinuria in group B (P < 0.01) but not in group A. There was no correlation between age or sex and Foxp3 expression in both groups (Tables 3 and 4).

Discussion
Foxp3+ Treg cells have been known as a major regulator of immune homeostasis through their immunosuppressive function. Th17 lineage is a CD4+ T cell subset that exerts its function by secreting pro-inflammatory cytokines and protecting host against microbial infections. The altered ratio between Foxp3+   (19). Recent studies of Foxp3 expression and function provided critical novel insights into biology of Treg cells and into the cellular mechanisms of the immune homeostasis (20). The majority of studies have investigated the role of Foxp3+ Treg cells in peripheral blood (21)(22)(23), with very few studies on immunohistochemical staining of these cells (13). In the present study, we confirmed that Foxp3 expression level in active proliferative LN (group B) was increased with a highly statistically significant difference in comparison to LN with minimal histological activity (group A). These results are in agreement with Abbass et al, (24) who found that Foxp3 gene expression is higher in patients with active lupus compared to those with inactive lupus. They were concerned in     T cells and the extent of proteinuria in patients with renal involvement. They observed a decrease in proportions of CD4+CD25−Foxp3+ T cells after the initiation of therapy with cyclophosphamide. This was paralleled by a decline in the extent of proteinuria. Similar to these results, Wang et al (29) proved that urinary Foxp3 mRNA is markedly up-regulated in patients with active LN, and the level of expression is closely associated with the clinical and histological disease activity. They detected higher level of urinary Foxp3 mRNA in patients with no response to therapy than those with partial response or complete response. These data highlight the role of medications targeted for Treg cells for amelioration of inflammatory process in active LN. In this study we did not evaluate the grade of immunohistochemical expression of Foxp3+ Treg cells after immunosuppressive therapy as it was a retrospective study. Further studies with patient follow-up are recommended to detect the effect of medications on Treg cells in renal tissue. Wang et al (29) recommended the use of Foxp3 mRNA in urine sediment as a non-invasive biomarker for assessing the severity. We also recommend the use of immunohistochemical expression of Foxp3 as a predictor of severity as there was significant positive correlation with serum creatinine, and NIH activity and chronicity indices in both groups in the current study.

Conclusions
Foxp3 expressing Treg cells in renal tissue were higher in proliferative versus non-proliferative LN and are associated with activity and severity of LN. Further studies are needed to determine prognostic value of Foxp3 expression in LN.

Limitations of the study
Evaluation of Foxp3 immunohistochemical expression before and after receiving the treatment of active LN was a limitation of our investigation.