The Expression and Effect of Helper IL-10-Producing B Cells in Human Minimal Change Nephrotic Syndrome

This  study  is  aimed  at  determining  the  putative  contribution  of  B  cell  types  in patients with newly diagnosed minimal change nephrotic syndrome (MCNS) before treatment  and  8-12  weeks  post-treatment.  Significantly  higher  percentages  of CD19+CD27+, CD19+CD38+, and CD19+CD86+ B cells were observed compared to healthy controls. Importantly, lower levels of Breg cells and intracellular expression of  IL-10  protein  were  detected  in  MCNS  patients.  24-h  urinary  proteins  were positively correlated with the percentage of CD19+CD27+ B cells and negatively correlated  with  the  percentage  of  Breg  cells.  A  positive  correlation  between the  percentage  of  CD19+CD5+CD1d+  cells  and  the  levels  of  serum  albumin was  observed  in  these  patients.  Treatment with  prednisone  (PDN)  significantly reduced the frequency of CD19+CD27+, CD19+CD38+, and CD19+CD86+ B cells, and elevated the levels of Breg cells and Breg cell-derived IL-10. This data indicates that several of the CD19+ B cell subtypes and IL-10+ Breg cells have a potentially crucial role in the MCNS patients.


Introduction
Minimal Change Nephrotic Syndrome (MCNS) is the major cause of the nephrotic syndrome (NS) in children, yet comprises only 10-15% of adult NS cases [1][2][3]. MCNS usually responds to glucocorticoids (steroids), and the long-term prognosis is generally good [4]. On kidney biopsy glomeruli appear normal by light microscopy and without immune deposits by immunofluorescence microscopy. The characteristic disease lesion, diffuse and generalized epithelial foot process effacement affecting glomerular albumin permeability, is only found by electron microscopy [5].
The pathophysiologic process of MCNS remains poorly understood but is frequently triggered by immunogenic stimuli such as infection or allergic reaction [6]. The release of cytokines by T cells has long been considered a key in the pathogenesis of MCNS [3]. However, the beneficial effects of Rituximab as a rescue therapy in children with steroid-dependent MCNS [7,8] suggests that B-lymphocytes may play an important role in this process.
Immunoglobulin production is the result of a complex interaction between B cells, T cells, and antigen-presenting cells such as as antibody-secreting cells, while CD27+CD19+ B cells serve as memory B cells, CD95+CD19+ B cells signal apoptotic induction, CD38+CD19+ B cells act as antibody-secreting cells following B cell activation, and CD1d hi CD5+CD19 hi serve as regulatory B cells (Breg) [11]. Importantly, these B-cell subsets may be involved at different stages of MCNS pathogenesis.
A recent study described a subset of regulatory B cells that do not participate in the pathogenesis of autoimmune diseases and act through the production of regulatory cytokines IL-10 and TGF-ß [10]. The potential of Bregs was first described in the regulation of autoimmunity, such as Experimental Autoimmune Encephalomyelitis (EAE), in which disease severity was increased in B-cell-deficient mice [12]. Later, antigen-induced IL-10 production from B cells was shown to play a critical role in the remission phase of EAE [13]. Similarly, B cell IL-10 production was shown to increase the severity of chronic colitis observed in TCR a-/-mice [14]. It has been demonstrated that transitional two marginal zone Bregs prevent collagen-induced arthritis in mice, but are fully dependent on CD40 and partially dependent on antigen [15]. Recently, a human counter-part of such Bregs has been characterized in peripheral blood. These Bregs are defective for CD40-dependent IL-10 production in lupus patients, indicating that this impaired function might contribute to the disease [16].
Currently, MCNS patients are treated with corticosteroids and other immunosuppressants such as cyclosporine (CyA), cyclophosphamide (CPA) and mycophenolate mofetil (MMF), in addition to anti-coagulation drugs, such as aspirin or dipyridamole. However, it is unclear how these therapeutic strategies affect B cells and immune measures in MCNS patients. Therefore we set out to examine the effects of various treatment strategies on B cell subpopulation ratios and IL-10 production in MCNS patients.

Patients
A total of 23 patients with MCNS who underwent renal biopsy were recruited for study from the Nephrology Department at the First Hospital of Jilin University (Jilin University, Changchun, China). The ethnicity of these subjects was Chinese. No patient had received corticosteroids or other immunosuppressive agents within 6 months before this study. The plasma laboratory tests of antinuclear antibody (ANA), anti-Sm antibody, anti-SSA, anti-SSB, anti-neutrophil cytoplasmic antibody (ANCA), antiphospholipid antibody, plasma complement C3 and C4, and rheumatoid factors were performed for all patients. Together with the renal histological and immunofluorescence results, the patients with potential autoimmune diseases were ruled out. None of the patients suffered from diabetes mellitus, neoplasia, active peptic ulcer disease, or viral hepatitis. At the time of blood collection and renal biopsy, no patients had fever or urinary, respiratory, or gastro-intestinal tract infections.
In addition, 12 sex-and age-matched healthy subjects were recruited from the medical examination center of the First Hospital of Jilin University. The study conformed to the guidelines of the Declaration of Helsinki and was approved by the Human Ethics Committee of Jilin University. Written informed consent was obtained from each participant.

Treatment and follow up
Nine patients of 23 MCNS patients were treated with prednisone (PDN, Tianyao Pharmaceuticals, Tianjin, China) at 1 mg/kg/d for the first two months, and gradually decreased to a maintenance dose of 10 mg/d over the next 6 months. In addition, individual patients were treated with aspirin (100 mg/d, Bayer, Germany) or dipyridamole (100 mg/d, Yunpeng Pharmaceutical, Shanxi, China) if the patient had a high risk of a hypercoagulable state. The patients visited the clinical office monthly and were followed up for at least 8-12 weeks after beginning treatment. Blood samples were collected at the time of kidney biopsy and after 8-12 weeks of treatment.

Blood sampling and analyses
The study participants were instructed to fast overnight and venous blood (10 mL) was collected into heparinized tubes the following morning. All samples were subjected to lymphocyte profiling and routine laboratory analyses within 24 h. The ADVIA 1650 biochemical analyzer (Bayer, Pittsburg, PA, USA) was used to measure triglycerides, cholesterol, proteinuria, serum uric acid, and serum albumin. The eGFR was calculated using the revised eGFR formula developed for Japanese subjects [17].

Detection of Breg cells and intracellular IL-10 protein expression
Isolated PBMCs were plated in 24-well plates (10 6 cells/well) and stimulated with 50 ng/mL phorbol myristate acetate (PMA), 500 ng/ml ionomycin, and 10 µg/ml lipopolysaccharide (LPS) (all from Sigma-Aldrich) in complete RPMI-1640 medium for 2 h at 37°C in 5% CO 2 . Brefeldin A (GolgiPlug; BD Biosciences, San Jose, CA, USA) was then added to each well and the cells were . The immunostained cells were fixed by incubating with 4% paraformaldehyde for 30 min at room temperature and permeabilized by incubating with 0.5% saponin in 10% fetal bovine serum in PBS for 30 min at room temperature. After PBS washes, the cells were stained with 5 µL FITC-anti-IL-10 (eBiosciences) and analyzed by flow cytometry as described above. At least 50,000 events per sample were analyzed.

Statistical analysis
All data are expressed as mean, median, and range. Multiple comparisons between the two groups were analyzed by the Kruskal-Wallis H non-parametric test, using the Bonferonni method to correct P value. Correlations between variables were evaluated by the Spearman's rank correlation test. All statistical analyses were performed by the SPSS software suite (v19.0 for Windows; SPSS, Inc., Chicago, IL, USA). A two-sided P-value of <0.05 was considered statistically significant.

A higher frequency of circulating CD19+CD27+ and CD19+CD38+ B cells in MCNS patients
There was no significant difference in the distribution of age and gender between MCNS patients and healthy controls (HC). Furthermore, there was no significant difference in the concentrations of uric acid, triglycerides, cholesterol and eGFR between the two groups. As expected, the 24-h urinary proteins were significantly higher in MCNS patients, yet albumin levels were lower compared to HC (Table 1).
Flow cytometry analysis indicated that the percentage of peripheral blood CD19+CD27+ in CD19+ B cells and lymphocytes was significantly higher in MCNS than in the HC (25 ( (Figure 2B and  2D). The frequency of CD19+CD38+CD86+ in CD19+ B cells was not significantly different between MCNS patients and controls (Table 2).

MCNS patients have a lower frequency of CD5+CD1d+CD19+ cells and lower expression of Breg cell-derived IL-10
As shown in Figure 3, the percentage of CD19+CD5+CD1d+

Influence of PDN treatment on B cells in MCNS patients
Nine of 23 MCNS patients in the study were followed up 8-12 weeks after initiation of PDN treatment. Treatment not only reduced the concentration of 24-h urinary proteins but also increased the level of serum albumin in these patients, although the treatment did not significantly alter the values of other measures ( Table 3). The percentages of CD19+CD27+, CD19+CD38+, and CD19+CD86+ cells was similar between HC and PDN, although they were significantly lower than that of MCNS patients (P<0.05) (Table 4, Figure 1 and 2).

Discussion
In this study, we examined distinct B cell subtype numbers in serum. Our study showed that the proportions of CD19+CD27+, CD19+CD38+, and CD19+CD86+ in peripheral blood of MCNS patients were elevated compared to normal subjects, and that all above cell numbers dramatically reduced after receiving PDN treatment. The percentage of CD19+CD5+CD1d+ Breg cells and the percentage of intracellular IL-10 were significantly lower in MCNS patients than in healthy controls. In addition, treatment with PDN for 8-12 weeks significantly reduced the frequency of CD19+CD27+, CD19+CD38+, and CD19+CD86+ B cells, and elevated the levels of Breg cells and Breg cell-derived IL-10. These findings support the hypothesis that B cell activation may contribute to the onset and/or progression of MCNS, and furthermore that the various B cell phenotypes may mediate disease pathogenesis.
Accumulating data points toward a strong contribution of B-cell immunity in patients with steroid-sensitive MCNS. Corticosteroids      memory B cells, and reconstitution of the B cell lineage after treatment in SLE patients [21]. Even once the total B cell count returns to normal a change in phenotype appeared to occur, with the B cells present being relatively deficient in the expression of CD27, a surface marker of memory B cells [22]. A study in adult Chinese patients also found elevated serum IgE levels in relapsing patients [23] explained by the fact that serum IgE levels reflected immune dysfunction as well as B and T cell activation [24,25]. Our study reveals that MCNS patients carry a higher frequency of memory B cells in peripheral blood, and that urinary proteins are positively correlated with the frequency of CD19+CD27+ B cells in these patients. Therefore, we speculate that CD19+CD27+ B cells may be involved in the pathogenesis of MCNS.
Extensive studies in mice have demonstrated that Breg cells play important roles in the suppression of experimentally-induced autoimmune diseases, including encephalomyelitis, inflammatory bowel disease, type 1 diabetes, SLE, and collagen-induced arthritis [12,13,16,[26][27][28][29]. Liang et al. reported that the numbers of CD19+TIM1+IL10+ and CD19+CD5+CD1d+IL10+ B cells were positively correlated with the numbers of CD4+CD25+Foxp3+ T cells in RA patients [30]. A previous study has suggested that the MCNS patients exhibit a significant increase in Th17 number,   Th17-related cytokines (IL-17 and IL-23) and transcription factor (RORγt) levels, in addition to an obvious decrease in Treg number, Treg-related cytokines (TGF-β1 and IL-10) and transcription factor (Foxp3) levels [31]. In our current study we showed that MCNS patients have a lower frequency of CD5+CD1d+CD19+ Breg cells. These finding support the notion that Breg cells may promote the activation of Treg cells in MCNS patients.
Breg cells are characterized by the CD1d hi CD5+CD19 hi phenotype and the production of regulatory cytokines IL-10 and TGF-β [11,12], which could influence T cell activation and inflammatory responses through IL-10 activity [32]. Our MCNS patient cohort showed significantly down-regulated Breg-derived IL-10, which was in accordance with these previous results. In addition, we found that treatment with PDN for 8-12 weeks not only significantly increased the frequency of circulating Breg cells but also elevated the levels of Breg cell-derived IL-10. Daien et al. demonstrated that the ability of B cells to produce IL-10 was altered in rheumatoid arthritis (RA), an impairment which influenced disease activity, biologic inflammation, and autoantibody levels, strongly suggesting a role of B10 cells in RA initiation [33]. Fillatreau et al. previously reported that B celldeficient mice developed EAE, which did not enter remission after immunization with myelin oligodendrocyte glycoprotein (MOG). However, CD40-stimulated B cells expression of IL-10 from the control group was able to relieve the EAE [13]. Hussai et al. reported that transfusion of BCR-stimulated IL-10-producing B cells protects NOD mice from type 1 diabetes in part by attenuating the severity of islet inflammation, suggested that this mechanism may play a greater role in protecting the animal from type 1 diabetes [27]. The human study presented here demonstrates Breg cell participation in MCNS pathogenesis, and suggests a potential mechanism related to IL-10 activity. of CD19+CD5+CD1d+ cells and the levels of serum albumin was observed in these patients. Collectively, these findings suggest that B cells may participate in the pathogenic process of MCNS.

Conclusion
Despite limitations of small sample size, IL-10-specific testing, one time measurements and a lack of longitudinal follow-up, our findings provide valuable new insights into understanding the pathogenesis of MCNS which may aid in developing new measures for prognosis. Future studies will further investigate the role of Breg cells in the pathogenic process of MCNS and the potential prognostic values.