A Lower Proportion of Regulatory B Cells in Patients with Henoch-Schoenlein Purpura Nephritis

Abstract

Background: Henoch-Schoenlein purpura is the one of most common types of systemic vasculitis that involves impaired renal function and Henoch-Schoenlein purpura nephritis (HSPN). The diagnosis of this condition is largely based on immunohistologic detection of immunoglobulin A1-containing immune complex in the glomerular deposits of mesangium. Despite clinical advances, the etiopathogenesis of HSPN is still largely unknown.

Methods: In this study, we enrolled 25 newly diagnosed HSPN patients and 14 healthy controls. Then, fractions of B cell subtypes were determined in venous blood using flow cytometry. The serum interleukin (IL)-10 concentration was determined by enzyme-linked immunosorbent assay.

Results: Compared to those in healthy controls, the numbers of CD38+CD19+, CD86+CD19+, CD38+CD86+CD19+, and CD95+CD19+ B cells per microliter of blood were significantly higher in HSPN patients. In contrast, the numbers of CD5+CD19+, IL-10+CD19+, CD5+CD1d+CD19+, and IL-10+CD5+CD1d+CD19+ B cells per microliter of blood and the serum IL-10 concentration were significantly lower in HSPN patients. Following treatment, the numbers of CD38+CD19+ and CD86+CD19+ B cells per microliter of blood were significantly reduced in HSPN patients. However, the numbers of CD5+CD1d+CD19+, CD5+CD1d+IL-10+CD19+, and IL-10+CD19+ B cells per microliter of blood and the serum IL-10 concentration were significantly increased in HSPN patients following treatment. The estimated glomerular filtration rate (eGFR) was negatively correlated with the number of CD38+CD19+ B cells but positively correlated with the numbers of IL-10+CD19+, CD1d+CD5+CD19+, and IL-10+CD1d+CD5+CD19+B cells per microliter of blood and the serum IL-10 concentration. The 24-h urinary protein concentration was positively correlated with the number of CD38+CD19+B cells but negatively correlated with the numbers of IL-10+CD19+, CD1d+CD5+CD19+, and IL-10+CD1d+CD5+CD19+B cells per microliter of blood and the serum IL-10 concentration.

Conclusion: Our results suggest that CD38+CD19+ and CD1d+CD5+CD19+ B cells (Bregs) contribute to the pathogenesis of HSPN.

Fig 1. Flow cytometric analysis of the numbers of different subsets of B cells.

PBMCs were collected from HSPN patients and healthy controls. Then, they were stained with PerCP-anti-CD19, PE-anti-CD38, APC-anti-CD86, or PerCP-anti-CD19, PE-anti-CD27, and APC-anti-CD95 (Biolegend, San Diego, CA, USA), or isotype-matched control IgG (Beckton Dickinson, San Jose, CA, USA). For further analysis of different subsets of B cells, the cells were gated initially on living lymphocytes and then on CD19⁺ B cells. (A) Flow cytometric analysis results. (B) The numbers of CD19⁺ B cells. (C) The numbers of CD38⁺CD19⁺ plasma cells. (D) The numbers of CD86⁺CD19⁺ B cells. (E) The numbers of CD38⁺CD86⁺CD19⁺ B cells. (F)The numbers of CD95⁺CD19⁺ B cells. Data are expressed as the means for individual subjects included in two separate experiments. (G) Mean fluorescence intensity of CD86 on B cells. (H) Mean fluorescence intensity of CD95 on B cells. (I) Mean fluorescence intensity of CD95 on CD27⁺ B cell subsets. (J) Mean fluorescence intensity of CD95 on CD27^- B cell subsets. The horizontal lines represent the median values. Data were analyzed by Mann-Whitney U-test.

Fig 2. Flow cytometric analysis of Bregs isolated from PBMCs of HSPN patients and healthy controls.

The cells were stained with anti-CD19, anti-CD5, anti-CD1d, and intracellular anti-IL-10 or isotype-matched IgG. The cells were characterized by flow cytometric analysis; living lymphocytes were gated initially, followed by gating on CD19⁺ B cells. Subsequently, we analyzed the numbers of CD5⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺ B cells among CD19⁺ B cells, and at least 30,000 events per sample were analyzed. (A) Flow cytometric analysis. (B) The numbers of CD5⁺CD19⁺ B cells. (C) The numbers of IL-10⁺CD19⁺ B cells. (D) The numbers of CD1d⁺CD5⁺CD19⁺ B cells. (E) The numbers of IL-10⁺CD1d⁺CD5⁺CD19⁺ B cells. (F) Serum concentrations of IL-10. Data are expressed as the means or concentrations for individual subjects that participated in two separate experiments. The horizontal lines represent the median values of each group. Data were analyzed by the Mann-Whitney U-test.

Fig 3. Correlation analysis of clinico-pathological features of HSPN patients, based on the percentages of CD19⁺ B cell subtypes.

(A) The eGFR was negatively correlated with the number of CD38⁺CD19⁺ B cells. (B) The eGFR was positively correlated with the numbers of IL-10⁺CD19⁺ B cells, (C) CD1d⁺CD5⁺CD19⁺ B cells (D), and IL-10⁺CD1d⁺CD5⁺CD19⁺ B cells (E) as well as the serum concentration of IL-10. (F) The 24-h urinary protein concentration was positively correlated with the number of CD38⁺CD19⁺ B cells. (G) The 24-h urinary protein concentration was negatively correlated with the numbers of IL-10⁺CD19⁺ B cells, (H) CD1d⁺CD5⁺CD19⁺ B cells, and (I) IL-10⁺CD1d⁺CD5⁺CD19⁺ B cells as well as the (J) serum concentration of IL-10. The potential correlations among the numbers of B cells of different subsets, Bregs, and the values of clinical parameters were analyzed by the Spearman correlation tests.

Fig 4. Correlations among different subsets of B cells and the serum concentration of IL-10 in HSPN patients.

Potential correlations among the numbers of B cells of different subsets and the serum concentration of IL-10 were analyzed by the Spearman correlation tests. Data are expressed as the means or concentrations for individual subjects that participated in two separate experiments. (A-B) The number of CD38⁺CD19⁺ B cells was negatively correlated with the serum IL-10 level and the percentage of IL-10⁺CD19⁺ B cells. (C-D) The number of CD86⁺CD19⁺ B cells was negatively correlated with the serum IL-10 level and the number of IL-10⁺CD19⁺ B cells. (E-F) The serum IL-10 level was positively correlated with the numbers of CD1d⁺CD5⁺CD19⁺ and IL-10⁺CD1d⁺CD5⁺CD19⁺ B cells.

Fig 5. The change in the frequency of B cell subtypes and the serum concentration of IL-10 in HSPN patients following treatment.

Differences in patients pre- and post-treatment were analyzed by the Wilcoxon test. Data are expressed as the means or concentrations for individual subjects who participated in two separate experiments. (A) Serum level IL-10 in individual patients’ pre- and post-treatment. (B-F) The numbers of CD38⁺CD19⁺, CD86⁺CD19⁺, IL-10⁺CD19⁺, CD5⁺CD1d⁺CD19⁺, and CD5⁺CD1d⁺IL-10⁺CD19⁺ B cells of individual patients in the pre- and post-treatment stages.

Fig 6. Correlation between the number of CD38⁺CD19⁺ B cells and the serum IgA concentration in HSPN patients.

(A) Serum IgA concentrations in HSPN patients and healthy controls. Data are expressed as the means or concentrations for individual patients who participated in two separate experiments. The horizontal lines indicate the median values for each group. (B) The serum IgA concentration was positively correlated with the number of CD38⁺CD19⁺ B cells. Analyzed by Spearman correlation tests.