Interleukin-10 produced by B cells is crucial for the suppression of Th17/Th1 responses, induction of T regulatory type 1 cells and reduction of collagen-induced arthritis

Abstract

Introduction: Interleukin-10 (IL-10) producing B cells, also known as regulatory B (Breg) cells, play a key role in controlling autoimmunity. Our laboratory and others have demonstrated a pivotal role for Bregs in rheumatological disorders, including experimental models of arthritis and lupus. The aim of this study was to identify the role of endogenous IL-10 secreting B cells in vivo in controlling the induction and disease progression of collagen-induced arthritis (CIA).

Methods: We generated chimeric mice that had IL-10 knocked-out specifically in the B cell population. These mice were compared with wild-type (WT) B cell chimeric mice for their susceptibility to CIA.

Results: Here we report that chimeric mice specifically lacking IL-10 producing B cells (IL-10-/- B cell) developed an exacerbated CIA compared to chimeric wild type B cell (WT B cell) mice. A marked increase in inflammatory Th1 and Th17 cells were detected in IL-10-/-B cell mice compared to WT B cell mice. Furthermore, there was a reduction in IL-10 secreting CD4+ Tr1 cells in these animals.

Conclusions: IL-10 producing B cells restrain inflammation by promoting differentiation of immuno-regulatory over pro-inflammatory T cells and, hence, act to maintain tolerance.

Figure 1

Lack of endogenous B cell-derived IL-10 results in exacerbated CIA. A. Chimeric mice were generated that lacked IL-10 specifically on B cells. Mean clinical score for CIA and numbers of diseased paws are shown. Data are expressed as mean ± SEM (n = 20, cumulative data from four separate experiments with n = 5 per experimental group). Data were compared by statistical analysis using the Fisher test. * P < 0.05. B. On Day 45 following immunization, mice were bled and the serum levels of total IgG1 and IgG2a CII-specific IgG antibodies were measured by ELISA. Data are expressed as mean ± SEM (n = 20, cumulative data from four separate experiments with n = 5 per experimental group). Data were compared by statistical analysis using the unpaired t test. * P < 0.05. C. The gradation of arthritis was scored from 0 to 4 according to the intensity of lining layer hyperplasia, mononuclear cell infiltration and pannus formation. Data are expressed as mean ± SEM (n = 20, cumulative data from four separate experiments with n = 5 per experimental group). Representative histological sections are also shown.

Figure 2

Lack of B cell-derived IL-10 results in increased Th17 and Th1 responses. A. A total of 35 days after CIA induction, draining lymph node cells were excised and cultured with PMA plus ionomycin in the presence of Brefeldin A for five hours. The intracellular levels of IFNγ and IL-17 were measured. Dot plots are gated on the CD4⁺ population. B. Numbers indicate percentages or absolute numbers of cells in the quadrants. Data show mean ± SEM (n = 4), representative of four experiments. Data were compared by statistical analysis using the unpaired t test. *** P < 0.001. C. Supernatant was collected from lymph node cells stimulated in vitro for 48 hours with anti-CD3, and then secreted IFNγ and IL-17 was measured by cytokine FlowCytomix kit. Data show mean ± SEM (n = 4), representative of four experiments. Data were compared by statistical analysis using the unpaired t test. ** P < 0.01. D. Additionally, a BD in vivo cytokine capture assay was used to assess in vivo levels of IFNγ over a 12-hour period. The mean levels of IFNγ-conjugated to antibody in serum are shown ± SEM (n = 5), representative of two separate experiments. Data were compared by statistical analysis using the unpaired t test. * P < 0.05.

Figure 3

Lack of B cell-derived IL-10 results in decreased Tr1 responses, however FoxP3⁺ Tregs are unaffected. A. A total of 35 days after CIA induction, draining lymph node cells were excised and cultured with PMA plus ionomycin in the presence of Brefeldin A for five hours. The CD4+ T cell population was identified using anti-CD4 FITC mAb and intracellular levels of IL-10 were measured. B. Numbers indicate percentages of cells in the quadrants. Data shown mean ± SEM (n = 4), are representative of four experiments. Data were compared by statistical analysis using the unpaired t test. *** P < 0.001. In addition, supernatant was collected from lymph node cells stimulated in vitro for 48 hours with anti-CD3, and then secreted IL-10 was measured by cytokine FlowCytomix kit. Data show mean ± SEM (n = 4), and are representative of four experiments. Data were compared by statistical analysis using the unpaired t test. *** P < 0.001. C. Tregs were assessed using anti-CD25, anti-CD4 and anti-FoxP3 mAb. Numbers indicate percentages of CD4 gated FoxP3⁺ cells or the absolute number of cells in a draining lymph node. Data show mean ± SEM (n = 4), and are representative of four experiments. D. Spleens were taken from IL-10^-/- B cell and WT B cell animals and used to isolate CD25+ Treg cells using Milteyi Biotech magnetic beads. T-effector cells were isolated from immunized WT B6 mice using the same process. The cells were cultured for 72 hours with anti-CD3 antibody (1 μg/ml). For 12 hours before harvesting cells were pulsed with (³H) thymidine. Data shown are mean ± SEM of triplicate wells and are representative of two independent experiments. E. Additionally, a BD in vivo cytokine capture assay was used to assess in vivo levels of IL-2 over a 12-hour period. The mean levels of IL-2-conjugated to antibody in serum are shown ± SEM (n = 4), are representative of two separate experiments. Data were compared by statistical analysis using the unpaired t test.