The atypical IκB protein IκB(NS) is important for Toll-like receptor-induced interleukin-10 production in B cells

Abstract

Although a major function of B cells is to mediate humoral immunity by producing antigen-specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). However, the mechanism by which IL-10 is induced in B cells has not been fully elucidated. Here, we report that IκBNS , an inducible nuclear IκB protein, is important for Toll-like receptor (TLR)-mediated IL-10 production in B cells. Studies using IκB(NS) knockout mice revealed that the number of IL-10-producing B cells is reduced in IκB(NS)(-/-) spleens and that the TLR-mediated induction of cytoplasmic IL-10-positive cells and IL-10 secretion in B cells are impaired in the absence of IκB(NS). The impairment of IL-10 production by a lack of IκB(NS) was not observed in TLR-triggered macrophages or T-cell-receptor-stimulated CD4(+) CD25(+) T cells. In addition, IκB(NS)-deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL-10(+) CD138(+) plasmablasts. These results suggest that IκB(NS) is selectively required for IL-10 production in B cells responding to TLR signals, so defining an additional role for IκB(NS) in the control of the B-cell-mediated immune responses.

Keywords: IκBNS; Toll-like receptors; interleukin-10-producing B cells.

Figure 1

Iκ B_NS ^−/− mice contain reduced numbers of interleukin‐10 (IL‐10)‐producing B cells in the spleen. (a) Flow cytometric analysis of CD1d^hi CD5⁺ B cells in spleen from wild‐type (Iκ B_NS ^+/+, WT) and Iκ B_NS‐deficient (Iκ B_NS ^−/−, KO) mice. Representative FACS profiles of CD19⁺ spleen B cells for CD1d and CD5 are shown. Numbers represent the percentage of cells in indicated gates. (b) The frequency of CD1d^hi CD5⁺ cells in CD19⁺ splenic B cells (left) and the numbers of CD1d^hi CD5⁺ B cells in wild‐type and Iκ B_NS ^−/− spleens (right) are shown. (c) Flow cytometric analysis of IL‐10‐producing B cells in spleen. Splenocytes from wild‐type and Iκ B_NS ^−/− mice were incubated with lipopolysaccharide (LPS) plus PMA, A23187 and GolgiPlug for 5 hr. The cells were then stained with anti‐CD19 and anti‐IL‐10. Representative dot plots show the frequencies of cytoplasmic IL‐10⁺ cells in CD19⁺ B cells. (d) The frequency of cytoplasmic IL‐10⁺ cells in CD19⁺ splenic B cells (left) and the number of IL‐10 producing B cells in the spleen (right) are shown. For (a) and (b), data are representative of at least three independent experiments. For (b) and (d), each symbol indicates an individual mouse. Horizontal bars represent the mean. n > 5; **P < 0·01.

Figure 2

B‐cell‐intrinsic deficiency in Iκ B_NS results in the impaired generation of interleukin‐10 (IL‐10) ‐producing B cells in vivo. bone marrow (BM) cells from B6.μMT mice were mixed with BM cells from wild‐type or Iκ B_NS ^−/− mice at a ratio of 4:1, and 1 × 10⁷ BM cells were transferred intravenously into irradiated B6.μMT mice. Lipopolysaccharide (LPS) ‐induced IL‐10 production in donor‐derived B cells was examined 8 weeks post transfer by the daily administration of LPS (10 μg, intraperitoneally) for 3 days. Controls received PBS alone. Two days after the last inoculum, splenocytes were stained with surface CD19 and intracellular IL‐10. (a) Representative FACS profiles of CD19⁺ splenocytes for the expression of cytoplasmic IL‐10 with the percentages of cells in gates. (b) The frequency of IL‐10‐producing cells in CD19⁺ splenic B cells. Data from four control and eight LPS‐treated mice are shown. Each symbol shows an individual mouse, and horizontal bars represent the mean. Two independent experiments were performed, and similar results were obtained. **P < 0·01.

Figure 3

Iκ B_NS‐deficient B cells fail to produce lipopolysaccharide (LPS) ‐induced interleukin‐10 (IL‐10). (a) Splenic B cells from wild‐type and Iκ B_NS ^−/− mice were cultured with various concentrations of LPS for 3 days. The cells were then FACS stained with anti‐CD19 and anti‐IL‐10. Representative FACS profiles of CD19⁺ B cells for cytoplasmic IL‐10 with the frequencies of cells in indicated gates are shown. (b) The amount of IL‐10 in the culture supernatant of LPS‐stimulated B cells. Splenic B cells were stimulated with LPS alone or LPS plus PMA, A23187 for 3 days. (c) Splenic B cells from wild‐type and Iκ B_NS ^−/− mice were activated with LPS (5 μg/ml) for 1 day, and they were co‐cultured with wild‐type T cells under stimulation with anti‐CD3ε and anti‐CD28 for 2 days. The expression level of IL‐10 in B cells and the expression levels of IL‐10 and interferon‐γ (IFN‐γ) in CD3ε ⁺ T cells were analysed by flow cytometry. Representative FACS plots with the frequencies of cells in indicated gates are shown. The bar graph shows the frequencies of IFN‐γ ⁺ cells in CD3ε ⁺ T cells (dot columns), IL‐10⁺ cells in CD19⁺ B cells (filled columns), and IL‐10⁺ cells in CD3ε ⁺ T cells (open columns) in the co‐culture. *P < 0·05, **P < 0·01. (d) Purified CD1d^hi CD5⁺ B cells from wild‐type and Iκ B_NS ^−/− spleens were incubated with or without LPS for 2 days, and the amount of IL‐10 in the culture supernatant was measured by ELISA. (e) Purified CD21^hi CD23^lo marginal zone (MZ) B cells and CD21⁺ CD23^hi follicular B cells from wild‐type and Iκ B_NS ^−/− spleens were incubated with LPS (5 μg/ml) for 4 days, and the amount of IL‐10 in the culture supernatant was measured by ELISA. The data shown were obtained from triplicate assay and represent at least two independent experiments. Error bars represent the standard deviation.

Figure 4

Toll‐like receptor (TLR) ‐induced interleukin‐10 (IL‐10) production is impaired in Iκ B_NS‐deficient B cells. (a) Splenic B cells isolated from wild‐type and Iκ B_NS ^−/− spleens were incubated for 2 days in the presence of the indicated reagents [5 μg/ml lipopolysaccharide (LPS), 5 μg/ml anti‐CD40, 10 μg/ml anti‐IgM, 10 ng/ml rIL‐4]. The proliferative activity of B cells was measured using a cell counting kit‐8 (top). Culture supernatant was used for ELISA to determine the amounts of IL‐10 (middle) and IL‐6 (bottom). (b) Purified splenic B cells were cultured with various TLR agonists for 2 days to collect supernatants or for 2 hr for the RT‐PCR analysis. The semi‐quantitative RT‐PCR analysis shows the TLR‐induced expression of Nfkbid in wild‐type B cells. β‐Actin was used as a loading control. The concentration of each TLR agonist is described in the Materials and methods. (c) Naive macrophages in the peritoneal cavity were collected from wild‐type and Iκ B_NS ^−/− mice and stimulated with the indicated amounts of LPS for 2 days. (d) CD4⁺ CD25⁻ conventional T cells (25⁻) and CD4⁺ CD25⁺ regulatory T cells (25⁺) from wild‐type and Iκ B_NS ^−/− lymph nodes were FACS sorted and stimulated with or without anti‐CD3ε and anti‐CD28 for 3 days. The amount of cytokines in the culture supernatants was determined by a triplicate ELISA. Data represent at least two independent experiments.

Figure 5

Expression of cytokine genes and nuclear factors in lipopolysaccharide (LPS) ‐activated Iκ B_NS‐deficient B cells. (a, b) The LPS‐induced transcription of Il10 is reduced in Iκ B_NS‐deficient B cells during the first 24 hr of induction. Total RNA was isolated from purified splenic B cells stimulated with 5 μg/ml of LPS for the indicated periods, and the semi‐quantitative RT‐PCR assessment of cytokine gene transcripts was performed. The LPS‐induced transcription kinetics of Nfkbid, Il10 and Il6 genes (a) and cytokine expression profiles within 24 hr (b) are shown. (c) The expression and nuclear localization of Iκ B_NS, NF‐κBp65, and p50 were analysed based on the immunoblotting of nuclear extracts prepared from LPS‐stimulated wild‐type and Iκ B_NS ^−/− B cells. The nuclear extracts were also used for DNA pull down with biotinylated probes for two interleukin‐10 (IL‐10) promoters containing a putative nuclear factor‐κB (NF‐κB)‐binding site. (d) Western blot analysis of DNA pull down for the nuclear lysates and IL‐10‐promoter probes containing proximal (−55/−46) or distal (−861/−851) NF‐κB motif. (e) The expression of NFATc1 was analysed based on the immunoblotting of nuclear extracts prepared from LPS‐stimulated wild‐type and Iκ B_NS ^−/− B cells. Iκ B_NS shows two and NFATc1 shows several bands depending on their phosphorylation status. Lamin B was used as a nuclear loading control. All data represent at least two independent experiments.

Figure 6

Toll‐like receptor (TLR) ‐induced differentiation of IL‐10⁺ CD138⁺ plasmablasts is impaired in the absence of Iκ B_NS. (a, b) Real‐time PCR analysis of the expression of Prdm1 and Irf4. FACS‐sorted CD1d^hi CD5⁺ B cells (a) and CD21^hi CD23^lo MZ B cells (b) from wild‐type and Iκ B_NS ^−/− spleens were cultured for the indicated periods in the presence of 5 μg/ml lipopolysaccharide (LPS), and total RNA was isolated and subjected to quantitative PCR analysis. All expression data were normalized to β‐actin expression. The data are representative of two independent experiments. *P < 0·05, **P < 0·01. (c) Splenic B cells isolated from wild‐type and Iκ B_NS ^−/− mice were stimulated with LPS (1·0 μg/ml) for 2 or 3 days, and the expression levels of surface CD138, CD44, and cytoplasmic interleukin 10 (IL‐10) on CD19⁺ B cells were analysed by flow cytometry. (d) Splenic B cells from wild‐type and Iκ B_NS ^−/− mice were incubated with various TLR agonists for 3 days, and the expression of CD138 and cytoplasmic IL‐10 were analysed by flow cytometry. Representative FACS profiles with the percentages of cells in quadrant gates are shown. The data represent three independent experiments.