IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases

Abstract

B lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has been associated primarily with interleukin 10 (IL-10) because B-cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens. Here we identify IL-35-producing B cells as key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T-cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a markedly improved resistance to infection with the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as shown by their superior containment of the bacterial growth and their prolonged survival after primary infection, and upon secondary challenge, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an increased function of B cells as antigen-presenting cells (APCs). During Salmonella infection, IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM(+)CD138(hi)TACI(+)CXCR4(+)CD1d(int)Tim1(int) plasma cells expressing the transcription factor Blimp1 (also known as Prdm1). During EAE, CD138(+) plasma cells were also the main source of B-cell-derived IL-35 and IL-10. Collectively, our data show the importance of IL-35-producing B cells in regulation of immunity and highlight IL-35 production by B cells as a potential therapeutic target for autoimmune and infectious diseases. This study reveals the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease.

Figure 1. B cells secrete IL-35 upon activation via TLR4 and CD40

a, EAE was induced in B-TLR2^−/− (grey squares; n=8), B-TLR4^−/− (black triangles; n=8), and B-WT mice (grey circles; n=16) by immunization with MOG_35-55 peptide in complete Freund’s adjuvant. Data show clinical EAE scores from two independent experiments (mean ± SEM). Cumulative disease scores were compared using unpaired t-test. b, Splenic B cells from IL-10.eGFP mice were stimulated for 72 h with LPS (1 μg/ml) or LPS (1 μg/ml)+αCD40 (10 μg/ml), and eGFP expression was measured by flow cytometry. Plots show eGFP expression by live CD19⁺ cells. Results are representative of three independent experiments. c, Hierarchical cluster analysis of secreted factors differentially expressed between B cells activated with LPS or LPS+αCD40 (Pearson correlation with average linkage). Affymetrix microarrays were performed in quadruplicates from splenic naïve B cells, and from B cells activated with LPS (1 μg/ml) or LPS (1 μg/ml)+αCD40 (10 μg/ml) for 24 h and 72 h. Expression levels of each gene is shown for each array compared to its average value for the 20 arrays, with a scale ranging from two-fold increase (yellow) to two-fold decrease (blue) compared to average. d, p35 mRNA expression was quantified by real-time PCR in LN and spleen from naïve C57BL/6 and B cell-deficient JHT mice, as well as in B cells purified from LN and spleen of C57BL/6 mice. Data show the compilation of three independent experiments (mean ± SEM). e, Splenic B cells were activated as indicated for 72 h, and treated with GolgiStop for the last 4 h of culture. B cell lysates were separated on SDS-PAGE gel and blotted with anti-EBi3 or anti-actin antibody. Data show representative result from three independent experiments. f, B cells from C57BL/6 or p35-deficient mice were activated for 72 h with LPS+αCD40 (clone FGK-45; 10 μg/ml). Culture supernatants were subjected to immunoprecipitation with anti-p35 followed by Western blot with anti-EBi3 antibody. Data shown are representative of two independent experiments.

Figure 2. IL-35 expression by B cells is required for recovery from EAE

a, EAE was induced in: left panel: B-p35^−/− (grey squares; n=17) and B-WT mice (black circles; n=16), middle panel: B-EBi3^−/− (grey squares, n=9) and corresponding B-WT mice (black circles; n=5), right panel: B-p40^−/− (grey squares; n=10) and B-WT mice (black circles; n=16) by immunization with MOG_35-55 peptide in Complete Freund’s adjuvant. Data show clinical EAE scores from two independent experiments (mean ± SEM). Cumulative disease scores were compared using unpaired t-test. b, Splenocytes were harvested from mice on day 10 after EAE induction, and pooled before re-stimulation for 48 h with MOG_35-55 in increasing concentrations. Culture supernatants were analyzed by ELISA to determine IFN-γ and IL-17 concentrations. Data show representative result from two independent experiments. c, EAE was induced in B-p35^−/− and corresponding B-WT mice by immunization with MOG_35-55 peptide in Complete Freund’s adjuvant. B cells and CD4⁺CD25⁻ T cells (Teff) were isolated from pooled draining LN and spleens on day 10 after immunization. 5×10⁵ B cells from B-p35^−/− or B-WT mice were then cultured with 1×10⁴ Teff cells from B-p35^−/− or B-WT mice in presence of MOG_35-55 in increasing concentrations, as indicated. Proliferation was assessed after 64 h by ³H-thymidine incorporation. CPM: counts per minute. Data show representative results from two independent experiments. d, Supernatants from cultures as described in (c) were harvested after 48 h, and analyzed by Bio-Plex to determine the concentrations of IL-17 and GM-CSF. Data shown (mean ± SEM) are pooled from two independent experiments. b-d, Graphs show mean ± SEM. Results were compared using a Two-way ANOVA followed by a Bonferroni post-test (* p<0.05, ** p<0.01; *** p<0.001).

Figure 3. B cell-derived IL-35 enhances susceptibility to Salmonella typhimurium

a, Top panel shows survival curves of B-p35^−/− (n=14) and their corresponding B-WT mice (n=16), B-EBi3^−/− (n=12) and their corresponding B-WT mice (n=13), B-p40^−/− (n=13) and their corresponding control B-WT mice (n=14) after i.v. infection with 100 colony-forming units (CFU) virulent Salmonella typhimurium strain (SL1344). Data are pooled from two independent experiments for each panel. Survival curves were compared using the Wilcoxon test. The bottom panel shows survival curves of B-p35^−/− (n=15) and their corresponding B-WT mice (n=16), B-EBi3^−/− (n=15) and their corresponding B-WT mice (n=15), B-p40^−/− (n=11) and their corresponding control B-WT mice (n=14), which were vaccinated with attenuated Salmonella (SL7207) and 90 days later re-challenged with 100 CFU virulent Salmonella (SL1344). Data are pooled from two independent experiments. Survival curves were compared using the Wilcoxon test. b, (left panel) representative FACS plot of mononuclear phagocytes (MP) gated as CD11b⁺Ly6C^hi cells among live splenocytes from a B-WT mouse at day 6 p.i. with SL1344; (right panel) frequencies of MP per spleen at day 6 p.i. in B-p35^−/−, B-EBi3^−/−, and B-p40^−/− mice together with their corresponding control B-WT mice. Numbers of mice analyzed: B-p35^−/− (n=6) and their corresponding B-WT mice (n=8), B-EBi3^−/− (n=7) and their corresponding B-WT mice (n=5), B-p40^−/− (n=8) and their corresponding control B-WT mice (n=8). Data are pooled from two independent experiments. Graphs show mean ± SEM. Data were analyzed with unpaired t-test. p-values > 0.05 are considered as non significant (ns). c, Indicated groups of mice were infected with attenuated Salmonella (SL7207). After 21 days cells from bone marrow were stained for surface CD4 and intracellular CD40L or IFN-γ after 6 h re-stimulation with heat-killed Salmonella. The left panel shows representative FACS plots of IFN-γ⁺ cells among CD4⁺ T cells from B-WT and B-p35^−/− mice. Middle and right panels show, respectively, frequencies of IFN-γ⁺ and CD40L⁺ cells among CD4⁺ T cells. Data shown are pooled from two independent experiments with the following total number of mice: B-p35^−/− (n=12) and their corresponding B-WT mice (n=10), B-EBi3^−/− (n=8) and their corresponding B-WT mice (n=9), B-p40^−/− (n=15) and their corresponding control B-WT mice (n=9). Graphs show mean ± SEM. Data were analyzed with an unpaired t-test.

Figure 4. IL-10 and IL-35 are expressed by CD138^hi plasma cells during Salmonella typhimurium infection

a, Splenic plasma cells (CD138^hi) and B cells (CD19⁺CD138⁻) were isolated from C57BL/6 mice on days 0, 1, 3, 5, and 8 after infection with 10⁷ CFU attenuated Salmonella (SL7207). EBi3 and IL-10 mRNA expression were then quantified by real-time PCR. Data show fold induction of EBi3 (left) and IL-10 (right) mRNA expression in plasma and B cells during infection compared to naïve B cells. A compilation of five independent experiments is shown (mean ± SEM). b, Single cells of CD138^intCD22⁺, CD138^hiCD22⁺, and CD138^hiCD22⁻ plasma cells were sorted by FACS from C57BL/6 mice on day 3 after infection with 10⁷ CFU attenuated Salmonella (SL7207). A total of 208 CD138^intCD22⁺, 206 CD138^hiCD22⁺, and 189 CD138⁺CD22⁻ single cells gave a positive signal for β-actin, and were included in data shown. Data show percentages of IL-10⁺ (left), p35⁺EBi3⁺ (middle), and IL-10⁺p35⁺EBi3⁺ (right) cells among each subset. c, Blimp1 mRNA expression in those single cells analyzed in (c) were also detected by single-cell PCR. Data show the percentages of Blimp1⁺ cells among IL-10-expressing (left), as well as p35 and EBi3 co-expressing cells (right). d, CD138^hi plasma cells and CD19⁺CD138⁻ B cells were isolated from spleen of C57BL/6 mice on day 3 after infection with attenuated Salmonella (SL7207; 10⁷ CFU) using a combination of magnetic and FACS methods. Naïve B splenic B cells were isolated from unchallenged C57BL/6 mice by magnetic selection. Isolated cells were activated for 24 h with PMA+ionomycin, and IL-10 concentrations in culture supernatants were determined by Bio-Plex. Data shown are pooled from 5 independent experiments. Results were compared suing unpaired t-test. d, Splenic CD19⁺CD138⁻ B cells (B cells) and CD138⁺ plasma cells (PC) were isolated from spleens of C57BL/6 (WT) and p35^−/−EBi3^−/−p40^−/− (KO) mice on day 3 p.i. with attenuated Salmonella (SL7207; 10⁷ CFU). B cell lysates were separated on SDS-PAGE gel and blotted with anti-EBi3, anti-p35, or anti-actin antibodies. Data show results from two independent cell preparations for WT samples, and one preparation for p35^−/−EBi3^−/−p40^−/− B and plasma cells.