B cell-derived IL-10 suppresses inflammatory disease in Lyn-deficient mice

Abstract

Lyn kinase deficient mice represent a well established genetic model of autoimmune/autoinflammatory disease that resembles systemic lupus erythematosus. We report that IL-10 plays a crucial immunosuppressive role in this model, modulating the inflammatory component of the disease caused by myeloid and T-cell activation. Double-mutant lyn(-/-)IL-10(-/-) mice manifested severe splenomegaly and lymphadenopathy, dramatically increased proinflammatory cytokine production, and severe tissue inflammation. Single-mutant lyn(-/-)mice showed expansion of IL-10-producing B cells. Interestingly, WT B cells adoptively transferred into lyn(-/-) mice showed increased differentiation into IL-10-producing B cells that assumed a similar phenotype to endogenous lyn(-/-) IL-10-producing B cells, suggesting that the inflammatory environment present in lyn(-/-) mice induces IL-10-producing B-cell differentiation. B cells, but not T or myeloid cells, were the critical source of IL-10 able to reduce inflammation and autoimmunity in double mutant lyn(-/-)IL-10(-/-) mice. IL-10 secretion by B cells was also crucial to sustain transcription factor Forkhead Box P3 (Foxp3) expression in regulatory T cells during disease development. These data reveal a dominant immunosuppressive function of B-cell-derived IL-10 in the Lyn-deficient model of autoimmunity, extending our current understanding of the role of IL-10 and IL-10-producing B cells in systemic lupus erythematosus.

Fig. 1.

Lyn^−/− mice have increased IL-10 serum levels and manifest dramatic splenomegaly and lymphadenopathy as a consequence of IL-10 deficiency. (A) ELISA of IL-10 serum levels in 2-, 4-, and 6-month-old WT or lyn^−/− mice. Each symbol represents the IL-10 serum level of an individual mouse. Data are pooled from two independent time-course experiments. (B) Representative images of spleen and lymph nodes from 5- to 6-mo-old WT, lyn^−/−, IL-10^−/−, or lyn^−/−IL-10^−/− animals. Data are representative of 10 to 12 mice for each genotype analyzed at end-point experiments. (C) Each symbol represents the weight of an individual spleen (Left) or lymph node (Right) from 2-, 4-, and 6- month old WT, lyn^−/−, IL-10^−/−, or lyn^−/−IL-10^−/− mice. Data are pooled from two independent time-course experiments. (D) Single-cell suspensions of spleens (Left) and lymph nodes (Right) from 6-mo-old WT, lyn^−/−, IL-10^−/−, and lyn^−/−IL-10^−/− mice were prepared, counted, and stained for flow cytometric analysis. The absolute number of total, myeloid (CD11b⁺), total B (CD19⁺ plus CD19/B220^low/−CD138⁺ cells), and T (TCRβ⁺) cells is reported. Data are pooled from two separate time-course experiments and are expressed as mean ± SEM (n = 8–12 mice per time point). Statistical differences of lyn^−/−IL-10^−/− double-mutant versus lyn^−/− or IL-10^−/− single-mutant mice are reported (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 2.

IL-10 deficiency increases T-cell activation in lyn^−/− mice. (A–C) Single-cell suspensions of spleens from 5- to 6-mo-old WT, lyn^−/−, IL-10^−/−, and lyn^−/−IL-10^−/− mice were counted and stained for flow cytometric analysis. (A) The absolute number of TCRβ⁺CD69⁺, TCRβ⁺CD44^highCD62L⁻ (effector cells), TCRβ⁺IFNγ⁺, and TCRβ⁺IL-17⁺ cells is reported as evidence of T-cell activation. (B) The frequency (Left) and absolute number (Right) of splenic CD4⁺CD25⁺Foxp3⁺ Tregs are reported. Data are pooled from three independent end-point experiments and are expressed as mean ± SEM (n = 8–12 mice per group). (C) Each symbol represents the mean fluorescence intensity (MFI) of Foxp3 expression in splenic CD4⁺CD25⁺Foxp3⁺ Tregs (calculated as percentage of MFI expression relative to WT levels for each experiment) from individual lyn^−/−, IL-10^−/−, or lyn^−/−IL-10^−/− animals. Data are pooled from two independent end-point experiments. (D) Representative FACS histograms showing the MFI expression of Foxp3 in splenic CD4⁺CD25⁺Foxp3⁺ Tregs from mice of the indicated genotype. Data are representative of five to 10 mice for each genotype. Statistical differences of lyn^−/−IL-10^−/− double-mutant versus lyn^−/− or IL-10^−/− single mutant mice are reported (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 3.

IL-10 deficiency exacerbates the production of proinflammatory cytokines in lyn^−/− mice. Serum levels of the indicated cytokines (A) and BAFF (B) in 5- to 6-mo-old WT, lyn^−/−, IL-10^−/−, or lyn^−/−IL-10^−/− mice, assessed by fluorescent bead-based Luminex assays or ELISA, respectively. Data are pooled from three independent end-point experiments and are expressed as mean ± SEM (n = 8–12 mice per group). (C) Single-cell suspensions of spleens from 5- to 6-mo-old WT, lyn^−/−, IL-10^−/−, and lyn^−/−IL-10^−/− mice were counted and stained for flow cytometric analysis. The absolute number of the different subsets of CD11b⁺ myeloid cells was obtained by analyzing the expression of the following markers: CD11b^high7/4^intGR-1^high (granulocytes), CD11b^high7/4^highGR-1^low (monocytes), CD11b⁺F4/80⁺ (macrophages), and CD11b⁺CD11c^high (myeloid DCs; mDCs). Data are from one end-point experiment and are expressed as mean ± SEM (n = 4–5 mice per group). (D) Each symbol represents BAFF or IL-6 mRNA expression in splenic macrophages and DCs purified from the spleens of individual 5- to 6-mo-old WT, lyn^−/−, IL-10^−/−, and lyn^−/−IL-10^−/− mice as described in Methods. Data are pooled from two end-point experiments. (E) The absolute number of IL-6⁺, TNF-α⁺, IL-12 p40⁺, and IL-10⁺ CD11b⁺ myeloid cells in the spleen of 5- to 6-mo-old WT, lyn^−/−, IL-10^−/−, or lyn^−/−IL-10^−/− mice was determined by FACS analysis as described in Methods. Data are pooled from two end-point experiments and are expressed as mean ± SEM (n = 4–5 mice per group). Statistical differences of lyn^−/−IL-10^−/− double-mutant vs. lyn^−/− or IL-10^−/− single mutant mice are reported (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 4.

The frequency of IL-10–producing B cells is increased in lyn^−/− mice. (A–C) Single-cell suspensions of spleens from 2-, 4-, and 6-mo-old control tiger or lyn^−/−tiger mice were stained for flow cytometric analysis. The gates designed to analyze GFP expression were set by using regular WT or lyn^−/− mice as GFP⁻ controls. (A) The frequency of total IL-10–GFP⁺ cells (among total splenocytes). (B) The frequency of IL-10–GFP⁺ B cells (among total CD19⁺ plus CD19/B220^low/−CD138⁺ cells), IL-10–GFP⁺ T cells (among total TCRβ⁺ cells), and IL-10–GFP⁺ myeloid cells (among total CD11b⁺ cells). Data are pooled from two separate time course experiments and are expressed as mean ± SEM (n = 6–8 mice per group). Statistical differences of lyn^−/−tiger vs. control tiger mice are reported (*P < 0.05; **P < 0.01). (C) Resting CD19⁺ B cells were sorted from single-cell suspensions of spleens and lymph nodes from control tiger (CD45.2⁺) mice, and 10 × 10⁶ were injected i.v. into 6-mo-old congenic CD45.1⁺ WT or lyn^−/− mice. Representative flow plots of the frequency (of total CD45.2⁺ CD19⁺ plus CD19/B220^low/−CD138⁺ donor B cells) and the phenotype (expression of CD138, CD21, and CD23) of the donor CD45.2⁺IL-10–GFP⁻ B cells or CD45.2⁺IL-10–GFP⁺ B cells detected in the spleen of host CD45.1⁺ WT (Left) or lyn^−/−mice (Right) 1 mo after the adoptive transfers. Data are representative of four mice for each condition. (D) IL-10 protein release, assessed by ELISA, by CD19⁺ B cells isolated from spleen and lymph nodes of 2-mo-old WT or lyn^−/− mice and stimulated for 72 h in the presence or absence of 5 μg/mL LPS, CpG oligonucleotide, or anti-CD40 Abs.

Fig. 5.

B-cell–derived IL-10 is sufficient to reduce disease development in lyn^−/−IL-10^−/− mice. Two-month-old lyn^−/−IL-10^−/− mice were injected with WT or IL-10^−/− B cells (10 × 10⁶ CD19⁺ B cells per mouse) isolated from spleen and lymph nodes of 2-mo-old WT or IL-10^−/− animals. Two to 3 mo after the adoptive transfer, mice were killed and analyzed for signs of disease development. (A) Each symbol represents the weight of an individual spleen from lyn^−/−IL-10^−/− mice untreated or treated by adoptive transfer of WT or IL-10^−/− B cells. (B–D) Single-cell suspensions of spleens were prepared, counted, and stained for flow cytometric analysis. (B) The absolute number of total myeloid (CD11b⁺), total B (CD19⁺ plus CD19/B220^low/−CD138⁺ cells), and T (TCRβ⁺) cells is reported. (C) The absolute number of CD19⁺CD69⁺ and CD19/B220^low/−CD138⁺ (plasma cells/plasmablasts), TCRβ⁺CD69⁺, and TCRβ⁺CD44^highCD62L⁻ (effector cells) is reported as evidence of B- and T-cell activation. Data from WT B-cell transfers are pooled from three independent end-point experiments, whereas data from IL-10^−/− B-cell transfers were pooled from two end-point experiment. Data are expressed as mean ± SEM (n = 4–15 mice per group). (D) Each symbol represents the MFI of Foxp3 expression in splenic CD4⁺CD25⁺Foxp3⁺ Tregs (calculated as percentage of MFI expression relative to WT levels for each experiment) from individual lyn^−/−IL-10^−/− mice untreated or treated by adoptive transfers of WT or IL-10^−/− B cells. Data are pooled from three independent end-point experiments. Statistical differences of untreated lyn^−/−IL-10^−/− mice vs. lyn^−/−IL-10^−/− mice treated with adoptive transfers of WT or IL-10^−/− B cells are reported (*P < 0.05).

Fig. P1.

Proposed model to explain the role of IL-10 and IL-10–producing B cells (i.e., Bregs) in Lyn-deficient autoimmunity. We recently showed that the Lyn-deficient model of SLE is characterized by hyperactivated B cells and pathogenic interactions between myeloid cells and T cells that sustain inflammation, disease progression, and kidney disease. This inflammatory loop is mediated, in part, by B-cell activating factor (BAFF) production by myeloid cells and IFN-γ production by T cells (5). In this study, we demonstrated that IL-10 plays a crucial role in suppressing disease development in lyn^−/− mice. IL-10 deficiency results in a strong exacerbation of myeloid cell proliferation and proinflammatory cytokine production, as well as T-cell activation and differentiation into T helper type 1 and T helper type 17 subsets. IL-10 is also crucial in sustaining Foxp3 expression in Tregs in lyn^−/− mice. In this study, we identified B cells as the crucial source of IL-10 that reduces disease development in this mouse model of autoimmunity.