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Interleukin-35 induces regulatory B cells that suppress autoimmune disease.

Wang RX, Yu CR, Dambuza IM, Mahdi RM, Dolinska MB, Sergeev YV, Wingfield PT, Kim SH, Egwuagu CE - Nat. Med. (2014)

Bottom Line: The mechanisms mediating the induction and development of Breg cells remain unclear.Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10.In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits.

View Article: PubMed Central - PubMed

Affiliation: 1] Molecular Immunology Section, Laboratory of Immunology, National Eye Institute (NEI), National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China.

ABSTRACT
Interleukin-10 (IL-10)-producing regulatory B (Breg) cells suppress autoimmune disease, and increased numbers of Breg cells prevent host defense to infection and promote tumor growth and metastasis by converting resting CD4(+) T cells to regulatory T (Treg) cells. The mechanisms mediating the induction and development of Breg cells remain unclear. Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10. Treatment of mice with IL-35 conferred protection from experimental autoimmune uveitis (EAU), and mice lacking IL-35 (p35 knockout (KO) mice) or defective in IL-35 signaling (IL-12Rβ2 KO mice) produced less Breg cells endogenously or after treatment with IL-35 and developed severe uveitis. Adoptive transfer of Breg cells induced by recombinant IL-35 suppressed EAU when transferred to mice with established disease, inhibiting pathogenic T helper type 17 (TH17) and TH1 cells while promoting Treg cell expansion. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits. As IL-35 also induced the conversion of human B cells into Breg cells, these findings suggest that IL-35 may be used to induce autologous Breg and IL-35(+) Breg cells and treat autoimmune and inflammatory disease.

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IL-35 preferentially induced CD5+CD19+B220lo Breg cells and a unique IL-35-producing Breg subpopulation (IL-35+Breg)(a) Purified CD19+ B cells were stimulated for 3 days in medium containing 50ng/ml pMIB or rIL-35 and numbers in quadrants indicate the percent of B220+ B cells expressing Ebi3, p35 and/or IL-10. (b) IL-10− or IL-10+ B cells enriched with a Breg Isolation Kit (see Methods section) were analyzed by intracellular cytokine staining assay. (c,d) Activated B-cells were stimulated with rIL-35 and subjected to chromatin immunoprecipitation (ChiP) assay (c) or RT-PCR (d). (e) Purified B cells were stimulated for 3 days in medium containing pMIB or IL-35 and expression of B220, CD19, CD5, Foxp3 or Tim-1 and/or IL-10 was analyzed by intracellular cytokine staining assay or cell surface FACS analysis. (f, g) C57BL/6 mice were injected with LPS (15 μg/mouse) and/or rIL-35 (1μg/mouse) and after 4 days splenic cells were analyzed by FACS. (h) Absolute numbers of B220loCD19+CD5+ or B220loCD19+CD5+IL-10+ cells in the spleen. Results are representative of at least 3 independent experiments (****P<0.05; **P<0.01; ***P < 0.001).
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Figure 2: IL-35 preferentially induced CD5+CD19+B220lo Breg cells and a unique IL-35-producing Breg subpopulation (IL-35+Breg)(a) Purified CD19+ B cells were stimulated for 3 days in medium containing 50ng/ml pMIB or rIL-35 and numbers in quadrants indicate the percent of B220+ B cells expressing Ebi3, p35 and/or IL-10. (b) IL-10− or IL-10+ B cells enriched with a Breg Isolation Kit (see Methods section) were analyzed by intracellular cytokine staining assay. (c,d) Activated B-cells were stimulated with rIL-35 and subjected to chromatin immunoprecipitation (ChiP) assay (c) or RT-PCR (d). (e) Purified B cells were stimulated for 3 days in medium containing pMIB or IL-35 and expression of B220, CD19, CD5, Foxp3 or Tim-1 and/or IL-10 was analyzed by intracellular cytokine staining assay or cell surface FACS analysis. (f, g) C57BL/6 mice were injected with LPS (15 μg/mouse) and/or rIL-35 (1μg/mouse) and after 4 days splenic cells were analyzed by FACS. (h) Absolute numbers of B220loCD19+CD5+ or B220loCD19+CD5+IL-10+ cells in the spleen. Results are representative of at least 3 independent experiments (****P<0.05; **P<0.01; ***P < 0.001).

Mentions: IL-35 induces an IL-35-producing regulatory T-cell population called iTR35 that suppresses inflammation16. We therefore examined whether rIL-35 could induce IL-35-producing B-cells. LPS-stimulated B-cells co-express Ebi3 and p35 (IL-35+) and following stimulation with rIL-35 the frequency of the Ebi3/p35-expressing B-cell population increased tremendously from 7.8% to 35.3% (Fig.2a; left panels) and 17.8% of the IL-35-producing B-cells also expressed IL-10 (Fig.2a; right panels). Henceforth we refer to this B-cell subset co-expressing p35 and Ebi3 as IL-35+Breg. Analysis of IL-10− and IL-10+ B-cells revealed that approximately 20% of the Breg-cells produced IL-35 and frequency of the IL-35+Bregs increased by >50% following stimulation with rIL-35 (Fig.2b). We further show that rIL-35 induced the binding of STAT1 to p35 or Ebi3 proximal promoter in B-cells (Fig.2c), up-regulated p35 and Ebi3 mRNA expression (Fig.2d, Supplementary Fig.2i) and secretion of IL-35 by Breg cells (Supplementary Fig.3). Interestingly, substantial percentage rIL-35-induced Breg cells express cell-surface CD5 and/or Tim-1 but not Foxp3 (Fig.2e). We examined effects of rIL-35 in vivo by injecting mice with LPS and/or rIL-35 and found that IL-35 induced a substantial decrease of B220+ B-cells (42.68% to 27.92%) and this was accompanied by appearance of a prominent population of CD5+B220lo B-cells in mice treated with rIL-35 but not with LPS alone (see red arrow in Fig.2f). Analysis of B220loCD5+ B-cell compartment further revealed that rIL-35 preferentially induces expansion of CD19+CD5+B220lo Breg cells (Fig.2g,2h). Together, these results suggest that IL-35 induces IL-35+Breg and while rIL-35 suppressed the proliferation of conventional B220+ B-cells, it selectively induced expansion of CD19+CD5+B220lo Breg cells in-vivo.


Interleukin-35 induces regulatory B cells that suppress autoimmune disease.

Wang RX, Yu CR, Dambuza IM, Mahdi RM, Dolinska MB, Sergeev YV, Wingfield PT, Kim SH, Egwuagu CE - Nat. Med. (2014)

IL-35 preferentially induced CD5+CD19+B220lo Breg cells and a unique IL-35-producing Breg subpopulation (IL-35+Breg)(a) Purified CD19+ B cells were stimulated for 3 days in medium containing 50ng/ml pMIB or rIL-35 and numbers in quadrants indicate the percent of B220+ B cells expressing Ebi3, p35 and/or IL-10. (b) IL-10− or IL-10+ B cells enriched with a Breg Isolation Kit (see Methods section) were analyzed by intracellular cytokine staining assay. (c,d) Activated B-cells were stimulated with rIL-35 and subjected to chromatin immunoprecipitation (ChiP) assay (c) or RT-PCR (d). (e) Purified B cells were stimulated for 3 days in medium containing pMIB or IL-35 and expression of B220, CD19, CD5, Foxp3 or Tim-1 and/or IL-10 was analyzed by intracellular cytokine staining assay or cell surface FACS analysis. (f, g) C57BL/6 mice were injected with LPS (15 μg/mouse) and/or rIL-35 (1μg/mouse) and after 4 days splenic cells were analyzed by FACS. (h) Absolute numbers of B220loCD19+CD5+ or B220loCD19+CD5+IL-10+ cells in the spleen. Results are representative of at least 3 independent experiments (****P<0.05; **P<0.01; ***P < 0.001).
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Figure 2: IL-35 preferentially induced CD5+CD19+B220lo Breg cells and a unique IL-35-producing Breg subpopulation (IL-35+Breg)(a) Purified CD19+ B cells were stimulated for 3 days in medium containing 50ng/ml pMIB or rIL-35 and numbers in quadrants indicate the percent of B220+ B cells expressing Ebi3, p35 and/or IL-10. (b) IL-10− or IL-10+ B cells enriched with a Breg Isolation Kit (see Methods section) were analyzed by intracellular cytokine staining assay. (c,d) Activated B-cells were stimulated with rIL-35 and subjected to chromatin immunoprecipitation (ChiP) assay (c) or RT-PCR (d). (e) Purified B cells were stimulated for 3 days in medium containing pMIB or IL-35 and expression of B220, CD19, CD5, Foxp3 or Tim-1 and/or IL-10 was analyzed by intracellular cytokine staining assay or cell surface FACS analysis. (f, g) C57BL/6 mice were injected with LPS (15 μg/mouse) and/or rIL-35 (1μg/mouse) and after 4 days splenic cells were analyzed by FACS. (h) Absolute numbers of B220loCD19+CD5+ or B220loCD19+CD5+IL-10+ cells in the spleen. Results are representative of at least 3 independent experiments (****P<0.05; **P<0.01; ***P < 0.001).
Mentions: IL-35 induces an IL-35-producing regulatory T-cell population called iTR35 that suppresses inflammation16. We therefore examined whether rIL-35 could induce IL-35-producing B-cells. LPS-stimulated B-cells co-express Ebi3 and p35 (IL-35+) and following stimulation with rIL-35 the frequency of the Ebi3/p35-expressing B-cell population increased tremendously from 7.8% to 35.3% (Fig.2a; left panels) and 17.8% of the IL-35-producing B-cells also expressed IL-10 (Fig.2a; right panels). Henceforth we refer to this B-cell subset co-expressing p35 and Ebi3 as IL-35+Breg. Analysis of IL-10− and IL-10+ B-cells revealed that approximately 20% of the Breg-cells produced IL-35 and frequency of the IL-35+Bregs increased by >50% following stimulation with rIL-35 (Fig.2b). We further show that rIL-35 induced the binding of STAT1 to p35 or Ebi3 proximal promoter in B-cells (Fig.2c), up-regulated p35 and Ebi3 mRNA expression (Fig.2d, Supplementary Fig.2i) and secretion of IL-35 by Breg cells (Supplementary Fig.3). Interestingly, substantial percentage rIL-35-induced Breg cells express cell-surface CD5 and/or Tim-1 but not Foxp3 (Fig.2e). We examined effects of rIL-35 in vivo by injecting mice with LPS and/or rIL-35 and found that IL-35 induced a substantial decrease of B220+ B-cells (42.68% to 27.92%) and this was accompanied by appearance of a prominent population of CD5+B220lo B-cells in mice treated with rIL-35 but not with LPS alone (see red arrow in Fig.2f). Analysis of B220loCD5+ B-cell compartment further revealed that rIL-35 preferentially induces expansion of CD19+CD5+B220lo Breg cells (Fig.2g,2h). Together, these results suggest that IL-35 induces IL-35+Breg and while rIL-35 suppressed the proliferation of conventional B220+ B-cells, it selectively induced expansion of CD19+CD5+B220lo Breg cells in-vivo.

Bottom Line: The mechanisms mediating the induction and development of Breg cells remain unclear.Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10.In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits.

View Article: PubMed Central - PubMed

Affiliation: 1] Molecular Immunology Section, Laboratory of Immunology, National Eye Institute (NEI), National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China.

ABSTRACT
Interleukin-10 (IL-10)-producing regulatory B (Breg) cells suppress autoimmune disease, and increased numbers of Breg cells prevent host defense to infection and promote tumor growth and metastasis by converting resting CD4(+) T cells to regulatory T (Treg) cells. The mechanisms mediating the induction and development of Breg cells remain unclear. Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10. Treatment of mice with IL-35 conferred protection from experimental autoimmune uveitis (EAU), and mice lacking IL-35 (p35 knockout (KO) mice) or defective in IL-35 signaling (IL-12Rβ2 KO mice) produced less Breg cells endogenously or after treatment with IL-35 and developed severe uveitis. Adoptive transfer of Breg cells induced by recombinant IL-35 suppressed EAU when transferred to mice with established disease, inhibiting pathogenic T helper type 17 (TH17) and TH1 cells while promoting Treg cell expansion. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits. As IL-35 also induced the conversion of human B cells into Breg cells, these findings suggest that IL-35 may be used to induce autologous Breg and IL-35(+) Breg cells and treat autoimmune and inflammatory disease.

Show MeSH
Related in: MedlinePlus