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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 mediates its biological effects on B-cells by activating STAT1 and STAT3 pathways through an IL-35 receptor comprising of IL-12Rβ2 and IL-27Rα(a) WEHI-279 B-cell line were transduced with control siRNA or IL-12Rβ1-, IL-12Rβ2-, IL-27Rα- or gp130-specific siRNA and after 3 days total RNA was analyzed by RT-PCR. (b, c) siRNA-treated B-cells were cultured in medium containing pMIB or rIL-35 for 3 days and B-cell proliferation (b) or rIL-35-induced production of IL-10 (c) was assessed by [3H]-thymidine incorporation assays or ELISA, respectively. (d, e) Primary B cells from WT, IL12Rβ2KO or IL27RαKO mice were stimulated by LPS in the presence of pMIB or rIL-35 and analyzed by [3H]-thymidine incorporation assay (d) or intracellular cytokine IL-10 staining assay (e). (f) WEHI-279 cells were cultured overnight in medium containing rIL-35 and co-expression of IL-27Rα and IL-12Rβ2 was detected by Western blotting and immunoprecipitation using anti-IL-27Rα anti-Ebi3 or control isotype-specific IgG Abs. (g) Primary B cells were transduced with IL-12Rβ1-, IL-12Rβ2-, IL-27Rα-, gp130 or both IL-12Rβ2- and IL-27Rα-siRNA. The cells were then stimulated in presence of pMIB or rIL-35 and expression of p35 and Ebi3 was detected by RT-PCR analysis. (h, i) Primary T cells (h) or B cells (i) from WT C57BL/6 mice were stimulated with anti-CD3/CD28 or LPS, respectively, and cells were then washed and starved for 2 h in serum free medium (0.5% BSA). Cells were then stimulated for 30 minutes with pMIB, rIL-35 or IL-12 and analyzed for STAT activation by Western blotting. Data represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001; ****P<0.0001).
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Figure 6: IL-35 mediates its biological effects on B-cells by activating STAT1 and STAT3 pathways through an IL-35 receptor comprising of IL-12Rβ2 and IL-27Rα(a) WEHI-279 B-cell line were transduced with control siRNA or IL-12Rβ1-, IL-12Rβ2-, IL-27Rα- or gp130-specific siRNA and after 3 days total RNA was analyzed by RT-PCR. (b, c) siRNA-treated B-cells were cultured in medium containing pMIB or rIL-35 for 3 days and B-cell proliferation (b) or rIL-35-induced production of IL-10 (c) was assessed by [3H]-thymidine incorporation assays or ELISA, respectively. (d, e) Primary B cells from WT, IL12Rβ2KO or IL27RαKO mice were stimulated by LPS in the presence of pMIB or rIL-35 and analyzed by [3H]-thymidine incorporation assay (d) or intracellular cytokine IL-10 staining assay (e). (f) WEHI-279 cells were cultured overnight in medium containing rIL-35 and co-expression of IL-27Rα and IL-12Rβ2 was detected by Western blotting and immunoprecipitation using anti-IL-27Rα anti-Ebi3 or control isotype-specific IgG Abs. (g) Primary B cells were transduced with IL-12Rβ1-, IL-12Rβ2-, IL-27Rα-, gp130 or both IL-12Rβ2- and IL-27Rα-siRNA. The cells were then stimulated in presence of pMIB or rIL-35 and expression of p35 and Ebi3 was detected by RT-PCR analysis. (h, i) Primary T cells (h) or B cells (i) from WT C57BL/6 mice were stimulated with anti-CD3/CD28 or LPS, respectively, and cells were then washed and starved for 2 h in serum free medium (0.5% BSA). Cells were then stimulated for 30 minutes with pMIB, rIL-35 or IL-12 and analyzed for STAT activation by Western blotting. Data represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001; ****P<0.0001).

Mentions: We used siRNA to specifically silence receptor subunits utilized by IL-12 family cytokines (Fig.6a) and examined effect of silencing each receptor subunit on rIL-35-mediated suppression of B-cell proliferation or IL-10 induction. Silencing IL-12Rβ1 or gp130 subunit did not affect IL-35-mediated inhibition of B-cell proliferation (Fig.6b) or IL-10 induction (Fig.6c). Furthermore, anti-gp130-neutralizing Abs did not block IL-35-induced inhibition of B-cell proliferation (Supplementary Fig.4a) or suppress IL-35-induced production of IL-10 (Supplementary Fig.4b). In contrast, silencing of IL-12Rβ2 and IL-27Rα completely abrogated the effects of IL-35 on B-cell proliferation and IL-10 production (Fig.6b,6c). We confirmed requirement of these receptors in IL-12Rβ2- or IL-27Rα-deficient B-cells as rIL-35 could not inhibit proliferation of IL-12Rβ2- or IL-27Rα deficient B-cells (Fig.6d) or induce expansion of Breg cells (Fig.6e). Moreover, reciprocal coimmunoprecipitation analysis confirmed the expression and interaction of IL-12Rβ2 and IL-27Rα on IL-35-stimulated B-cells (Fig.6f). Furthermore, generation of IL-35+Bregs required IL-35-induced signaling through IL-12Rβ2 or IL-27Rα chains (Fig.6g). Western blot analysis also revealed that rIL-35 preferentially activates STAT1, STAT3 and STAT4 in T-cells (Fig.6h) while in B-cells it signals primarily through STAT1 and STAT3 but not STAT4 (Fig.6i; Supplementary Fig.4c,4d). Taken together, these results support the notion that IL-35 signals in B-cells through IL-12Rβ2 and IL-27Rα and suggest that IL-35 mediates its biological activities in T- and B-cells through differential activation of overlapping and distinct STAT pathways.


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 mediates its biological effects on B-cells by activating STAT1 and STAT3 pathways through an IL-35 receptor comprising of IL-12Rβ2 and IL-27Rα(a) WEHI-279 B-cell line were transduced with control siRNA or IL-12Rβ1-, IL-12Rβ2-, IL-27Rα- or gp130-specific siRNA and after 3 days total RNA was analyzed by RT-PCR. (b, c) siRNA-treated B-cells were cultured in medium containing pMIB or rIL-35 for 3 days and B-cell proliferation (b) or rIL-35-induced production of IL-10 (c) was assessed by [3H]-thymidine incorporation assays or ELISA, respectively. (d, e) Primary B cells from WT, IL12Rβ2KO or IL27RαKO mice were stimulated by LPS in the presence of pMIB or rIL-35 and analyzed by [3H]-thymidine incorporation assay (d) or intracellular cytokine IL-10 staining assay (e). (f) WEHI-279 cells were cultured overnight in medium containing rIL-35 and co-expression of IL-27Rα and IL-12Rβ2 was detected by Western blotting and immunoprecipitation using anti-IL-27Rα anti-Ebi3 or control isotype-specific IgG Abs. (g) Primary B cells were transduced with IL-12Rβ1-, IL-12Rβ2-, IL-27Rα-, gp130 or both IL-12Rβ2- and IL-27Rα-siRNA. The cells were then stimulated in presence of pMIB or rIL-35 and expression of p35 and Ebi3 was detected by RT-PCR analysis. (h, i) Primary T cells (h) or B cells (i) from WT C57BL/6 mice were stimulated with anti-CD3/CD28 or LPS, respectively, and cells were then washed and starved for 2 h in serum free medium (0.5% BSA). Cells were then stimulated for 30 minutes with pMIB, rIL-35 or IL-12 and analyzed for STAT activation by Western blotting. Data represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001; ****P<0.0001).
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Figure 6: IL-35 mediates its biological effects on B-cells by activating STAT1 and STAT3 pathways through an IL-35 receptor comprising of IL-12Rβ2 and IL-27Rα(a) WEHI-279 B-cell line were transduced with control siRNA or IL-12Rβ1-, IL-12Rβ2-, IL-27Rα- or gp130-specific siRNA and after 3 days total RNA was analyzed by RT-PCR. (b, c) siRNA-treated B-cells were cultured in medium containing pMIB or rIL-35 for 3 days and B-cell proliferation (b) or rIL-35-induced production of IL-10 (c) was assessed by [3H]-thymidine incorporation assays or ELISA, respectively. (d, e) Primary B cells from WT, IL12Rβ2KO or IL27RαKO mice were stimulated by LPS in the presence of pMIB or rIL-35 and analyzed by [3H]-thymidine incorporation assay (d) or intracellular cytokine IL-10 staining assay (e). (f) WEHI-279 cells were cultured overnight in medium containing rIL-35 and co-expression of IL-27Rα and IL-12Rβ2 was detected by Western blotting and immunoprecipitation using anti-IL-27Rα anti-Ebi3 or control isotype-specific IgG Abs. (g) Primary B cells were transduced with IL-12Rβ1-, IL-12Rβ2-, IL-27Rα-, gp130 or both IL-12Rβ2- and IL-27Rα-siRNA. The cells were then stimulated in presence of pMIB or rIL-35 and expression of p35 and Ebi3 was detected by RT-PCR analysis. (h, i) Primary T cells (h) or B cells (i) from WT C57BL/6 mice were stimulated with anti-CD3/CD28 or LPS, respectively, and cells were then washed and starved for 2 h in serum free medium (0.5% BSA). Cells were then stimulated for 30 minutes with pMIB, rIL-35 or IL-12 and analyzed for STAT activation by Western blotting. Data represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001; ****P<0.0001).
Mentions: We used siRNA to specifically silence receptor subunits utilized by IL-12 family cytokines (Fig.6a) and examined effect of silencing each receptor subunit on rIL-35-mediated suppression of B-cell proliferation or IL-10 induction. Silencing IL-12Rβ1 or gp130 subunit did not affect IL-35-mediated inhibition of B-cell proliferation (Fig.6b) or IL-10 induction (Fig.6c). Furthermore, anti-gp130-neutralizing Abs did not block IL-35-induced inhibition of B-cell proliferation (Supplementary Fig.4a) or suppress IL-35-induced production of IL-10 (Supplementary Fig.4b). In contrast, silencing of IL-12Rβ2 and IL-27Rα completely abrogated the effects of IL-35 on B-cell proliferation and IL-10 production (Fig.6b,6c). We confirmed requirement of these receptors in IL-12Rβ2- or IL-27Rα-deficient B-cells as rIL-35 could not inhibit proliferation of IL-12Rβ2- or IL-27Rα deficient B-cells (Fig.6d) or induce expansion of Breg cells (Fig.6e). Moreover, reciprocal coimmunoprecipitation analysis confirmed the expression and interaction of IL-12Rβ2 and IL-27Rα on IL-35-stimulated B-cells (Fig.6f). Furthermore, generation of IL-35+Bregs required IL-35-induced signaling through IL-12Rβ2 or IL-27Rα chains (Fig.6g). Western blot analysis also revealed that rIL-35 preferentially activates STAT1, STAT3 and STAT4 in T-cells (Fig.6h) while in B-cells it signals primarily through STAT1 and STAT3 but not STAT4 (Fig.6i; Supplementary Fig.4c,4d). Taken together, these results support the notion that IL-35 signals in B-cells through IL-12Rβ2 and IL-27Rα and suggest that IL-35 mediates its biological activities in T- and B-cells through differential activation of overlapping and distinct STAT pathways.

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