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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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rIL-35 induced in vivo expansion of Breg cells and suppressed EAU developmentEAU was induced in C57BL/6 mice and eyes were analyzed at day-21 post-immunization by funduscopy or histology. (a) Fundus images of the retina (top): Black-arrow, inflammation with blurred optic-disc margins (papilledema); Blue-arrows, retinal vasculitis; White-arrows, retinal/choroidal infiltrates. H&E histological sections: Scale bar, 500 μM. OPN, optic nerve; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE/CH retinal pigmented epithelial and choroid. Open white-arrow, lymphocytes; black-arrowhead, enlarged retinal blood vessels; Blue-asterisk, retinal-folds. EAU scores were based on changes at the retina, optic nerve disc and choroid (ONLINE METHODS). (b-d) Intracellular-cytokine analysis of IL-17- or IFN-γ-expressing T cells in draining LN (b) or IL-10-producing B cells in spleen (c, d) on day-21 post-immunization. Spleen cells isolated from control, pMIB, rIL-35-treated EAU mice were also analyzed for IL-35 (p35 and Ebi3) expression by RT-PCR (e) or intracellular cytokine staining assay (f). (g) B cells from untreated, pMIB-treated or rIL-35-treated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 and CD19+ B-cells were isolated on a cell sorter. The purified CD19+ B cells were co-cultured with uveitogenic LN and spleen cells for 3 days in medium containing IRBP and the cells (1×107) were then transferred into naïve syngeneic mice. Fundus images of the eyes and EAU scores recorded 10 days after adoptive transfer. Data represents at least 3 independent experiments (**P< 0.01; ***P <0.001; ****P< 0.0001).
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Figure 3: rIL-35 induced in vivo expansion of Breg cells and suppressed EAU developmentEAU was induced in C57BL/6 mice and eyes were analyzed at day-21 post-immunization by funduscopy or histology. (a) Fundus images of the retina (top): Black-arrow, inflammation with blurred optic-disc margins (papilledema); Blue-arrows, retinal vasculitis; White-arrows, retinal/choroidal infiltrates. H&E histological sections: Scale bar, 500 μM. OPN, optic nerve; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE/CH retinal pigmented epithelial and choroid. Open white-arrow, lymphocytes; black-arrowhead, enlarged retinal blood vessels; Blue-asterisk, retinal-folds. EAU scores were based on changes at the retina, optic nerve disc and choroid (ONLINE METHODS). (b-d) Intracellular-cytokine analysis of IL-17- or IFN-γ-expressing T cells in draining LN (b) or IL-10-producing B cells in spleen (c, d) on day-21 post-immunization. Spleen cells isolated from control, pMIB, rIL-35-treated EAU mice were also analyzed for IL-35 (p35 and Ebi3) expression by RT-PCR (e) or intracellular cytokine staining assay (f). (g) B cells from untreated, pMIB-treated or rIL-35-treated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 and CD19+ B-cells were isolated on a cell sorter. The purified CD19+ B cells were co-cultured with uveitogenic LN and spleen cells for 3 days in medium containing IRBP and the cells (1×107) were then transferred into naïve syngeneic mice. Fundus images of the eyes and EAU scores recorded 10 days after adoptive transfer. Data represents at least 3 independent experiments (**P< 0.01; ***P <0.001; ****P< 0.0001).

Mentions: We next induced EAU, a rodent model of human uveitis25,26 and investigated whether rIL-35 can be used to treat EAU. Mice were treated with pMIB or rIL-35 (100ng/mouse) and disease severity was assessed on day-21 post-immunization as described (Supplementary Methods). Histology and fundus images of control mice (no treatment) or mice that received pMIB revealed severe inflammation characterized by papilledema, retinal vasculitis, retinal folds, substantial infiltration of inflammatory cells into the vitreous and chorio-retinal infiltrates (Fig.3a). In contrast, rIL-35-treated mice had trace or very mild EAU with significantly lower EAU scores (Fig.3a). Amelioration of EAU was accompanied by significant reduction of Th17 and Th1 cells in the draining LN (Fig.3b) with concomitant increase of Breg cells (Fig.3c). Breg cells comprised ~5.18% of the B-cells in spleen and draining LNs of rIL-35-treated EAU mice compared to ~0.91% in mice treated with pMIB (Fig.3d) and the Breg population in rIL-35-treated mice contained IL-35+Breg-cells (Fig.3e), suggesting that IL-35 induces both Breg and IL-35+Breg cells in vivo. Although there is no unique marker, or set of markers, that exclusively identifies Breg cells8, we observed a >4-folds increase of CD1dhiCD5+ B-cells during EAU (Fig.3f), a Breg cell phenotype previously shown to suppress inflammation in a contact hypersensitivity model27. Importantly, administration of Breg cells from rIL-35-treated mice suppressed EAU induced by adoptive transfer of IRBP-specific pathogenic T cells (Fig. 3g), consistent with recent reports showing that Breg cells restrain excessive inflammation during autoimmune diseases8,28. Collectively, these observations provide evidence that IL-35 induced Breg and IL-35+Breg cells that suppress and ameliorate uveitis.


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)

rIL-35 induced in vivo expansion of Breg cells and suppressed EAU developmentEAU was induced in C57BL/6 mice and eyes were analyzed at day-21 post-immunization by funduscopy or histology. (a) Fundus images of the retina (top): Black-arrow, inflammation with blurred optic-disc margins (papilledema); Blue-arrows, retinal vasculitis; White-arrows, retinal/choroidal infiltrates. H&E histological sections: Scale bar, 500 μM. OPN, optic nerve; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE/CH retinal pigmented epithelial and choroid. Open white-arrow, lymphocytes; black-arrowhead, enlarged retinal blood vessels; Blue-asterisk, retinal-folds. EAU scores were based on changes at the retina, optic nerve disc and choroid (ONLINE METHODS). (b-d) Intracellular-cytokine analysis of IL-17- or IFN-γ-expressing T cells in draining LN (b) or IL-10-producing B cells in spleen (c, d) on day-21 post-immunization. Spleen cells isolated from control, pMIB, rIL-35-treated EAU mice were also analyzed for IL-35 (p35 and Ebi3) expression by RT-PCR (e) or intracellular cytokine staining assay (f). (g) B cells from untreated, pMIB-treated or rIL-35-treated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 and CD19+ B-cells were isolated on a cell sorter. The purified CD19+ B cells were co-cultured with uveitogenic LN and spleen cells for 3 days in medium containing IRBP and the cells (1×107) were then transferred into naïve syngeneic mice. Fundus images of the eyes and EAU scores recorded 10 days after adoptive transfer. Data represents at least 3 independent experiments (**P< 0.01; ***P <0.001; ****P< 0.0001).
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Related In: Results  -  Collection

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Figure 3: rIL-35 induced in vivo expansion of Breg cells and suppressed EAU developmentEAU was induced in C57BL/6 mice and eyes were analyzed at day-21 post-immunization by funduscopy or histology. (a) Fundus images of the retina (top): Black-arrow, inflammation with blurred optic-disc margins (papilledema); Blue-arrows, retinal vasculitis; White-arrows, retinal/choroidal infiltrates. H&E histological sections: Scale bar, 500 μM. OPN, optic nerve; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE/CH retinal pigmented epithelial and choroid. Open white-arrow, lymphocytes; black-arrowhead, enlarged retinal blood vessels; Blue-asterisk, retinal-folds. EAU scores were based on changes at the retina, optic nerve disc and choroid (ONLINE METHODS). (b-d) Intracellular-cytokine analysis of IL-17- or IFN-γ-expressing T cells in draining LN (b) or IL-10-producing B cells in spleen (c, d) on day-21 post-immunization. Spleen cells isolated from control, pMIB, rIL-35-treated EAU mice were also analyzed for IL-35 (p35 and Ebi3) expression by RT-PCR (e) or intracellular cytokine staining assay (f). (g) B cells from untreated, pMIB-treated or rIL-35-treated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 and CD19+ B-cells were isolated on a cell sorter. The purified CD19+ B cells were co-cultured with uveitogenic LN and spleen cells for 3 days in medium containing IRBP and the cells (1×107) were then transferred into naïve syngeneic mice. Fundus images of the eyes and EAU scores recorded 10 days after adoptive transfer. Data represents at least 3 independent experiments (**P< 0.01; ***P <0.001; ****P< 0.0001).
Mentions: We next induced EAU, a rodent model of human uveitis25,26 and investigated whether rIL-35 can be used to treat EAU. Mice were treated with pMIB or rIL-35 (100ng/mouse) and disease severity was assessed on day-21 post-immunization as described (Supplementary Methods). Histology and fundus images of control mice (no treatment) or mice that received pMIB revealed severe inflammation characterized by papilledema, retinal vasculitis, retinal folds, substantial infiltration of inflammatory cells into the vitreous and chorio-retinal infiltrates (Fig.3a). In contrast, rIL-35-treated mice had trace or very mild EAU with significantly lower EAU scores (Fig.3a). Amelioration of EAU was accompanied by significant reduction of Th17 and Th1 cells in the draining LN (Fig.3b) with concomitant increase of Breg cells (Fig.3c). Breg cells comprised ~5.18% of the B-cells in spleen and draining LNs of rIL-35-treated EAU mice compared to ~0.91% in mice treated with pMIB (Fig.3d) and the Breg population in rIL-35-treated mice contained IL-35+Breg-cells (Fig.3e), suggesting that IL-35 induces both Breg and IL-35+Breg cells in vivo. Although there is no unique marker, or set of markers, that exclusively identifies Breg cells8, we observed a >4-folds increase of CD1dhiCD5+ B-cells during EAU (Fig.3f), a Breg cell phenotype previously shown to suppress inflammation in a contact hypersensitivity model27. Importantly, administration of Breg cells from rIL-35-treated mice suppressed EAU induced by adoptive transfer of IRBP-specific pathogenic T cells (Fig. 3g), consistent with recent reports showing that Breg cells restrain excessive inflammation during autoimmune diseases8,28. Collectively, these observations provide evidence that IL-35 induced Breg and IL-35+Breg cells that suppress and ameliorate uveitis.

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