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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-signaling is required for suppressive functions of Breg and i35-Breg cells(a) EAU was induced in WT, p35KO, IL-12Rβ2KO or IL-10KO mice. Fundus images (top panels) and H&E-stained sections (bottom panels) of eyes enucleated 21 days after disease induction. Scale bar, 500 μM. (b) Analysis of IL-17- or IFN-γ-expressing T cells in the draining LN. (c) B cells from indicated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 in medium containing pMIB or rIL-35 and percentage of Breg cells was determined by intracellular cytokine assay. (d) CD19+ B cells from (c) were co-cultured with draining LN cells from WT EAU mice (1:5) for 4 days in medium containing IRBP or pMIB and analyzed by [3H]-thymidine incorporation assay. (e, f) Ex-vivo activated B-cells from (c) were co-cultured with uveitogenic LN T cells (1:5) for 3 days and cells were then transferred to naïve congenic CD45.1+ mice. Fundus images and EAU scores were obtained 10 days after adoptive transfer (e) and the percentage of CD4+CD45.2+ T cells, IFN-γ- or IL-17-expressing CD4+CD45.2+ T cells in the draining LNs was determined by cell surface and intracellular cytokine staining (f). (g, h) Balb/c or Ebi3KO (Balb/c background) mice were immunized with IRBP/CFA and eyes enucleated on day-21 post-immunization were analyzed by fundoscopy or histology and red-arrowheads indicate blood vessels. Scale bar, 500 μM (g). (h) IL-17, IFN-γ or IL-10-expressing LN CD4+ T cells or IL-10-producing splenic B cells were analyzed by intracellular cytokine staining assay. (i) EAU was induced in WT or muMT mice by active immunization with IRBP/CFA and the mice were treated with rIL-35 as described (Supplementary Methods) and eyes examined by fundoscopy on day 21 post-immunization. Results represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001).
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Figure 5: IL-35-signaling is required for suppressive functions of Breg and i35-Breg cells(a) EAU was induced in WT, p35KO, IL-12Rβ2KO or IL-10KO mice. Fundus images (top panels) and H&E-stained sections (bottom panels) of eyes enucleated 21 days after disease induction. Scale bar, 500 μM. (b) Analysis of IL-17- or IFN-γ-expressing T cells in the draining LN. (c) B cells from indicated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 in medium containing pMIB or rIL-35 and percentage of Breg cells was determined by intracellular cytokine assay. (d) CD19+ B cells from (c) were co-cultured with draining LN cells from WT EAU mice (1:5) for 4 days in medium containing IRBP or pMIB and analyzed by [3H]-thymidine incorporation assay. (e, f) Ex-vivo activated B-cells from (c) were co-cultured with uveitogenic LN T cells (1:5) for 3 days and cells were then transferred to naïve congenic CD45.1+ mice. Fundus images and EAU scores were obtained 10 days after adoptive transfer (e) and the percentage of CD4+CD45.2+ T cells, IFN-γ- or IL-17-expressing CD4+CD45.2+ T cells in the draining LNs was determined by cell surface and intracellular cytokine staining (f). (g, h) Balb/c or Ebi3KO (Balb/c background) mice were immunized with IRBP/CFA and eyes enucleated on day-21 post-immunization were analyzed by fundoscopy or histology and red-arrowheads indicate blood vessels. Scale bar, 500 μM (g). (h) IL-17, IFN-γ or IL-10-expressing LN CD4+ T cells or IL-10-producing splenic B cells were analyzed by intracellular cytokine staining assay. (i) EAU was induced in WT or muMT mice by active immunization with IRBP/CFA and the mice were treated with rIL-35 as described (Supplementary Methods) and eyes examined by fundoscopy on day 21 post-immunization. Results represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001).

Mentions: We induced EAU in p35KO, IL-12Rβ2KO or IL-10KO mice and examined whether loss of IL-35 or its signaling component29 would compromise generation of Breg-cells and exacerbate uveitis. Compared to WT, IL-12Rβ2KO mice developed severe EAU (Fig.5a) with marked expansion of Th17 (Fig.5b). Also, B-cells from IL-12Rβ2KO or IL-10KO could not induce Breg cells in response to rIL-35 (Fig.5c) or suppress proliferation of uveitogenic T-cells (Fig.5d). Disease in p35KO mice was comparable to WT and it is not clear whether the modest effect of p35-deficiency stemmed from the fact that IL-12 (utilizes p35 subunit) is required for the induction and exacerbation of EAU30. Use of p35KO or IL-12Rβ2KO mice to examine requirement of IL-35 for in-vivo Breg generation or Breg/IL-35+Breg-mediated EAU suppression is complicated because IL-12Rβ2KO and p35KO mice also have defective IL-12 signaling. We therefore sorted B-cells from WT, p35KO, IL-12Rβ2KO or IL-10KO EAU mice, stimulated them ex-vivo with rIL-35 and investigated whether they could suppress EAU induced by adoptive transfer of uveitogenic cells. Mice that received pMIB-treated B-cells from WT or rIL-35-treated B-cells from p35KO, IL-12Rβ2KO or IL-10KO mice developed EAU with relatively high EAU scores while mice that received rIL-35-treated B cells from WT mice were protected from EAU pathology (Fig.5e) and had marked reduction of Th17 cells (Fig. 5f). Although the Balb/c mouse strain is very resistant to EAU induction31, Ebi3KO mice on a Balb/c background developed EAU characterized by optic neuritis, papilledema, retinal vasculitis and hemorrhage (Fig.5g) and had 3.6-folds less Breg cells (Fig.5h). Nonetheless, Ebi3KO mice produce Breg cells, albeit at very low frequency, suggesting existence of alternative pathways that induce Breg cells expansion13,14. Requirement of Breg/IL-35+Breg-mediated suppression of uveitis is further underscored by development of severe EAU by B-cell deficient mice (muMT) that have intact Treg compartment. While rIL-35 suppressed EAU in WT mice, rIL-35 treatment could not effectively ameliorate uveitis in muMT mice (Fig.5i). Although the data indicates that Breg/IL-35+Breg cells possess intrinsic immunosuppressive activities, it also suggests that there is a meaningful non-B-cell IL-35 driven suppressor response 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-signaling is required for suppressive functions of Breg and i35-Breg cells(a) EAU was induced in WT, p35KO, IL-12Rβ2KO or IL-10KO mice. Fundus images (top panels) and H&E-stained sections (bottom panels) of eyes enucleated 21 days after disease induction. Scale bar, 500 μM. (b) Analysis of IL-17- or IFN-γ-expressing T cells in the draining LN. (c) B cells from indicated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 in medium containing pMIB or rIL-35 and percentage of Breg cells was determined by intracellular cytokine assay. (d) CD19+ B cells from (c) were co-cultured with draining LN cells from WT EAU mice (1:5) for 4 days in medium containing IRBP or pMIB and analyzed by [3H]-thymidine incorporation assay. (e, f) Ex-vivo activated B-cells from (c) were co-cultured with uveitogenic LN T cells (1:5) for 3 days and cells were then transferred to naïve congenic CD45.1+ mice. Fundus images and EAU scores were obtained 10 days after adoptive transfer (e) and the percentage of CD4+CD45.2+ T cells, IFN-γ- or IL-17-expressing CD4+CD45.2+ T cells in the draining LNs was determined by cell surface and intracellular cytokine staining (f). (g, h) Balb/c or Ebi3KO (Balb/c background) mice were immunized with IRBP/CFA and eyes enucleated on day-21 post-immunization were analyzed by fundoscopy or histology and red-arrowheads indicate blood vessels. Scale bar, 500 μM (g). (h) IL-17, IFN-γ or IL-10-expressing LN CD4+ T cells or IL-10-producing splenic B cells were analyzed by intracellular cytokine staining assay. (i) EAU was induced in WT or muMT mice by active immunization with IRBP/CFA and the mice were treated with rIL-35 as described (Supplementary Methods) and eyes examined by fundoscopy on day 21 post-immunization. Results represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001).
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Figure 5: IL-35-signaling is required for suppressive functions of Breg and i35-Breg cells(a) EAU was induced in WT, p35KO, IL-12Rβ2KO or IL-10KO mice. Fundus images (top panels) and H&E-stained sections (bottom panels) of eyes enucleated 21 days after disease induction. Scale bar, 500 μM. (b) Analysis of IL-17- or IFN-γ-expressing T cells in the draining LN. (c) B cells from indicated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 in medium containing pMIB or rIL-35 and percentage of Breg cells was determined by intracellular cytokine assay. (d) CD19+ B cells from (c) were co-cultured with draining LN cells from WT EAU mice (1:5) for 4 days in medium containing IRBP or pMIB and analyzed by [3H]-thymidine incorporation assay. (e, f) Ex-vivo activated B-cells from (c) were co-cultured with uveitogenic LN T cells (1:5) for 3 days and cells were then transferred to naïve congenic CD45.1+ mice. Fundus images and EAU scores were obtained 10 days after adoptive transfer (e) and the percentage of CD4+CD45.2+ T cells, IFN-γ- or IL-17-expressing CD4+CD45.2+ T cells in the draining LNs was determined by cell surface and intracellular cytokine staining (f). (g, h) Balb/c or Ebi3KO (Balb/c background) mice were immunized with IRBP/CFA and eyes enucleated on day-21 post-immunization were analyzed by fundoscopy or histology and red-arrowheads indicate blood vessels. Scale bar, 500 μM (g). (h) IL-17, IFN-γ or IL-10-expressing LN CD4+ T cells or IL-10-producing splenic B cells were analyzed by intracellular cytokine staining assay. (i) EAU was induced in WT or muMT mice by active immunization with IRBP/CFA and the mice were treated with rIL-35 as described (Supplementary Methods) and eyes examined by fundoscopy on day 21 post-immunization. Results represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001).
Mentions: We induced EAU in p35KO, IL-12Rβ2KO or IL-10KO mice and examined whether loss of IL-35 or its signaling component29 would compromise generation of Breg-cells and exacerbate uveitis. Compared to WT, IL-12Rβ2KO mice developed severe EAU (Fig.5a) with marked expansion of Th17 (Fig.5b). Also, B-cells from IL-12Rβ2KO or IL-10KO could not induce Breg cells in response to rIL-35 (Fig.5c) or suppress proliferation of uveitogenic T-cells (Fig.5d). Disease in p35KO mice was comparable to WT and it is not clear whether the modest effect of p35-deficiency stemmed from the fact that IL-12 (utilizes p35 subunit) is required for the induction and exacerbation of EAU30. Use of p35KO or IL-12Rβ2KO mice to examine requirement of IL-35 for in-vivo Breg generation or Breg/IL-35+Breg-mediated EAU suppression is complicated because IL-12Rβ2KO and p35KO mice also have defective IL-12 signaling. We therefore sorted B-cells from WT, p35KO, IL-12Rβ2KO or IL-10KO EAU mice, stimulated them ex-vivo with rIL-35 and investigated whether they could suppress EAU induced by adoptive transfer of uveitogenic cells. Mice that received pMIB-treated B-cells from WT or rIL-35-treated B-cells from p35KO, IL-12Rβ2KO or IL-10KO mice developed EAU with relatively high EAU scores while mice that received rIL-35-treated B cells from WT mice were protected from EAU pathology (Fig.5e) and had marked reduction of Th17 cells (Fig. 5f). Although the Balb/c mouse strain is very resistant to EAU induction31, Ebi3KO mice on a Balb/c background developed EAU characterized by optic neuritis, papilledema, retinal vasculitis and hemorrhage (Fig.5g) and had 3.6-folds less Breg cells (Fig.5h). Nonetheless, Ebi3KO mice produce Breg cells, albeit at very low frequency, suggesting existence of alternative pathways that induce Breg cells expansion13,14. Requirement of Breg/IL-35+Breg-mediated suppression of uveitis is further underscored by development of severe EAU by B-cell deficient mice (muMT) that have intact Treg compartment. While rIL-35 suppressed EAU in WT mice, rIL-35 treatment could not effectively ameliorate uveitis in muMT mice (Fig.5i). Although the data indicates that Breg/IL-35+Breg cells possess intrinsic immunosuppressive activities, it also suggests that there is a meaningful non-B-cell IL-35 driven suppressor response 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