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Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation.

Lin CC, Bradstreet TR, Schwarzkopf EA, Sim J, Carrero JA, Chou C, Cook LE, Egawa T, Taneja R, Murphy TL, Russell JH, Edelson BT - Nat Commun (2014)

Bottom Line: Bhlhe40 is required in vivo in a T cell-intrinsic manner, where it positively regulates the production of GM-CSF and negatively regulates the production of IL-10.In vitro, GM-CSF secretion is selectively abrogated in polarized Bhlhe40(-/-) TH1 and TH17 cells, and these cells show increased production of IL-10.These findings identify Bhlhe40 as a critical regulator of autoreactive T-cell pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri 63110, USA.

ABSTRACT
TH1 and TH17 cells mediate neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Pathogenic TH cells in EAE must produce the pro-inflammatory cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). TH cell pathogenicity in EAE is also regulated by cell-intrinsic production of the immunosuppressive cytokine interleukin 10 (IL-10). Here we demonstrate that mice deficient for the basic helix-loop-helix (bHLH) transcription factor Bhlhe40 (Bhlhe40(-/-)) are resistant to the induction of EAE. Bhlhe40 is required in vivo in a T cell-intrinsic manner, where it positively regulates the production of GM-CSF and negatively regulates the production of IL-10. In vitro, GM-CSF secretion is selectively abrogated in polarized Bhlhe40(-/-) TH1 and TH17 cells, and these cells show increased production of IL-10. Blockade of IL-10 receptor in Bhlhe40(-/-) mice renders them susceptible to EAE. These findings identify Bhlhe40 as a critical regulator of autoreactive T-cell pathogenicity.

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T cells require Bhlhe40 for normal cytokine production after immunizationa, b, ELISPOT assays for the quantitation of cells secreting (a) IL-2, IFN-γ, and IL-17A or (b) GM-CSF and IL-10 performed on DLN cells 7 days after immunization of WT and Bhlhe40−/− mice. Data for IL-2, IFN-γ, IL-17A, and GM-CSF are combined from 3 independent experiments (n=9 mice per group). Data for IL-10 is from one representative experiment of two (n=4 mice per group). c, e, DLN cells from immunized WT and Bhlhe40−/− mice (n=14 per group) were cultured with or without MOG(35-55) and with or without IL-1β, IL-23, and/or IL-12 as indicated. (c) GM-CSF or (e) IL-10 was measured in the supernatant at day 4. Data are combined from 5 independent experiments. Cells from all mice were not used in all conditions in each of the 4 experiments. d, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with MOG(35-55) with or without IL-1β for 4 days, followed by ICS. Representative plots are gated on CD4+ T cells. f, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with MOG(35-55) with or without IL-12 for 4 days, followed by ICS. Representative plots are gated on CD4+ T cells. g, Frequencies of GM-CSF+ and IL-17A+ γδ T cells in DLNs 7 days after immunization of WT and Bhlhe40−/− mice (n=3 per group) as determined by ICS. h, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with or without MOG(35-55) and with or without IL-1β and/or IL-23 as indicated for 4 days. Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 4 hours and then analyzed for IL-17A and GM-CSF by intracellular staining (i.e. our normal ICS protocol). Representative plots are gated on γδ T cells.
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Figure 3: T cells require Bhlhe40 for normal cytokine production after immunizationa, b, ELISPOT assays for the quantitation of cells secreting (a) IL-2, IFN-γ, and IL-17A or (b) GM-CSF and IL-10 performed on DLN cells 7 days after immunization of WT and Bhlhe40−/− mice. Data for IL-2, IFN-γ, IL-17A, and GM-CSF are combined from 3 independent experiments (n=9 mice per group). Data for IL-10 is from one representative experiment of two (n=4 mice per group). c, e, DLN cells from immunized WT and Bhlhe40−/− mice (n=14 per group) were cultured with or without MOG(35-55) and with or without IL-1β, IL-23, and/or IL-12 as indicated. (c) GM-CSF or (e) IL-10 was measured in the supernatant at day 4. Data are combined from 5 independent experiments. Cells from all mice were not used in all conditions in each of the 4 experiments. d, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with MOG(35-55) with or without IL-1β for 4 days, followed by ICS. Representative plots are gated on CD4+ T cells. f, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with MOG(35-55) with or without IL-12 for 4 days, followed by ICS. Representative plots are gated on CD4+ T cells. g, Frequencies of GM-CSF+ and IL-17A+ γδ T cells in DLNs 7 days after immunization of WT and Bhlhe40−/− mice (n=3 per group) as determined by ICS. h, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with or without MOG(35-55) and with or without IL-1β and/or IL-23 as indicated for 4 days. Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 4 hours and then analyzed for IL-17A and GM-CSF by intracellular staining (i.e. our normal ICS protocol). Representative plots are gated on γδ T cells.

Mentions: Draining lymph nodes (DLNs) from immunized Bhlhe40−/− mice had reduced cellularity relative to WT mice (Supplementary Fig. 3a). Flow cytometry of these DLNs indicated normal frequencies of lymphoid and myeloid cell populations, indicating that the decreased cellularity was not due to the reduction in a particular cell type (Supplementary Fig. 3b). Similar frequencies of interleukin 2 (IL-2)- and IFN-γ-producing T cells and a modest reduction in the frequency of IL-17A-producing T cells were observed in DLNs from Bhlhe40−/− mice relative to WT mice in response to stimulation with MOG(35-55) or concanavalin A (ConA) as measured by ELISPOT assays (Fig. 3a). Consistent with their decreased IL-17A production, the frequency of RORγt+ CD4+ T cells was moderately decreased in DLNs of Bhlhe40−/− mice (Supplementary Fig. 4a, b). DLNs from Bhlhe40−/− mice showed a markedly reduced frequency of MOG(35-55)-specific GM-CSF-producing T cells relative to WT mice, and an increased frequency of MOG(35-55)-specific IL-10-producing T cells (Fig. 3b). Intracellular cytokine staining (ICS) of DLN cells also showed diminished GM-CSF (Supplementary Fig. 4c, d) and increased IL-10 production (Supplementary Fig. 4e, f) from Bhlhe40−/− CD4+ T cells. The defect in GM-CSF production by Bhlhe40−/− CD4+ T cells could not be fully restored in vitro by culture with IL-1β or IL-23, cytokines that are reported to promote GM-CSF secretion10-13 (Fig. 3c, d). The increased IL-10 production by Bhlhe40−/− CD4+ T cells was significantly augmented by in vitro culture with IL-12 (Fig. 3e, f).


Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation.

Lin CC, Bradstreet TR, Schwarzkopf EA, Sim J, Carrero JA, Chou C, Cook LE, Egawa T, Taneja R, Murphy TL, Russell JH, Edelson BT - Nat Commun (2014)

T cells require Bhlhe40 for normal cytokine production after immunizationa, b, ELISPOT assays for the quantitation of cells secreting (a) IL-2, IFN-γ, and IL-17A or (b) GM-CSF and IL-10 performed on DLN cells 7 days after immunization of WT and Bhlhe40−/− mice. Data for IL-2, IFN-γ, IL-17A, and GM-CSF are combined from 3 independent experiments (n=9 mice per group). Data for IL-10 is from one representative experiment of two (n=4 mice per group). c, e, DLN cells from immunized WT and Bhlhe40−/− mice (n=14 per group) were cultured with or without MOG(35-55) and with or without IL-1β, IL-23, and/or IL-12 as indicated. (c) GM-CSF or (e) IL-10 was measured in the supernatant at day 4. Data are combined from 5 independent experiments. Cells from all mice were not used in all conditions in each of the 4 experiments. d, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with MOG(35-55) with or without IL-1β for 4 days, followed by ICS. Representative plots are gated on CD4+ T cells. f, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with MOG(35-55) with or without IL-12 for 4 days, followed by ICS. Representative plots are gated on CD4+ T cells. g, Frequencies of GM-CSF+ and IL-17A+ γδ T cells in DLNs 7 days after immunization of WT and Bhlhe40−/− mice (n=3 per group) as determined by ICS. h, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with or without MOG(35-55) and with or without IL-1β and/or IL-23 as indicated for 4 days. Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 4 hours and then analyzed for IL-17A and GM-CSF by intracellular staining (i.e. our normal ICS protocol). Representative plots are gated on γδ T cells.
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Figure 3: T cells require Bhlhe40 for normal cytokine production after immunizationa, b, ELISPOT assays for the quantitation of cells secreting (a) IL-2, IFN-γ, and IL-17A or (b) GM-CSF and IL-10 performed on DLN cells 7 days after immunization of WT and Bhlhe40−/− mice. Data for IL-2, IFN-γ, IL-17A, and GM-CSF are combined from 3 independent experiments (n=9 mice per group). Data for IL-10 is from one representative experiment of two (n=4 mice per group). c, e, DLN cells from immunized WT and Bhlhe40−/− mice (n=14 per group) were cultured with or without MOG(35-55) and with or without IL-1β, IL-23, and/or IL-12 as indicated. (c) GM-CSF or (e) IL-10 was measured in the supernatant at day 4. Data are combined from 5 independent experiments. Cells from all mice were not used in all conditions in each of the 4 experiments. d, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with MOG(35-55) with or without IL-1β for 4 days, followed by ICS. Representative plots are gated on CD4+ T cells. f, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with MOG(35-55) with or without IL-12 for 4 days, followed by ICS. Representative plots are gated on CD4+ T cells. g, Frequencies of GM-CSF+ and IL-17A+ γδ T cells in DLNs 7 days after immunization of WT and Bhlhe40−/− mice (n=3 per group) as determined by ICS. h, DLN cells from immunized WT and Bhlhe40−/− mice were cultured with or without MOG(35-55) and with or without IL-1β and/or IL-23 as indicated for 4 days. Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 4 hours and then analyzed for IL-17A and GM-CSF by intracellular staining (i.e. our normal ICS protocol). Representative plots are gated on γδ T cells.
Mentions: Draining lymph nodes (DLNs) from immunized Bhlhe40−/− mice had reduced cellularity relative to WT mice (Supplementary Fig. 3a). Flow cytometry of these DLNs indicated normal frequencies of lymphoid and myeloid cell populations, indicating that the decreased cellularity was not due to the reduction in a particular cell type (Supplementary Fig. 3b). Similar frequencies of interleukin 2 (IL-2)- and IFN-γ-producing T cells and a modest reduction in the frequency of IL-17A-producing T cells were observed in DLNs from Bhlhe40−/− mice relative to WT mice in response to stimulation with MOG(35-55) or concanavalin A (ConA) as measured by ELISPOT assays (Fig. 3a). Consistent with their decreased IL-17A production, the frequency of RORγt+ CD4+ T cells was moderately decreased in DLNs of Bhlhe40−/− mice (Supplementary Fig. 4a, b). DLNs from Bhlhe40−/− mice showed a markedly reduced frequency of MOG(35-55)-specific GM-CSF-producing T cells relative to WT mice, and an increased frequency of MOG(35-55)-specific IL-10-producing T cells (Fig. 3b). Intracellular cytokine staining (ICS) of DLN cells also showed diminished GM-CSF (Supplementary Fig. 4c, d) and increased IL-10 production (Supplementary Fig. 4e, f) from Bhlhe40−/− CD4+ T cells. The defect in GM-CSF production by Bhlhe40−/− CD4+ T cells could not be fully restored in vitro by culture with IL-1β or IL-23, cytokines that are reported to promote GM-CSF secretion10-13 (Fig. 3c, d). The increased IL-10 production by Bhlhe40−/− CD4+ T cells was significantly augmented by in vitro culture with IL-12 (Fig. 3e, f).

Bottom Line: Bhlhe40 is required in vivo in a T cell-intrinsic manner, where it positively regulates the production of GM-CSF and negatively regulates the production of IL-10.In vitro, GM-CSF secretion is selectively abrogated in polarized Bhlhe40(-/-) TH1 and TH17 cells, and these cells show increased production of IL-10.These findings identify Bhlhe40 as a critical regulator of autoreactive T-cell pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri 63110, USA.

ABSTRACT
TH1 and TH17 cells mediate neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Pathogenic TH cells in EAE must produce the pro-inflammatory cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). TH cell pathogenicity in EAE is also regulated by cell-intrinsic production of the immunosuppressive cytokine interleukin 10 (IL-10). Here we demonstrate that mice deficient for the basic helix-loop-helix (bHLH) transcription factor Bhlhe40 (Bhlhe40(-/-)) are resistant to the induction of EAE. Bhlhe40 is required in vivo in a T cell-intrinsic manner, where it positively regulates the production of GM-CSF and negatively regulates the production of IL-10. In vitro, GM-CSF secretion is selectively abrogated in polarized Bhlhe40(-/-) TH1 and TH17 cells, and these cells show increased production of IL-10. Blockade of IL-10 receptor in Bhlhe40(-/-) mice renders them susceptible to EAE. These findings identify Bhlhe40 as a critical regulator of autoreactive T-cell pathogenicity.

Show MeSH
Related in: MedlinePlus