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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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Transcriptional analysis of Bhlhe40-deficient TH cellsa, Heat map of gene expression for selected TH1, TH2, and TH17 lineage-specific genes in polarized WT and Bhlhe40−/− TH cells. b, Pairwise comparison of WT and Bhlhe40−/− TH1, TH2, and TH17 cells. Probes that are overexpressed ≥2-fold in the indicated populations are shown in red or blue. Numbers in corners indicate the total number of probes meeting these conditions. Probes for selected relevant genes are shown in green. Coefficients of determination (R2) are indicated for each comparison. c, Venn diagrams showing the overlap of probes differentially expressed by WT and KO TH cell lineages. Numbers in regions indicate the number of probes with greater than or equal to a 2-fold difference in expression (WT>KO or WT<KO). d, Quantitative RT-PCR analysis of Ifng, Il4, and Il17a expression in WT and Bhlhe40−/− TH1, TH2, and TH17 cells (n=3 per group). e, Quantitative RT-PCR analysis of the expression of the indicated 9 genes in WT and Bhlhe40−/− TH1, TH2, and TH17 cells (n=3 per group).
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Figure 5: Transcriptional analysis of Bhlhe40-deficient TH cellsa, Heat map of gene expression for selected TH1, TH2, and TH17 lineage-specific genes in polarized WT and Bhlhe40−/− TH cells. b, Pairwise comparison of WT and Bhlhe40−/− TH1, TH2, and TH17 cells. Probes that are overexpressed ≥2-fold in the indicated populations are shown in red or blue. Numbers in corners indicate the total number of probes meeting these conditions. Probes for selected relevant genes are shown in green. Coefficients of determination (R2) are indicated for each comparison. c, Venn diagrams showing the overlap of probes differentially expressed by WT and KO TH cell lineages. Numbers in regions indicate the number of probes with greater than or equal to a 2-fold difference in expression (WT>KO or WT<KO). d, Quantitative RT-PCR analysis of Ifng, Il4, and Il17a expression in WT and Bhlhe40−/− TH1, TH2, and TH17 cells (n=3 per group). e, Quantitative RT-PCR analysis of the expression of the indicated 9 genes in WT and Bhlhe40−/− TH1, TH2, and TH17 cells (n=3 per group).

Mentions: We performed expression microarray analysis on WT and Bhlhe40−/− CD4+ T cells cultured in TH1, TH2, and TH17 conditions and validated these results by quantitative RT-PCR for selected transcripts (Fig. 5). This analysis confirmed that Bhlhe40−/− T cells were able to acquire characteristics largely reflective of normal TH1, TH2, or TH17 differentiation, including their expression of signature cytokines and transcription factors (Fig. 5a, d). However, a common set of genes was affected by the loss of Bhlhe40 across two or more TH lineages (Fig. 5b, c, e), including reduced expression of Csf2, Il3, Il1a, Ccl1, Ifitm3, and Ptgs2. Notably, expression levels of Il10, Ikzf3 (Aiolos), and Xcl1 were increased in Bhlhe40−/− T cells. We confirmed reduced secretion of IL-3 by Bhlhe40−/− CD4+ T cells following stimulation with anti-CD3 alone or anti-CD3 and anti-CD28 (Supplementary Fig. 7d).


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)

Transcriptional analysis of Bhlhe40-deficient TH cellsa, Heat map of gene expression for selected TH1, TH2, and TH17 lineage-specific genes in polarized WT and Bhlhe40−/− TH cells. b, Pairwise comparison of WT and Bhlhe40−/− TH1, TH2, and TH17 cells. Probes that are overexpressed ≥2-fold in the indicated populations are shown in red or blue. Numbers in corners indicate the total number of probes meeting these conditions. Probes for selected relevant genes are shown in green. Coefficients of determination (R2) are indicated for each comparison. c, Venn diagrams showing the overlap of probes differentially expressed by WT and KO TH cell lineages. Numbers in regions indicate the number of probes with greater than or equal to a 2-fold difference in expression (WT>KO or WT<KO). d, Quantitative RT-PCR analysis of Ifng, Il4, and Il17a expression in WT and Bhlhe40−/− TH1, TH2, and TH17 cells (n=3 per group). e, Quantitative RT-PCR analysis of the expression of the indicated 9 genes in WT and Bhlhe40−/− TH1, TH2, and TH17 cells (n=3 per group).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016562&req=5

Figure 5: Transcriptional analysis of Bhlhe40-deficient TH cellsa, Heat map of gene expression for selected TH1, TH2, and TH17 lineage-specific genes in polarized WT and Bhlhe40−/− TH cells. b, Pairwise comparison of WT and Bhlhe40−/− TH1, TH2, and TH17 cells. Probes that are overexpressed ≥2-fold in the indicated populations are shown in red or blue. Numbers in corners indicate the total number of probes meeting these conditions. Probes for selected relevant genes are shown in green. Coefficients of determination (R2) are indicated for each comparison. c, Venn diagrams showing the overlap of probes differentially expressed by WT and KO TH cell lineages. Numbers in regions indicate the number of probes with greater than or equal to a 2-fold difference in expression (WT>KO or WT<KO). d, Quantitative RT-PCR analysis of Ifng, Il4, and Il17a expression in WT and Bhlhe40−/− TH1, TH2, and TH17 cells (n=3 per group). e, Quantitative RT-PCR analysis of the expression of the indicated 9 genes in WT and Bhlhe40−/− TH1, TH2, and TH17 cells (n=3 per group).
Mentions: We performed expression microarray analysis on WT and Bhlhe40−/− CD4+ T cells cultured in TH1, TH2, and TH17 conditions and validated these results by quantitative RT-PCR for selected transcripts (Fig. 5). This analysis confirmed that Bhlhe40−/− T cells were able to acquire characteristics largely reflective of normal TH1, TH2, or TH17 differentiation, including their expression of signature cytokines and transcription factors (Fig. 5a, d). However, a common set of genes was affected by the loss of Bhlhe40 across two or more TH lineages (Fig. 5b, c, e), including reduced expression of Csf2, Il3, Il1a, Ccl1, Ifitm3, and Ptgs2. Notably, expression levels of Il10, Ikzf3 (Aiolos), and Xcl1 were increased in Bhlhe40−/− T cells. We confirmed reduced secretion of IL-3 by Bhlhe40−/− CD4+ T cells following stimulation with anti-CD3 alone or anti-CD3 and anti-CD28 (Supplementary Fig. 7d).

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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Related in: MedlinePlus