The Transcription Factor Bhlhe40 Sustains T H 17 Cell Pathogenicity - - PowerPoint PPT Presentation
The Transcription Factor Bhlhe40 Sustains T H 17 Cell Pathogenicity in EAE Brian T. Edelson Dept. of Pathology and Immunology Div. of Laboratory and Genomic Medicine Washington University School of Medicine St. Louis, MO USA 1 February 2018
Brian T. Edelson
Washington University School of Medicine
1 February 2018
The Transcription Factor Bhlhe40 Sustains TH17 Cell Pathogenicity in EAE
Disclosure: Honorarium, Pfizer, Inc (June 2017)
Basic helix-loop-helix (bHLH) family, member e40 (aka DEC1, Stra13) – first cloned in 1997 in differentiated chondrocytes and as an RA-induced gene in P19 embryonal carcinoma cells Evolutionarily conserved: present in zebrafish, xenopus. Expression: non-hematopoietic cells; some macrophages, dendritic cells, granulocytes, activated T cells, iNKT cells Function: generally transcriptional repression, some examples of transcriptional activation; binds class B E box (CACGTG) In vivo: circadian rhythm, muscle repair, neuronal excitability, immune cell function Key question: what drives the pathogenicity of autoreactive CD4+ T cells in EAE and MS
DNA binding & dimerizationBhlhe40
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Bhlhe40-/- Mice Are Protected from EAE
c d
IFN-γ IL-17A GM-CSF IL-10
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Lin et al., 2014, Nat. Commun.
Pertussis Toxin (PTX) Induces Bhlhe40 Expression in TH Cells During EAE
±MOG35-55/CFA ±Pertussis toxin (WT or mutant) Bhlhe40GFP reporter mice GFP (Bhlhe40) and cytokine expression by CD4+ T cells in the lymph nodes 7 days Lin et al., 2016, J. Exp Med.
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A
IL-1β Enhances Bhlhe40 Expression in Polarized TH17 Cells
B
DLN cell cultures O/N (taken day 7 post EAE induction) Lin et al., 2016, J. Exp Med.
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Bhlhe40flox/flox Mice
Bhlhe40fl/fl (n=10) LysMCreBhlhe40fl/fl (n=4) Bhlhe40fl/fl (n=6) MRP8CreBhlhe40fl/fl (n=7)
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Deletion of Bhlhe40 in T Cells or IL-17Apos Cells
Bhlhe40fl/fl (n=12) Cd4CreBhlhe40fl/fl (n=14) Bhlhe40fl/fl (n=14) Il17aCreBhlhe40fl/fl (n=10)
Note – Delayed onset and reduction in EAE severity seen here using Il17aCre- mediated deletion of Bhlhe40 is more significant than seen after Il17aCre-mediated deletion of Tbx21 or Rorc (Brucklacher-Waldert et al., 2016, J. Immunol.)
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Il17aCre x R26-LoxP-Stop-LoxP-tdTomato x Bhlhe40GFP mice Bhlhe40 Expression in IFN-γposIL-17Aneg “exTH17” and IFN-γposIL-17Apos cells
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D7 DLN, ICS after P/I
IL-17Apos Triple neg IL-17AposGM-CSFpos IL-17AposIFNγpos IL-17AposIFNγposGM-CSFpos IFNγpos IFNγposGM-CSFpos GM-CSFpos
IL-17A fateposBhlhe40-GFPnegCD4+ T Cells IL-17A fateposBhlhe40-GFPposCD4+ T Cells
Bhlhe40 Expression in IL-17A Fate-Mapped Cells
D7 DLN, ICS after P/I (n=4)
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10 kb
WT KO
10 kb
WT KO
10 kb
WT KO
Bhlhe40 Il10 Csf2/Il3
B A C
3355 peaks
Bhlhe40 ChIP-Seq on WT and KO TH17 cells
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BHLHE40 Expression in Human TH Subsets (RNA-Seq) – Sorted from PBMCs (healthy donors)
Arlehamn C. L. et al., 2014, J. Immunol.
CCR6+CXCR3+ = TH1/17
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Role of Bhlhe40 in Pathogenic CD4+ T Cells
Lin et al., 2014, Nat. Commun. Lin et al., 2016, J. Exp. Med.
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Ronchi et al., 2016, Nat. Commun. Mufazalov et al., 2016, EMBO
Chih-Chung “Jerry” Lin Tara R. Bradstreet Elizabeth A. Schwarzkopf Nicholas Jarjour Melissa Cook Washington University School of Medicine Maxim Artyomov Takeshi Egawa John Russell Gregory Wu National University of Singapore Reshma Taneja
Acknowledgments
Transcriptional Analysis of Polarized Bhlhe40-/- TH Cells
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Bhlhe40-Expressing CD4+ T Cells Are Abundant in the CNS During EAE
MOG35-55/CFA Pertussis toxin (co-adjuvant) Bhlhe40GFP reporter mice GFP (Bhlhe40) expression by CD4+ T cells in the spleen and CNS Active EAE induction 14-15 days
Spinal cord (d15) CD4 GFP
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Lin et al., 2016, J. Exp. Med.
IL-1β Enhances Bhlhe40 Expression in Polarized TH17 Cells
EAE Pathogenic gene signature
A B C
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Pertussis Toxin (PTX) Induces IL-1β Production by Myeloid Cells
B A (Monocytes/monocyte-derived cells) DLN, Day 7 post EAE induction
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1: TB-specific (tetramer-positive) T cells 2: naïve CD4 T cells 3: “TH1/17” cells (6+X3+) 4: TH17 cells 5: TH1 cells 6: TH2 cells
Modified from Arlehamn C. L. et al. J Immunol 2014
Identification of human TH subsets (gating strategy)
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Zhang H et al., J Allergy Clin Immunol 2013 (GSE43005) Arlehamn C. L., et al. J Immunol 2014 (GSE56179)