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 Disclosure: Honorarium, Pfizer, Inc (June 2017)
DNA binding & dimerization Bhlhe40 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 2
Bhlhe40 -/- Mice Are Protected from EAE c d IFN- γ IL-17A GM-CSF IL-10 3 Lin et al., 2014, Nat. Commun.
Pertussis Toxin (PTX) Induces Bhlhe40 Expression in T H Cells During EAE ± MOG 35-55 /CFA GFP (Bhlhe40) and 7 days ± Pertussis toxin cytokine expression by CD4 + T cells (WT or mutant) in the lymph nodes Bhlhe40 GFP reporter mice 4 Lin et al., 2016, J. Exp Med.
IL- 1β Enhances Bhlhe40 Expression in Polarized T H 17 Cells A B DLN cell cultures O/N (taken day 7 post EAE induction) 5 Lin et al., 2016, J. Exp Med.
Bhlhe40 flox/flox Mice Bhlhe40 fl/fl (n=10) Bhlhe40 fl/fl (n=6) LysM Cre Bhlhe40 fl/fl (n=4) MRP8 Cre Bhlhe40 fl/fl (n=7) 6
Deletion of Bhlhe40 in T Cells or IL-17A pos Cells Bhlhe40 fl/fl (n=12) Bhlhe40 fl/fl (n=14) Cd4 Cre Bhlhe40 fl/fl (n=14) Il17a Cre Bhlhe40 fl/fl (n=10) Note – Delayed onset and reduction in EAE severity seen here using Il17a Cre - mediated deletion of Bhlhe40 is more significant than seen after Il17a Cre -mediated deletion of Tbx21 or Rorc (Brucklacher-Waldert et al., 2016, J. Immunol. ) 7
Bhlhe40 Expression in IFN- γ pos IL-17A neg “exT H 17” and IFN - γ pos IL-17A pos cells Il17a Cre x R26-LoxP-Stop-LoxP-tdTomato x Bhlhe40 GFP mice D7 DLN, ICS after P/I 8
Bhlhe40 Expression in IL-17A Fate-Mapped Cells IL-17A fate pos Bhlhe40- GFP neg CD4 + T Cells IL-17A fate pos Bhlhe40- GFP pos CD4 + T Cells IFN γ pos IFN γ pos GM-CSF pos IL-17A pos Triple neg GM-CSF pos IL-17A pos GM-CSF pos IL-17A pos IFN γ pos D7 DLN, ICS after P/I (n=4) IL-17A pos IFN γ pos GM-CSF pos 9
A 3355 peaks B Bhlhe40 Promoter (<1kb) Promoter (1-2kb) ChIP-Seq on WT Promoter (2-3kb) 3'UTR Other Exon and KO T H 17 cells 1st Intron Other Intron Downstream ( 3kb) Distal Intergenic C 10 kb Bhlhe40 WT KO Il10 10 kb WT KO 10 kb Csf2/Il3 WT KO 10
BHLHE40 Expression in Human T H Subsets (RNA-Seq) – Sorted from PBMCs (healthy donors) CCR6+CXCR3+ = TH1/17 11 Arlehamn C. L. et al., 2014, J. Immunol.
Role of Bhlhe40 in Pathogenic CD4+ T Cells Lin et al., 2014, Nat. Commun . Ronchi et al., 2016, Nat. Commun. Lin et al., 2016, J. Exp. Med. Mufazalov et al., 2016, EMBO 12
Acknowledgments 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
BACKUP SLIDES
Transcriptional Analysis of Polarized Bhlhe40 -/- T H Cells 16
Bhlhe40-Expressing CD4 + T Cells Are Abundant in the CNS During EAE GFP (Bhlhe40) MOG 35-55 /CFA 14-15 days expression Active EAE by CD4 + T cells Pertussis toxin induction in the spleen and CNS (co-adjuvant) Bhlhe40 GFP reporter mice Spinal cord (d15) CD4 GFP Lin et al., 2016, J. Exp. Med. 17
IL- 1β Enhances Bhlhe40 Expression in Polarized T H 17 Cells A B C EAE Pathogenic gene signature 18
Pertussis Toxin (PTX) Induces IL- 1β Production by Myeloid Cells A B (Monocytes/monocyte-derived cells) DLN, Day 7 post EAE induction 19
Identification of human T H subsets (gating strategy) 1: TB-specific (tetramer-positive) T cells 2: naïve CD4 T cells 3: “T H 1/17” cells (6 + X3 + ) 4: T H 17 cells 5: T H 1 cells 6: T H 2 cells Modified from Arlehamn C. L. et al. J Immunol 2014
Zhang H et al., J Allergy Clin Immunol 2013 (GSE43005) Arlehamn C. L., et al. J Immunol 2014 (GSE56179) 22
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