Autoregulation of Th1-mediated inflammation by twist1

Uwe Niesner, Inka Albrecht, Marko Janke, Cornelia Doebis, Christoph Loddenkemper, Maria H. Lexberg, Katharina Eulenburg, Stephan Kreher, Juliana Koeck, Ria

Baumgrass, Kerstin Bonhagen, Thomas Kamradt, Philipp Enghard, Jens Y. Humrich, Sascha Rutz, Ulf Schulze-Topphoff, Orhan Aktas, Sina Bartfeld, Helena Radbruch, Ahmed N. Hegazy, Max Löhning, Daniel C. Baumgart, Rainer Duchmann, Martin

Rudwaleit, Thomas Häupl, Inna Gitelman, Veit Krenn, Joachim Gruen, Jochen Sieper, Martin Zeitz, Bertram Wiedenmann, Frauke Zipp, Alf Hamann, Michal Janitz,

Alexander Scheffold, Gerd R. Burmester, Hyun D. Chang, and Andreas Radbruch

Journal of Experimental MedicineVolume 205(8):1889-1901

August 2, 2008

© 2008 Niesner et.al.

Th cell balance

naive T cell

TGFβIL-1/IL-6/IL-23

IFNγ

IL-17A-FIL-22

IL-4IL-10IL-13IL-5

Activation STAT4Production IFNγActivation Tbet

Activation RORC2

Activation STAT6Activation GATA3

IL-4

IL-12

Th1

Th17

Th2

Twist1: a basic helix-loop-helix transcription factor

basic helix-loop-helix transcription factorbinds to a consensus sequence called e-box: CANNTG

Transcriptional regulation of bHLH transcription factors

Repeated Th1 stimulation

naive CD4+ CD62L+ T cellsfrom DO11.10 mice (carrying a TCR specific for Ovalbumin)

splenic APCOvalbuminIL-12anti-IL-4

6d

restimulation

gene array

...

38 fold upregulation of twist1 expression in 4 times restimulated Th1 cells

PMA/iono CD3/CD28

quantitative real-time PCR

Twist1 expression is induced in Th1, but not Th2 or Th17 cells

Twist1 induction requires IL-12 signaling via STAT4, but not IFN-γ or T-bet

Signaling through NFAT and NF-κB is required for induction of twist1 expression

PDTC blocksNF-κBBTP blocks NFATCSA blocks IL-2 signaling

Ex vivo isolated memory Th cells express twist1

Generation of Th1 cells constitutively expressing twist1

• d0: splenic DO11.10 cells activated in vitro

• d2: infection with twist1 encoding or control retrovirus

• d5: sort of GFP+

Genes differentially expressed upon ectopic twist1 overexpression

Twist1 suppresses the expression of effector cytokines

NF-κB driven luciferase reporter

• d0: splenic DO11.10 cells activated in vitro

• d2: infection with twist1 or I-κBαM encoding retrovirus

• d3: nucleoporation with a mixture of luciferase encoding plasmids

• d5: sort of GFP+

Inhibition of NF-κB–mediated signaling by twist1 is promoter-specific

DTH model

• 6d-old-Th1 cells overexpressing twist1 injected i.v. in BALB/c

• 1d later OVA + IFA injection in footpad

• messure of swelling of the footpad

Ectopic twist1 overexpression controls DTH

Twist1 knockdown increases inflammatory response in murine arthritis

Conclusions

• repeatedly stimulated Th1 cells express twist1• twist1 expression is NF-κB, NFAT and STAT4

dependent• twist1 is expressed in humans and mice

– in Th1 effector memory cells – on CD4+ T cells of inflammed tissue

• Tbet is not needed for twist1 expression• twist1 binds to e-boxes in the regulatory regions

of specific target genes• twist1 reduces activation-induced expression IL-2,

IFN-γ, TNF-α on the protein level and theirby dampening an inflammation