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Departments of Ophthalmology and Internal Medicine Program of - - PowerPoint PPT Presentation

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Update on Immunopathogenesis of Graves' Ophthalmopathy Terry J. Smith, M.D Departments of Ophthalmology and Internal Medicine Program of Cellular and Molecular Biology University of Michigan PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE

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Update on Immunopathogenesis of Graves' Ophthalmopathy

Terry J. Smith, M.D

Departments of Ophthalmology and Internal Medicine Program of Cellular and Molecular Biology University of Michigan

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Speaker Disclosures

Inventor of the following issued US Patents 6936426 7998681 8153121 8178304 Consultant: Novartis Consultant: Merck Consultant: Lithera Previously paid consultant: Riverview

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Learning Objectives Review recent developments in solving the pathogenesis of TAO Introduce the concept that bone marrow derived fibrocytes express high levels of TSHR and infiltrate the orbit in TAO. Discuss the concept that fibrocytes express several “thyroid- specific’ proteins that become suppressed by native orbital fibroblasts Introduce the concept that the orbit in TAO represents a recapitulation of the thyroid

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Thyrotoxicosis Ophthalmopathy Dermopathy

Graves’ Disease

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Disease

Time

Ideal Immunomodulatory Therapy

Active Phase Stable Phase 18-36 months 5-7years Surgery

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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HA, HAS-2 Cao et al JBC 1998 HAS 1, HAS-2, HAS 3 Kaback et al JCEM 1999 IL-16, RANTES Sciaky et al J Immunol 2000 PPAR-ᴕ Smith et al JCEM 2002 PGHS-2, mPGS Han et al JBC 2002 IL-IRA Cao et al AJP 2003 IGF-IR Pritchard et al J Immunol 2003 TIMP-1 Han & Smith J Immunol 2005 15-Lipoxygenase Chen et al JBC 2006 UDP-glucose dehydrogenase Tsui et al JBC 2011

Orbital fibroblasts exhibit a unique phenotype

Differential Gene Expression in Orbital Fibroblasts

Gene/Response Reference

PAI-I Smith et al AJP 1992 HLA-DR Heufelder et al JCEM 1991 Endothelin-I Smith et al AJP 1993 Actin- Rearrangement Smith et al PNAS 1994 HA Smith et al AJP 1995 Adipogenesis Sorisky et al JCEM 1996 COX-2, PGE 2 Wang et al JBC 1996 IL-6, IL-8, CD40 Sempowski et al AJP 1998 Actin Neighbor Protein Young et al PNAS 1998

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Identifying an Orbital Antigen Explaining TAO

  • While TSHR represents the central antigen in GD,

the mechanisms through which orbital tissues become activated remains uncertain.

  • Multiple antigens have been proposed as potentially

important to the pathogenesis of TAO

  • TSHR (Fenzi et al, Bahn et al, several others)
  • Tg (Kriss, Marino et al)
  • Muscle antigens (Wall et al)
  • IGF-1R (Kendall-Taylor, Smith)

To date, no convincing evidence substantiates antigen- specific T cell infiltration of the orbit in TAO

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Electroporation of TSHR-Expressing Plasmid Results in Orbital Inflammation

  • Electroporation with TSHR A subunit containing plasmid

leads to a phenotype both typical and atypical of TAO

  • Orbital congestion
  • Muscle enlargement
  • Orbital fat expansion
  • Infiltration of T cells, F4/80+ macrophages, and mast cells
  • Predominance of TSHR blocking Abs and hypothyroidism
  • Dramatic mononuclear infiltration of the optic nerve
  • IGF-1R containing plasmids failed to elicit pathology
  • Anti-IGF-1R Abs became detectable in many animals

immunized with TSHR.

Moshkelgosha et al Endocrinology 2013

+ + + +

  • PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)
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Electroporation of TSHR-Expressing Plasmid Results in Orbital Inflammation

Moshkelgosha et al Endocrinol 2013

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Conundrum: TSHR or IGF-1R. Which is the relevant orbital antigen?

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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IGF-1R Mediates Induction of T Cell Chemokines TAO Fibroblasts

  • IGF-1
  • GD-IgG
  • Anti-IGF-1R Abs

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Tsui et al J Immunol.(2008)

TSHR and IGF-1R Co-Localize and Form a Signaling Complex

IGF-1Rb (red) TSHR (green) demonstrates Co-localization (yellow/orange) (A–C) TAO orbital fibroblasts (D–F) Thyrocytes (G-I) TAO orbital fibroblasts (J-L) TAO orbital fibroblasts IGF- 1Ra vs TSHR (M-O) TAO orbital fat

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Tsui et al J Immunol (2008)

IGF-1R Inhibition Attenuates TSHR Signaling

  • Inhibition of IGF-1R with 1H7, a blocking

mAb, attenuates signaling through both IGF-1R and TSHR

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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TAO Fibroblast Heterogeneity-Divergent Capacity to Differentiate into Adipocytes and Myofibroblasts

Myofibroblast

TGF-b PPAR

Smith and Phipps 1995

Adipocyte Thy-1+ Thy-1-

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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TAO Orbital Fibroblasts Can be Further Segregated into CD34+ and CD34- Cells

CD34 PERCP

10 10

1

10

2

10

3

10

4

3 5 8 10 13

CD34

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Hematopoietic Stem cells Circulating CD14+ Precursors Multipotent progenitors Circulating Fibrocytes

CD34+ Fibrocytes Bucala et al 1994

Migrate to sites of injury

Myofibroblast Fibroblast Fibroadipocyte

Recruitment Chemokine signals

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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p<0.001

Control Graves’

n=25 n=70

Fibrocyte Count

Peripheral Blood Fibrocytes Increased in GD

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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CD34 CXCR4 CD11b CD14 Col1

GD ISO a-actin GD ISO GD ISO GD ISO GD ISO

TAO fibrocytes display a typical phenotype

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Unlike TAO Fibroblasts, IGF-1 Receptor Levels

  • n Fibrocytes are Relatively Low

IGF-1R

ISO

TAO Fibroblast TAO Fibrocyte

IGF-1R

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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But, TSHR Levels on Fibrocytes Exceed Those on TAO Orbital Fibroblasts

Gillespie et al JCEM (2012)

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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100 200 300 400 500

IL-6 (pg/ml)

n=6 n=6 n=8

Control IL-1b TSH

* * 50 100 150 200 250 300 350 400 450 500

TNF-a (pg/ml)

n=6 n=6 n=8

Control IL-1b TSH

* * *

*p<0.01 *p<0.01 Douglas et al. 2010

Fibrocytes Express Cytokines in Response to TSH

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Healthy LSP E. A . Graves’ CD34 B . Healthy CD34 F. D. CD 31 C. Graves’ LSP

Fibrocytes Infiltrate the Orbit in TAO

Healthy LSP

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Fibrocyte Thyroid Fibrocyte Anti-Tg IgG Anti-Tg IgG Anti-Tg IgG

25 mm 25 mm 100 mm

Isotype IgG Isotype IgG

25 mm 25 mm

CD45 Isotype

Do Fibrocytes Express Other “Thyroid-Specific” Proteins?

Fernando et al PNAS 2012

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Relative Luciferase Activity (Fold-Change)

2 4 6 8 Orbital fibroblast Fibrocyte

Luc Luc Luc

  • 181/+16

Tg Fibrocyte OF

D

**

Fibrocytes Fibroblasts

Tg mRNA (fold - increase)

Thyroid

H GD GD-OF H-OF DF

Tg mRNA Expression Results from an Active Gene Promoter

Fernando et al PNAS 2012

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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5 10 15 225 NIS mRNA (fold - change) 1 10 100 10×108 TPO mRNA (fold - change) 69 kDa NIS 103 kDa TPO

TPO NIS

Fibrocytes Express NIS and TPO

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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New Graves’ Paradigm

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Circulation Orbital Tissue

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Orbital Tissue Circulation

  • TSHR

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Conclusions

Orbital fibroblasts in TAO exhibit unique phenotypes. TSHR/IGF-1R complexes form in fibroblasts and

  • fibrocytes. Interrupting IGF-1R function attenuates Erk

and Akt signaling initiated by TSHR and blocks cytokine induction. CD34+ fibrocytes express functional TSHR, Tg, NIS, and TPO and infiltrate the orbit in TAO where levels drop. This results from negative influence from native CD34-

  • rbital fibroblasts

These insights might lead to more specific therapies Could the TAO orbit represent a recapitulation of the thyroid? .

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)

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Acknowledgements

UCLA

Rui Han Shanli Tsui Beiling Chen Andrew Gianoukakis Jane Pritchard Nikoo Afifiyan Robert Goldberg Raymond Douglas Mei-Ju Shih Chieh Chih Tsai Vibha Naik Shweta Kamat Chris S. King Cathy J. Hwang Kelvin K-L Chong

Michigan

Raymond Douglas Stephen Atkins Roshini Fernando Bin Li Nupur Raychauduri Erin Gillespie Ying Lu Tunde Meister Fox Lab David Fox Steve Lundy

Odense

Tom Brix Laszlo Hegedus

PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Terry J. Smith)