Outline Predisposition to Idiopathic Pulmonary Fibrosis (IPF) - - PDF document

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Outline Predisposition to Idiopathic Pulmonary Fibrosis (IPF) - - PDF document

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11/7/2018 Pathobiology of Idiopathic Pulmonary Fibrosis Paul Wolters,MD Professor University of California, San Francisco Outline Predisposition to Idiopathic Pulmonary Fibrosis (IPF) Epidemiology of IPF Genetic predisposition

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Pathobiology of Idiopathic Pulmonary Fibrosis

Paul Wolters,MD Professor University of California, San Francisco

Outline

  • Predisposition to Idiopathic Pulmonary Fibrosis

(IPF)

– Epidemiology of IPF – Genetic predisposition

  • Initiation of IPF

– Epithelial cell dysfunction

  • Telomere dysfunction
  • Propagation of IPF

– Pathologic Matrix

  • Relevance of IPF pathobiology to other fibrotic

lung diseases

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IPF: Pathology IPF: Pathology

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IPF Survival

Bjoraker et al, Am J Resp Crit Care Med ‘98 /IPF

Telomeres

  • Telomere: Repetitive DNA sequence at end of chromosomes,

which protect the end of chromosomes from deterioration

  • Maintained by telomerase enzyme complex

– TERT (reverse transcriptase), TERC (RNA template)

Calado and Young, NEJM 2009

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Replication Replication Replication TTAGGG

Telomeres shorten with cell division Cell Death or Senescence

Predisposition to IPF: Epidemiology

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Risk Factors for IPF

  • Tobacco smoking
  • Working in dusty environments
  • Gastroesophageal reflux disease
  • Aging
  • Genetic predisposition

IPF is a Disease Associated With Aging

Ley, Clin Epidemiol 2013

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IPF Genetic Predisposition: Summary

Genes Associated with Familial Pulmonary Fibrosis Genes Associated with Sporadic IPF SPA MUC5B SPC DSP TERT TERT TERC TERC RTEL1 OBFC1 PARN DPP9 ABCA3 FAM13A TINF2 TOLLIP DKC1 RTEL1 NAF1 PARN AKAP13

Pulmonary fibrosis genetic studies overwhelmingly implicate epithelial cells and telomere dysfunction in disease process

Telomeres are Shortened in Type II Cells (AECII) of IPF Patients

Alder J K et al. PNAS 2008 Kropski et al. Am J Resp Crit Care Med 2015

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Peripheral Blood Leukocyte Telomere Length Predicts IPF Progression

Dressen et al, Lancet Resp Med 2018

Peripheral Blood Leukocyte Telomere Length Predicts IPF Patient Survival

Stuart et al, Lancet Resp Med 2014

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IPF Epithelial Cells Express Senescence markers

IPF Normal

Disayabutr et al, PLoS One 2016 Zhang et al Histopathology 2018

IPF Lung p16

SA‐Gal

Initiation of IPF: Epithelial cell Telomere Dysfunction

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Modeling Telomere Dysfunction in Mice

  • Mouse telomeres are 5‐10x longer than human

telomeres

  • Deletion of TERT or TERC does not successfully

model diseases of telomere dysfunction

  • Mice become sterile before diseases manifest
  • Deletion of shelterin proteins TRF1, TRF2

Cell Specific Deletion of TRF1

SPC‐cre ERT/+ TRF1flox/flox

TRF1flox/flox

X

SPC‐creERT SPC‐cre +/+ TRF1flox/flox

Cre Expressed Cre Not Expressed

Tamoxifen (i.p) Tamoxifen (i.p)

LOXP LOXP LOXP LOXP

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  • Cell selective deletion of TRF1
  • Progressive telomere shortening
  • K5: delete from skin
  • Hyperpigmentation
  • Mx1: delete from BM
  • Bone marrow failure

Genes Dev 2009

Telomere Dysfunction in Surfactant Protein C (SPC) Expressing Cells

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TRF1 β-actin TRF1F/F SPC-creTRF1F/F

SPC-creTRF1F/F TRF1F/F

γH2AX SPC DAPI

SPC‐cre TRF1 F/F mice

  • Cross TRF1 F/F mice

with SPC‐Cre ERT2 rtTA mice

  • Treat mice with

tamoxifen weekly

  • Activate DNA damage

response within 2 weeks

SPC‐cre TRF1 F/F mice: 2 Weeks Tamoxifen

2 4 6 8 10 20 40 60 80 100

TRF1F/F SPC-creTRF1F/F

**

Days (post influenza virus infection) Percent survival

4x 20x TRF1F/F SPC-creTRF1F/F

  • Normal Lung Histopathology
  • Susceptible to Influenza
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SPC‐cre TRF1 F/F mice: Spontaneously Develop Lung Fibrosis

4x 20x SPC-creTRF1F/F

2 4 6 8 10 20 40 60 80 100

TRF1F/F SPC-creTRF1F/F

***

Tamoxifen (Months) Percent survival

3 M Tamox 8 M Tamox

10 20 30 40 50

TRF1F/F SPC-creTRF1F/F

*

3M 8M Tamox Active TGF-1 (pg/ml)

Lung Remodeling is Associated with Increased Levels of Active TGF

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SPC‐cre TRF1 F/F mice: Accumulate SAgal Epithelial Cells SPC‐cre TRF1 F/F mice: Accumulate SMA Immunoreactive Cells

SMA

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SPC‐cre TRF1 F/F mice: Develop Short Telomeres in AECII Cells Similarities between SPC‐cre TRF1 F/F mice and IPF

Feature IPF SPC‐cre TRF1 F/F mice Subpleural Fibrosis Yes yes Fibroblast accumulation Yes yes Macrophage accumulation Yes Yes Progressive/terminal Yes Yes AECII cell hyperplasia Yes Yes Epithelial reprogramming Yes Yes Senescent AECII cells Yes Yes Age/Time Dependent Yes Yes Short telomeres in AECII cells Yes Yes

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Propagation of IPF : Pathologic Matrix

IPF Fibroblast Biology

  • IPF fibroblasts are different from “normal” fibroblasts.

– Resistant to apoptosis – Make more collagen – More invasive

  • Fibroblast phenotype differs when cultured on matrix of

different stiffness (Young’s elastic modulus).

– Stiffness of lung: 1 kPa – Stiffness of plastic: 2‐4 GPa

  • Fibroblasts alter phenotype when cultured on matrix

derived from IPF lung vs. normal lung

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Decellularized lung

Booth et al, AJRCCM, 2012

IPF Extracellular Matrix Has a Unique Composition

Higher in IPF Matrix Lower in IPF Matrix Booth et al, AJRCCM, 2012

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IPF Matrix is Stiffer than Normal

Booth et al, AJRCCM, 2012

Fibrotic Matrix Activates a Positive Feedback

Parker et al JCI 2014

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Wolters et al, Lancet Resp Med 2018

Telomere Dysfunction in Fibrotic Lung Diseases other than IPF

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Telomere Mediated Lung Fibrosis is Diagnostically Heterogeneous

Newton et al ERJ 2016

Survival is Poor Regardless of Clinical Subtype

Newton et al ERJ 2016

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Pathologic Classification of Pulmonary Fibrosis

Usual Interstitial Pneumonia Nonspecific Interstitial Pneumonia Hypersensitivity Pneumonitis

Normal

Pathologic Pattern by Clinical Diagnosis

IPF

  • UIP 100%

RA

  • UIP 70%
  • NSIP 20%
  • Bronchiolitis

10% Scleroderma

  • UIP 25%
  • NSIP 75%

HP

  • NSIP 85%
  • UIP 15%
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5 10 15

N IPF non‐IPF UIP NSIP

n=12 n=11 n=11 n=4

Telomere Signal/Cell

Telomeres are Short in Type II cells of patients with non‐IPF UIP

** **

p‐value N vs IPF 0.0045 N vs non‐IPF UIP 0.0022

SPC DAPI QFISH

p16 Immunoblot

(n=12) (n=12) (n=15)

p16 P‐value N vs. IPF <0.01 N vs. non‐IPF UIP 0.04 IPF vs non‐IPF UIP <<0.01 p16 β‐actin

0.5 1 1.5 2 2.5 N IPF non‐IPF UIP

p16 N IPF non‐IPF UIP

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Hypersensitivity Pneumonitis

PBL Telomere Length Predicts Survival In Hypersensitivity Pneumonitis

Ley et al, Lancet Resp Med 2017

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MUC5B Minor Allele and Short Telomere Length predict Histopathologic and Radiologic Features Consistent with IPF

Ley et al, Lancet Resp Med 2017

Deletion of TRF1 in Club cells

2 4 6 8 10 12 20 40 60 80 100

Tamoxifen (Months) Percent survival

TRF1F/F SCGB1a1-creTRF1F/F

***

Scgb1a1‐cre TRF1 flox/flox TRF1flox/flox

Club cells secrete CCSP / CC10 / CC16 / Uteroglobin

  • Cross TRF1 F/F mice with SPC‐Cre ERT2 rtTA

mice

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TRF1flox/flox

4X 20X 40X

SCGB1a1-cre TRF1 flox/flox

Collagen deposition around airways

Conclusion

  • Telomere dysfunction isolated to lung epithelial cells is

sufficient to cause lung fibrosis and remodeling.

– Type II cell hyperplasia, macrophage accumulation, myofibroblast accumulation, fibrosis. – Histopathologic phenotype appears to be cell specific

  • Short telomeres contribute to lung remodeling in other

clinical contexts.

– Hypersensitivity Pneumonitis – CTD‐ILD (RA‐ILD, SSc‐ILD) – CLAD

  • Histopathologic appearance of disease depends on the

cellular subtype that has short telomeres.

  • Prediction: In the future pulmonary fibrosis cases will be

classified/treated based on their molecular phenotype

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Acknowledgements

Lab Natalia Achtar‐Zadeh Supparerk Dysabutyr Gary Green Janet La Shuo Liu Ram Naikawadi Meenal Sinha Yingwei Zhang Academic Collaborators Hal Collard Matthew Donne Hilary Faust Christine Garcia Jeff Golden John Greenland Brett Ley Joyce Lee Mark Looney Benat Mallavia Michael Matthay Jason Rock David Schwartz UCSF Lung Transplant team

Funding: Nina Ireland Program for Lung Health, Harroun Family Foundation, NIH