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

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 • Initiation of IPF – Epithelial cell dysfunction • Telomere dysfunction • Propagation of IPF – Pathologic Matrix • Relevance of IPF pathobiology to other fibrotic lung diseases 1

11/7/2018 IPF: Pathology IPF: Pathology 2

11/7/2018 IPF Survival /IPF Bjoraker et al, Am J Resp Crit Care Med ‘ 98 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 3

11/7/2018 Telomeres shorten with cell division TTAGGG Replication Replication Replication Cell Death or Senescence Predisposition to IPF: Epidemiology 4

11/7/2018 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 5

11/7/2018 IPF Genetic Predisposition: Summary Genes Associated with Familial Genes Associated with Sporadic IPF Pulmonary Fibrosis 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 6

11/7/2018 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 7

11/7/2018 IPF Epithelial Cells Express Senescence markers IPF Normal IPF Lung p16 SA‐  Gal Disayabutr et al, PLoS One 2016 Zhang et al Histopathology 2018 Initiation of IPF: Epithelial cell Telomere Dysfunction 8

11/7/2018 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 X LOXP LOXP LOXP LOXP TRF1 flox/flox SPC‐cre ERT SPC‐cre ERT/+ TRF1 flox/flox SPC‐cre +/+ TRF1 flox/flox Cre Not Cre Expressed Expressed Tamoxifen (i.p) Tamoxifen (i.p) 9

11/7/2018 Genes Dev 2009 • Cell selective deletion of TRF1 • Progressive telomere shortening • K5: delete from skin • Hyperpigmentation • Mx1: delete from BM • Bone marrow failure Telomere Dysfunction in Surfactant Protein C (SPC) Expressing Cells 10

11/7/2018 SPC‐cre TRF1 F/F mice SPC-creTRF1 F/F TRF1 F/F • Cross TRF1 F/F mice TRF1 β-actin with SPC‐Cre ERT2 rtTA mice • Treat mice with TRF1 F/F SPC-creTRF1 F/F tamoxifen weekly • Activate DNA damage γH2AX SPC response within 2 DAPI weeks SPC‐cre TRF1 F/F mice: 2 Weeks Tamoxifen TRF1 F/F SPC-creTRF1 F/F • Normal Lung Histopathology • Susceptible to Influenza 4x TRF1 F/F SPC-creTRF1 F/F 100 Percent survival ** 80 60 20x 40 20 0 0 2 4 6 8 10 Days (post influenza virus infection) 11

11/7/2018 SPC‐cre TRF1 F/F mice: Spontaneously Develop Lung Fibrosis SPC-creTRF1 F/F 3 M Tamox 8 M Tamox TRF1 F/F SPC-creTRF1 F/F *** 100 Percent survival 4x 80 60 40 20 0 0 2 4 6 8 10 Tamoxifen (Months) 20x Lung Remodeling is Associated with Increased Levels of Active TGF  TRF1 F/F SPC-creTRF1 F/F * Active TGF-  1 (pg/ml) 50 40 30 20 10 0 Tamox 3M 8M 12

11/7/2018 SPC‐cre TRF1 F/F mice: Accumulate SA  gal Epithelial Cells SPC‐cre TRF1 F/F mice: Accumulate  SMA Immunoreactive Cells  SMA 13

11/7/2018 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 Yes Yes cells 14

11/7/2018 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 15

11/7/2018 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 16

11/7/2018 IPF Matrix is Stiffer than Normal Booth et al, AJRCCM, 2012 Fibrotic Matrix Activates a Positive Feedback Parker et al JCI 2014 17

11/7/2018 Wolters et al, Lancet Resp Med 2018 Telomere Dysfunction in Fibrotic Lung Diseases other than IPF 18

11/7/2018 Telomere Mediated Lung Fibrosis is Diagnostically Heterogeneous Newton et al ERJ 2016 Survival is Poor Regardless of Clinical Subtype Newton et al ERJ 2016 19

11/7/2018 Pathologic Classification of Pulmonary Fibrosis Usual Interstitial Pneumonia Nonspecific Interstitial Pneumonia Hypersensitivity Pneumonitis Normal Pathologic Pattern by Clinical Diagnosis HP IPF RA Scleroderma • UIP 100% • NSIP 85% • UIP 70% • UIP 25% • UIP 15% • NSIP 20% • NSIP 75% • Bronchiolitis 10% 20

11/7/2018 Telomeres are Short in Type II cells of patients with non‐IPF UIP 15 ** SPC ** DAPI Telomere Signal/Cell 10 QFISH 5 p‐value N vs IPF 0.0045 N vs non‐IPF UIP 0.0022 0 N IPF non‐IPF UIP NSIP n=12 n=11 n=11 n=4 p16 Immunoblot N IPF non‐IPF UIP p16 β‐actin p16 2.5 2 1.5 p16 P‐value N vs. IPF <0.01 1 N vs. non‐IPF UIP 0.04 IPF vs non‐IPF UIP <<0.01 0.5 0 N IPF non‐IPF UIP (n=12) (n=12) (n=15) 21

11/7/2018 Hypersensitivity Pneumonitis PBL Telomere Length Predicts Survival In Hypersensitivity Pneumonitis Ley et al, Lancet Resp Med 2017 22

11/7/2018 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 Club cells secrete CCSP / CC10 / CC16 / Uteroglobin Cross TRF1 F/F mice with SPC‐Cre ERT2 rtTA • mice TRF1 flox/flox Scgb1a1‐cre TRF1 flox/flox TRF1 F/F SCGB1a1-creTRF1 F/F *** 100 Percent survival 80 60 40 20 0 0 2 4 6 8 10 12 Tamoxifen (Months) 23

11/7/2018 Collagen deposition around airways 4X 20X 40X TRF1 flox/flox SCGB1a1-cre TRF1 flox/flox 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 24

11/7/2018 Acknowledgements Academic Collaborators Lab Hal Collard Natalia Achtar‐Zadeh Matthew Donne Supparerk Dysabutyr Hilary Faust Gary Green Christine Garcia Janet La Jeff Golden Shuo Liu John Greenland Ram Naikawadi Brett Ley Meenal Sinha Joyce Lee Yingwei Zhang Mark Looney Benat Mallavia Michael Matthay Funding: Nina Ireland Program for Lung Health, Harroun Family Jason Rock Foundation, NIH David Schwartz UCSF Lung Transplant team 25