The Mechanism of Epithelial Injury and Repair: An Overview

Normal Type 1 and Type 2 Alveolar Epithelial Cells, Small

Used with permission from Medical Histology Atlas. Pennsylvania State University College of Medicine.

Through the past decade, substantial progress has been made elucidating pathways involved in lung injury repair and remodeling in IPF. However, many fundamental questions remain. For example, while the prevailing hypothesis focuses on AEC injury and repair mechanisms driving IPF pathogenesis, and animal models14 suggest that epithelial cell injury can lead to lung fibrosis, there is uncertainty as to the source(s) of AEC injury in most patients with IPF.5

In this context, the study of patients with familial pulmonary fibrosis, a subgroup of patients in which 2 or more first-degree relatives are affected with the disease, led to the identification of new potential disease mechanisms. In patients with mutations in surfactant proteins,1112 defects in protein folding activate endoplasmic reticulum (ER) stress response pathways,15 which leads to epithelial cell death. In patients with telomerase mutations, the proliferative capacity of alveolar progenitor cells may be limited in response to injury.1617 Other factors, such as gastroesophageal reflux disease,18 tobacco smoke exposure,19 respiratory viruses,20 and a variety of other factors have been proposed as a source of recurrent/repetitive injury to the alveolar epithelium.1821 However, the mechanisms through which they promote lung fibrosis remain incompletely understood.

While sources of AEC injury may be diverse, subsequent steps in the injury response have been characterized in some detail (Figure 1).22 Following injury to AECs, there is a loss of epithelial integrity accompanied by death of AECs through apoptosis and other pathways.23 Epithelial cell death triggers release of a variety of cytokines and chemokines by surviving epithelial cells and inflammatory cells, including transforming growth factor-beta (TGFβ) and platelet-derived growth factor (PDGF).10 These growth factors activate repair programs in epithelial cells and promote accumulation of myofibroblasts in fibroblast foci and an increase in extracellular matrix secretion and deposition.10 Although these mechanisms are involved in normal wound healing and epithelial repair processes, it is hypothesized that in the setting of recurrent or ongoing epithelial injury, repeated cycles of injury/incomplete repair lead to progressive scar formation, which over time leads to clinically evident lung fibrosis (Figure 2).5

Figure 1.
Proposed mechanism for the pathogenesis of IPF. Adapted from Maher TM. Clin Chest Med. 2012; 33:69-83.22

Proposed Mechanism for IPF Pathogenesis

Figure 2.
Micrograph derived from hematoxylin and eosin stains from 2 patients with IPF demonstrating a fibroblastic focus. Note the loose architecture of connective tissue and fibroblasts. Activated hyperplastic epithelial cells are frequently found in close proximity to the cluster of fibroblasts. Reprinted with permission from the Annual Review of Pathology: Mechanisms of Disease, Volume 9 © 2014 by Annual Reviews www.annualreviews.org

Hematoxylin and Eosin Stains in IPF Micrograph, Fibroblastic Focus

Normal Injury Repair Mechanisms

In the normal adult alveolus, the gas exchange surface is comprised of Type I AECs in close interface with the pulmonary capillary network.24 Type II AECs are secretory cells found in the “corners” of alveolar spaces. (Figure 3) A key function of type II AECs is surfactant production, which is crucial for reducing surface tension within alveoli and maintaining the gas exchange surface; loss of function mutations in surfactant proteins25 or key trafficking components26 leads to severe interstitial lung disease in childhood.

Figure 3.
Normal Type I and Type 2 AECs in the adult alveoli. Used with permission from Medical Histology Atlas. Pennsylvania State University College of Medicine. medicalhistology.us/wiki/Main/LungImages. Accessed April 25, 2014.

Normal Type 1 and Type 2 Alveolar Epithelial Cells

Another proposed key function of type II AECs is to serve as progenitor cells for both type I and type II AECs.27 Elegant animal studies using cell-fate mapping strategies suggest that type II AECs are long-lived and are capable of differentiating into Type I AECs during normal alveolar maintenance27 and following injury.28 Turnover of AECs during maintenance appears to be most prominent in the periphery of the lung,27 where the earliest radiographic changes in IPF typically occur,29 suggesting a possible relationship between epithelial repair mechanisms and IPF.

The Current Model of IPF Pathogenesis

The current model of IPF pathogenesis suggests multiple factors and pathways interact through various stages of disease development to produce the histopathological and clinical features of IPF.5 As depicted in Figure 4, predisposing risk factors that foster epithelial cell dysfunction create a susceptible lung epithelium.5 Following injurious stimuli, activation of injury repair mechanisms initiates pathogenic changes. Subsequently, disease progression is mediated through ongoing extracellular matrix deposition, accumulation of myofibroblasts, and aberrant tissue remodeling.5

Figure 4.
The clinical course of IPF pathogenesis consists of predisposition, initiation, and progression. Adapted from: Wolters PJ, Collard HR, Jones KD. Annu Rev Pathol Mech Dis. 2014;9:157-179.5

The Course of IPF Progression

Predisposition

In recent years there has been significant progress in our understanding of the genetic predisposition to IPF, and both common and rare genetic variants have been linked to IPF risk.1112131617303132 As summarized in Table 1, association with a common genetic variant has been found in a large proportion of patients with sporadic IPF.32 In 2011, a genome-wide linkage study32 identified a polymorphism in the promoter of the gene encoding mucin 5B (MUC5B) that was associated with a 6-fold increased risk of IPF. Among patients with IPF, the minor allele frequency was 34% compared to 9% in control subjects; multiple subsequent studies in independent cohorts have confirmed this association.333435 The MUC5B promoter polymorphism has been associated with increased MUC5B mRNA expression,32 and with risk of asymptomatic interstitial lung abnormalities on CT scan.36 Although MUC5B has a role in host defense responses in the airways,37 its role in IPF pathogenesis is not yet clear.

As summarized in Table 1, more than 10 additional loci have been associated with IPF and cumulatively may be responsible for as much as 31% of the genetic risk for IPF.30 Common variants in 3 telomere-related genes (TERT, TERC, and OBFC1) have also been associated with IPF.30 Interestingly, rare mutations in 3 components of the telomerase complex telomerase reverse transcriptase (TERT),1617 telomerase RNA component (TERC)1617 and dyskerin (DKC1)38 have been identified in familial pulmonary fibrosis. Additionally, up to a third of IPF patients have short telomeres in peripheral blood mononuclear cells3940 and AECs,3839 suggesting that defects in telomere maintenance may underlie one path to lung fibrosis. Similarly, in a small proportion of families with IPF, dominant mutations in 2 surfactant proteins, surfactant protein C (SFTPC)1112 and surfactant protein A (SFTPA2)13 have been identified, leading to interest in the role of protein misfolding and ER stress in lung fibrosis.

Table 1.
Genes and genetic loci associated with IPF

Rare Variants Common Variants
SFTPC1112 MUC5B31323334
SFTPA213 TERT30
TERT1617 DSP30
TERC1617 7q2230a
DKC138 MUC230
  15q14-1530a
  MAPT30
  DPP930
  TERC30
  FAM13A30
  OBFC130
  ATP11A30
  TOLLIP31
  MDGA231
  SPPL2C31

a Chromosome region

In addition, human and animal studies suggest a variety of other mechanisms and pathways contribute to predisposition and cellular dysfunction in IPF. Oxidative stress contributes to epithelial injury and fibroblast differentiation.4142434445 Aging leads to alterations in epithelial repair46 through autophagy pathways,47 and worsens several animal models of lung fibrosis.4648 Additionally, mechanical factors including stretch/stress injury alter epithelial cell phenotype and cytokine production.495051

Initiation

Following injury to the alveolar epithelium, repair pathways are activated. There is intralveolar activation of procoagulant mechanisms,52535455 including platelet activation, that lead to alterations in epithelial cell phenotypes mediated through proteinase-activated receptors (PARs). This leads to production of chemokines and cytokines from epithelial cells56 which promote fibroblast recruitment and activation.57 In addition to release of active transforming-growth factor beta (TGFβ) from platelet granules, there is enhanced localized production and activation of TGFβ by epithelial cells.5859 Levels of active TGFβ are increased in the lungs of IPF patients,60 and both in vitro59 and in vivo studies14 have suggested that overexpression of TGFβ can lead to lung fibrosis. Latent TGFβ is produced constitutively by AECs5859 but is maintained in an inactive state bound to one of a variety of TGFβ binding proteins including latency associated peptide (LAP).61 In response to injurious stimuli, TGFβ is activated through binding to integrins expressed on the surface of AECs.61 TGFβ has broad effects on epithelial cells and fibroblasts, including alterations in microRNA expression.62 While the full extent of the role of TGFβ in lung remodeling is not yet fully understood, its expression in epithelial cells has been shown to cause epithelial apoptosis and lung fibrosis in genetically modified animals.63

In addition, other factors promote the accumulation of fibroblasts to the injured region of the lung and extracellular matrix deposition, including cytokine-,646566 chemokine-,67 and lipid-68 mediated chemotaxis and proliferation, and possible recruitment of circulating fibrocytes.69 Localized production of TGFβ and other growth factors leads to activation of fibroblasts and myofibroblast differentiation. This process appears to be mediated at least in part through activation of developmental pathways, including the Wnt/β-catenin axis707172 and the sonic hedgehog (SHH) pathway,74 and altered microRNA expression.6273

Progression

The progression stage is characterized by accelerated fibroblast differentiation, deposition of collagen and other extracellular matrix proteins, and extracellular matrix remodeling.5 Epithelial cells, fibroblasts, and macrophages produce a variety of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs)7576777879 that participate in matrix remodeling. Localized production of TGFβ1480 and PDGF81 promotes differentiation of fibroblasts into myofibroblasts,1480 which then increase production of collagen and other extracellular matrix proteins. This differentiation process appears to be mediated at least in part through NADPH oxidase generated hydrogen peroxide.45 In addition to enhanced proliferation and activation, fibroblasts become relatively resistant to apoptosis through enhanced periostin expression8283 and activation of the PI3K/AKT pathway.84

Enhanced collagen production and matrix remodeling then contributes to a “feed-forward” cycle, promoting alterations in gene expression patterns,84 further myofibroblast differentiation,85 and TGFβ activation through stretch-mediated integrin signaling86 which increases matrix remodeling. Further epigenetic8788 and transcriptional regulatory changes,89 along with alterations in microRNA expression,739091 lead to large-scale alterations in cellular gene expression patterns, culminating in a pro-fibrotic phenotype.

The progression phase is also marked by acute exacerbations92 that are associated with rapid decline of lung function. The etiology of acute exacerbations is uncertain, although in some cases microaspiration may be a precipitant.93 There is evidence of increased AEC injury,94 and while clinical and radiographic features suggest the possibility of viral infection, extensive efforts have not confirmed viruses as etiologic agents in patients with acute exacerbations.95

Over time, this process leads to progression from microscopic to macroscopic disease, including the gross architectural destruction and honeycomb-like cystic changes that characterize advanced IPF.96

Content contributed by:
Jonathan A. Kropski, MD
Vanderbilt University