Existencia de soluci´on

1.1.4 Molecular Mechanisms of IPF

1.1.4.1 IPF may arise from a dysregulated wound healing process

IPF is believed to arise as a result of dysregulated wound healing process following alveolar epithelial injury caused by a persistent irritant. It is proposed that initial and repetitive injury occurs to the alveolar epithelium, and most likely causes damage to the type I alveolar epithelial cells (AEC1s) that line the alveolar surface²³. Under homeostatic conditions, these cells play a role in regulating structural mesenchymal cells through cell-cell contact and mediator secretion. When AEC1s are injured or lost, it is thought that the underlying AEC2s undergo hyperplastic proliferation to cover the exposed basement membranes. If this process is inefficient, alveoli can collapse and consolidate.

In normal conditions, AEC2 undergo steady state turnover (apoptosis), and the remainder of the cells will spread and differentiate into AEC1s. Under pathological conditions and in the presence of profibrotic mediators, including Transforming Growth Factor (TGF)-β however, fibroblasts

accumulate at the site of injury and differentiate into α-Smooth Muscle Actin (α-SMA) expressing myofibroblasts that have increased expression of ECM components, including collagen to form specialised structures called fibrotic foci^21,22. AEC2 surrounding the fibrotic foci are hyperplastic and abnormal rather than undergoing normal repair²⁴, and may relate to cellular stress and failure to regenerate AEC1s lost by injury²⁵.

Several lines of evidence support the hypothesis that insults to the alveolar epithelium can serve as a trigger of IPF. Early ultrastructural studies by electron microscopy have demonstrated alveolar epithelial injury in lung biopsies from IPF patients²⁶. Immunohistochemical studies have shown the upregulation of various mediators indicative of alveolar cell apoptosis, including pro-apoptotic Fas, p53 and p21, B-cell lymphoma (Bcl)-2 associated X protein (Bax) and Caspase-3, whereas anti-apoptotic Bcl-2 is downregulated^27–30.BAL from IPF patients also show soluble mediators indicative of increased apoptosis³¹. Inhibiting epithelial cell apoptosis with various approaches, including blocking the Fas-Fas ligand pathway and blocking caspase activation, abrogates bleomycin (BLM)-induced pulmonary fibrosis^32–35. On the other hand, direct stimulation of Fas via anti-Fas antibody or injury to type II pneumocytes via a diphtheria toxin (DTR) approach also leads to pulmonary fibrosis^36,37. The various factors that may lead to AEC damage is summarised in figure 1.2.

Epithelial injury may occur through insults from environmental factors, viral infections or genetic mutations in AECs. Enviromental factors such as cigarette smoking and microaspiration

(asymptomatic aspiration of oropharyngeal secretions or gastric fluids into the lung)^1,38,39 may lead to repeated injury to the lung. Other environmental factors, such as asbestos or silica, may persist in the lungs due to their large size and failure of antigen presenting cells (APCs) to effectively

phagocytose and remove them, resulting in prolonged lung injury. Both of these events may lead to that pathological chronic inflammation and subsequent dysregulated wound-healing that may ultimately result in pulmonary fibrosis.

Chronic viral infections, mainly herpes virus infections, may contribute to the pathogenesis of IPF^38,40–43. There is correlation between certain latent viral infections, such as Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus (HHV)-7 and HHV-8 and human IPF25,40–42,44–47. Such viruses are known to infect and remain latent in pulmonary epithelial cells⁴⁸, thereby rendering them less able to repair subsequent injury. Infection may also potentially change the transcriptional program of these cells such that they secrete profibrotic factors and recruit inflammatory cells and fibrocytes (bone marrow derived cells that express both hematopoietic and stromal markers) to the lung¹².

Genetic predisposition may also contribute to some forms of familial pulmonary fibrosis as identified in early studies^49–52. Pulmonary fibrosis developed in a subset of patients with defined clinical syndromes, such as Hermansky-Pudlak Syndrome (HPS) and dyskeratosis congenita (DKC)^49,53–55. HPS patients carry autosomal recessive mutations that lead to defective trafficking of intracellular Figure 1.2 IPF may arise from persistent injury of the alveolar epithelium, leading to alveolar epithelial cell (AEC)1 apoptosis and AEC2 hyperplasia. Persistent injury may be caused by endogenous genetic mutations in familial IPF, environmental factors such as cigarette smoke, microaspiration, asbestos or silica, or viruses including Epstein Barr Virus (EBV), Cytomegalovirus (CMV) and Human Herpes Virus (HHV)-7 and -8.

Chapter 1 | General Introduction

32 organelles. HPS associated pulmonary fibrosis (HPSPF) is thought to be caused by defective secretion of pulmonary surfactant by AEC2s that leads to tissue injury, or lysosomal accumulation that leads to aberrent activation of pulmonary macrophages, and will be discussed in greater detail in section 1.4.

DKC involves a group of mutations that directly or indirectly affects the functionality of telomerase RNA component (TERC) that extends telomeres. Shortened telomeres may lead to cellular apoptosis or permanent cell cycle damage due to the deoxyribonuclease nucleic acid (DNA) damage response.

In the context of IPF, AECs may not be able to self-renew and differentiate effectively following tissue injury⁵⁶ and thereby fail to efficiently repair and cover the denuded basement membrane, thus perpetuating the release of pro-fibrotic mediators and promoting fibrosis⁵⁶.

Genetic analysis of IPF patients have since identified mutations in several genes that may be involved in pathogenesis of the disease. A common theme to all mutations identified to date is that they affect genes that are expressed in lung epithelial cells [e.g. Surfactant Protein A (SPA)⁵⁷, Surfactant Protein C (SPC)12,49,58–62, and Mucin 5B (MUC5B)^63,64] or lead to molecular changes in AECs [e.g. Telomerase Reverse Transcriptase (TERT)^56,65]. Pulmonary surfactants are released by AEC2s to reduce surface tension within the lung. Missense or short deletion mutations in the SPC gene gives rise to the variant misfolded protein that acts as a persistent irritant through accumulation or complex formation and causes alveolar epithelial injury12,49,58–62. SPC mutations account for ~1% of sporadic IPF^61,62. Missense mutations in the SPA gene give rise to protein instability and retention in the endoplasmic reticulum (ER)⁵⁷. Reduced SPA secretion may affect lung homeostasis and lead to tissue injury. MUC5B is essential for the production of mucus, which is a pulmonary lubricant that serves as a chemical barrier. A common single nucleotide polymorphism (SNP) in the MUC5B promoter region is associated with familial interstitial fibrosis and also sporadic IPF^63,64. The SNP is present in 34-38% of sporadic IPF patients compared with 9-11% of healthy controls⁶⁴. The exact mechanism the variant MUC5B in promoting pulmonary fibrosis is unclear, although two hypotheses have been proposed. First, the variant protein that may alter the local cell environment, perhaps by altering the mucosal host defence and/or local cytokine production, such that these changes affect the capability of bronchiolar or alveolar epithelial cells to self-renew or differentiate in response to injury. Second, the variant MUC5B may also lead to excessive mucus production that may provide a physical barrier that compromises normal epithelial repair of denuded basal lamina⁶³. Mutations in essential telomerase genes, including human telomerase transcriptase (hTERT), telomerase RNA (hTR), and the associated machinery [e.g. dyskerin gene in DKC] lead to accelerated telomere shortening^65–69, and as aforementioned affects the ability of AEC2 to repair efficiently following tissue injury⁵⁶. 8-15% of patients with FPF^56,70 and 1-3% of patients with sporadic IPF^71,72 have one or more telomere-related mutations.

There are two non-exclusive hypotheses on the inducers for such dysregulated wound healing, including pathological chronic inflammation, and also an aberrantly activated epithelial cell

mediated process. Pathological chronic inflammation may lead to prolonged tissue injury that results in a dysregulated wound healing process. Immune cells that are recruited to the site of tissue injury, such as monocytes, macrophages, and lymphocytes, accumulate and may drive fibrosis through the extended release of of pro-fibrotic mediators that induce the migration, proliferation and activation of fibroblasts. In the aberrantly activated epithelial cell model, it was proposed that injury to the alveolar epithelium directly activated AECs to promote fibrosis through the release of pro-fibrotic mediators and also epithelial-to-mesenchymal (EMT) transition. The mechanisms for chronic inflammation and aberrantly activated epithelial cells in the initiation and progression of IPF will be discussed in greater detail in sections 1.1.4.2 and 1.1.4.3 respectively.

Chapter 1 | General Introduction

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