Investigations into the role of pulmonary innate immunity during fibrotic lung disease

Abstract

Interstitial lung diseases (ILD) incorporate a broad range of lung pathologies characterised by abnormalities in the pulmonary interstitium. The most common ILD is idiopathic pulmonary fibrosis (IPF), a progressive fibrotic lung disease with poor patient prognosis [1]. The pathological mechanisms and mediators driving IPF remain to be elucidated. The lung microbiome of IPF patients has been reported as altered compared to healthy controls [2, 3], with increased bacterial burden correlating with poor patient prognosis [2]. Our understanding as to why there are differences, and whether bacteria are contributing toward the pathology, has not yet been investigated. This project investigated the hypothesis that changes in specific bacterial genera in IPF may occur as a result of defective response by innate immune cells. This defective clearance of bacteria may lead to a pro-fibrotic response driven by repeat bacterial stimulation. To examine differences in immune composition, inflammatory profiles of the bronchial alveolar lavage (BAL) from healthy, IPF and other ILDs were compared with focus on the function and phenotype of airway macrophages (AMs). Furthermore, pro-inflammatory/pro-fibrotic responses by primary bronchial epithelial cells (BECs) and fibroblasts from IPF and healthy controls were examined post bacterial and TLR stimulation. Characterisation of the inflammatory and leukocyte profiles of the BAL showed significant increases in monocytes, AMs, and mediators CCL2 and M-CSF in IPF, which inversely correlated with forced vital capacity (FVC). Phenotypic analysis of AMs revealed two distinct phenotypes based on CD71+/- expression. The CD71+/- AMs exhibited significantly different surface marker and gene expression, transferrin uptake, phagocytosis and nitric oxide (NO) production. Interestingly, CD71- AMs were increased in IPF, correlating inversely with FVC decline, but were virtually absent from healthy control BAL. Supernatants from isolated and cultured IPF CD71- and CD71+ AMs differentially increased collagen IV secretion, with CD71- cells increasing collagen IV compared to CD71+. Supernatants from LPS stimulated CD71-/+ cells increased collagen IV deposition further. No differences were found comparing healthy and IPF AM ability to phagocytose bacteria, however, NO production appeared significantly higher in healthy compared to IPF. Stimulation of primary healthy and IPF BECs with various TLR ligands revealed significant increases in pro-inflammatory and pro-fibrotic gene expression post viral ligand Poly: IC stimulation. Moreover, epithelial cell supernatants collected after Poly: IC stimulation increased the release of pro-inflammatory mediators by fibroblasts. However, direct bacterial stimulation, or after the TLR4 ligand, LPS, induced limited or no changes in BECs or fibroblasts, indicating viral infection, rather than bacterial, may influence disease pathogenesis. Collectively, this body of data suggests that viral stimulation contributes toward fibrosis by activating BECs, inducing an upregulation of pro-fibrotic mediators including CCL2, IL-6, IL-8 and TGFβ1. CD71- AMs may enhance fibrotic progression in IPF patients increasing the deposition of collagen by epithelial cells and fibroblasts. Specific targeting of CD71- AM may represent a novel therapeutic strategy to alleviate fibrotic progression in patients.