Cigarette smoke exposure and impaired endoplasmic reticulum stress responses underlie altered pulmonary macrophage composition and function during lung injury, fibrosis, and repair.

Abstract

RATIONALE. Fibrotic interstitial lung diseases (ILDs) comprise a wide array of heterogeneous disorders of known and unknown etiology. Pulmonary fibrosis constitutes the late phase of ILDs. A variety of extrinsic and intrinsic risk factors are implicated in fibrotic ILD development and pathogenesis, with macrophages considered central orchestrators of disease pathogenesis. Particularly, cigarette smoking, protein misfolding, endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR), have been associated with impaired macrophage activation and function. However, a comprehensive understanding of the impact of these processes on the composition and function of pulmonary macrophage subpopulations and subsequently on tissue remodelling in pulmonary fibrosis is yet to be elucidated. In this Ph.D. thesis, we assessed the impact of cigarette smoke (CS) exposure and the myeloid-specific deletion of Atf6α, one of the UPR mediators, on pulmonary macrophage subpopulation composition and function during lung injury, tissue remodelling, and fibrogenesis. METHODS. Current literature to date demonstrates conflicting evidence regarding the impact of smoking status on long-term outcomes in the setting of fibrotic ILDs. Therefore, to further understand the impact of smoking status on fibrotic ILD patients’ survival, we began by assessing a prospective observational cohort, the Canadian Registry for Pulmonary Fibrosis (CARE-PF). This included 3062 patients with fibrotic ILD who were considered smokers or never smokers. Next, we used a preclinical experimental mouse model of concurrent bleomycin-induced lung injury and CS exposure to investigate the effect of CS on macrophage subpopulation composition and function during tissue remodelling processes. Lastly, given that CS is known to stimulate the UPR and that an impaired UPR is a potential mechanism for macrophage dysfunction, we specifically addressed the impact of altered UPR on pulmonary macrophage composition and function during bleomycin-induced lung injury. To achieve that, we utilized the myeloid-specific deletion of Atf6α, one of the UPR mediators, experimental approach. MAIN RESULTS. Findings from subgroup analysis of CARE-PF patient cohort demonstrated that overall, there was a significant interaction between smoking and fibrotic ILD diagnosis (p-value for interaction is 0.039), with respect to mortality. Specifically, the subgroup analysis has shown that smoking was an effect modifier and significantly increased mortality in connective tissue disease-associated ILD and unclassifiable ILD patients, but did not have a significant effect on mortality in idiopathic pulmonary fibrosis and chronic hypersensitivity pneumonitis patients. Furthermore, preclinical findings demonstrated that cigarette smoke exposure impaired the composition of pulmonary macrophages increasing CD11b+ subpopulations including monocyte-derived alveolar macrophages (Mo-AM) as well as interstitial macrophage (IM)1, -2 and -3, at multiple CS exposure timepoints. The expansion of Mo-AM and IM3 was dependent on IL-1α and likely reflective of increased cell recruitment. Compositional changes in macrophage subpopulations were associated with impaired induction of fibrogenesis including decreased α-smooth muscle actin positive myofibroblast following intratracheal bleomycin treatment. Mechanistically, in vivo and ex vivo assays demonstrated predominant macrophage M1 functional status and reduced matrix metallopeptidase 9 activity in cigarette smoke-exposed mice. Lastly, following bleomycin administration, the myeloid-specific deletion of Atf6α altered pulmonary macrophage composition, expanding CD11b+ populations with dual polarized CD38+CD206+ expressing macrophages. Compositional changes were associated with an aggravation of fibrogenesis including increased myofibroblast and collagen deposition. Further mechanistic ex vivo investigation revealed that ATF6α was required for CHOP induction and for the apoptotic death of bone marrow-derived CD11b+ macrophages during chronic ER stress, a process we speculate to be crucial for the attenuation of fibrogenesis. CONCLUSION. CS and aberrant ER stress/UPR disturbed pulmonary macrophage subpopulation composition and function, expanding CD11b+ macrophages, and resulted in alterations in wound healing and repair processes. Further investigation of CD11b+ macrophages in clinical samples obtained from fibrotic ILD patients enrolled in CARE-PF is required. Targeting these populations through the UPR might offer a potential therapeutic approach to halt fibrotic ILD progression. We believe that a better understanding of the complex interplay of CS, UPR, and macrophage will identify potential intervention strategies to restore conventional macrophage and UPR functions and mitigate disease exacerbation.