The Ferocious Role of Iron in Alveolar Type II Epithelial Cell Function

Abstract

Alveolar type 2 (AT2) cells are specialized epithelial cells in the lung with critical roles in maintaining alveolar function and integrity. These metabolically active cells are primarily responsible for synthesizing, secreting, and recycling pulmonary surfactant; a complex mixture of lipids and proteins that reduces surface tension at the air-liquid interface. Beyond surfactant production, AT2 cells are pivotal in lung repair, possessing the ability to self-renew or transdifferentiate into alveolar type 1 (AT1) cells to restore epithelial integrity following injury. AT2 cells are central to alveolar health, with loss or dysfunction of AT2 cells contributing to several chronic lung diseases. The functional identity of AT2 cells is intimately linked to mitochondrial homeostasis, reflected by their reliance on mitochondrial activity to sustain the metabolic demands of their specialized roles. Mitochondria require a variety of nutrients for optimal function, with iron being one of the most essential. Mitochondrial energy production and iron metabolism are interdependent processes, each crucial to the other's function. Together, they lie at the centre of cellular and tissue iron homeostasis and both have been implicated in the pathogenesis of several chronic lung diseases. Yet mitochondrial iron (MITOIRON) regulation is an underappreciated and understudied function in AT2 cells of the lung. We hypothesized that iron metabolism, specifically MITOIRON metabolism influences AT2 cell-specific functions and contributes to their dysfunction in disease. In this thesis this hypothesis was tested using both in vitro and in vivo models, whereby iron and MITOIRON levels were manipulated in AT2 cells and their effects on surfactant homeostasis and alveolar repair examined. Collectively, these approaches revealed that both excessive and insufficient iron levels negatively impact AT2 cell function, with modulation of MITOIRON particularly detrimental. Specifically, disruptions in MITOIRON metabolism in AT2 cells profoundly impaired all aspects of surfactant production and AT2 cell mediated repair processes both in vitro and in vivo. CRISPR/Cas9-mediated knockdown of the mitochondrial iron transporters Mitoferrin 1 (Mfrn1) and Mitoferrin 2 (Mfrn2), significantly decreased intracellular iron, heme and MITOIRON levels in vitro. Reduced MITOIRON levels stunted wound healing and impaired surfactant synthesis, exocytosis and uptake in vitro. Conditional loss of Mfrn1/2 in murine AT2 cells in vivo resulted in a loss of AT2 cell markers and disruptions in surface tension. Additionally, these mice presented with severe alveolar neutrophilia, inflammation and failed to activate the integrated stress response, leading to lung-iron dyshomeostasis, acute lung injury, a loss of lung integrity and ultimately mortality. In the above models, such AT2 cell iron deficiency unexpectedly correlated with the presence of a senescent-like phenotype, a recognized characteristic of AT2 cells in lung disease but commonly linked to excessive iron accumulation. The development of an in vitro model of the injured lung epithelium using bleomycin (BLM) or cigarette smoke (CS) extract revealed that AT2 cell iron-deficiency is coupled with pre-senescent characteristics. This may act as a trigger for AT2 cells to acquire more iron, leading to AT2 cell iron accumulation and sustained cellular senescence. These findings were recapitulated in a murine BLM-induced fibrosis model whereby AT2 cells iron deficiency precedes elevated senescence markers and iron accumulation. This thesis provides the most in-depth analysis of how iron and MITOIRON metabolism contributes to the specialized functions of AT2 cells, including the first-ever targeted genetic manipulation of MITOIRON specifically in AT2 cells. Together, the findings uncover a previously unrecognized role for iron in sustaining AT2 cell function, implicating MITOIRON metabolism as a potential contributor to cellular dysfunction in lung disease. By illuminating the link between mitochondrial iron and AT2 cell integrity, this work opens new avenues for therapeutic intervention in disorders marked by AT2 cell dysfunction and senescence.