UCLA

Iron is an essential trace mineral for normal biological function, and systemic iron homeostasis is tightly regulated via complex systemic mechanisms and iron transporters. While there has been much focus on systemic iron regulation and homeostasis, iron regulation in the lung has not been well characterized. In addition, little is understood about the regulation of iron transporters and their role in specific cell types and under different pathophysiological conditions in the lung. Altered iron levels have also been associated with various lung pathologies, with iron overload associated with acute lung iron injury and idiopathic pulmonary fibrosis severity and iron deficiency correlating with worse pulmonary arterial hypertension (PAH). Our goals were to elucidate the effect of iron dysregulation in multiple lung pathologies and to further explore the role of iron transporters in the lung.

For Chapters One and Two, we utilized hepcidin knockout mice (HKO) as a model of severe iron overload. In Chapter One, we induced acute lung injury (ALI) in HKO mice and wild-type (WT) littermates via oropharyngeal aspiration (OP) of lipopolysaccharide. While we did not observe any major differences in systemic inflammatory response or airway neutrophil infiltration, we did notice a mild and transient increase in vascular leakage and increased neutrophil activity, potentially due to increased lung tissue apoptosis. In Chapter Two, we treated HKO mice and WT littermates with bleomycin OP to induce pulmonary fibrosis and did not observe any effect of iron overload on lung collagen levels nor any differences in overall disease severity. Together, these data indicate that despite increased lung iron levels in human patients with ALI or idiopathic pulmonary fibrosis, iron overload may not play a significant role in the progression of either disease.

In Chapter Three, we examined the role of ZIP8, a transmembrane divalent metal ion importer that is most highly expressed in the lung and inducible by inflammatory stimuli. We generated and characterized a novel global inducible ZIP8 knockout (KO) mouse and observed an unexpected phenotype of elevated spleen iron levels and decreased serum iron in ZIP8 KO mice. These data suggest that ZIP8 plays a role in iron recycling during homeostasis. However, we did not see any difference in response to the stress states of hemolytic anemia or iron deficiency in ZIP8 KO versus wild-type mice, suggesting that Zip8 may be redundant in this system. We also showed that ZIP8 is expressed on lung distal airspace epithelial cells and transports iron from the airway into lung tissue. ZIP8 deletion, however, had no detrimental effect on the severity of LPS-induced acute lung injury or on the outcomes of Klebsiella pneumoniae lung infection. Thus, ZIP8 plays a role in systemic iron homeostasis but does not modulate the severity of inflammatory lung injury or the host defense against a common bacterial cause of pneumonia.

In Chapter Four, our goal was to establish a mouse model of iron deficiency in bone morphogenetic protein (BMP) type II receptor (Bmpr2)-associated PAH, as mutations in Bmpr2 are the most common genetic cause of PAH. We placed mice heterozygous for a Bmpr2-null allele and littermate controls on a low-iron diet to induce iron deficiency. Surprisingly, we found that iron-deficient Bmpr2 heterozygous mice may have less severe PAH than corresponding controls as evidenced by hemodynamics. We determined that while there are associations between iron deficiency and PAH potentially through Bmpr2, the Bmpr2 mutant mouse model is not the appropriate model for elucidating this relationship.

Chapter Five explored the role of erythroferrone (ERFE) in a mouse model of -thalassemia. ERFE is an erythroid hormone that increases iron availability by functioning as a BMP trap and inhibiting the master iron regulatory hormone hepcidin. Humans with -thalassemia and other forms of ineffective erythropoiesis produce very high levels of ERFE. To define the contribution of excessive ERFE levels on the severity of -thalassemia, we generated a “humanized” mouse model of -thalassemia by crossing mice overexpressing ERFE with Th3/+ mice, a -thalassemia mouse model that does not expresses high levels of ERFE. We found that elevated ERFE levels impair pup survival in a mouse model of -thalassemia during early life and greatly increase iron loading in the context of ineffective erythropoiesis. These findings suggest that targeting ERFE in β-thalassemia should be further studied for potential therapeutic applications.

In conclusion, we have examined the effect of iron overload and deficiency as well as the role of iron regulators and transporters on lung iron homeostasis and pathologies. Using various mouse models, we have characterized the effects of iron during ALI, pulmonary fibrosis and pulmonary vascular disease, the role of ZIP8 in iron pathophysiology, and the role of ERFE in β-thalassemia.