Immunopathogenesis in fungal asthma

The objective of this competitive renewal R01 is to continue to uncover mechanisms that contribute to the severity of disease in the 30%+ of asthmatics who are sensitized to fungi. Leukotrienes, immunopathogenic mediators derived from the metabolism of arachidonic acid (AA) by 5-lipoxygenase (5-LOX), are often elevated in severe asthma. As such, this pathway is a therapeutic target of the FDA-approved anti-asthmatic drugs Zileuton®/Zyflo® (inhibits 5-LOX) and Singulair®/Montelukast® (leukotriene receptor antagonist). A second enzyme involved in AA metabolism is 12/15-lipoxygenase (12/15-LOX). We show that asthmatic C57BL/6 wild-type mice have increased lung expression of Alox15 mRNA as well as increased levels of the 12/15-LOX product 12-HETE compared to naïve or vehicle treated mice. Asthmatic 12/15-LOX deficient mice (Alox15-/-) demonstrated lower airway hyperactivity (AHR) in the presence of lower type 1 and type 2-associated mediators, lower chemokine levels and decreases in multiple myeloid cell types. Bone marrow chimera studies identified 12/15-LOX expression in hematopoietic cells as contributing to exacerbation of allergic fungal asthma. Intriguingly, the absence of 12/15-LOX expression in dendritic cells and/or macrophages (via Cd11c Cre/Alox15 floxed mice) had no effect on AHR, implicating other hematopoietic cells in driving 12/15-LOX mediated AHR during allergic fungal asthma. In other data, we demonstrate the development of a 12/15-LOX reporter mouse in which 12/15- LOX is HA-tagged and show feasibility of using these mice to identify 12/15-LOX expressing cells in the lung by flow cytometry. Thus, 12/15-LOXHA mice will serve as a novel tool to identify hematopoietic/myeloid cell type(s) expressing 12/15-LOX during allergic fungal asthma. In other studies, we found that mice deficient in immune response gene 1 (Irg1-/-) (also known as aconitate decarboxylase, Acod1), which generates the immunomodulatory TCA cycle metabolite itaconate, clear A. fumigatus from the lung more efficiently, seemingly as a result of increased proinflammatory responses and augmented macrophage and neutrophil antifungal activity. In contrast, Irg1/Acod1-/- subjected to allergic fungal asthma had increased AHR, indicating a protective role for itaconate. Collectively, we hypothesize that the severity of allergic fungal asthma is dictated by the balance between specific lipidomic (i.e. 12/15-LOX) and metabolomic (itaconate) responses. The specific aims of the proposal are: (1) to define hematopoietic-associated 12/15-lipoxygenase dependent mechanisms promoting AHR during allergic fungal asthma, (2) to define mechanisms of itaconate-mediated regulation of AHR during allergic fungal asthma and (3) to define the therapeutic potential of 12/15-LOX and itaconate on AHR during allergic fungal asthma.