Characterizing the Alveolar Macrophage and Neutrophil Responses to Inhaled Coccidioides Spores using Ce3D-cleared Precision-Cut Lung Slices

Introduction/Rationale: Coccidioidomycosis (Valley Fever) is a respiratory disease caused by inhalation of spores from Coccidioides spp, dimorphic fungi endemic to arid regions of the Americas. Although thousands of cases are reported annually, the true incidence is likely far higher. Most infections are cleared asymptomatically, but a subset progresses to severe pulmonary or disseminated disease, suggesting that early host-pathogen interactions at the respiratory barrier critically shape disease outcome.

Methods: To understand the roles of the various cells coordinating this rapid response, we infected mice with fluorescently-labelled Coccidioides posadasii Δcts2/ard1/cts3 and prepared 1-mm thick clearing-enhanced 3D (Ce3D) precision cut lung slices (PCLS) to investigate 3D spatial aspects of the lung. We utilized Imaris-based 3D spatial analyses to quantify the early cellular spatial dynamics of alveolar macrophages and neutrophils and their interactions with inhaled spores.

Results: We observed that inhaled spores deposited near the broncho-alveolar duct junction (BADJ). Alveolar macrophages (AM) rapidly migrated locally to phagocytose spores in alveolar spaces and interestingly, in terminal bronchiolar spaces, as well. At low spore doses, AMs were highly effective at capturing most spores, and neutrophils were not recruited to the lung. Conversely, at higher spore doses, AMs no longer effectively phagocytosed spores, suggesting they could be overwhelmed by the increased fungal load. Instead, significant amounts of neutrophils were recruited and contributed to phagocytosis of most of the spores. Finally, we also demonstrated that neutrophils migrated to the site of BADJ infection via both alveolar capillaries and through the pulmonary arteriole, which is an uncharacterized route of neutrophil entry.

Conclusion: Our work provides a novel spatial perspective of the early dynamics of pulmonary fungal infections by leveraging robust 3D imaging and analyses.