Figure 2. A. fumigatus germination at 24 hours following inhalation of conidia is increased in absence of alveolar macrophage NOX2, especially with neutropenia.

(A) Schema to assess germination into hyphae of inhaled AF293 conidia at 24 hpi without or with antibody-mediated neutrophil depletion. (B) Peripheral blood obtained by retro-orbital puncture was used to determine the absolute neutrophil count (ANC) at time of challenge (day 0) and at 24 hpi. (C) Total leukocyte counts from 1 ml BAL fluid. The percentage of neutrophils were identified by cytospin and used to calculate total BAL neutrophils. (D) Enumeration of hyphae in 1 ml BAL fluid. Each point represents an individual mouse. (E) Aspergillus CFU in 1 ml BAL fluid and in homogenate of right lung for the indicated mice. (F) Hyphae length measured on BAL cytospins from indicated groups, combined data from at least three mice per group. (G) Photomicrographs of hyphae (indicated with black arrowheads). i. 28 μm hypha from Ncf2^LysMCre mouse treated with isotype (63X). ii. 44 μm hypha from Ncf2^LysMCre mouse treated with anti-Ly6G (100X). iii. 66 μm hypha from Ncf2^fl/fl mouse treated with anti-Ly6G (100X); iv. tiled photomicrographs showing 244 μm hypha from Ncf2^LysMCre mouse treated with isotype (40 X). (H) Phagocytosis of YFP-labeled AF293 conidia by AM from indicated Ncf2 genotypes at two hours after inhalation. Ncf2^fl/−Ex3 mice are heterozygous for a floxed and an Exon 3-deleted Ncf2 allele. CD45⁺ AM were identified as SiglecF⁺CD11c⁺ and gated for YFP using BFL1 channel to identify AM with ingested conidia. Data represents at least 2 independent sets of experiments with n≥4 per group. Graphical data shown as mean ± SD. Student’s ‘t’ test was performed for comparisons between 2 groups and *P<0.05, **P<0.01, ***P<0.001 ****P< 0.0001 were considered as significant. For (F), distribution of hyphae length is non-normal per the Kolmogorov-Smirnov test and statistical comparisons between 2 groups used the Mann-Whitney test.