C57BL/6J (Stock #000664), C57BL/6NJ (Stock #005304), B6.129S2-Alox5^tm1Fun (Alox5^−/−; Stock #004155), and B6.129S4-Ltb4r1^tm1Adl (Ltb4r1^−/−; Stock #008102) were purchased from Jackson Laboratories. Mice with a targeted deletion of Hif1a in myeloid cells were created via crosses into a background of lysozyme M-driven cre-recombinase (Hif1a^LysM/LysM), as previously done (30). All mice were 8–10 weeks of age at the time of infection. All animal experiments were approved by the Montana State University Institutional Animal Care and Use Committee or Dartmouth College Institutional Animal Care and Use Committee.

Aspergillus fumigatus strains CEA10 and Af293 were grown and harvested as previously described (10). For fungal inoculation, mice were anesthetized with isoflurane and challenged by the intratracheally (i.t.) route with 4–7 × 10⁷ A. fumigatus conidia in 100 µl sterile PBS. At the indicated time after challenge, mice were euthanized using an overdose of pentobarbital. Samples were collected and analyzed for inflammatory cell recruitment, fungal growth, lung damage, and vascular/epithelial leakage as previously described (10). For survival studies, mice were challenged with 4–7 × 10⁷ conidia of either CEA10 or Af293 and monitored daily using a humane endpoint scoring system. Mice were humanely euthanized once they met endpoint criteria.

Bone marrow chimeric mice were made by lethal irradiation of C57BL/6 mice followed by intravenous reconstitution with either C57BL/6 bone marrow or Alox5^−/− bone marrow. Mice were rested 6–8 weeks prior to challenge with 4 × 10⁷ conidia of CEA10 i.t. At the indicated time-points, mice were euthanized using an overdose of pentobarbital, and samples collected and analyzed for inflammatory cell recruitment and fungal growth as previously described (10).

Lipids were extracted from bronchoalveolar lavage fluid (BALF) using a hot-methanol extraction. Briefly, three parts HPLC-grade methanol were added to one part BALF sample. Samples were then vortexed for 30 s and placed into an 80°C water bath for 2 min. Tubes were spun at 14,000 RPM for 15 min and supernatant was collected then dried using a vacuum concentrator. Pellets were resuspended in HPLC-grade water in a volume equal to the starting volume of BALF sample. Extracted samples were then analyzed using enzyme immunoassay kits for LTB₄, cysteinyl leukotrienes (cysLT) (Cayman Chemical). Plates were read using a SpectraMax^® Paradigm^® plate reader (Molecular Devices).

Statistical significance between experimental groups was determined using a Mann–Whitney U test (comparison of two experimental groups that are not normally distributed) or an one-way ANOVA with a Dunn’s post-test (comparison of greater than two experimental groups that are not normally distributed), using the GraphPad Prism 6 software. For survival studies, Mantel–Cox log-rank test was used to determine whether there were significant differences in survival between C57BL/6, Hif1a^LysM/LysM, Ltb4r1^−/−, and Alox5^−/− mice for each A. fumigatus strain.

In order to determine whether leukotrienes are produced following A. fumigatus challenge, we challenged C57BL/6 wild-type mice with the CEA10 strain of A. fumigatus. Throughout a time course of 6–48 h post-infection (hpi), we collected BALF and measured leukotriene production. We found that LTB₄ and cysLT showed increased production after A. fumigatus CEA10 challenge. The production of these inflammatory lipid mediators followed a similar trend early after infection in which their expression peaked at 6 hpi, followed by decreased levels at 12 hpi. At 24 and 48 hpi LTB₄ increased from the 12 hpi levels, while the cysLT levels continue to decrease (Figure 1). These data demonstrate that both LTB₄ and cysLTs are synthesized following challenge of immunocompetent mice with A. fumigatus.

LTB₄ is known to be important in the recruitment of neutrophils in numerous inflammatory settings (14), but whether it is crucial in regulating the innate immune response following A. fumigatus challenge is unknown. To address whether LTB₄ was critical for neutrophil recruitment and resistance against IPA, we challenged Ltb4r1^−/− and C57BL/6 mice with A. fumigatus. At 12 hpi, we analyzed BALF via differential cytospins stained with Diff-Quik™ to assess early inflammatory cell recruitment to the airways. Compared with the C57BL/6 mice, Ltb4r1^−/− mice had a significant defect in neutrophil and eosinophil numbers, while macrophage numbers was similar (Figure 2A). Because the early recruitment of neutrophils is needed for host resistance to invasive A. fumigatus infection (6), we next addressed whether fungal growth was enhanced in the Ltb4r1^−/− animals. At 24 hpi, Grocott-Gomori methenamine silver (GMS) staining of lung sections revealed the presence of an increased proportion of germinated A. fumigatus in Ltb4r1^−/− mice compared with C57BL/6 mice (Figure 2B) demonstrating Ltb4r1^−/− mice were impaired in their ability to clear the fungi (Figure 2C). Lastly, lung damage and endothelial/epithelial leakage induced by A. fumigatus challenge were assessed by quantifying lactate dehydrogenase (LDH) and albumin in the BALF, respectively. Albumin levels were significantly elevated in Ltb4r1^−/− mice compared with C57BL/6 mice, indicating an increase in protein leakage from the vascular system (Figure 2D). In contrast, LDH levels were only mildly elevated suggesting induction of similar degrees of cell damage (Figure 2D). Taken together, these data demonstrate that LTB₄ signaling through its high-affinity receptor LTB4R1 is important in mediating neutrophil and eosinophil recruitment to the airways, which was ultimately necessary for host resistance to A. fumigatus growth.

Aspergillus fumigatus itself is known to be capable of producing eicosanoids (25), which results in an infection system in which both the mammalian and fungal cells could be the source of bioactive LTB₄. Thus, to address whether LTB₄ production coming from the murine cells was necessary for host resistance against A. fumigatus, we challenged 5-lipoxygenase (5-LO) (Alox5^−/−) deficient mice with A. fumigatus. Alox5^−/− mice cannot convert arachidonic acid to LTA₄ and, therefore, lack all leukotriene synthesis (22). After A. fumigatus challenge, inflammatory cell recruitment to the airways was quantified at 12 hpi via cytospins and Diff-Quik™ staining. Similar to what we found with the Ltb4r1^−/− mice, Alox5^−/− mice had a significant defect in both neutrophil and eosinophil recruitment, while macrophage accumulation remained largely similar to C57BL/6 (Figure 3A). This defect in neutrophil and eosinophil recruitment correlated with an impairment in the ability of Alox5^−/− mice to control fungal growth within the lung, demonstrated by a significantly higher germination rate (Figures 3B,C). We also measured LDH and albumin levels in the BALF of the Alox5^−/− mice to assess lung damage and vascular/epithelial permeability, respectively. Interestingly, unlike the Ltb4r1^−/− mice, LDH and albumin levels in BALF from Alox5^−/− mice were not significantly different than C57BL/6 levels (Figure 3D). Together, these data indicate that leukotriene synthesis by host cells is critical for neutrophil and eosinophil recruitment, as well as host resistance against A. fumigatus growth.

To begin to determine the cells that contribute to 5-LO activity after A. fumigatus challenge, we utilized a bone marrow chimera approach. C57BL/6 mice were lethally irradiated then reconstituted with either C57BL/6 or Alox5^−/− bone marrow intravenously to develop the following groups: C57BL/6 mice possessing C57BL/6 bone marrow and C57BL/6 mice possessing Alox5^−/− bone marrow. Mice were then rested for 6–8 weeks prior to challenge with 4 × 10⁷ conidia of CEA10. At 36 hpi, mice were euthanized, BAL collected for analysis of leukocyte recruitment to the airways, and lungs saved for histological analysis to assess fungal growth by GMS staining. Compared with C57BL/6 mice possessing C57BL/6 bone marrow, C57BL/6 mice possessing Alox5^−/− bone marrow had a significant defect in neutrophil and eosinophil recruitment to the airways at 36 hpi, while macrophage accumulation was not significantly altered (Figure 4A). Moreover, at 36 hpi, C57BL/6 mice possessing Alox5^−/− bone marrow were impaired in their ability to control fungal germination compared with C57BL/6 mice possessing C57BL/6 bone marrow (Figure 4B). Taken together, these data suggest that radiosensitive cells contribute to 5-LO activity which is needed for neutrophil and eosinophil recruitment to the airways and control of fungal germination following pulmonary A. fumigatus challenge.

Thus far, we have shown that in response to the CEA10 strain of A. fumigatus, host-derived leukotriene synthesis is critical for timely neutrophil recruitment and control of fungal germination within the lung. Due to studies that demonstrate fungal-strain dependency on the host immune response, we next sought to determine if leukotriene-dependent immunity was reliant on the strain of A. fumigatus used for challenge (12, 13, 26). Inflammatory responses against the hypervirulent CEA10 isolate are highly inflammatory and heavily dependent on IL-1α for maintaining host resistance (12). Additionally, we showed that IL-1α release from macrophages following CEA10 challenge required calpain, which is a Ca²⁺-dependent protease. Since, cPLA2 release of arachidonic acid is also a Ca²⁺-dependent event, we hypothesized that strain-specific LTB₄ responses might be likely. To test this, we challenged C57BL/6 and Alox5^−/− mice intratracheally with 5 × 10⁷ conidia of either CEA10 or Af293. We monitored health and survival of mice in the 192 h following fungal challenge. Alox5 (5-LO) was dispensable for survival of mice challenged with the Af293 strain of A. fumigatus, which is demonstrated by 100% survival of all mice challenged with Af293 (Figure 5). Conversely, Alox5^−/− mice challenged with CEA10 displayed significantly higher mortality than C57BL/6 mice. Similar mortality trends were observed in the Ltb4r1^−/− mice (data not shown). Overall, these data are in agreement with several other studies demonstrating that innate immune responses necessary for host resistance against A. fumigatus are highly dependent on the fungal strains used.

Under hypoxic conditions, the transcription factor hypoxia inducible factor-1α (HIF-1α) is activated, which allows translocation to the nucleus where it can bind to hypoxia response elements in the promoter region of target genes (27). HIF-1α is involved in controlling expression of 5-LO activating protein, a protein critical for the biosynthesis of LTB₄ and cysLTs, after hypoxic challenge (28). Previous work has shown that hypoxic microenvironments form within the lung in multiple murine models of IPA (29). In an immune competent model of fungal bronchopneumonia, myeloid-derived HIF-1α was shown to be critical for neutrophil recruitment and ultimately, survival of mice challenged with A. fumigatus CEA10 strain (30). Given the link between HIF-1α and leukotriene biosynthesis, we sought to determine if HIF-1α contributes to LTB₄ production following A. fumigatus challenge. To test this, C57BL/6 or myeloid-specific lysozyme-M cre-recombinase driven HIF-1α null mice (Hif1a^LysM/LysM) mice were challenged with 5 × 10⁷ conidia of either CEA10 or Af293, and LTB₄ protein levels measured in the BALF at 8 hpi. Following challenge with CEA10, there was a significant decrease in LTB₄ protein released into the airways of Hif1a^LysM/LysM mice compared with C57BL/6 mice (Figure 6A). Interestingly, following Af293 challenge LTB₄ levels remained unchanged between C57BL/6 and Hif1a^LysM/LysM mice (Figure 6A). Overall, this suggests that HIF-1α contributes to control of LTB₄ release following pulmonary A. fumigatus challenge in a fungal-strain-dependent manner.

Due to strain-specific release of LTB₄ in Hif1a^LysM/LysM mice (Figure 6A), we next sought to determine if Hif1a^LysM/LysM mice displayed a strain-specific susceptibility to A. fumigatus challenge. To test this, we challenged C57BL/6 and Hif1a^LysM/LysM mice intratracheally with 7 × 10⁷ conidia of either CEA10 or Af293. We monitored health and survival of mice in the 11 days following fungal challenge. HIF-1α expression in LysM-expressing cells was dispensable for survival of mice challenged with the Af293 strain of A. fumigatus, which is demonstrated by 90% survival of all mice challenged with Af293 (Figure 6B). Conversely, Hif1a^LysM/LysM mice challenged with CEA10 displayed significantly higher mortality (10% survival), as previously reported (Figure 6B). Overall, these data are in agreement with the survival analysis of the Alox5^−/− mice and reiterate that the innate immune responses necessary for host resistance against highly virulent A. fumigatus isolates, such as CEA10, are highly dependent on a robust inflammatory response.