Early Neutrophil Recruitment and Aggregation in the Murine Lung Inhibit Germination ofAspergillus fumigatusConidia

Abstract

Several types of polymorphonuclear neutrophil (PMN) deficiency are a predisposing condition for fatalAspergillus fumigatus infection. In order to study the defensive role of PMNs in the lungs, with particular reference to PMN recruitment and antimicrobial oxidant activity, responses to pulmonary instillation of A. fumigatus conidia were examined. Responses in BALB/c and C57BL/6 mice were compared with those in CXCR2 ؊/؊ and gp91 phox؊/؊ mice, which are known to have delayed recruitment of PMN to the lungs in response to inflammatory stimuli and inactive NADPH oxidase, respectively. In BALB/c mice, PMNs were recruited to the lungs and formed oxidase-active aggregates with conidia, which inhibited germination. In C57BL/6, gp91 phox؊/؊ , and CXCR2 ؊/؊ mice, PMN recruitment was slower and there was increased germination compared to that in BALB/c mice at 6 and 12 h. In gp91 phox؊/؊ mice, germination was extensive in PMN aggregates but negligible in alveolar macrophages (AM). Lung sections taken at 6 and 48 h from BALB/c mice showed PMN accumulation at peribronchiolar sites but no germinating conidia. Those from C57BL/6 and CXCR2 ؊/؊ mice showed germinating conidia at 6 h but not at 48 h and few inflammatory cells. In contrast, those from gp91 phox؊/؊ mice showed germination at 6 h with more-extensive hyphal proliferation and tissue invasion at 48 h. These results indicate that when the lungs are exposed to large numbers of conidia, in addition to the phagocytic activity of AM, early PMN recruitment and formation of oxidative-active aggregates are essential in preventing germination of A. fumigatus conidia.Despite life-long exposure to Aspergillus fumigatus, very low morbidity is seen in immunocompetent individuals, indicating that a rapid and effective resistance to this organism has evolved. Individuals with normal immune systems rarely develop invasive pulmonary aspergillosis (IPA) even when exposed to high environmental concentrations of A. fumigatus conidia arising from, for example, disturbance of moldy wood chip piles, sludge, or compost (20) but may develop allergic bronchopulmonary aspergillosis or extrinsic allergic alveolitis (13). However, in recent decades there has been an increasing number of invasive and often fatal A. fumigatus infections in patients with immunosuppressive disorders (5, 16, 39), and A. fumigatus is now recognized as the leading airborne fungal pathogen in immunocompromised individuals (12,35).Resident alveolar macrophages (AM) phagocytose and subsequently destroy inhaled conidia (8, 38). However, the importance of polymorphonuclear neutrophils (PMN) in innate immunity to aspergillosis is indicated by the observation that patients and mice with PMN deficiency due to a variety of causes are susceptible to IPA (9, 30, 34, 37). The current study with mice was undertaken since there is limited information on PMN recruitment in response to inhalation of conidia and the mechanism by which PMN protect against aspergillosis.Our hypothesis was that following exposure of the lung to conidia, ea...

Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Independent Resistance to Aspergillus fumigatus in Alveolar Macrophages

“…13073; American Type Culture Collection) of A. fumigatus was grown on Sabouraud dextrose agar slants in 75-cm 2 culture flasks at 37°C for 5 days and conidia were collected in 0.1% Tween 20 in HBSS (no. 10-547F; Cambrex Bio Science) by gentle rocking as described (13). Conidia were then diluted in HBSS without Tween 20 to obtain either 10 6 or 10 7 conidia in 40 l for intrapharyngeal administration.…”

“…Human patients with defects in this system suffer from chronic granulomatous disease characterized by recurrent infections, extensive granuloma formation, and frequently, IPA (11). Although it is generally accepted that NADPH oxidase is necessary for killing A. fumigatus conidia by PMN (12,13), there is conflicting information on the role of this mechanism in AM-mediated resistance to IPA (12, 14 -17). Therefore, in addition to transcriptional studies on AM exposed to A. fumigatus conidia, we have examined functional aspects of AM for evidence of NADPH oxidase involvement following contact with conidia.…”

Human Polymorphonuclear Leukocytes Inhibit Aspergillus fumigatus Conidial Growth by Lactoferrin-Mediated Iron Depletion

“…Later, Levitz and Diamond (15) found that swollen conidia are indeed killed by human PMN, but through in vitro experiments implicated oxidative products as the mechanism. Recent studies in mice have demonstrated the importance of PMN recruitment to the lung in response to A. fumigatus conidia (16). Interestingly, whereas somewhat greater conidial germination was noted in CGD mice (knockouts in the gp91 subcomponent of the NADPH oxidase), between 60 and 70% of conidia in airway fluids remained ungerminated even after 12 h (16).…”

“…Recent studies in mice have demonstrated the importance of PMN recruitment to the lung in response to A. fumigatus conidia (16). Interestingly, whereas somewhat greater conidial germination was noted in CGD mice (knockouts in the gp91 subcomponent of the NADPH oxidase), between 60 and 70% of conidia in airway fluids remained ungerminated even after 12 h (16). Effective orchestration of the host response is complicated by the rapid developmental transformation of Aspergillus upon inhalation and clearly requires several distinct pattern recognition pathways (17)(18)(19)(20).…”

Fungal killing by mammalian phagocytic cells

“…Bonnet et al have shown that neutrophils form aggregates around A. fumigatus conidia and that this aggregation inhibited conidial germination in a NADPH oxidasedependent manner [49]. Another mechanism, named neutrophil extracellular trap (NET), has been described in defense against bacteria [50].…”