Absence of Respiratory Burst in X-linked Chronic Granulomatous Disease Mice Leads to Abnormalities in Both Host Defense and Inflammatory Response to Aspergillus fumigatus

Animals.

X-CGD mice had previously been generated by targeted disruption of the gene encoding gp91^phox, the large subunit of the NADPH respiratory burst oxidase complex (15). Phagocytes from these mice are unable to generate superoxide as measured by both the nitroblue tetrazolium test and cytochrome c reduction assays. Mice were housed in microisolator cages under specific pathogen-free conditions and fed autoclaved food and acidified water ad libitum. For experiments involving X-CGD mice generated by backcrossing carrier females with C57Bl/6J males for generations up to N₇, wild-type littermates from the same backcross generation were used to minimize any potential bias contributed by differences in original 129-SV × C57Bl/6J genetic backgrounds. For experiments after generation N₇, agematched wild-type C57Bl/6J mice were used. Mice studied in these experiments were from 6 to 10 wk old.

Isolation and Preparation of Aspergillus Inocula.

A clinical isolate of A. fumigatus (ATCC No. 90240; American Type Culture Collection, Rockville, MD) was cultured on Sabouraud dextrose agar (Becton Dickinson Labware, Lincoln Park, NJ) for 4 d at room temperature before use. Conidia were removed by flooding the culture dish with 5–10 ml of sterile PBS and gently scraping the mycelia with a sterile razor blade. Suspensions consisting mostly of single conidia were prepared by vortexing the mixture vigorously for 1 min followed by filtration through three-ply cotton gauze. Conidia were quantitated using a hemocytometer and diluted to the appropriate concentration in PBS. Quantitation was confirmed by plate culture of serial dilutions on Sabouraud dextrose agar. Conidia suspensions were always used on the day of harvest and kept on ice until needed. Viability as determined by quantitative culture was typically >95%.

Killing of A. fumigatus Conidia by Alveolar Macrophages.

For each experiment, alveolar macrophages were harvested from at least three wild-type and three X-CGD mice as previously described (18). Briefly, mice were killed by ketamine overdose and exsanguinated by inferior vena cava puncture. The thoracic cavity was opened and the trachea exposed by dissection. An 18-g angiocath (Becton Dickinson Vascular Access, Sandy, UT) was inserted in the trachea and the lungs were lavaged with 20 ml of prewarmed (37°C) PBS containing penicillin (50 U/ml; GIBCO BRL, Gaithersburg, MD) and streptomycin (50 μg/ml; GIBCO BRL) in 1 ml vol. Viability was always >95% and lavaged cells were >97% macrophages as determined by differential counts of Wright-Giemsa stained cytospins.

Assays for macrophage-mediated killing of A. fumigatus conidia were adapted from the method of Schaffner (13). Alveolar lavage cells were pooled, centrifuged at 600 g, washed once with PBS, and resuspended in RPMI 1640 containing 2 mM L-glutamine (GIBCO BRL), 50 U/ml penicillin, 50 μg/ml streptomycin, and 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO) at a concentration of 2.5 × 10⁶ cells/ml. Freshly isolated conidia were suspended at a concentration of 12.5 × 10⁶ conidia/ml in PBS and combined with an equal volume of macrophage cell suspension (1:5 macrophage/conidia). Macrophage/conidia mixtures were tumbled at ∼10 rpm for 45 min at 37°C, layered over 2 ml Histopaque-1119 (Sigma Chemical Co.), and centrifuged at 700 g for 20 min at 4°C. Interface cells were removed, washed once in PBS, and resuspended at 1 × 10⁵ cells/ml. 1-ml aliquots were placed in polypropylene tubes (Falcon #2059; Becton Dickinson Labware) in triplicate and incubated for 0, 6, or 20 h at 37°C. Cells were lysed by addition of 9 ml distilled water containing 1% Triton X-100 (Sigma Chemical Co.) with vortexing for 3 min. The number of colony-forming units recovered was counted from serial dilutions of lysates plated on Sabouraud agar and incubated for 48 h at 37°C. Counted plates contained between 5 and 50 conidia. The number of viable conidia recovered at 6 h from control tubes containing no macrophages was not significantly different from the number of conidia recovered at 0 h.

Experimental Infection with A. fumigatus.

Mice were infected with A. fumigatus conidia as previously described (15). The trachea was exposed, a 24-g angiocath (Becton Dickinson Vascular Access) was inserted, and the inoculum was instilled in 35 μl of sterile saline containing 5% colloidal carbon (Eberhard Faber, Inc., Lewisburg, TN) to allow localization of the inoculum in each lung. The challenge dose was confirmed by both quantitative culture of homogenized lung tissue obtained from at least two wild-type mice 1–2 h after instillation and plate culture of the inoculum suspension. As prophylaxis against secondary bacterial infection, mice were given 1.25 mg ceftriaxone (Rocephin; Hoffman-La Roche, Nutley, NJ) immediately after infection, and again 24 h later, followed by oral tetracycline (5 mg/ml in drinking water) for the remainder of the experiment. Mice were examined daily and killed by halothane overdose when symptoms suggested advanced disease (lethargy, hunched posture, ruffled fur, and labored respirations) or at 2–3 wk. Lungs were removed and fixed in neutral buffered formalin for histologic examination of paraffin-embedded sections obtained from carbon-stained regions of lung. Sections were stained with hematoxylin and eosin for assessment of pathological changes or Grocott methamine silver for assessment of hyphae. Histologic sections were analyzed in regions containing colloidal carbon, which is a marker for regions receiving conidia. Mice were scored as positive for Aspergillus infection if they had pathological indications of disease, e.g., abscesses, extensive bronchopneumonia, or inflammatory nodules, on gross or histologic examination.

Administration of Sterile A. fumigatus Hyphal Cell Walls.

Mice were prepared as described above except that, instead of A. fumigatus conidia, 5 μg of sterile cell wall material isolated from A. fumigatus hyphae (see below) was administered in 35 μl of sterile saline containing 5% colloidal carbon. This dose was chosen after pilot experiments demonstrated that this dose produced a mild, selflimited, pneumonia with no mortality in wild-type mice. Higher doses from 50 to 500 μg caused severe acute pulmonary inflammation and occasional deaths in wild-type mice. Except where noted, at least five mice were used per experiment. Mice were given antibiotic prophylaxis as described above. Mice were killed at 24 h, 72 h, 1, 3, and 6 wk by halothane overdose, and lungs were prepared as described above for histologic examination. Peripheral blood counts were performed as described (19).

A. fumigatus hyphal cell walls were prepared from an overnight culture grown on Sabouraud dextrose agar at 37°C. The mycelia were scraped from a culture dish and suspended in 50 ml of PBS. The hyphae were washed five times in 50 ml of PBS, and then autoclaved to sterilize them. Sterilized hyphae were dried, frozen in liquid nitrogen, and ground to a fine powder with a mortar and pestle. The powder was washed three times in PBS, and then sonicated for 10 min at a setting of 100 with a 50% duty cycle (Vibra-cell; Sonics & Materials Inc., Danbury, CT). The resulting suspension was washed with PBS and centrifuged at 15,700 g to pellet. The pellet was lyophilized, weighed, and resuspended at a concentration of 3 mg/ml in double-distilled water. Sterile technique and sterile solutions were used throughout the procedure. Cultures of the final hyphal preparation for bacterial and fungal growth were negative.

Morphometric Quantitation of Lung Inflammatory Infiltrate.

The number of inflammatory cells (PMNs, large mononuclear cells, and small mononuclear cells) were counted in randomly selected alveoli containing colloidal carbon as previously described (20). Data were expressed as number of cells per 100 alveoli. Groups were compared using a two-tailed Student's t test for normally distributed data or a Mann-Whitney test for nonparametric data.

RNA Isolation and Northern Analysis.

Lungs from untreated wild-type and X-CGD mice were removed and frozen immediately in liquid nitrogen then stored at −70°C. Lungs from mice given sterile A. fumigatus hyphae were removed 24 h, 72 h, or 1 wk after treatment and stored similarly. Lung tissue was ground to a powder with a mortar and pestle chilled with liquid nitrogen, dissolved in Solution D (4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% N-lauryl sarcosine, and 0.1 M 2-mercaptoethanol; all from Sigma Chemical Co.) and total RNA extracted as described (21). 10 μg of RNA was run on a 1% agarose/formaldehyde gel and transferred to Magnacharge nylon membranes (Micron Seps Inc., Westboro, MA) according to the manufacturer's instructions. Radiolabeled cDNA probes were prepared by random priming (Prime-a-Gene kit; Promega Corp., Madison, WI) according to the manufacturer's instructions. The murine IL-1β cDNA was provided by Dr. Patrick Gray (Genentech, Inc., South San Francisco, CA), the TGFβ₁ cDNA by Dr. Rik Derynck (Genentech, Inc.), and the JE (murine monocyte chemotactic protein-1), KC (murine homologue of the human groα gene), and TNF-α cDNAs were from the American Type Culture Collection. To correct for differences in loading, the Northern blots were probed for β-actin. Differences in expression were quantitated by densitometry (GS-700 Densitometer; Bio Rad Labs., Hercules, CA) and were normalized to β-actin expression.

Killing of A. fumigatus Conidia by Alveolar Macrophages from X-CGD Mice.

To evaluate the role of phagocyte-derived oxidants in resistance to the conidial form of A. fumigatus, we compared the ability of alveolar macrophages from wild-type and X-CGD mice to kill conidia in vitro. No differences were observed in the phagocytic potential of X-CGD versus wild-type macrophages as measured by the phagocytic index, i.e., the ratio of ingested conidia to macrophages, or the percentage of macrophages phagocytosing at least one conidia (Table 1). 6 h after phagocytosis, the majority of ingested conidia in both wild-type and X-CGD macrophages had been killed, with a similar percentage of conidia killed in cultures containing wild-type macrophages (86 ± 18%) as were killed in cultures containing macrophages from X-CGD mice (92 ± 10%). By 20 h of incubation, both wild-type and X-CGD mice had effected more than a log reduction in viable conidia. However, we are unable to draw any firm conclusion regarding macrophage killing at this time point since control cultures containing no macrophages also show a similar loss in viable conidia, presumably through germination and subsequent adherence and aggregation of hyphae.

Experimental Infection with A. fumigatus.

Wild-type and X-CGD mice were challenged with A. fumigatus conidia at doses ranging from 1 × 10⁵ to 48 per animal, as administered by intratracheal instillation. No mortality was observed in wild-type mice, even at the highest dose tested. In contrast, pulmonary disease occurred in all X-CGD mice after Aspergillus exposure, with 24 of the 28 mice dead or moribund within 11–21 d due to progressive lung disease (Fig. 1). X-CGD mice given 1 × 10⁵ conidia developed a rapidly progressing and uniformly fatal bronchopneumonia with all mice dead or moribund by the fourth day after infection, similar to what was previously observed with exposure to 3 × 10⁶ conidia (15). Administration of lower doses resulted in a longer asymptomatic period and survival of a small number of mice (Fig. 1).

Fungal cultures of lung homogenates obtained at autopsy showed persistence of viable colony-forming units (cfu) of A. fumigatus in 27 out of 28 X-CGD mice after respiratory challenge (Table 2). Only one X-CGD mouse, who was studied at 21 d after exposure to 48 spores, did not have fungus cultured from lung tissue, although both abscesses and chronic inflammatory lesions with rare hyphal forms visible by silver stain were seen in lung sections prepared from this mouse (not shown). In contrast, very few, if any, viable A. fumigatus cfu were cultured from lungs obtained from wild-type mice, even after 3–4 d of challenge with 1 × 10⁵ conidia (Table 2).

In agreement with previous studies (8, 15), the histologic appearance of lungs from wild-type mice challenged intratracheally with 1 × 10⁵ conidia and killed 3 d after infection, appeared either normal or showed only a mild peribronchial pneumonia (data not shown). No hyphae were visible in silver-stained sections (data not shown).

Gross and histologic abnormalities after A. fumigatus respiratory challenge were observed in the lungs of all X-CGD mice at all doses of conidia studied. X-CGD mice that died or were moribund within the first week after Aspergillus challenge had similar pathology. Histologic findings, which included extensive peribronchiolar and alveolar inflammation consisting of neutrophils, necrotic debris, and edema, were similar to that previously described for X-CGD mice challenged with 3 × 10⁶ conidia (not shown) (15). In X-CGD mice that survived beyond 1 wk, chronic inflammatory changes became more evident. Areas of almost complete neutrophil consolidation surrounded by a more diffuse mononuclear cell infiltrate and edema were a common finding (Fig. 2 a), as was the formation of wellcircumscribed abscesses (not shown). Intact hyphae, as visualized by silver staining, were detected in all X-CGD mice challenged with Aspergillus. Regions with an extensive neutrophil infiltrate typically contained numerous hyphae (Fig. 2 b), while areas of chronic inflammation had few hyphae (not shown). However, occasional regions with numerous focal collections of neutrophils did not contain detectable hyphae (Fig. 2 c). In X-CGD mice who survived for more than 2 wk after Aspergillus challenge, chronic inflammatory lesions were typically distributed in a nodular pattern and were comprised largely of mononuclear cells (Fig. 2 d) along with other cell types frequently associated with granulomatous inflammation including large, highly vacuolated macrophages (foam cells; not shown) and giant cells (Fig. 2 e). Neutrophils were often present in the centers of these nodules (Fig. 2 e), and occasionally, necrotic debris (not shown).

Granulomatous Inflammation in X-CGD Mice Elicited by Sterile A. fumigatus Hyphae.

The observation of intense acute and chronic inflammation in X-CGD mice with no or small numbers of hyphae (Fig. 2, a and c) led us to speculate that the defect in phagocyte oxidant production was associated with a prolonged inflammatory response that was independent of active fungal infection. To separate the effects of persistent infection in X-CGD mice from the inflammatory response to Aspergillus, we administered sterile hyphal fragments to wild-type and X-CGD mice.

Striking differences in the appearance of the alveolar inflammatory infiltrate in wild-type versus X-CGD mice were evident even during the first 72 h after instillation of hyphae. At 24 h, an intense neutrophil alveolar infiltrate was seen in X-CGD mice, with foci of neutrophils often found in tight clusters around an eosinophilic center, with many cells assuming a spindle-shaped orientation towards the center of the inflammatory focus (Fig. 3 a). In contrast, in wild-type mice, the predominantly neutrophilic inflammatory infiltrate was diffusely scattered among alveoli without this focal pattern (Fig. 3 b). By 72 h, increased numbers of large mononuclear cells, debris, and a fibrinous exudate were seen in wild-type lung samples, again in a diffuse distribution (not shown). In X-CGD lung, focal accumulations of neutrophils were still numerous and were often surrounded by mononuclear cells (not shown).

Morphometric analysis of the inflammatory cell infiltrate elicited by intratracheal administration of sterile hyphal fragments was also performed to obtain a quantitative measure of the differences between wild-type and X-CGD mice. At both 24 and 72 h, the neutrophils were the predominant cell type in both X-CGD and wild-type alveoli (Fig. 4). However, X-CGD mice had almost five times the amount of neutrophils as wild-type controls. The number of mononuclear cells was also significantly higher (almost double) in X-CGD mice compared to wild-type mice 72 h after administration of hyphae (Fig. 4). No differences in the peripheral leukocyte counts were observed between wild-type and X-CGD mice at either of these time points (data not shown).

One week after administration of 5 μg of sterile hyphal fragments, 11 out of 13 wild-type mice still had evidence of a mild, diffuse alveolar pneumonia, consisting of neutrophils and mononuclear cells (not shown). In two wildtype mice, a more severe chronic inflammatory response developed, characterized by extensive infiltration of alveoli with large mononuclear cells and a fibrinous exudate (not shown). In contrast, at 1 wk, 8 out of 8 X-CGD mice were found to have a distinctive granuloma-like inflammatory infiltrate consisting of neutrophil microabscesses surrounded by large mononuclear cells with an epithelioid morphology (not shown). By 21 d, the contrast between wild-type and X-CGD mice was even more striking. The pneumonia in 3 out of 3 wild-type mice had almost entirely resolved (Fig. 5 a), whereas 3 out of 3 X-CGD mice still had numerous inflammatory nodules (Fig. 5 b) which often become confluent (Fig. 5 c). These foci continued to show a characteristic granuloma-like structure, with a central area of neutrophils surrounded by epithelioid mononuclear cells (Fig. 5 d). Lungs obtained from 3 of 3 X-CGD mice 6 wk after administration of sterile hyphal fragments showed persistence of these inflammatory lesions. No evidence of hyphal growth was evident in Grocott methamine silver–stained sections (not shown), and cultures of lungs from X-CGD mice killed at 1 wk showed no growth of fungi and grew similar numbers of bacteria to that observed in wild-type mice (<1 × 10⁴ cfu/lung).

Expression of Cytokines in Lungs after Intratracheal Administration of Sterile A. fumigatus Hyphae.

To determine if the differences in the inflammatory response to sterilized A. fumigatus hyphae between X-CGD and wild-type mice observed by histologic examination were associated with differences in cytokine expression, we examined Northern blots of total lung RNA from X-CGD and wild-type mice using probes for several proinflammatory mediators, as well as TGFβ₁, a potent antiinflammatory cytokine (22).

Of the cytokines examined, IL-1β, TNF-α, JE, and KC showed very low, if any, levels of expression in untreated mice, with no significant differences observed between X-CGD and wild-type mice (Figs. 6 and 7). TGFβ₁ expression was detectable in untreated mice at similar levels in wild type and X-CGD (Figs. 6 and 7). The most striking differences in mRNA expression after instillation of hyphae were observed with the proinflammatory cytokines IL-1β and TNF-α. Although markedly increased in both X-CGD and wild-type mice by 24 h after instillation, the expression of IL-1β and TNF-α in X-CGD mice was nearly 5- and 8.5-fold, respectively, higher than levels seen in wild-type animals (Fig. 7, a and b). By 72 h, expression of both cytokine mRNAs had dropped nearly to baseline levels in wild-type mice, while expression in X-CGD mice was even higher than at 24 h and remained persistently elevated for at least a week (Fig. 7, a and b). In addition, we also examined expression of two chemokines, KC and JE. KC, the murine groα homologue, binds to the murine orphan IL-8 receptor and is a potent neutrophil chemoattractant and activator (23), while JE, the murine homologue of monocyte chemoattractant protein-1 has similar activity for monocytes (24). Expression of KC and JE peaked at 24 h in both wild-type and X-CGD mice, and then declined (Fig. 6). Expression of both KC (Fig. 7 c) and JE (data not shown) were significantly higher at 24 h in X-CGD mice relative to wild-type controls, and KC remained significantly elevated in X-CGD versus wild-type mice at 72 h and 1 wk (three- to fivefold, respectively). TGFβ₁ was significantly elevated in X-CGD mice relative to wild-type controls at 24 h, but not at later time points (Fig. 7 d).