Severe Impairment in Early Host Defense against Candida albicans in Mice Deficient in Myeloperoxidase

Cloning of the mouse Mpo gene and construction of a targeting vector.

A 396-bp DNA fragment containing exons 5 and 6 of the mouse Mpo gene was amplified from ES cell genomic DNA with primers designed from the published sequence (46). This fragment was used as a probe to screen a λ phage library made from strain 129 mouse genomic DNA. A clone containing a part of the Mpo gene was isolated, and its restriction map and a partial nucleotide sequence were determined. For constructing the targeting vector (Fig. 1A), a 2.2-kb fragment containing exons 1 to 5 and a 5.9-kb fragment containing exons 8 to 11 were used as the two homologous arms flanking the neo gene. The herpes simplex virus (HSV) thymidine kinase (TK) gene was positioned downstream of the longer arm (32).

Gene targeting and production of mice by use of modified ES cells.

BK4 cells, a subclone of E14TG2a derived from strain 129/Ola mice, were cultured on feeder cells as described previously (44). The targeting vector mentioned above was linearized with NotI and introduced into ES cells by electroporation. Colonies doubly resistant to G418 (200 μg/ml) and ganciclovir (2 μM) were screened for homologous recombination by Southern blot analysis following digestion of genomic DNA with BglII by use of the 722-bp PCR-amplified fragment containing exon 5 of the Mpo gene as a probe. The resulting cells with a disrupted Mpo gene were injected into blastocysts to obtain chimeras as described previously (25, 47). Animals classified as chimeric by coat color were mated with strain C57BL/6 mice, and F₁ animals heterozygous for the disrupted Mpo gene were obtained. Interbreeding of heterozygous offspring was used to produce mice homozygous for the modified MPO allele.

Northern blotting of bone marrow cells.

Total RNA was isolated from bone marrow cells with TRIzol (Gibco BRL) as recommended by the manufacturer. Each sample (10 μg) was electrophoresed in a 2.2 M formaldehyde–1% agarose gel, transferred to a Hybond nylon membrane (Amersham Corp.), and probed with a 2.2-kb PstI fragment of a human MPO cDNA vector (pH17) (17) containing exons 2 through 12 of the MPO gene. This fragment is 85% identical to that in mouse cDNA. A cDNA clone for chicken β-actin was used as a probe to evaluate the amount of mRNA loaded. mRNA levels were estimated by densitometric analysis of autoradiograms after serial exposures.

Cell counting and measurement of MPO activity.

Blood was drawn from the retro-orbital plexus of mice into EDTA-containing tubes. Blood cell analysis was carried out by flow cytometry with Technicon H-1 by the method recommended by the manufacturer. Briefly, separation of leukocytes was performed by peroxidase staining and simultaneous measurement of light scattering (Perox-Channel). For peroxidase staining, erythrocytes were lsyed in the Technicon H-1-integrated Perox-Chamber, and leukocytes were fixed with formalin and stained for peroxidase with H₂O₂ and 4-chloro-1-naphthol as a chromogen. The cell distribution pattern was plotted by a so-called leukogram with peroxidase activity on the x axis and light scattering on the y axis and was analyzed by a multispecies software program developed for animal blood samples. If intracellular MPO activity decreases, neutrophils and monocytes shift to the left.

Thioglycolate-induced peritonitis.

Mice were injected intraperitoneally with 1 ml of 3% fluid thioglycolate medium (Difco). After 4 h, peritoneal exudate cells were harvested by peritoneal lavage with 20 ml of phosphate-buffered saline (PBS). Total cell numbers were determined with a hemocytometer. The percentage of neutrophils was determined by microscopic examination of Wright-Giemsa-stained samples.

Cytochemical and biochemical determination of MPO activity.

Isolated neutrophil-rich peritoneal exudate cells were stained for MPO activity with the 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate system from Sigma (St. Louis, Mo.) following formalin-acetone fixation of the cells. Briefly, oxidation of the substrate TMB in MPO-positive cells yields a blue insoluble reaction product which is visualized by light microscopy. MPO activity was quantitatively measured by the method of Suzuki et al. (45) with some modifications. Peritoneal exudate neutrophils were adjusted to 5 × 10⁶ cells/ml and incubated with N-formyl-Met-Leu-Phe (1 μM) and cytochalasin B (5 μg/ml) for 10 min at 37°C. Triton X-100 (final concentration, 0.1%) was added to the cell suspensions for total MPO release. Aliquots of the cell extracts were incubated with the TMB liquid substrate system, and the oxidized product was detected spectrophotometrically. The activity was expressed as the initial rate of increase in the absorbance at 655 nm.

Generation of HOCl and O₂⁻ from neutrophils.

HOCl generation by peritoneal exudate neutrophils was measured by the chlorination of monochlorodimedon (MCD) (21). Peritoneal exudate cells (2 × 10⁶/ml) were incubated in PBS containing 1 mM CaCl₂, 0.5 mM MgCl₂, 5 mM glucose, 100 ng of phorbol myrisate acetate (PMA) per ml, and 20 μM MCD at 37°C for 20 min. At the end of this period, samples were ice chilled and centrifuged at 12,000 × g for 5 min. The activity in supernatants of reaction mixtures with or without cells was measured at 290 nm. The amount of HOCl generated was calculated by use of a molar linear absorption coefficient of 19,000 M⁻¹/cm (16).

O₂⁻ generation was determined as the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (14). Cytochrome c (40 μM) with or without 20 μg of SOD per ml was added to peritoneal exudate cells (2 × 10⁶/ml) that had been stimulated with 100 ng of PMA per ml at 37°C for 5 min, and O₂⁻ generation in the samples was continuously measured for another 5 min at 550 nm with a spectrophotometer. O₂⁻ generated from the cells was calculated as the difference between levels in SOD-containing samples and those in samples not containing SOD by use of an absorption coefficient of 21,000 M⁻¹/cm (14).

Experimental infections with C. albicans and Staphylococcus aureus.

Stock cultures of C. albicans (ATCC 18804) were cultured on 2% agar slant medium (pH 6.4) containing 83 mM glucose, 2 mM MgSO₄ · 7H₂O, 7.4 mM KH₂PO₄, 0.5% Polypeptone, and 0.2% yeast extract for 10 days at 37°C. Blastoconidia of C. albicans grown on the slant were transferred to 1.2% agar plates containing 28 mM glucose and 0.2% Polypeptone. After cultivation for 2 days at 37°C, the blastoconidia were harvested in sterile saline. The number of fungi was counted with a hemocytometer and adjusted to 2 × 10⁸ cells/ml; the viable number was also determined by plating the diluted samples on agar plates. Wild-type, heterozygous, and homozygous mutant mice were injected by the intratracheal route with 0.02 ml of the fungal suspension. At 0.5, 20, 80, and 120 h after the challenge, the organs were removed aseptically and homogenized in sterile saline. At least five mice were used per group. Aliquots of the homogenates were plated in duplicate on agar plates with Guanofuracin-Sabouraud medium (Eiken Chemical Co., Tokyo, Japan) and incubated for 48 h at 37°C. The number of viable C. albicans was calculated from the number of colonies grown on the plates and was expressed in CFU. Data were recorded as the mean log₁₀ CFU per organ.

In studies of systemic infections, wild-type and homozygous mutant mice were injected intraperitoneally with 4 × 10⁶ cells of C. albicans. Seven days later, the organs were removed, homogenized, and plated on agar plates as described above. Five mice were used per group.

For intraperitoneal infection with S. aureus, mice were injected intraperitoneally with 0.1 ml of a suspension (7 × 10⁸/ml) of S. aureus (FDA209P) that had been grown for 24 h on a plate with Trypticase soy agar at 37°C. Twenty-four and 48 h later, peritoneal exudate fluid was harvested with PBS, diluted, and cultured for viable S. aureus on Trypticase soy agar at 37°C. Three to five mice of different genotypes were studied at each time point.

Preparation of sections and slides.

Lungs were fixed in a buffered 4% paraformaldehyde solution, dehydrated in ethanol, and embedded in paraffin for sectioning. Sections were prepared, and hematoxylin and eosin (H&E) staining and Grocott staining were carried out by standard protocols.

Targeted disruption of the mouse Mpo gene.

The targeting strategy used to disrupt the Mpo coding sequence is illustrated in Fig. 1A. The targeting vector was constructed by removing a 0.7-kb XbaI-EcoRI fragment containing exon 6 through a part of intron 7 and replacing it with the neo gene. In addition, a copy of the HSV TK gene was placed at the 3′ end of the construct. Both of the selectable marker genes were inserted in the same transcriptional orientation as the Mpo gene. Male chimeric mice generated by use of correctly targeted ES cells transmitted the mutation of the Mpo gene through the germ line when mated with C57BL/6 female mice (Fig. 1B). Heterozygote (Mpo^+/−) mating then produced homozygous mutant mice (Mpo^−/−) at the expected Mendelian frequency. The mutant mice were viable, exhibited normal growth and development, and were fertile. As shown in Fig. 1C, Northern blot analysis of mRNA isolated from the bone marrow of homozygous mutant mice showed no significant hybridization to the human MPO cDNA probe, whereas the bone marrow of wild-type mice (Mpo^+/+) contained mRNA for Mpo, indicating the absence of any detectable transcript for the Mpo gene in the mutants. The heterozygous mice showed the expected 50% reduction in mRNA levels.

Identification of MPO-deficient neutrophils and monocytes.

Figure 2 shows the leukograms for the peroxidase activity of peripheral blood cells from wild-type and homozygous mutant mice determined with Technicon H-1. When stained vesicles are lacking, the light scattering changes characteristically, leading to a cluster location upward on the y axis and a shift to the left on the x axis. The neutrophil clusters from homozygous mutant mice clearly shifted to the left, unstained cell area, and no stained cells were observed in the normal neutrophil area. The mutant mice also showed no clusters in the monocyte area. These results strongly suggested that the neutrophils and the monocytes of the homozygous mice that we generated are completely deficient in MPO. However, the clusters of their eosinophils did not shift to the left, demonstrating that eosinophil peroxidase is genetically distinct from MPO.

MPO activity in peritoneal exudate neutrophils.

In order to explore MPO deficiency in further detail, neutrophils were isolated from the thioglycolate-injected peritoneal cavity of wild-type, heterozygous, and homozygous mice. Judging from the microscopic examination of Wright-Giemsa-stained samples, more than 95% of the exudate cells recovered 4 h after injection were neutrophils, regardless of their genotypes. Cytochemical staining with TMB revealed that more than 95% of the exudate cells in wild-type and heterozygous mice were peroxidase positive (Fig. 3A). In contrast, the majority of homozygous mutant mice were peroxidase negative. Quantitative analysis by spectrophotometry demonstrated that the average MPO activity in wild-type mice was 1.7 nmol/min/10⁶ cells (Fig. 3B). Heterozygous animals showed the expected 50% reduction in MPO activity. Homozygous mutant mice showed only a low level of MPO activity. This low level of activity likely represents peroxidase activity supplied by eosinophils, because eosinophil peroxidase is normally active in homozygous mutant mice (Fig. 2) and is able to oxidize TMB. From these results, we conclude that homozygous mutant mice have no detectable MPO activity, while heterozygous mice have half the level of MPO activity present in wild-type littermates.

HOCl and O₂⁻ generation from normal and MPO-deficient neutrophils.

During the respiratory burst, neutrophils generate O₂⁻ via the NADPH oxidase system. O₂⁻ is then reduced to H₂O₂, from which HOCl is generated by the MPO reaction.

The rapid reaction of MCD with HOCl to form dichlorodimedon (21) was used to determine the level of HOCl generation from the peritoneal exudate neutrophils (Fig. 4). The average level of HOCl generation from wild-type neutrophils was 0.47 nmol/min/10⁶ cells. On the other hand, as anticipated from the absence of peroxidase activity (Fig. 2 and 3), the neutrophils of homozygous mutant mice produced HOCl at an almost undetectable level. Because EPO also produces HOCl from H₂O₂, a low level of HOCl detected in homozygous mutant mice could be supplied from eosinophils. The neutrophils of heterozygous mutant mice generated from half the normal level of HOCl. These results demonstrate that the levels of HOCl generated from the neutrophils in each genotype correlate well with the MPO activities measured with TMB as a substrate (Fig. 3).

O₂⁻ generation by peritoneal exudate neutrophils was measured as SOD-inhibitable cytochrome c reduction. The average level of O₂⁻ generation from neutrophils of wild-type mice was 0.39 nmol/min/10⁶ cells (Fig. 4) and was completely inhibited in the presence of SOD (data not shown). The neutrophils of homozygous mutant mice generated a slightly higher level of O₂⁻ (0.50 nmol/min/10⁶ cells) than did those of wild-type mice (P, <0.05) (Fig. 4). A slight enhancement of O₂⁻ generation was also seen in heterozygous mutant mice, although it did not reach statistical significance (P, 0.075).

Clearance of S. aureus in vivo.

To assess whether MPO-deficient mice have enhanced susceptibility to S. aureus infection in vivo, mice were intraperitoneally challenged with 7 × 10⁷ cells of S. aureus. Most of the cells injected disappeared by 48 h both in wild-type and in homozygous mutant mice, and there was no difference in the rates of clearance between those mice (Fig. 5). Gross anatomical experiments revealed no obvious inflammation of the lungs, liver, heart, kidneys, and intestines.

Pulmonary infection with C. albicans.

To investigate the susceptibility of MPO-deficient mice to C. albicans compared to that of wild-type littermates, 26 wild-type, 18 heterozygous mutant, and 27 homozygous mutant mice were challenged with 4 × 10⁶ fungi intratracheally. This is a relatively low dose, since the 50% lethal dose for mice challenged by the intratracheal route was estimated to be in excess of 10⁸ yeast cells by Sawyer (42). On the following day, signs of severe infection, including ruffled fur and hunched posture, were observed only in the homozygous mutant mice. The wild-type mice were able to eliminate the fungi effectively, and approximately 2,000 fungi were recovered from their lungs 120 h after infection (Fig. 6A). In contrast, in the mutant mice, the clearance of viable C. albicans was significantly delayed, and nearly 500 times as many viable fungi were cultured from their lungs (Fig. 6A). In addition, the survival of the mutant mice was dramatically decreased, and 18 of the 27 mice died by 5 days after the challenge. We also determined the numbers of viable C. albicans cultured from other tissues. In the kidneys (Fig. 6B), the number of C. albicans in the wild-type mice was almost below the detectable level, whereas that in the homozygous mutant mice was time dependently increased, and about 6,000 fungi were detected at 120 h. In the brain, heart, and liver of both wild-type and homozygous mutant mice, the numbers of C. albicans were all below 100 fungi per each organ from 20 to 120 h. C. albicans was not detectable in the spleen. There was no difference in the numbers of viable fungi obtained from the mutant mice that had died or that had been weakened. Although the clearance of fungi from the lungs in the heterozygous mutant mice was slightly delayed at 80 h compared to that in the wild-type mice, almost the same number of fungi disappeared by 120 h (Fig. 6A).

Only one wild-type mouse died 3 days after the challenge as a consequence of lung bleeding, but no gross or histological abnormalities were observed in the lungs of this mouse or those of the wild-type mice still alive by 120 h (Fig. 7A and B). In contrast, the lungs of the mutant mice 120 h after the challenge showed diffuse enlargement and brownish discoloration (Fig. 7A). Additionally, patchy white, soft, and spherical masses 1 to 5 mm in diameter were observed on the surfaces of every lobe (Fig. 7A). H&E-stained sections of the lungs revealed massive infiltration of alveolar and peribronchiolar spaces with neutrophil polymorphonuclear leukocytes, and fairly extensive edema filled many other alveolar spaces (Fig. 7C). In peribronchiolar spaces around neutrophil infiltrates, mucus infiltration was also observed. Furthermore, some of the peribronchial epithelial cells were observed to be peeling off (Fig. 7D). By Grocott staining, hyphae of C. albicans were detected in several alveolar spaces of the mutant mice (Fig. 7E) but not the wild-type mice (data not shown). These clinical features are characteristic of alveolar and peribronchiolar pneumonia, and the most likely explanation for the death of the mutant mice infected with C. albicans is severe impairment of lung functions caused by myeloid cell infiltration and edema.

Systemic infection with C. albicans via the intraperitoneal route.

To evaluate the susceptibility of wild-type and MPO-deficient mice, C. albicans was administered intraperitoneally at a dose of 4 × 10⁶ fungi/mouse. Seven days later, the lungs, brain, kidneys, spleen, and liver were removed from five animals in each group and pooled to determine the dissemination of the fungi into these organs. Gross anatomical evaluation revealed no obvious inflammation in any of those organs. In the wild-type mice, the highest and lowest numbers of fungi were found in the liver and the brain, respectively (Fig. 8), consistent with a previous report (20). In contrast, significantly higher numbers of fungi were disseminated into every organ in the homozygous mutant mice, and approximately 290, 11, 110, 17, and 10 times as many viable fungi were cultured from the lungs, brain, kidneys, spleen, and liver, respectively (Fig. 8). Unlike the results of the intratracheal infection, no obvious signs of severe infection were observed in either wild-type or homozygous mutant mice during a period of 7 days after the challenge. When mice were challenged intraperitoneally at a high dose (10⁸ fungi/mouse), all homozygous mutant mice died by 2 days, while all wild-type mice survived for 7 days without many signs of distress. These results demonstrate that MPO is very important for the murine host defense against C. albicans and that the lack of MPO enhances C. albicans dissemination, especially into the lungs and kidneys.