Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase

Abstract

Myeloperoxidase (MPO) catalyzes the reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid (HOCl), which is used for microbial killing by phagocytic cells. Despite the important role of MPO in host defense, however, MPO deficiency is relatively common in humans, and most of these individuals are in good health. To define the in vivo role of MPO, we have generated by gene targeting mice having no MPO activity in their neutrophils and monocytes. The mice without MPO developed normally, were fertile, and showed normal clearance of intraperitoneal Staphylococcus aureus. However, they showed increased susceptibility to pneumonia and death following intratracheal infection with Candida albicans. Furthermore, the lack of MPO significantly enhanced the dissemination of intraperitoneally injected C. albicans into various organs during the first 7 days. Thus, MPO is important for early host defense against fungal infection, and the inability to generate HOCl cannot be compensated for by other oxygen-dependent systems in vivo in mice. The mutant mice serve as a model for studying pulmonary and systemic candidiasis.

FIG. 1

Targeted disruption of the mouse Mpo gene and germ line transmission of the disrupted allele. (A) Structures of the wild-type Mpo locus, targeting vector, and mutant allele generated by homologous recombination. Exons are shown as black boxes and numbered. The targeting vector contains the neo gene (NEO) in place of the XbaI-EcoRI region containing exons 6 and 7. The HSV TK gene is attached to the end of the region of homology. The broken line indicates the vector sequence. The lengths of diagnostic BglII restriction fragments and the location of a probe used for Southern blot analysis are shown. B, BamHI; Bg, BglII; E, EcoRI; N, NotI; X, XbaI. (B) Southern blot analysis. Genomic DNA was isolated from tail snips of the offspring of a heterozygous cross, digested with BglII, and analyzed by Southern hybridization with the DNA probe indicated in panel A. Genotypes are indicated as wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. (C) Northern blot analysis of bone marrow mRNA. Total RNA (10 μg) isolated from bone marrow of wild-type (+/+), heterozygous mutant (+/−), and homozygous mutant (−/−) mice was electrophoresed, blotted, and hybridized to a human MPO cDNA probe. The amounts of RNA loaded are indicated by hybridization to a cDNA probe for chicken β-actin.

FIG. 2

Leukograms of Technicon H-1 analysis of wild-type (+/+) and homozygous mutant (−/−) mice. Light scattering is plotted on the y axis, and peroxidase (PEROX) activity is plotted on the x axis. The numbers indicate the following cells: 1, lymphocytes; 2, monocytes; 3, neutrophils; 4, eosinophils. The lower the peroxidase activity of a single cell, the more the cell is located toward the left on the x axis. Histograms of peroxidase activity are shown below the graphs.

FIG. 3

MPO activity of neutrophils. Wild-type (+/+), heterozygous mutant (+/−), and homozygous mutant (−/−) mice were injected with thioglycolate in the peritoneal cavity, and peritoneal exudate cells were collected 4 h later. (A) The cells were fixed and stained with TMB for peroxidase activity. (B) Peroxidase activities in total peritoneal exudate cells were measured with TMB as a substrate. Three animals of different genotypes, each tested in duplicate, were used. Results represent means ± standard deviations.

FIG. 4

HOCl and O₂⁻ generation from neutrophils of wild-type (+/+), heterozygous mutant (+/−), and homozygous mutant (−/−) mice. The levels of HOCl (closed bars) and O₂⁻ (open bars) generated from PMA-stimulated peritoneal exudate neutrophils (2 × 10⁵ cells) were determined by the chlorination of MCD and the SOD-inhibitable reduction of cytochrome c, respectively. Three animals of each genotype were used in each experiment, and assays were performed in triplicate. Results represent means ± standard deviations. The asterisk indicates a P value of <0.05 for mutant versus wild-type mice, as determined by Student’s t test.

FIG. 5

Clearance of viable S. aureus from the peritoneal cavity. Peritoneal exudate fluid was cultured 24 and 48 h after intraperitoneal injection of 7 × 10⁷ CFU of S. aureus. Results represent mean log₁₀ CFU ± standard deviations obtained from three to five wild-type mice (closed circles) and homozygous mutant mice (open circles).

FIG. 6

Pulmonary infection with C. albicans in wild-type, heterozygous mutant, and homozygous mutant mice. Wild-type (closed circles), heterozygous mutant (open triangles), and homozygous mutant (open circles) mice were injected intratracheally with 4 × 10⁶ CFU of C. albicans. At the indicated times after the challenge, whole lungs (A) and kidneys (B) were homogenized, and aliquots of the homogenates were plated on Guanofuracin-Sabouraud agar plates. Five mice or more were used in each group. Results represent mean log₁₀ CFU per organ ± standard deviations.

FIG. 7

Lung pathology observed in wild-type and homozygous mutant mice 120 h after intratracheal challenge with C. albicans. (A) Representative gross appearance of lungs from wild-type (+/+) and homozygous mutant (−/−) mice. (B) H&E-stained section from a wild-type mouse. Magnification, ×40. (C) H&E-stained section from a representative homozygous mutant mouse. Magnification, ×40. (D) Same sample as C but at a magnification of ×400. (E) Grocott-stained section from a homozygous mutant mouse. Magnification, ×400.

FIG. 8

Numbers of C. albicans cultured from various organs of mice after intraperitoneal challenge. Wild-type mice (closed bars) and homozygous mutant mice (open bars) were injected intraperitoneally with 4 × 10⁶ cells of C. albicans. Seven days after the challenge, the organs were removed and homogenized, and aliquots of the homogenates were plated on Guanofuracin-Sabouraud agar plates. Five mice were used in each group. Results represent mean log₁₀ CFU per organ ± standard deviations.