Mutation of the Cyba gene encoding p22phox causes vestibular and immune defects in mice

Abstract

In humans, hereditary inactivation of either p22(phox) or gp91(phox) leads to chronic granulomatous disease (CGD), a severe immune disorder characterized by the inability of phagocytes to produce bacteria-destroying ROS. Heterodimers of p22(phox) and gp91(phox) proteins constitute the superoxide-producing cytochrome core of the phagocyte NADPH oxidase. In this study, we identified the nmf333 mouse strain as what we believe to be the first animal model of p22(phox) deficiency. Characterization of nmf333 mice revealed that deletion of p22(phox) inactivated not only the phagocyte NADPH oxidase, but also a second cytochrome in the inner ear epithelium. As a consequence, mice of the nmf333 strain exhibit a compound phenotype consisting of both a CGD-like immune defect and a balance disorder caused by the aberrant development of gravity-sensing organs. Thus, in addition to identifying a model of p22(phox)-dependent immune deficiency, our study indicates that a clinically identifiable patient population with an otherwise cryptic loss of gravity-sensor function may exist. Thus, p22(phox) represents a shared and essential component of at least 2 superoxide-producing cytochromes with entirely different biological functions. The site of p22(phox) expression in the inner ear leads us to propose what we believe to be a novel mechanism for the control of vestibular organogenesis.

Figure 1. Missense mutation in the Cyba gene of the nmf333 mouse strain.

(A) The p22phox-encoding Cyba gene is located near the distal telomere of mouse chromosome 8 (top ideogram). Cyba contains 6 exons (numbered black boxes). A deoxythymidine nucleotide (T) in exon 5 of WT Cyba (upper chromatogram) is replaced by a deoxycytosine (C) in the nmf333 mutant strain, as indicated by an arrow in the lower chromatogram. The point mutation changed the 121st amino acid of p22phox from tyrosine (Tyr) to histidine (His), as shown in the translation lines. (B) The tyrosine to histidine amino acid substitution (Y121H) is localized to the second predicted transmembrane helix of p22phox. (C) Genotyping for the nmf333 mutation with PCR amplification and subsequent BslI digestion of a fragment of the Cyba gene. Lanes show a 100-bp ladder and the genotyping results using DNA samples from WT, heterozygous nmf333 (nmf333/+), and homozygous nmf333 (nmf333/nmf333) mice. Fragment sizes are given in bp.

Figure 2. Lack of p22phox expression and superoxide production in the neutrophil granulocytes of homozygous nmf333 mice.

(A) Immunoblot detection of p22phox in the SDS-PAGE–separated protein lysate of WT neutrophils. The p22phox band is absent from the lysate of nmf333 neutrophils (top). Comparable protein loading is demonstrated by the actin signal present in both samples (bottom). Arrowheads and numbers indicate the positions of MW standards. (B) Rate of superoxide production by nonstimulated (–) and PMA-activated (+) neutrophil granulocytes isolated from the bone marrow of WT and homozygous nmf333 mice, as measured using the superoxide dismutase–sensitive cytochrome c reduction assay. Bars represent mean ± SEM (n = 3–5). ***P = 0.0005; unpaired 2-tailed t test. (C) Superoxide production of neutrophil granulocytes isolated from the bone marrow of WT (open circles) and homozygous nmf333 (filled circles) mice and stimulated with PMA (arrow), as detected by luminol-amplified chemiluminescence. Traces are representative of n = 3. RLU, relative light units.

Figure 3. Necrotizing pneumonia in homozygous nmf333 mice following B. cepacia lung infection.

(A) Probability of survival after inoculation with various CFU of B. cepacia (10⁴ CFU, filled squares; 10⁵ CFU, filled triangles; 10⁶ CFU, open circles and filled circles) into the tracheas of WT (open circles; n = 8) and nmf333 (filled circles, filled triangles, filled squares; n = 5 or 6) mice. P = 0.0009 between the 10⁶ CFU infected WT and nmf333 groups; log-rank test. (B) Number of B. cepacia recovered from the lung and spleen homogenates of WT and homozygous nmf333 mice 3.5 days after intratracheal inoculation with 10⁴ B. cepacia, as evaluated by quantitative culture; bacteria were not detected in lung and spleen homogenates from WT mice. Bars represent mean ± SEM (n = 3). **P = 0.029, *P = 0.043; t tests with 2 degrees of freedom. (C) Histopathological appearance of lung and spleen sections obtained from WT and homozygous nmf333 mice 3.5 days after intratracheal inoculation with 10⁴ CFU B. cepacia. Whereas WT tissue samples had minimal or no inflammation, necrotizing pneumonia and splenitis were apparent in the nmf333 strain. Hematoxylin and eosin staining were used. Scale bars: 100 μm.

Figure 5. Expression of p22phox in the apical membrane of endolymphatic duct and sac.

(A) Endolymphatic duct sections of a WT and a homozygous nmf333 mouse at E14 were immunostained with an anti-p22phox antibody and visualized by confocal microscopy. Top: localization of the immunofluorescence signal (p22phox). Bottom: superimposition of the fluorescence signal and the differential interference contrast image (merged). Asterisks indicate endolymphatic duct lumens. (B) Immunostaining of WT endolymphatic sac (ES) and duct (asterisk) with the anti-p22phox antibody at E17.5. Top: red fluorescence signal of p22phox immunolocalization. Bottom: superimposition of the fluorescence signal and the differential interference contrast image (merged). (C) In P1 WT mice, the lower region of the endolymphatic duct exhibits p22phox immunofluorescence (left). Right: both the red fluorescence signal and the differential interference contrast image. The asterisk indicates the lumen of the endolymphatic duct. Scale bars: 20 μm.

Figure 4. Balance organ dysfunction and a lack of otoconia in nmf333 mice.

(A) Balance in WT and homozygous nmf333 mice was examined using swimming tests. Homozygous mutants were unable to orient themselves in water and swirled under the surface. (B) Balance function based on VsEP waveforms; 2 representative replicates of VsEP waveforms are shown for WT, heterozygous, and homozygous mutant nmf333 mice exposed to linear acceleration stimuli from +6 to –12 dB relative to reference intensity (re) 1.0 g/ms in 3-dB increments, with auditory masking (M) and without auditory masking. Positive-response peaks are labeled with arrowheads. VsEP responses were absent in nmf333/nmf333 mice. (C) Summarized VsEP data. Individual VsEP thresholds are shown for WT (open circles; n = 6), heterozygous (open squares; n = 6), and nmf333/333 (filled squares; n = 4) mice. Mean thresholds are indicated by solid horizontal lines ± 1 SD (dashed lines). P < 0.0001, 1-way ANOVA; post-hoc Dunnett’s test between WT and heterozygous (P > 0.05) and between WT and nmf333 groups (**P < 0.01). (D) Otoconial presence determined by von Kossa staining. The von Kossa method revealed the calcium salt content characteristic of otoconia (arrows) in the saccule (S) and utricle (U) of a WT mouse. No calcium salts are present in the utricle and saccule of nmf333 mice. n = 3 per group. Scale bars: 100 μm. (E) Otoconial presence was investigated by scanning electron microscopy in the saccules of WT and homozygous nmf333 mice. The otoconial crystal layer is completely absent from the nmf333 saccule.

Figure 6. The Cyba transgene reduces the B. cepacia susceptibility of the nmf333 strain.

(A) The Cyba transgene consists of a CMV immediate-early enhancer, a chicken β-actin promoter, a chimeric intron, the p22phox-encoding Cyba sequence, and an SV40 polyadenylation (polyA) site. (B) Blue colorimetric test for superoxide production in PMA-stimulated neutrophil granulocytes isolated from homozygous nmf333 mice and from Cyba-transgenic homozygous nmf333 [Tg(Cyba)-nmf333] mice. Two neutrophils are visible in each. Nuclei were stained with Safranin O. Original magnification, ×63. Images are representative of n = 38. (C) Quantitative analysis of superoxide production by PMA-activated neutrophil granulocytes isolated from homozygous nmf333 mice, Cyba-transgenic homozygous nmf333, and WT mice. Superoxide production was assessed using the superoxide dismutase–sensitive ferricytochrome c reduction assay. Horizontal lines indicate the mean rate of superoxide production; each circle represents a single mouse (n = 5 to 8 mice). One-way ANOVA, P < 0.0001. post hoc Tukey’s test: *P < 0.05, **P < 0.01. (D) Probability of survival of homozygous nmf333 mice (filled squares; n = 5) and of Cyba-transgenic homozygous nmf333 mice (open triangles; n = 7) after intratracheal inoculation with 10⁴ CFU B. cepacia (log-rank test, P = 0.0004). (E) Typical histological appearance of lung sections prepared from Cyba-transgenic homozygous nmf333 mice 3.5 days after intratracheal inoculation with 10⁴ CFU B. cepacia (n = 3). Left: low-magnification overview. Right: higher-magnification view of peribronchial inflammation in the boxed region. Scale bars: 100 μm.

Figure 7. The Cyba transgene rescues the balance disorder characteristic of the nmf333 strain.

(A) Immunostaining of the endolymphatic duct (asterisk) of an embryo from a Cyba-transgenic homozygous nmf333 mouse and from a homozygous nmf333 mouse at E15 using an anti-p22phox antibody. Left: localization of the red immunofluorescence signal (p22phox). Right: superimposition of the fluorescence signal and the differential interference contrast image (merged). SC, semicircular canal lumen. Scale bars: 20 μm. (B) Von Kossa staining of calcium salts in the saccule of a Cyba-transgenic homozygous nmf333 mouse (arrow indicates otoconia). n = 3. Scale bar: 100 μm. (C) The Cyba transgene restored the ability of nmf333 mice to orient themselves in water as demonstrated by the swimming test. (D) Time spent on fixed-speed rotarod (10 rpm) before falling by WT, homozygous nmf333, and Cyba-transgenic homozygous nmf333 mice during 3 assays over 3 days (d1, d2, d3). The maximum duration of the test was 180 s (horizontal line; n = 5 per group). P < 0.0001 for the genotype variable and P = 0.0042 for the time variable, 2-way ANOVA; **P < 0.01, ***P < 0.001, post-hoc Bonferroni test. (E) The Cyba transgene restores the VsEP threshold in mice homozygous for nmf333. Individual VsEP thresholds are shown for nmf333/333 (filled squares, n = 4) and Cyba-transgenic homozygous nmf333 (filled circles, n = 7) mice. Mean thresholds are indicated by solid horizontal lines ± 1 SD (dashed lines). ***P < 0.0001; 1-tailed t test against the theoretically possible lowest threshold in the nmf333 group (6 dB re 1.0 g/ms).