NBEAL2 is required for neutrophil and NK cell function and pathogen defense

Abstract

Mutations in the human NBEAL2 gene cause gray platelet syndrome (GPS), a bleeding diathesis characterized by a lack of α granules in platelets. The functions of the NBEAL2 protein have not been explored outside platelet biology, but there are reports of increased frequency of infection and abnormal neutrophil morphology in patients with GPS. We therefore investigated the role of NBEAL2 in immunity by analyzing the phenotype of Nbeal2-deficient mice. We found profound abnormalities in the Nbeal2-deficient immune system, particularly in the function of neutrophils and NK cells. Phenotyping of Nbeal2-deficient neutrophils showed a severe reduction in granule contents across all granule subsets. Despite this, Nbeal2-deficient neutrophils had an enhanced phagocyte respiratory burst relative to Nbeal2-expressing neutrophils. This respiratory burst was associated with increased expression of cytosolic components of the NADPH oxidase complex. Nbeal2-deficient NK cells were also dysfunctional and showed reduced degranulation. These abnormalities were associated with increased susceptibility to both bacterial (Staphylococcus aureus) and viral (murine CMV) infection in vivo. These results define an essential role for NBEAL2 in mammalian immunity.

Figure 1. Immunophenotyping shows reduced granularity in Nbeal2^–/– granulocytes.

(A) Flow plots of BM neutrophils showing forward scatter (FSC) and side scatter (SSC), together with the geometric mean of SSC in Gr-1^hiCD11b⁺ neutrophils from BM, blood, and spleen (n = 6–7). (B) Proportion of neutrophils in the BM, blood, and spleen and absolute numbers of splenic neutrophils (n = 6–7). (C) Transmission electron micrographs of BM neutrophils from whole BM sections. Representative images of neutrophils (top) (original magnification, ×3,500) and a section from the image (bottom) (original magnification, ×6,500). Scale bars: 500 nm (all images). Electron-dense granules (arrowheads) were counted by an investigator blinded to genotype for the WT cells (n = 13) and Nbeal2^–/– cells (n = 23) across 3 biological replicates. (D) Volcano plot shows the 3,485 proteins identified in the BM neutrophil proteome of Nbeal2^+/+ and Nbeal2^–/– mice. The y axis shows FDR-corrected P values, the x axis displays the fold change (log₂) of Nbeal2^+/+ protein expression compared with Nbeal2^–/– expression, and the horizontal line shows the cutoff at P = 0.05. Granule components, NADPH oxidase subunits, and Nbeal2 are indicated in red. Error bars indicate the mean and SD. Data are representative of 2 independent experiments (A and B) or a pooled analysis from 3 independent MS runs (D). *P < 0.05, **P < 0.01, and ***P < 0.001, by Kruskal-Wallis (A and B) and Mann-Whitney U (C) tests.

Figure 2. Nbeal2^–/– neutrophils have altered effector functions and show susceptibility to S. aureus in vivo.

(A) Extracellular elastase release of BM neutrophils in response to cytochalasin B (Cyt. B) and fMLP (n = 4). (B–D) BM neutrophils stimulated with fMLP (B), PMA (C), or zymosan (D) and luminol cleavage fluorescence were quantified over time and the AUC calculated (n = 3). (E–G) Mice were infected i.v. with the sh100 strain of S. aureus and (E) monitored daily for weight changes during the initial 6 days after infection (n = 5–7). (F) Survival curve of mice infected and monitored for 22 days. Mice were sacrificed if their weight dropped more than 20% from their starting weight (n = 9–10). (G) Pooled bacterial counts in the kidney, liver, and spleen on day 6 after infection from 2 independent experiments (n = 5–7). Data are presented as the mean and SD (A), the mean and SEM (B–E), or the median (G). Data are representative of 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by Mann-Whitney U test (A and G), Kruskal-Wallis test (B–D), 2-way ANOVA (E), or log-rank test (F). RLU, relative light units.

Figure 3. Nbeal2^–/– mice have dysfunctional NK cells in vitro and an impaired response to mCMV in vivo.

(A) Proportion and absolute numbers of CD3^–B220^–CD49b⁺NKp46⁺ splenic NK cells for the indicated genotypes. (B) Expression of CD11b and CD27 (maturation markers) on CD3^–B220^–NK1.1⁺NKp46⁺ NK cells. Maturation was measured from gates R1 to R4 (with R4 being the most mature). Shown are the proportions and absolute numbers for each subset (n = 5–7). (C) Surface expression of LAMP-1/isotype of splenic NK cells 0 and 2 hours after PMA/ionomycin stimulation. Representative FACS gating and LAMP-1 histogram showing isotype control antibody staining (gray area) and LAMP-1 in Nbeal2^+/+ (black line) and Nbeal2^–/– mice (red line) (n = 4–5). max, maximum; MFI, mean fluorescence intensity. (D–F) Nbeal2^+/+ or Nbeal2^–/– mice were infected (i.p.) with 3 × 10⁴ salivary gland–propagated Smith strain mCMV, and BW was monitored for 4 days after infection (D). On day 4, mice were sacrificed, and virus PFU were quantified in the spleen (E) and lungs (F) (n = 11). (G) Splenic NK cells were isolated and cultured in 1,000 U IL-2 for 4 days before cytotoxicity (LDH release) was tested on YAC-1 cells (n = 3). Data are presented as the mean and SD (A–D), median (E and F), or SEM (G) and are representative of 3 (A, E, and F) or 2 (B, C, and G) independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by Kruskal-Wallis test (A), Mann-Whitney U test (B, C, E, and F), or 2-way ANOVA (D and G).