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. 2012 Dec 28;287(53):44603-18.
doi: 10.1074/jbc.M112.414029. Epub 2012 Oct 31.

MUNC13-4 Protein Regulates the Oxidative Response and Is Essential for Phagosomal Maturation and Bacterial Killing in Neutrophils

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Free PMC article

MUNC13-4 Protein Regulates the Oxidative Response and Is Essential for Phagosomal Maturation and Bacterial Killing in Neutrophils

Jlenia Monfregola et al. J Biol Chem. .
Free PMC article

Abstract

Neutrophils use diverse mechanisms to kill pathogens including phagocytosis, exocytosis, generation of reactive oxygen species (ROS), and neutrophil extracellular traps. These mechanisms rely on their ability to mobilize intracellular organelles and to deliver granular cargoes to specific cellular compartments or into the extracellular milieu, but the molecular mechanisms regulating vesicular trafficking in neutrophils are not well understood. MUNC13-4 is a RAB27A effector that coordinates exocytosis in hematopoietic cells, and its deficiency is associated with the human immunodeficiency familial hemophagocytic lymphohistiocytosis type 3. In this work, we have established an essential role for MUNC13-4 in selective vesicular trafficking, phagosomal maturation, and intracellular bacterial killing in neutrophils. Using neutrophils from munc13-4 knock-out (KO) mice, we show that MUNC13-4 is necessary for the regulation of p22(phox)-expressing granule trafficking to the plasma membrane and regulates extracellular ROS production. MUNC13-4 was also essential for the regulation of intracellular ROS production induced by Pseudomonas aeruginosa despite normal trafficking of p22(phox)-expressing vesicles toward the phagosome. Importantly, in the absence of MUNC13-4, phagosomal maturation was impaired as observed by the defective delivery of azurophilic granules and multivesicular bodies to the phagosome. Significantly, this mechanism was intact in RAB27A KO neutrophils. Intracellular bacterial killing was markedly impaired in MUNC13-4 KO neutrophils. MUNC13-4-deficient cells showed a significant increase in neutrophil extracellular trap formation but were unable to compensate for the impaired bacterial killing. Altogether, these findings characterize novel functions of MUNC13-4 in the innate immune response of the neutrophil and have direct implications for the understanding of immunodeficiencies in patients with MUNC13-4 deficiency.

Figures

FIGURE 1.
FIGURE 1.
Translocation of the p22phox-expressing organelles at the exocytic active zone is impaired in MUNC13-4 KO neutrophils. A and B, MUNC13-4 colocalizes and cofractionates with flavocytochrome b558-expressing vesicles. A, neutrophils were fixed, and the subcellular localization of endogenous MUNC13-4 and gp91phox at the exocytic active zone was analyzed by immunofluorescence using TIRF microscopy (scale bars, 10 μm). The colocalization of MUNC13-4 and gp91phox in neutrophil granules is shown in the inset. B, neutrophils were lysed and fractionated using sucrose gradients, and endogenous proteins were analyzed by Western blot. C, decreased number of p22phox-expressing organelles at the exocytic active zone in MUNC13-4 KO neutrophils. The distribution of p22phox-expressing organelles at the exocytic active zone was analyzed by immunofluorescence using TIRF microscopy. Representative images of unstimulated or fMLP-stimulated wild type and MUNC13-4 KO neutrophils are shown (scale bars, 10 μm). D, quantitative analysis of the distribution of p22phox-expressing vesicles at the exocytic active zone per area unit of MUNC13-4 KO and wild type neutrophils. A total of 80 wild type and 40 MUNC13-4 KO cells from three independent experiments were analyzed. Results are expressed as mean ± S.E. (error bars). E, the level of p22phox expression in wild type and MUNC13-4 KO neutrophils was analyzed by Western blot and subsequently quantified. The data are representative of three independent experiments with similar results. Quantitative analysis represents the mean ± S.E. (error bars).
FIGURE 2.
FIGURE 2.
MUNC13-4 KO neutrophils are characterized by an impaired integration of p22phox into the plasma membrane and defective extracellular ROS production. A, confocal microscopy analysis of the distribution of endogenous p22phox in relationship to the plasma membrane. Wild type and MUNC13-4 KO neutrophils were left untreated or stimulated with 10 μm fMLP and fixed, and immunofluorescence analysis was performed using a polyclonal antibody specific for the detection of endogenous p22phox and rhodamine-WGA to label the plasma membrane. The samples were analyzed by confocal microscopy, and representative z-sections for each condition are shown. Inset, the arrowheads point to p22phox-positive granules, which are integrated into the plasma membrane in wild type cells but not in MUNC13-4 KO cells (scale bars, 10 μm). B, quantitative analysis showing the percentage of the p22phox-containing vesicles in the exocytic active zone that are integrated into the plasma membrane. The results are representative of three independent experiments with similar results (mean ± S.E. (error bars)). C, ROS production was measured using bone marrow-derived neutrophils from MUNC13-4 KO and WT mice treated with the cell-impermeant probe isoluminol in the presence of horseradish peroxidase and stimulated with 10 μm fMLP. The representative graph shows the kinetics of extracellular ROS production, which is defective in MUNC13-4 KO cells (red line and symbols). D and E, quantitative determination of the total ROS produced during the time of analysis (calculated as the integral or area under the curve). Results are expressed as mean ± S.E. (error bars); statistical analysis was performed using a non-parametric Mann-Whitney test (D, n = 8; E, n = 3). RLU, relative light units.
FIGURE 3.
FIGURE 3.
MUNC13-4 KO neutrophils have impaired intracellular ROS production. A, neutrophils from MUNC13-4 KO and wild type mice were incubated with serum-opsonized P. aeruginosa at a ratio of 10:1 (bacteria/neutrophil), and intracellular ROS production was analyzed by the luminol-dependent chemiluminescence reaction. Reactions were started by the addition of bacteria to a prewarmed (37 °C) neutrophil suspension, and chemiluminescence was continuously monitored from this time point. Where indicated, the inhibitor of phagocytosis cytochalasin D (CytD; 10 μg/ml) was used as control. B, quantitative determination of the total ROS produced during the time of analysis (calculated as the integral or area under the curve). Results are expressed as mean ± S.E. (error bars) from six independent experiments. C, undetectable extracellular ROS production induced by P. aeruginosa. The extracellular oxidative response was measured using cell-impermeant isoluminol in the presence of horseradish peroxidase. Different from fMLP, live P. aeruginosa was unable to induce an extracellular oxidative response under these assay conditions. RLU, relative light units.
FIGURE 4.
FIGURE 4.
MUNC13-4 KO neutrophils have normal phagocytic activity. Confocal microscopy analysis was performed to quantify live P. aeruginosa internalization in wild type and MUNC13-4 KO neutrophils. Cells were incubated with opsonized, tetramethylrhodamine-conjugated P. aeruginosa for 45 min at 37 °C. Fluorescent extracellular bacteria were quenched with trypan blue, and internalized bacteria were counted manually from up to 10 section fields for each sample. No significant differences were observed in the percentage of cells containing phagosomes (A) between wild type and MUNC13-4 KO cells. B, the distribution of the cell populations containing increasing numbers of phagosomes was also similar in wild type and MUNC13-4 KO cells. C, no significant differences were observed in the average number of phagosomes/cell between MUNC13-4 KO and wild type neutrophils. The results are mean ± S.E. (error bars) from three independent experiments. D, representative image of confocal sections used to count the number of bacteria present in each cell population. * indicates bacteria already counted in the previous z-section. The thickness of each stack is 0.5 μm (scale bars, 10 μm). Individual intracellular bacteria are indicated with numbers.
FIGURE 5.
FIGURE 5.
Phagosomal maturation is impaired in MUNC13-4 KO neutrophils. Wild type and MUNC13-4 KO neutrophils were incubated with opsonized, tetramethylrhodamine-conjugated P. aeruginosa at 4 °C, and synchronized phagocytosis was started by incubation of the neutrophil/bacteria mixture at 37 °C for 15 (A and B) or 45 min (C–F). A, the subcellular localization of endogenous p22phox was analyzed by immunofluorescence. B, the percentage of p22phox-positive phagosomes was calculated by analyzing 187 and 282 phagosomes from wild type and MUNC13-4 KO cells, respectively. C, the subcellular localization of endogenous MPO was analyzed by immunofluorescence. D, the percentage of the phagosomes that were positive for MPO staining was calculated by analyzing 880 and 657 phagosomes from wild type and MUNC13-4 KO cells, respectively. E, analysis of endogenous LAMP1 localization was performed as described above. F, the percentage of LAMP1-positive phagosomes was determined after counting a total of 759 and 633 phagosomes in wild type and MUNC13-4 KO cells, respectively. Results are expressed as mean ± S.E. (error bars) from three independent experiments. Scale bars, 10 μm.
FIGURE 6.
FIGURE 6.
RAB27A is not required for bacterium-induced intracellular ROS production or phagosomal maturation. A and B, neutrophils from RAB27A-deficient and WT mice were incubated with opsonized P. aeruginosa, and the luminol-dependent chemiluminescence reaction was monitored for the indicated time. The kinetics are representative of three independent experiments. B, quantitative analysis of P. aeruginosa-induced ROS production. The results are expressed as mean ± S.E. (error bars). C, confocal microscopy analysis of the distribution of endogenous MPO and LAMP1 in wild type and RAB27A-deficient cells after phagocytosis of serum-opsonized, tetramethylrhodamine-conjugated P. aeruginosa for 45′ at 37 °C. Scale bars, 10 μm. RLU, relative light units.
FIGURE 7.
FIGURE 7.
MUNC13-4 KO neutrophils have impaired bacterial killing. A, the ability of wild type and MUNC13-4 KO neutrophils to kill opsonized P. aeruginosa was analyzed by incubating freshly isolated neutrophils or heat-killed neutrophils (negative control) in the presence of opsonized P. aeruginosa for 3 h at 37 °C followed by release of internalized live bacteria by alkaline lysis of neutrophils at pH 11 and subsequent quantification of live bacteria by the XTT reaction as described under “Experimental Procedures.” B, the ability of WT and MUNC13-4 KO neutrophils to kill extracellular bacteria was measured as described above except that alkaline lysis was omitted, and therefore, only extracellular bacteria were quantified. Where indicated, the cells were treated with DNase I to destroy neutrophil extracellular traps after the 3-h incubation period. The results are expressed as mean ± S.E. (error bars) (n = 6). Quantitative analysis was performed using a non-parametric Mann-Whitney test. C, P. aeruginosa killing by gp91phox KO neutrophils was analyzed as described in A. The results are expressed as mean ± S.D. (error bars) (n = 2).
FIGURE 8.
FIGURE 8.
NET production is increased in MUNC13-4 KO neutrophils. A, wild type and MUNC13-4 KO neutrophils were incubated for 3 h in the presence of P. aeruginosa, stained with DAPI, and analyzed by confocal microscopy. Representative images of NETs produced by neutrophils are shown. B, NETs were counted from 10 independent fields for each sample using ImageJ software, and the percentages of NET-producing cells were calculated. Results are mean ± S.E. (error bars) from three independent experiments. Similar results were obtained using the cell-impermeant DNA dye SYTOX Green (C). D, confocal microscopy analysis of neutrophil NETs after incubation with opsonized, tetramethylrhodamine-conjugated P. aeruginosa (red) shows a similar distribution of MPO (green; arrowheads) along NETs in wild type and MUNC13-4 KO cells. Scale bars, 10 μm.

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