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MUNC13-4 protein regulates the oxidative response and is essential for phagosomal maturation and bacterial killing in
Jlenia Monfregola1, Jennifer Linda Johnson, Michael M Meijler
1Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
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.
Insights
MUNC13-4 is crucial for neutrophil antimicrobial functions, regulating vesicle trafficking, bacterial killing, and reactive oxygen species (ROS) production. Its deficiency impairs these processes, impacting innate immunity and potentially causing immunodeficiencies.
Area of Science:
- Immunology
- Cell Biology
- Molecular Mechanisms of Innate Immunity
Background:
- Neutrophils combat pathogens via phagocytosis, ROS generation, and NETs, relying on regulated vesicular trafficking.
- Molecular regulators of neutrophil vesicular trafficking, essential for pathogen clearance, remain incompletely understood.
- MUNC13-4, a RAB27A effector, is implicated in hematopoietic cell exocytosis and linked to familial hemophagocytic lymphohistiocytosis type 3.
Purpose of the Study:
- To elucidate the role of MUNC13-4 in neutrophil-mediated defense mechanisms.
- To investigate MUNC13-4's function in vesicular trafficking, phagosomal maturation, and intracellular bacterial killing.
- To understand the implications of MUNC13-4 deficiency in neutrophil innate immune responses.
Main Methods:
- Utilized neutrophils from munc13-4 knock-out (KO) mice for functional assays.
- Assessed granule trafficking to the plasma membrane and ROS production (extracellular and intracellular).
- Evaluated phagosomal maturation, bacterial killing, and neutrophil extracellular trap (NET) formation.
Main Results:
- MUNC13-4 deficiency impaired p22(phox)-expressing granule trafficking and extracellular ROS production.
- Intracellular ROS production against Pseudomonas aeruginosa was defective in MUNC13-4 KO neutrophils.
- Phagosomal maturation was compromised by defective azurophilic granule and multivesicular body delivery; bacterial killing was significantly impaired.
- MUNC13-4 KO neutrophils showed increased NET formation, but this did not rescue impaired bacterial killing.
Conclusions:
- MUNC13-4 plays a critical role in selective vesicular trafficking, phagosomal maturation, and intracellular bacterial killing in neutrophils.
- MUNC13-4 regulates both extracellular and intracellular ROS production, contributing to neutrophil antimicrobial efficacy.
- Findings highlight novel MUNC13-4 functions in innate immunity and offer insights into MUNC13-4-related immunodeficiencies.
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