Intracellular vesicular transport is essential for all aspects of neutrophil physiology and defects in this mechanism leads to disease in humans. In neutrophils, vesicular trafficking is associated with the processes of exocytosis, phagocytosis, signaling and transmigration; however, the molecular mechanisms that regulate mobilization of the different neutrophil secretory organelles require further elucidation. We have identified several key regulators of neutrophil granule trafficking, including the small GTPase Rab27a and its effectors JFC1 and Munc13-4. We have also identified WASH as a neutrophil factor that regulates cytoskeleton remodeling, vesicular trafficking and exocytosis. Furthermore, we have pioneered the development of systems biology approaches to analyze vesicular dynamics and actin remodeling in granulocytes. Finally, we have identified a novel mechanism of late endosomal maturation that involves the interaction between the calcium sensor Munc13-4 and the late endosomal SNARE protein syntaxin 7 (STX7), to regulate TLR9 signaling and downstream neutrophil functions. Here, we use innovative quantitative methods to elucidate the mechanisms regulating vesicular transport associated with exocytosis, phagocytosis and late endosomal maturation in neutrophils. We also propose to use novel small-molecule inhibitors of Rab27a-JFC1 and Munc13-4-STX7 binding to investigate mechanisms of vesicular transport and to elucidate neutrophil function in disease using in vivo models of systemic inflammation. The central goal of this grant is to elucidate the vesicular transport mechanisms that govern neutrophil pro-inflammatory processes, develop translational approaches to interfere with these processes and provide preclinical validation for their use to attenuate systemic inflammation. Since dysregulated neutrophil activation is injurious to the host and neutrophil secretory proteins play fundamental roles in the damage to the endothelium associated with endotoxemia, sepsis and sterile inflammation, these studies have important physiological significance and potential clinical applications. We hypothesize that the differential regulation of vesicular transport by Rab27a and its effectors is an essential mechanism to determine specific neutrophil functions and responses to insult. We also propose that small-molecule modulators of specific vesicular transport pathways will prevent some of the deleterious consequences of neutrophil activation during systemic inflammation. To test our hypotheses we propose the following Specific Aims: 1) Define the mechanisms that differentially regulate vesicular trafficking, actin-dependent propulsion and blockage, and exocytosis of neutrophil granule subsets; 2) Establish the molecular mechanisms regulating endosomal maturation, endosomal function and nucleic acid-sensing TLR-signaling in neutrophils; 3) Develop mechanistic and translational approaches to regulate neutrophil vesicular trafficking pathways and migration for the control of systemic inflammation. The proposed research will elucidate the molecular mechanisms regulating vesicular transport in neutrophils and lead to new strategies to treat inflammatory diseases.

Public Health Relevance

Neutrophils are white cells that circulate in the blood and play an essential role in protecting us from microbial infections through the production and release of substances that are toxic to invading microorganisms. These toxic products, when released in large amounts or in an uncontrolled manner, can be harmful to the host (humans). The study of the mechanisms that control the release of toxic products by neutrophils will lead to the design of therapeutic strategies for the control of inflammatory disorders and infectious processes including cardiovascular disease and sepsis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Molecular and Cellular Hematology Study Section (MCH)
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Zou, Shimian
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Scripps Research Institute
La Jolla
United States
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Kurz, Angela R M; Catz, Sergio D; Sperandio, Markus (2018) Noncanonical Hippo Signalling in the Regulation of Leukocyte Function. Trends Immunol 39:656-669
Zhang, Jinzhong; Johnson, Jennifer L; He, Jing et al. (2017) Cystinosin, the small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the chaperone-mediated autophagy receptor LAMP2A. J Biol Chem 292:10328-10346
Ramadass, Mahalakshmi; Johnson, Jennifer Linda; Catz, Sergio D (2017) Rab27a regulates GM-CSF-dependent priming of neutrophil exocytosis. J Leukoc Biol 101:693-702
Johnson, Jennifer L; Ramadass, Mahalakshmi; Haimovich, Ariela et al. (2017) Increased Neutrophil Secretion Induced by NLRP3 Mutation Links the Inflammasome to Azurophilic Granule Exocytosis. Front Cell Infect Microbiol 7:507
Catz, Sergio D (2017) Editorial: The secrets of secretion. J Leukoc Biol 102:4-6
Kurz, Angela R M; Pruenster, Monika; Rohwedder, Ina et al. (2016) MST1-dependent vesicle trafficking regulates neutrophil transmigration through the vascular basement membrane. J Clin Invest 126:4125-4139
He, Jing; Johnson, Jennifer L; Monfregola, Jlenia et al. (2016) Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses. Mol Biol Cell 27:572-87
Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi et al. (2016) Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane. J Biol Chem 291:3423-38
Ramadass, Mahalakshmi; Catz, Sergio D (2016) Molecular mechanisms regulating secretory organelles and endosomes in neutrophils and their implications for inflammation. Immunol Rev 273:249-65
Napolitano, Gennaro; Johnson, Jennifer L; He, Jing et al. (2015) Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. EMBO Mol Med 7:158-74

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