Intracellular vesicular transport is essential for nearly all aspects of neutrophil physiology and defects in this mechanism leads to disease in humans. In neutrophils, vesicular trafficking is directly associated with the processes of exocytosis, phagocytosis, adhesion and chemotaxis;however, the molecular mechanisms that regulate mobilization of the different neutrophil secretory organelles during secretion, pathogen internalization or migration are incompletely understood. We have identified several key regulatory factors essential for the control of granule trafficking in neutrophils including the smll GTPase Rab27a that controls the mobilization and secretion of neutrophil granules in vitro and in vivo. This function is regulated by two Rab27a effectors, JFC1 and Munc13-4. Furthermore, we have recently identified previously unrecognized neutrophil secretory factors that interact with JFC1 and regulate cytoskeleton remodeling, vesicular trafficking and exocytosis and have pioneered the development of systems biology approaches to analyze vesicular dynamics and actin remodeling in granulocytes. In this application, we propose to use innovative analytical and quantitative methods to elucidate the mechanisms regulating vesicular transport associated with exocytosis, phagocytosis and chemotaxis in neutrophils. We also propose to use novel small molecule inhibitors of Rab27a-effector binding to interrogate mechanisms of vesicular transport and elucidate Rab27a-dependent processes in disease using an in vivo model of endotoxin-induced systemic inflammation. Since the toxic content of neutrophil granules is injurious to the host and neutrophil secretory proteins play a fundamental role in the damage to endothelium associated with endotoxemia and sepsis, these studies have important physiological significance and potential clinical applications in disease. 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 vesicular transport pathways will prevent some of the deleterious consequences of neutrophil secretion during systemic inflammation. To test our hypotheses we propose the following Specific Aims: 1) To define the mechanisms that regulate vesicular trafficking and actin remodeling during exocytosis in granulocytes and establish the role of the Rab27a effector JFC1 in this mechanism;2) To establish the mechanisms regulated by Munc13-4 in vesicular transport and vesicular fusion associated with exocytosis, phagocytosis and bacterial killing;3) To test the hypothesis that novel small molecule inhibitors of Rab27a-effector binding modulate neutrophil granule trafficking and exocytosis and attenuate inflammation. The results of the proposed research plan should uncover the molecular mechanisms regulating vesicular transport in neutrophils and lead to effective molecular strategies for the treatment of 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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL088256-06
Application #
8703748
Study Section
Special Emphasis Panel (ZRG1-VH-D (02))
Program Officer
Zou, Shimian
Project Start
2007-04-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
6
Fiscal Year
2014
Total Cost
$464,275
Indirect Cost
$219,275
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Catz, Sergio D (2014) The role of Rab27a in the regulation of neutrophil function. Cell Microbiol 16:1301-10
Catz, Sergio Daniel (2013) Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors. J Leukoc Biol 94:613-22
Johnson, Jennifer L; Napolitano, Gennaro; Monfregola, Jlenia et al. (2013) Upregulation of the Rab27a-dependent trafficking and secretory mechanisms improves lysosomal transport, alleviates endoplasmic reticulum stress, and reduces lysosome overload in cystinosis. Mol Cell Biol 33:2950-62
Johnson, Jennifer L; Monfregola, Jlenia; Napolitano, Gennaro et al. (2012) Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase-activating protein Gem-interacting protein. Mol Biol Cell 23:1902-16
Johnson, Jennifer L; Hong, Hong; Monfregola, Jlenia et al. (2011) Increased survival and reduced neutrophil infiltration of the liver in Rab27a- but not Munc13-4-deficient mice in lipopolysaccharide-induced systemic inflammation. Infect Immun 79:3607-18
Johnson, Jennifer L; Hong, Hong; Monfregola, Jlenia et al. (2011) Munc13-4 restricts motility of Rab27a-expressing vesicles to facilitate lipopolysaccharide-induced priming of exocytosis in neutrophils. J Biol Chem 286:5647-56
Johnson, Jennifer L; Brzezinska, Agnieszka A; Tolmachova, Tanya et al. (2010) Rab27a and Rab27b regulate neutrophil azurophilic granule exocytosis and NADPH oxidase activity by independent mechanisms. Traffic 11:533-47
Munafo, Daniela B; Johnson, Jennifer L; Brzezinska, Agnieszka A et al. (2009) DNase I inhibits a late phase of reactive oxygen species production in neutrophils. J Innate Immun 1:527-42