Vesicular trafficking mechanisms regulating granulocyte function

Project Summary: 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 NETs production; 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 now identified WASH as a neutrophil factor that regulates cytoskeleton
remodeling, vesicular trafficking and phagosomal maturation. 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, late endosomal maturation
and NETs production in neutrophils. We also propose to use our newly designed 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 and infection. 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 of neutrophil granule subsets during phagocytosis and NETosis; 2)
Establish the molecular mechanisms of mitochondrial nucleic acid-sensing by endosomal TLR signaling in
neutrophils; 3) Develop mechanistic and translational approaches to decrease neutrophil-mediated systemic
inflammation without affecting the innate immune response. The proposed research will elucidate the molecular
mechanisms regulating neutrophil vesicular transport and lead to new strategies to treat inflammatory diseases.

Grant Number: 5R01HL088256-15
NIH Institute/Center: NIH
Principal Investigator: Sergio Catz