Macrophage phagocytosis is the first line of defense against infection. Normally, Fc?R-mediated phagocytosis efficiently clears pathogens and presents antigen to the adaptive immune system. However, dysregulation of FcR signaling contributes to chronic inflammatory diseases such as rheumatoid arthritis and atherosclerosis. Thus, understanding the molecular mechanisms of macrophage phagocytosis is essential to the discovery of novel targets for regulation of chronic inflammation. Mice lacking protein kinase C-epsilon (PKC-?) are highly susceptible to bacteria infections and fail to mount an effective inflammatory response. We have shown that PKC-? is necessary for efficient Fc?R-mediated phagocytosis and for production of pro-inflammatory genes. The mechanism of PKC-? action requires localization to phagosomes and catalytic activity. However, how PKC-? is activated and signals for phagocytosis and gene induction are unknown. Functionally, we have shown that PKC-? is involved in pseudopod extension, a process that requires fusion of intracellular vesicles into the phagosome. Structurally, we have identified the pseudosubstrate domain as critical for PKC-? translocation and shown that it preferentially interacts with polyphosphoinositides. This novel finding has implications for the mechanism of PKC-? activation, which is currently unknown. Based on our research and that of others, we propose that catalytically active PKC-? is necessary for the focal delivery of vesicles into the forming phagosome. PKC-? is activated by diacylglycerol and a phosphoinositide monophosphate (PIP). Active PKC-? phosphorylates proteins involved in the vesicle fusion necessary for membrane delivery and pseudopod extension. PKC-? is also necessary for the induction of genes required for resolution of infection. This model will be tested using bone marrow-derived macrophages from wild type and PKC-? null mice. Specifically, we will I) Identify the PIP required for PKC-? translocation to phagosomes, II) Determine the role of PKC-? in vesicle trafficking and fusion, III) Use conventional and traceable PKC techniques to identify PKC-? substrates, and IV) Use an unbiased, qPCR array approach to identify PKC-?-regulated genes. Defining the mechanisms by which PKC-? transduces Fc?R-initiated signals in phagocytosis and gene induction is critical to our understanding of host defense and the defects that contribute to chronic inflammatory diseases. The proposed studies use cutting edge technologies, coupled with classical approaches, in primary macrophages. We will rigorously test hypotheses supported by preliminary data and reach beyond current paradigms to identify PKC-?-regulated genes and substrates. The information gained will provide insight into the role of PKC-? in Fc?R-mediated signal transduction and how its loss impacts innate immunity.

Public Health Relevance

IgG-mediated phagocytosis is an essential function of the innate immune system. Dysregulation can lead to chronic inflammatory diseases including rheumatoid arthritis and atherosclerosis. Phagocytosis requires the focal delivery of intracellular membranes to the site of target binding. How this occurs and what directs that focal delivery is unknown. We have evidence that protein kinase C-epsilon plays a major role in membrane dynamics during phagocytosis but the mechanism by which that occurs is unknown. Understanding membrane remodeling using phagocytosis as a model system will advance our knowledge of the innate immune system and may be applicable to other cell processes, including tumor metastisis and wound healing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM090325-09
Application #
8287133
Study Section
Innate Immunity and Inflammation Study Section (III)
Program Officer
Dunsmore, Sarah
Project Start
2002-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
9
Fiscal Year
2012
Total Cost
$304,890
Indirect Cost
$110,703
Name
Albany Medical College
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
190592162
City
Albany
State
NY
Country
United States
Zip Code
12208
Wood, Tiffany R; Chow, Rachel Y; Hanes, Cheryl M et al. (2013) PKC-ýý pseudosubstrate and catalytic activity are necessary for membrane delivery during IgG-mediated phagocytosis. J Leukoc Biol 94:109-22
Harmon, Erin Y; Fronhofer, Van; Keller, Rebecca S et al. (2012) Ultrasound biomicroscopy for longitudinal studies of carotid plaque development in mice: validation with histological endpoints. PLoS One 7:e29944
Lennartz, Michelle R; Aggarwal, Ankur; Michaud, Tanya M et al. (2011) Ligation of macrophage Fc? receptors recapitulates the gene expression pattern of vulnerable human carotid plaques. PLoS One 6:e21803