Macrophages and neutrophils play major roles in host defense against microbial infections. In order to perform this function, these cell types must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H+ into the organelle. Alongside the pumps are various channels that shunt the transmembrane potential generated by movement of protons;in different organelles these comprise H+ channels, K+ channels and Cl- channels. Nevertheless, the contribution of these pathways to maintenance of intraorganellar pH is poorly studied. Recently, we demonstrated that murine alveolar macrophages (AMs) but not neutrophils employ the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) Cl- channel as a major shunt mechanism. Lysosomes and phagosomes in murine cftr/- AMs failed to acidify and the cells were deficient in bacterial killing compared to wild-type controls. We have also shown that AMs lacking CFTR are deficient in stimulus-induced secretion. Here we propose to extend these observations by investigating the role of Cl- flux in a common set of phagocyte core functions namely, organellar acidification, granule secretion, and microbicidal activity. We will compare the phagocytic and secretory activities of alveolar and peritoneal macrophages as well as neutrophils and the anion channels that are involved in the regulation of these activities. CFTR and ClC-3 chloride channels are the most reasonable channels with which to begin our studies given our preliminary data on CFTR in murine AMs and that of others in human and mouse neutrophils showing a differential functional dependence on either ClC-3 or CFTR depending upon the species. Utilizing primary cells obtained from normal, Cftr-deficient (or mutant) and ClC3-deficient mice as well as human cells from non-CF and CF patients, we will use a variety of molecular, immunochemical, microscopic and electrophysiological techniques, well-established in our laboratories, to provide a multi-faceted systems approach to the problem.

Public Health Relevance

Patients with CF are highly susceptible to chronic bacterial infection. To date, lung dysfunction in CF has been largely attributed to depletion of the liquid layer covering the upper airway epithelium with a consequent accumulation of mucus that is thought to contribute to the persistence of bacteria in the airway tree. We propose that an additional defect may be attributable to a failure of CFTR-deficient alveolar macrophages to exhibit vigorous bactericidal activity. Defects in the behavior of the innate immune system could have important consequences for microbial defense in CF patients.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036823-21
Application #
8027723
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Hagan, Ann A
Project Start
1986-07-01
Project End
2013-06-30
Budget Start
2011-02-01
Budget End
2013-06-30
Support Year
21
Fiscal Year
2011
Total Cost
$293,526
Indirect Cost
Name
University of Chicago
Department
Biology
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Meijer, Laurent; Nelson, Deborah J; Riazanski, Vladimir et al. (2016) Modulating Innate and Adaptive Immunity by (R)-Roscovitine: Potential Therapeutic Opportunity in Cystic Fibrosis. J Innate Immun 8:330-49
Riazanski, Vladimir; Gabdoulkhakova, Aida G; Boynton, Lin S et al. (2015) TRPC6 channel translocation into phagosomal membrane augments phagosomal function. Proc Natl Acad Sci U S A 112:E6486-95
Domingue, Jada C; Ao, Mei; Sarathy, Jayashree et al. (2014) HEK-293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl- transport. Physiol Rep 2:
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Di, Anke; Brown, Mary E; Deriy, Ludmila V et al. (2006) CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol 8:933-44
Wang, Xue Qing; Deriy, Ludmila V; Foss, Sarah et al. (2006) CLC-3 channels modulate excitatory synaptic transmission in hippocampal neurons. Neuron 52:321-33

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