The neutrophil serine proteases (NSPs) (elastase, cathepsin G and proteinase-3) released at inflammatory sites by degranulating or necrotic neutrophils are major contributors to the pathology of cystic fibrosis and chronic obstructive pulmonary disease (COPD). SerpinB1/MNEI (Monocyte neutrophil elastase inhibitor) is naturally occurring in lungs and blood cells and is a highly efficient inhibitor of the three NSPs. We have produced a mouse deficient for mnei, which replicates the protease excess of inflammatory lung disease and provides a model for studying the currently underestimated contribution of mnei/SerpinB1 to pulmonary protection. The mnei deficient mice fail to clear Pseudomonas aeruginosa and have increased proinflammatory cytokines and depletion of surfactant protein-D (SP-D), a known target of NSPs. SP-D, which is required for bacterial uptake and clearance of apoptotic neutrophils by alveolar macrophages, is found as inactive fragments in lungs of Pseudomonas-infected mice lacking mnei/serpinb1. Necrotic neutrophils also accumulate.
Aim 1 will test the hypothesis that mnei provides broad pulmonary host defense protection against both Gram-negative (P.aeruginosa, Haemophilus influenzae) and Gram-positive (Staphylococcus aureus) organisms and in two genetic backgrounds (C57BL/6 and129S6). Mnei-/- and wild type mice will be evaluated for survival, bacteriology, and recruitment and survival of neutrophils;lavage samples will be analyzed for cytokines, proteases, SP-A and SP-D.
Aim 2 will address the cellular defects that contribute to the defective anti-Pseudomonas response during the critical time block (6-24 hr) during which antimicrobial defense deteriorates. Parenchymal cells, neutrophils and alveolar macrophages of infected mice will be examined for activation, cell injury, and surface receptor cleavage. Systemic response (blood cytokines) will be assessed. Neutrophils will be analyzed ex vivo for survival and bacterial killing activity, and alveolar macrophages for ability to engulf necrotic cells. Since mnei is expressed at high level in myeloid cells, but is also expressed in lung parenchyma, chimeric mice will be generated to determine whether non-hematopoietic cells contribute to the phenotype.
Aim 3 will test whether the defective anti-Pseudomonas defense and increased inflammation of mnei deficient mice can be reconstituted by intranasal delivery of recombinant MNEI. To test the role of SP-D depletion, we will determine whether the defective response can be (partially) corrected by lung-specific overexpression of SP-D. These studies in mice to delineate mechanisms by which an aggressive host response leads to lung injury as occurs in patients with cystic fibrosis and COPD (chronic bronchitis, emphysema) will provide understanding that may lead to improved treatment. Indeed, mnei, the central molecule under study in the project, is a candidate therapeutic for inflammatory lung disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL066548-07
Application #
7585712
Study Section
Lung Injury, Repair, and Remodeling Study Section (LIRR)
Program Officer
Croxton, Thomas
Project Start
2000-09-29
Project End
2011-03-31
Budget Start
2009-04-01
Budget End
2010-03-31
Support Year
7
Fiscal Year
2009
Total Cost
$525,000
Indirect Cost
Name
Immune Disease Institute, Inc.
Department
Type
DUNS #
059709394
City
Boston
State
MA
Country
United States
Zip Code
02115
Burgener, Sabrina S; Baumann, Mathias; Basilico, Paola et al. (2016) Myeloid conditional deletion and transgenic models reveal a threshold for the neutrophil survival factor Serpinb1. Biol Chem 397:897-905
El Ouaamari, Abdelfattah; Dirice, Ercument; Gedeon, Nicholas et al. (2016) SerpinB1 Promotes Pancreatic ? Cell Proliferation. Cell Metab 23:194-205
Hou, Lifei; Cooley, Jessica; Swanson, Richard et al. (2015) The protease cathepsin L regulates Th17 cell differentiation. J Autoimmun 65:56-63
Zhao, Picheng; Hou, Lifei; Farley, Kalamo et al. (2014) SerpinB1 regulates homeostatic expansion of IL-17+ ?? and CD4+ Th17 cells. J Leukoc Biol 95:521-30
Stolley, J Michael; Gong, Dapeng; Farley, Kalamo et al. (2012) Increased surfactant protein D fails to improve bacterial clearance and inflammation in serpinB1-/- mice. Am J Respir Cell Mol Biol 47:792-9
Farley, Kalamo; Stolley, J Michael; Zhao, Picheng et al. (2012) A serpinB1 regulatory mechanism is essential for restricting neutrophil extracellular trap generation. J Immunol 189:4574-81
Cooley, J; Sontag, M K; Accurso, F J et al. (2011) SerpinB1 in cystic fibrosis airway fluids: quantity, molecular form and mechanism of elastase inhibition. Eur Respir J 37:1083-90
Gong, Dapeng; Farley, Kalamo; White, Mitchell et al. (2011) Critical role of serpinB1 in regulating inflammatory responses in pulmonary influenza infection. J Infect Dis 204:592-600
Benarafa, Charaf; LeCuyer, Tessa E; Baumann, Mathias et al. (2011) SerpinB1 protects the mature neutrophil reserve in the bone marrow. J Leukoc Biol 90:21-9
Davies, Philip L; Spiller, O Brad; Beeton, Michael L et al. (2010) Relationship of proteinases and proteinase inhibitors with microbial presence in chronic lung disease of prematurity. Thorax 65:246-51

Showing the most recent 10 out of 21 publications