Project

Discipline

Neutrophils; Proteases; Serpins; Apoptosis; Inflammation; Lung

Lead
Lay summary
Neutrophils are blood cells that are rapidly recruited to sites of inflammation and infection. These cells carry a wide range of antimicrobial enzymes and molecules stored in granules. The activity of these proteins is required to kill invading pathogens and to mount an inflammatory response. However, in circumstances where excessive inflammation occurs, these proteins turn against the host by destroying protective molecules and by inducing an inflammatory vicious cycle that can be fatal to the host. This excessive neutrophilic response is a hallmark of pulmonary inflammation in cystic fibrosis and chronic obstructive pulmonary disease. Among the neutrophil weapons that have this dual protective/pathologic role are neutrophil serine proteases, potent enzymes that hydrolyze other proteins. While these proteases directly kill microbes that have been taken up by neutrophils, they have pathologic functions by fueling the inflammatory response and by destroying extracellular matrix proteins and immune defense proteins. The regulation of neutrophil proteases by specific inhibitors is therefore crucial in maintaining homeostasis. The proposed studies aim to increase understanding of the role of neutrophil proteases and their inhibitor SerpinB1 in inflammation, infection and homeostasis. SerpinB1 is a member of the serpin (SERine Protease INhibitor) family of proteins and is arguably one of the best inhibitors of the neutrophil serine proteases, including neutrophil elastase, cathepsin G and proteinase-3. We have previously shown that serpinB1 preserves the innate immune response to lung bacterial infection by protecting neutrophils from an early death and by inhibiting the destruction of protective molecules found in the lung. Building on these findings, we will investigate the role of serpinB1 in neutrophil and lung cell homeostasis in steady state and during inflammatory injury. Overall, these studies will provide significant advances in understanding the regulation of neutrophil serine proteases in health and disease and may lead to the identification of new targets for therapeutic intervention.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

Number	Title	Start	Funding scheme

SerpinB1 is a member of the serpin (SERine Protease INhibitor) family of proteins, which function as irreversible inhibitors of serine proteases. SerpinB1, also known as MNEI (monocyte neutrophil elastase inhibitor), is arguably one of the best inhibitors of the three neutrophil serine proteases (NSPs), including neutrophil elastase, cathepsin G and proteinase-3. NSPs are key components of the innate defenses by directly killing invading microbes, a process that is largely occuring inside phagosomes. However, as a double edged sword, NSPs can turn against the host if released in excess of inhibitors in the extracellular milieu at infection or inflammatory sites. Indeed, excess NSPs can digest extracellular matrix, destroy innate immune defense molecules and antibodies and induce the production of inflammatory mediators.To explore a physiological role for serpinB1 in controlling excess NSPs in infectious and inflammatory disease, we have generated mice deficient for serpinB1 (serpinB1-/-) by targeted mutagenesis. These mice failed to clear, and succumbed to, Pseudomonas aeruginosa lung infection. This innate immune defect was due in part to increased proteolysis of lung surfactant protein-D and to early death of lung recruited neutrophils. These findings were highly significant because (i) it demonstrated that serpinB1, a cytoplasmic inhibitor of NSPs carried at high levels in neutrophils, provides a physiological and vital shield against extracellular pathologic actions of NSPs, and (ii) it indicated that serpinB1 has an additional function in neutrophil homeostasis at inflammatory sites. Building on these findings, neutrophil development and homeostasis in the bone marrow of serpinB1-/- mice was investigated further and preliminary evidence strongly suggests that serpinB1 is required to preserve the size of the neutrophil reservoir bone marrow. In addition, preliminary data also indicate that serpinB1 has a role in protecting lung epithelial cells against excess neutrophil serine proteases.To test the hypothesis that serpinB1 provides a cytoprotective shield in neutrophils and lung epithelial cells, we will focus on the following specific aims. In specific aim 1, we will investigate the mode, pathways and timing of cell death of bone marrow neutrophils in serpinB1-/- and wild type mice in steady state granulopoiesis using in vitro culture of neutrophils and mixed bone marrow chimera. In specific aim 2A, we will test whether the size of the bone marrow reservoir affects the kinetics of neutrophil recruitment, survival and function of neutrophils during acute lung injury. In specific aim2B, we will use a transgenic approach to test the hypothesis that intracellular levels of serpinB1 directly affect the size of the bone marrow neutrophil reservoir in steady state and the mobilization of neutrophils during inflammation. In aim 3, we will explore the regulation of serpinB1 expression in human lung epithelial cell lines and investigate a cytoprotective role for serpinB1 in primary culture of lung epithelial and mesenchymal cells of WT and serpinB1-/- mice.Overall, these studies will provide highly significant advances in understanding the regulation of neutrophil serine proteases in homeostasis of neutrophils and lung inflammation.