3R-Project 126-11

Model development and validation to investigate myeloid cell homeostasis

Mathias Baumann and Charaf Benarafa
Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland
charaf.benarafa@tki.unibe.ch

Keywords: mice; transgenic mice; neutrophils; inflammation; cell cultures: transgenic; reduction; infectiosity

Duration: 2 years Project Completion: 2013

Background and Aim
Neutrophils develop in the bone marrow from hematopoietic stem cells and play a critical role in infection and inflammation. Understanding the molecular mechanisms that regulate neutrophil homeostasis is therefore crucial in designing new therapeutic strategies to modulate inflammatory and immune responses. Mature neutrophils are short-lived and thus poorly amenable to molecular manipulation. In the present project, we will validate the use of immortalized mouse neutrophil progenitors that can be conditionally induced to differentiate in vitro into unlimited numbers of neutrophils at different stages of differentiation (1). We propose the following specific aims to validate this methodology to study neutrophil death mechanisms regulated by serine proteases and serpinB1 (2, 3).
Specific Aim 1. To reproduce our in vivo findings that serpinB1 regulates neutrophil homeostasis and survival upon differentiation using an alternative method to primary cells isolated from experimental animals.
Specific Aim 2. To investigate the role of specific serine proteases in neutrophil homeostasis.

Method and Results
in progress (present status)
We will first characterize immortalized wild-type and serpinB1-/- myeloid progenitors generated by estrogen receptor (ER)-mediated conditional expression of hoxb8 in the presence of stem cell factor (SCF). Markers for proliferation, differentiation, pattern of granule protein expression and cell death pathways will then be investigated at several points in time in differentiation induced by ER-signaling withdrawal.
We will then generate myeloid progenitor lines with deficiency in neutrophil serine proteases and investigate death pathways in neutrophils differentiated in vitro.

Conclusions and Relevance for 3R
By achieving these aims, we will extensively validate an alternative method for studying neutrophil development, homeostasis and cell death pathways using large numbers of cells at a defined differentiation stage in the neutrophil lineage generated in vitro.

References
1. Wang, G.G., K.R. Calvo, M.P. Pasillas, D.B. Sykes, H. Hacker, and M.P. Kamps. 2006. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat Methods 3:287-293.

2. Benarafa, C., T.E. Lecuyer, M. Baumann, J.M. Stolley, T.P. Cremona, and E. Remold-O'Donnell. 2011. SerpinB1 protects the mature neutrophil reserve in the bone marrow. J Leukoc Biol (in press).

3. Benarafa, C., G.P. Priebe, and E. Remold-O'Donnell. 2007. The neutrophil serine protease inhibitor serpin B1 preserves lung defense functions in Pseudomonas aeruginosa infection. J Exp Med 204:1901-1909.