3R-Project 126-11

Development and validation of a model 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

Duration: 2 years Project Completion: 2013

Background and Aim

Every single day, the bone marrow of an adult human produces 100 billion (10¹¹) neutrophils to replace the rapidly aging circulating population. Differentiating from haematopoietic stem cells and developing in the bone marrow, neutrophils play a critical role in combating infections: they are recruited en masse to kill and eliminate microbes. The neutrophil-killing machinery includes powerful enzymes, such as proteases, whose activities are indiscriminate and, if unregulated, lead to substantial inflammatory responses, which may be injurious to the host cells and tissues.

An understanding of the molecular mechanisms underlying neutrophil homeostasis is thus of importance in the development of new therapeutic strategies that aim to modulate inflammatory and immune responses.

A major challenge in the field of neutrophil biology is posed by the extremely short lifespan of these cells. Ex vivo, they survive no more than a few days; under these conditions, genetic and molecular manipulations are not conceivable. Studies that have been conducted with transgenic and knock-out mice have yielded fundamental information appertaining to the functions of individual proteins in neutrophil biology under steady-state and pathological conditions. Since mice have but a small number of circulating neutrophils, functional assays have been largely performed either in vivo or on mature cells that have been isolated from the bone marrow of transgenic and knock-out animals, for which purposes many mice are consumed.

It was the purpose of this project to test the utility of in-vitro-immortalized line of neutrophil progenitors (MyPh8) (Wang et al. 2006), which can be conditionally induced under culturing conditions to differentiate into unlimited numbers of mature cells. The availability of an approach of this kind would reduce the need for isolating primary neutrophils from living mice and thus help to cut down on the consumption of animals for experimental purposes. It was also our aim to validate the methodology by investigating the function of the intracellular serine protease inhibitor serpinB1 in neutrophil homeostasis, as previously demonstrated not only in vitro for isolated bone-marrow cells, but also in vivo (Benarafa et al. 2007; 2011).

Method and Results

We first established and characterized immortalized wild-type (WT) and serpinB1-deficient myeloid progenitors (MyPh8), which were generated by the lentiviral transduction of the oestrogen-receptor-mediated expression of HoxB8 in the presence of stem cell factor. Upon withdrawal of oestrogen and treatment with G-CSF, myeloid progenitors differentiated into cells that manifested the characteristic features of mature neutrophils, including the expression of Ly-6G and MMP-9 and the ability to produce reactive oxygen species. SerpinB1-defcient MyPh8-cells were more susceptible to death than their WT-counterparts, as previously reported for primary neutrophils (Benarafa et al. 2011).

Our studies traced the premature death of serpinB1-deficient neutrophils to a cell-intrinsic death pathway that is mediated by cathepsin G, a known target of serpinB1 (Baumann et al. 2013). To further validate these data, we generated MyPh8-cell lines from cathepsin-G-deficient (CG-/-) and double-knock-out (CG.sB1-/-) mice. However, the rates of spontaneous death of the MyPh8-cells prior to induction increased in proportion to the duration of culturing, thereby compromising the interpretation of the data gleaned at different passaging times. When generated in parallel, the cell-death kinetics of the CG.sB1-double-deficient MyPh8-derived neutrophils and their WT-counterparts were similar.

Conclusions and Relevance for 3R

We have investigated an approach involving the differentiation of myeloid progenitors into cells bearing the characteristic features of mature neutrophils with the objective of helping to reduce the number of mice that are used for neutrophil studies. We have confirmed existing in-vitro and in-vivo data appertaining to bone-marrow-derived neutrophils, which reveal the regulation of neutrophil death to be mediated by cathepsin G and to be inhibited by serpinB1. However, as an alternative to primary neutrophils, MyPh8-cells that are derived from various genetically-modified mice are of limited potential, owing to the discovery that the rates of spontaneous cell death increase as function of culturing duration, which compromises the interpretation of data gleaned at different passaging times. This is a particularly inconvenient caveat when studying cell-death pathways.

In conclusion, the potential of the investigated approach to help in reducing the number of experimental mice used in neutrophil studies is not as great as originally hoped. The methodology will nevertheless be valuable in studies involving mice with severely-compromised late-embryonic or postnatal phenotypes. In such animals, the foetal liver could be used as a source of haematopoietic stem cells for the derivation of an MyPh8-line. It has been recently shown that by combining oestrogen-regulated Hoxb8 with the Flt3-ligand instead of SCF, the pluripotent progenitor status of the MyPh8-cells could be enhanced (Redecke et al. 2013).

References

1. Baumann, M., Pham, C.T.N. & Benarafa, C. (2013) SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G. Blood 121, 3900–3907.

2. Benarafa, C., LeCuyer, T.E., Baumann, M., Stolley, J.M., Cremona, T.P. & Remold-O'Donnell, E. (2011) SerpinB1 protects the mature neutrophil reserve in the bone marrow. Journal of leukocyte biology 90, 21–29.

3. Benarafa, C., Priebe, G.P. & Remold-O'Donnell, E. (2007) The neutrophil serine protease inhibitor serpinb1 preserves lung defense functions in Pseudomonas aeruginosa infection. The Journal of experimental medicine 204, 1901–1909.

4. Redecke, V., Wu, R., Zhou, J., Finkelstein, D., Chaturvedi, V., High, A.A. & Häcker, H. (2013) Hematopoietic progenitor cell lines with myeloid and lymphoid potential. Nature methods 10, 795–803.

5. Wang, G.G., Calvo, K.R., Pasillas, M.P., Sykes, D.B., Häcker, H. & Kamps, M.P. (2006) Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nature methods 3, 287–293.