Proteinase 3 Is Expressed in Stem Cells and Regulates Bone Marrow Hematopoiesis

Previously published data from our lab identified proteinase 3 (PR3) as a key protein in regulating spontaneous death of neutrophils. Our lab showed that PR3 could cleave caspase 3 in a caspase 9-independent manner in ageing neutrophils (Loison et al . JCI. 2014). Meanwhile, the maintenance of stem cell pool in the bone marrow and the development of mature blood cells are tightly regulated processes. In an effort to understand the hematopoietic consequences of absence of PR3, we analyzed stem and progenitor cell compartments in wild-type (WT) and PR3 knock-out (PR3-/-) mice.

Of note, PR3 is thought to be a neutrophil-specific serine protease. Surprisingly, we observed PR3 expression in sorted Lin- Sca-1+ c-Kit+ (LSK) cells by gene expression analysis at mRNA level as well as Western blotting at protein level. Conventional flow cytometry methods also confirmed this expression.

While bone marrow cellularity was similar between WT and PR3-/- mice, conventional flow cytometry studies showed that bone marrow from PR3-/- mice contained a higher frequency of LSK and Lin- Sca-1- c-Kit+ (LK) cells. Colony forming cell assays also suggested that both bone marrow cells and splenocytes from PR3-/- mice contained a higher number of progenitor cells compared to their WT counterparts. Histological analysis of bone marrow smears revealed that bone marrow from PR3-/- mice had a higher number of immature myeloid cells and a reduced number of mature leukoyctes and lymphocytes.

Next, we asked if the enhanced stem and progenitor cell compartments in PR3-/- mice were functional in vivo. Toward this aim, we performed competitive reconstitution experiments using total bone marrow cells as well as sublethal irradiation experiments. PR3-/- donor-derived cells gave rise to a higher number of cells in the recipient mice compared to WT donor-derived cells in competitive reconstitution experiments. Also, after WT and PR3-/- mice were irradiated with 4 Gy, peripheral blood cell counts recovered faster in PR3-/- mice. Similarly, when mice were irradiated with 6 Gy, PR3-/- mice had a higher survival rate.  These data suggest that enhanced stem and progenitor cell compartments in PR3-/- mice are functional. To understand if the enhanced hematopoietic activity in PR3-/- mice is an intrinsic feature of stem cells, another set of competitive reconstitution experiments were performed using equal numbers of sorted LSK cells. Again, PR3-/- donor-derived cells were more dominant than WT donor-derived cells in the recipient mice.

Then, we wanted to understand the underlying mechanism. The rate of proliferation and senescence in LSK cells were analyzed by BrdU incorporation studies and C12FDG staining, respectively. No difference in proliferation or senescence was observed between WT and PR3-/- mice. We also looked at the apoptosis rate of LSK cells in vivo and in vitro. Untreated LSK cells from WT and PR3-/- mice exhibited similar levels of viability both in vivo and in vitro. However, when bone marrow macrophages responsible for clearance of apoptotic cells were depleted using clodronate liposomes, PR3-/- LSK cells exhibit enhanced viability compared to WT LSK cells as evidenced by annexin V and 7-AAD staining. Using a fluorogenic substrate of caspase 3, we also observed that the enhanced viability seen in PR3-/- LSK cells after depletion of macrophages is due to a reduction in caspase 3 activation.

Additionally, we asked whether LSK cells from PR3-/- mice represent features associated with stem cells from aged mice such as an increase in DNA damage accumulation and reduced polarity. Immunofluorescent microscopy studies suggested that long-term hematopoietic stem cells from PR3-/- mice exhibit higher levels of p-gH2AX foci, a marker of DNA damage, and a reduced polarized distribution of a-tubulin. Finally, we saw a reduced lifespan in PR3-/- mice. These data indicate that PR3 regulates bone marrow hematopoiesis by contributing to the maintenance of a healthy stem cell pool in the bone marrow.