In this study we tested whether the beneficial effects of in vivo PGE2 treatment, which induced bone microarchitectural changes and caused specific expansion of the ST-HSC and MPP populations without loss of LT-HSCs11
, are recapitulated in the setting of myeloablative injury in which the entire hematopoietic system, including HSPCs, are subjected to apoptotic stress. Our central hypothesis was that in vivo treatment, by targeting both the HSPCs and their niche, would afford additional benefits to the actions of PGE2 demonstrated on HSPCs ex vivo, which have been elucidated by studies in both murine models10
and non-human primates12
We first determined that PGE2 treatment in naïve mice inhibits apoptosis in HSPCs. This finding not only provides a mechanism for the observed PGE2-dependent HSPC expansion in vivo, since HSPC differentiation and proliferation rates were not altered, but also strongly anticipated a beneficial effect of in vivo PGE2 in instances of increased apoptotic stress. Apoptosis has been previously identified as a significant cellular fate of HSPCs, which can alter their pool size when manipulated38
. While apoptosis was decreased in both LT-HSCs and ST-HSCs/MPPs of PGE2-treated mice, we suspect that, in the absence of injury, PGE2 treatment decreases apoptosis only in cycling LT-HSCs, rather than in dormant LT-HSCs39
, both of which are contained in the populations we define as LT-HSCs in our studies.
To increase HSPC apoptosis in vivo, mice were exposed to radiation injury. In spite of their quiescence, HSPCs are susceptible to even low doses of acute radiation injury, which reduces the number of engraftable HSCs as demonstrated in murine models as well as in non-human primates and in humans40–42
. Prostaglandins have been implicated in protection of intestinal clonogenic cells when given prior to lethal radiation doses and when given in combination to bone marrow transplantation43,44,45
. However, to our knowledge, increased HSPC survival with in vivo stimulation of prostaglandin signaling following sub-lethal radiation injury has not yet been defined.
In these studies, we detected a general reversal in the overall expression pattern of apoptosis-related genes in response to TBI with dmPGE2 treatment, indicating activation of a robust pro-survival program in HSPCs. Notably, in the setting of sublethal TBI, dmPGE2 did not selectively protect a population of HSPCs with limited self-renewal, since superior engraftment of BMMCs from dmPGE2-treated irradiated mice persisted for at least 22 weeks post-transplantation. This difference between the injured and non-injured in vivo models may be due to the use of the more sustained PGE2 analogue, dmPGE2.
In addition to decreased apoptotic rates, the increase in repopulating activity of HSPCs from dmPGE2-treated mice post-TBI could also be explained by further qualitative effects of dmPGE2 on HSPCs, such as changes in homing or retention in the niche, especially since some reports have suggested that ex vivo exposure to dmPGE2 prior to transplantation may increase homing of HSCs10, 20
. Further studies are needed to determine if dmPGE2 in our model improves HSPC homing.
Several early reports demonstrated that PGE2 treatment can specifically inhibit myelopoiesis. However, there was no decrease in myeloid or erythroid bone marrow progenitors with in vivo dmPGE2 treatment at any time point analyzed. In fact, our data identified an increase in hematopoietic progenitors, particularly at 14 days post-TBI, 11 days following the final dmPGE dose. The lack of PGE2-depended progenitor inhibition may be specifically due to the injury setting. Another explanation may be that the injected dmPGE2 would be expected to have dissipated by the time in which the progenitors are expanding.
The accelerated hematopoietic recovery afforded by in vivo dmPGE2 treatment suggests that PGE2-dependent inhibition of apoptosis in the context of injury preserves HSPCs that are not severely damaged and are thus able to properly differentiate. Moreover, the short term benefits of in vivo dmPGE2 treatment are not at the expense of long term-repopulating cells, as demonstrated by superior long-term engraftment of BMMCs from dmPGE2-treated mice.
Since we observed anti-apoptotic effects of PGE2 on HSPCs in vitro, a component of the beneficial action of PGE2 in vivo is likely to be directly on the HSPCs. However, the unexpected acceleration of hematopoietic recovery with its delayed effects strongly suggests a contribution of PGE2-induced microenvironmental changes as well. Thus, we next focused on TBI-induced microenvironmental changes in PGE2 regulation and how they are modulated by in vivo dmPGE2 treatment.
Following TBI, bone marrow PGE2 is rapidly increased, likely by up-regulation of Cox-2 in CD45−
bone marrow microenvironmental cells, and remains elevated for at least 6 days after injury. This observation provides a scenario in which increased PGE2 could be an endogenous physiologic signal protecting injured HSPCs. This hypothesis is in line with data implicating PGE2 in a central evolutionarily conserved mechanism for tissue repair after injury29
. Moreover, these results are consistent with the previous analysis of Cox2−/−
mice, which based on our data would be expected to have defects in marrow PGE2 production in response to injury. Mice with global lack of Cox-2 have in fact decreased rates of hematopoietic recovery after treatment with the chemotherapeutic agent 5-Fluorouracil46
. In this context, the observed increase in endogenous PGE2 post-TBI in our model, even in the setting of low dose radiation, would caution against the inhibition of cyclo-oxygenases via anti-inflammatory therapies during marrow recovery.
The induction of microenvironmental Cox-2 following sub-lethal TBI was modulated by dmPGE2, particularly in CD45+ cells. This result demonstrates that 1) PGE2 produced in response to marrow injury can act via the microenvironment to amplify and prolong its own effects and 2) that the microenvironmental response to injury can be further augmented by treatment with PGE2 agonists with the goal of accelerating hematopoietic recovery. Macrophages in the bone marrow are known to be relatively radioresistant, and to up-regulate Cox-2 expression in response to PGE2 signaling35
. Thus, we examined the prevalence of macrophages in the bone marrow before and following radiation injury by immunohistochemistry. The increase in F4/80+ cells after TBI suggests that macrophages could be a microenvironmental population mediating the delayed effects of dmPGE2 after injury. Further, the α-SMA-expressing population of macrophages was specifically increased in the bone marrow of dmPGE2-treated mice post-TBI. This cell population was recently shown to support HSCs following sub-lethal radiation injury via up-regulation of Cox-237
. Taken together, the microenvironmental effects of dmPGE2 treatment following radiation injury may be mediated by a specific subpopulation of macrophages in the bone marrow. This hypothesis, if confirmed, may add to the role of marrow macrophages, which have lately been implicated as a regulatory component of the HSPC niche47–49
. This dmPGE2-dependent activation of CD45+ cells could explain some of the delayed beneficial effects of dmPGE2 on hematopoietic recovery. Additional studies are required to further define the contribution of specific subsets of marrow macrophages to the dmPGE2 effect on marrow recovery, and to determine if other populations within the bone marrow are participating as well.
Based on the current results, our working model summarizing the observed effects of in vivo dmPGE2 after TBI includes both direct and indirect cellular mechanisms (Supplemental Fig. 3A
). Expression of EP2 and EP4 receptors during injury supports direct actions of PGE2 on HSPCs, and therefore all the mechanisms implicated in the HSPC response to PGE2 ex vivo are likely at play (Supplemental Fig. 3A
). dmPGE2 administration in vivo also modulates the bone marrow microenvironmental response to injury likely through specific subpopulations of macrophages persisting in the bone marrow (Supplemental Fig. 3B,C
), accelerating recovery of hematopoietic progenitors and blood counts. Therefore, in vivo manipulation of HSPC function may provide an exciting potential treatment strategy to remedy myelosuppression.
This PGE2-dependent improvement of hematopoietic recovery has significant therapeutic implications. While bone marrow injury from radiation exposure or chemotherapeutic treatment, which can cause significant anemia, thrombocytopenia and leukopenia, can be treated with G-CSF and GM-CSF, megakaryopoiesis is minimally stimulated by these treatments50
, thus thrombocytopenia continues to be a serious clinical issue. In this context, our studies raise the prospect that the use of PGE2 agonists may represent a novel approach to meaningfully accelerate recovery of peripheral blood counts in patients following myelosuppressive treatments or injuries during a vulnerable time when few therapeutic options are currently available.