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Exposure to 20 mGy radiation decreases HSC functions
Discussion
Here we show that exposure to a single 20 mGy LDIR alters the functional properties of human HSPC. No defect in HSPC differentiation potential tested in primary cul- tures was detected after exposure to LDIR. However, HSPC irradiated at 20 mGy in vivo in the NSG mouse BM harbored a defect in human hematopoietic reconstitution potential. This defect was cell-intrinsic since 20 mGy-irra- diated CD34+CD38low cells isolated after in vivo irradiation failed to serially reconstitute NSG mice as efficiently as non-irradiated cells; the same was observed with in vivo 20 mGy-irradiated bulk BM cells. This in vivo phenotype was also observed in vitro when using LDIR-exposed CD34+CD38-/lowCD45RA–CD90+ HSPC in serial CFU-C and LTC-IC assays, supporting the fact that these effects are cell-autonomous and not limited to transplantation conditions. Likewise, in vitro, HSPC exposure to 20 mGy induced a loss of secondary CFU-C potential as well as a decrease in secondary LTC-IC frequency. Altogether, based on the use of in vivo assays and in vitro surrogate assays to evaluate the self-renewal potential,25-27 these functional results strongly argue for an effect of 20 mGy LDIR on the long-term HSC functional properties, most likely through a loss of self-renewal potential. Of note, 20 mGy LDIR has been shown to decrease self-renewal capacity in murine HSC as well.22
High-dose ionizing radiations (HDIR) (2.5 Gy) are known to induce DNA DSB in human HSPC, rejoining is delayed, and H2AX foci persist leading to a loss of HSC functions partly related to apoptosis and activation of p53 pathway.23 Despite the publication of several studies over the past few years,36-38 little is known about which path- way is used to repair DNA DSB in human HSPC.16 In the present work, we tested if a single 20 mGy LDIR can alter cell cycle and induce apoptosis, and cause DNA DSB in HSPC. Surprisingly, and in contrast to HDIR, 20 mGy irra- diation did not induce obvious cell cycle defects nor pro- mote apoptosis in HSPC, since no increased cleaved caspase 3 protein was detected after exposure to 20 mGy LDIR. Moreover, no significant increase in H2AX and 53BP1 foci numbers was revealed, suggesting that 20 mGy LDIR does not produce DNA DSB. Finally, neither p53 nor ATM pathway was activated after 20 mGy exposure. However, and similarly to HDIR, 20 mGy irradiation led to 8-oxo-dG lesions in HSPC DNA. No such lesion was observed in sham-irradiated cells. Altogether, 20 mGy LDIR does not induce classic DNA damage and repair pathways usually activated by γ-irradiation but rather triggers 8-oxo-dG-dependent DNA damage that can be linked to uncontrolled increase in ROS levels. Moreover, NRF2 protein was found in the nucleus of 20 mGy-irradi- ated HSPC. Indeed, increased ROS levels were detected immediately after HSPC exposure to 20 mGy and, in line with this, we could observe that 20 mGy-irradiated HSPC had a delay in mitochondrial activation compared to con- trol cells. Our results and those from Romeo’s lab22 sug- gest that the transient increase in ROS levels is likely to be responsible for HSPC defects after LDIR exposure. We tested this hypothesis using antioxidant treatment of HSPC prior to exposure to LDIR. Importantly, pre-treat- ment of HSPC with NAC or Catalase prior to LDIR expo- sure did rescue the loss of in vitro serial clonogenic poten- tial of HSPC. In mouse, irradiation can induce p38MAPK activation through increased levels of ROS.19,35 Prevention
of p38MAPK activation leads to decreased IR toxicity in HSC.35 It is also known that dormant HSC have little or no p38MAPK activation, and that p38MAPK activation in HSC is associated with differentiation and loss of HSC self-renewal.17,39 In humans, the function of the p38MAPK pathway is still not fully understand but preventing p38MPAK activation allows HSC maintenance/expansion in vitro.40,41 Interestingly, in our model, we observed a ROS- dependent p38MAPK activation in human HSPC after exposure to both 2.5 Gy and 20 mGy IR. The involvement of the p38MAPK pathway in the LDIR-mediated HSC self-renewal defects was then confirmed in serial replating CFU-C assays. Indeed, pre-treatment of HSPC with a spe- cific inhibitor of p38MAPK prior to LDIR rescued their serial replating capacities. This is in agreement with the fact that HSC treatments either with NAC or p38MAPK inhibitor increase LTC-IC frequency and promote higher hematopoietic reconstitution upon serial transplanta- tion.18,19 It is important to highlight that two other studies on the effect of LDIR have also shown that LDIR did not induce classic DNA damage and repair pathways, but rather an oxidative stress (increase in ROS level and NRF2 nuclear localization).22,42 Therefore, oxidative stress induc- tion seems to be a feature of exposure to LDIR, leading either to a differentiation defect in the case of cycling stem cells42 or a self-renewal defect in the case of quiescent stem cells, as we observed for human HSPC; this is also the case for mouse HSC.22
Increased ROS levels as well as p38MAPK activation in HSC are associated with aging and stress during serial trans- plantation.17,18,43 The aging phenomenon is clearly a strong driver of differentiation and expansion of myeloid-biased HSPC.44 Here we were not able to detect any bias toward myelopoiesis when analyzing the progeny of the surviving LDIR-treated human HSC after serial transplantation, maybe due to the NSG mouse model, as the NSG BM microenvironment is more supportive of B-cell rather than myeloid-cell differentiation.45 Moreover, all experiments were performed with HSPC from CB, i.e. young HSPC. Thus, although it is tempting to speculate that exposure to LDIR may induce early/accelerated aging of the human HSC, we have no formal proof of that. Since radiation sen- sitivity and transplantation efficiency are highly dependent on the ontogenic origin of HSPC,46-48 aged HSPC may be more sensitive to LDIR. Another feature of HSC aging is higher risk of leukemic transformation, especially in the presence of an oncogenic-initiating event such as a muta- tion of the epigenetic modifiers DNMT3a or TET2, as observed in blood from elderly people.49 A very interesting and important question for the future would be to deter- mine if aged HSC exposed to LDIR are more prone to (pre)leukemic transformation, especially when HSC con- tain primary oncogenic mutations.
To sum up, in contrast to HDIR, 20 mGy does not induce DNA DSB, nor apoptosis and a defect in the cell cycle. However, both 20 mGy and 2.5 Gy IR induce 8-oxo-dG lesions in DNA, increase ROS levels, and acti- vate the p38MAPK pathway leading to HSC self-renewal defects. Nevertheless, only 20 mGy-LDIR effects were counteracted by use of antioxidants prior to irradiation exposure, indicating there are major differences between these two IR doses. These results show for the first time that a dose as low as 20 mGy can have a huge impact on human HSC through both similar and also different molecular mechanisms to those of high IR doses.
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