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E. Henry et al.
Low doses of ionizing radiations increase reactive oxygen species levels, 8-oxo-dG lesions, induce NRF2 translocation into the nucleus, activate p38MAPK pathway and delay mitochondrial activation
Since irradiation is known to promote ROS produc- tion,10,18,30 we next quantified ROS levels after LDIR expo- sure. ROS levels in CD34+CD38lowCD45RA– CD90+ HSPC after exposure to LDIR were measured immediately or 3 h after irradiation of CD34+ cells. Menadione and NAC treatments were used to respectively induce and inhibit ROS production. Increased ROS levels in HSPC were observed immediately after exposure to 20 mGy LDIR and to a lesser extent after exposure to 2.5 Gy, as com- pared to no irradiation (Figure 5A and Online Supplementary Figure S4A and B). These ROS increased levels were transient as no further difference in ROS lev- els could be detected 3 h after irradiation (Online Supplementary Figure S4C). NAC pretreatment of HSPC significantly decreased this early burst of ROS after 20 mGy and 2.5 Gy exposure. As increased ROS levels can lead to 8-oxo-dG lesions, as well as NRF2 translocation into the nucleus, we looked for 8-oxo-dG lesions in DNA of irradiated versus sham-irradiated HSPC30,31 and NRF2 location into HSPC.22,24 As expected sham-irradiated and H2O2-treated (control) cells exhibited respectively no and highly detectable anti-8-oxo-dG nuclear labeling. After exposure to 20 mGy, 8-oxo-dG staining was detected in the HSPC nucleus showing that 20 mGy LDIR can induce 8-oxo-dG lesions in DNA (Figure 5B and C). Similarly, the NRF2 protein was found in the nucleus of 20 mGy- and 2.5 Gy-irradiated HSPC compared to sham- irradiated cells (Figure 5D). As an increase in ROS is also associated with a delay in mitochondrial activation,32 we used mitotracker green (MTG) and TMRE probes to study respectively mitochondrial mass and membrane potential. Of note, CB CD34+ cells and CB HSPC are mainly quiescent, therefore there is very little mitochon- drial activation (TMREneg) (Figure 5E, first left panel, and data not shown). HSPC exposure to LDIR did not alter the mitochondrial mass (MTG) of CD34+ cells in short-term culture (Online Supplementary Figure S5A). However, a delay in mitochondrial activation occurred (MTG+ TMRE+ HSPC) as soon as 3 h post IR (Figure 5E and Online Supplementary Figure S5B), suggesting that LDIR affect mitochondrial activity. In line with mitochondria activation, autophagy activation was monitored after IR (Online Supplementary Figure S6). The CytoID probe was used to follow autophagy in HSPC.33,34 As expected, after treatment with chloroquine and rapamycine, autophagy was detected in CD34+ cells (Online Supplementary Figure S6A). Besides, LDIR did not induce autophagy in HSPC after a different culture time (Online Supplementary Figure S6B and C). Finally, we investigated whether the observed increase of ROS can lead to p38MAPK activa- tion as previously documented.19 Thr180/Tyr18 phos- phorylation was used as a marker of p38MAPK activa- tion. As a positive control of p38MAPK activation, increased p38MAPK phosphorylation (P-p38MAPK) can be detected in PMA-treated HSPC (Online Supplementary Figure S5C). In irradiated HSPC, we observed an increase of P-p38MAPK after exposure to 20 mGy and 2.5 Gy IR compared to sham-irradiated controls, suggesting that LDIR can activate p38MAPK pathway in HSPC similarly to high irradiation doses (2.5 Gy)35 (Figure 5F). To further confirm that p38MAPK acti-
vation was due to the early transient increase in ROS lev- els, HSPC were treated with NAC or SB203580, a p38MAPK inhibitor, prior to 20 mGy irradiation. As expected, SB203580 prevented increased p38MAPK phosphorylation in 20 mGy-irradiated HSPC (Figure 5G). NAC treatment resulted in the same decrease in p38MAPK phosphorylation in 20 mGy-irradiated HSPC (Figure 5G). Altogether, these results show that LDIR increase ROS levels leading to DNA 8-oxo-dG lesions, NRF2 translocation into the nucleus and p38MAPK acti- vation in 20 mGy-irradiated HSPC.
20 mGy-dependent reactive oxygen species increase and p38MAPK activation lead to defects in the serial clonogenic potential of hematopoietic stem/progeni- tor cells
As increased ROS levels can lead to HSC loss of poten- tials,18 we then asked if ROS-dependent pathways could explain the HSPC functional defects after LDIR exposure. To this end, serial CFU-C assays were performed using sorted CD34+CD38lowCD45RA–CD90+ HSPC pre-treated or not with NAC before exposure to LDIR and cultures. 20 mGy-irradiated HSPC generated the same number of primary CFU-C compared to sham-irradiated HSPC with or without NAC treatment (Figure 6A and Online Supplementary Figure S7A). However, 20 mGy-irradiated HSPC treated with NAC before IR, but not 2.5 Gy-irra- diated cells, were capable of generating equivalent num- bers of secondary CFU-C compared to sham-irradiated HSPC, showing that NAC treatment prior to exposure to 20 mGy protected HSPC from the loss of in vitro serial clonogenic potential (Figure 6B and Online Supplementary Figure S7B). This result was obtained when the serial plating assays were performed with the whole cell pop- ulation harvested from primary CFU cultures (Figure 6B), and also after picking up and replating individual pri- mary CFU-GM colonies (Online Supplementary Figure S7C). Rescue of secondary replating properties of HSPC after 20 mGy LDIR was also obtained using HSPC pre- treatment with Catalase, another antioxidant enzyme (Online Supplementary Figure S7D). These results show that preventing ROS production with antioxidants before LDIR exposure rescues the in vitro serial clono- genic potentials of HSPC.
Finally, we wondered whether ROS-mediated p38MAPK activation was involved in LDIR-induced HSC self-renewal defects. HSPC were pre-treated with SB203580, a specific inhibitor of p38MAPK, prior to 20 mGy irradiation and serial CFU-C assays. No difference in the number of primary CFU-C was detected with SB203580 pretreated HSPC regardless of the irradiation dose used (Figure 6C and Online Supplementary Figure S7E). However, whereas SB203580-untreated 20 mGy- irradiated HSPC generated very few secondary CFU-C, SB203580 treatment of HSPC protected their capacity to generate secondary CFU-C as efficiently as sham-irradi- ated HSPC (Figure 6D and Online Supplementary Figure S7F), suggesting that p38MAPK pathway activation par- ticipates in LDIR-mediated HSPC defects. Based on all these results, we propose a model in which 20 mGy LDIR rapidly increases ROS amounts in HSPC that induce p38MAPK activation altogether leading to a defect in the long-term maintenance of the clonogenic potential of CD34+CD38lowCD45RA–CD90+ HSPC (Figure 6E).
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haematologica | 2020; 105(8)