Sublethal Dose Of Total Body Irradiation Research Articles

Pharmacological Inhibition of Luteinizing Hormone Signaling Promotes HSC Quiescence and Hematopoietic Regeneration

During hematopoiesis there is a balance between divergent functions such as self-renewal, differentiation, and proliferation. This process allows for continued blood formation throughout life as well as hematopoietic regeneration after periods of stress. It was recently shown that suppression of sex steroids, in addition to promoting T and B cell lymphopoiesis, promotes hematopoietic rejuvenation by mediating functional changes to HSC pool (Khong et al., 2015).Although those changes suggested a direct impact of testosterone on HSC function, when we examined the expression of hormone receptors on the hematopoietic compartment, we found almost no detectable expression of the androgen receptor. In contrast, expression of luteinizing hormone (LHR) was highly enriched on HSCs with nearly absent expression in downstream hematopoietic progenitor cells as well as in the stromal cell compartment. Consistent with the mouse data, high expression of LHR was also found in human HSCs (Fig. 1A). Given the peculiar expression of the LHR on HSCs, we sought to examine the direct effect of LH on HSC function. LH significantly enhanced mouse HSC colony formation in cobblestone area-forming cell and colony-forming cell assays. In addition, LH significantly expand human HSCs in a stroma-free culture system, without impacting on their colony-forming potential (Fig. 1B).Given the direct effects of LH on HSCs, we investigated if blocking LH could attenuate HSC proliferation in two different models of hematopoietic stress that force HSCs out of their quiescent status. Pharmacological suppression of LH using a luteinizing hormone-releasing hormone-antagonist (LHRH-Ant) inhibits HSCs from entry into cell cycle after Poly I:C and sub-lethal dose of total body irradiation (SL-TBI) exposure. Importantly, LH inhibition also significantly improved the proportion of live HSCs after SL-TBI (Fig. 1C). To gain some insights into the direct impact of LH on HSCs, we generated a hematopoietic chimera using KiLHRD582G mice, which have a constitutively-active D582G mutation in the Lhr gene. While there was no difference in primary reconstitution in mice receiving WT or KiLHRD582G HSCs, we found that mice reconstituted with KiLHRD582G and subsequently exposed to SL-TBI showed significantly higher proportion of apoptotic HSC compared to mice reconstituted with WT HSCs (Fig. 1D). To elucidate the molecular changes induced by LH inhibition, we assessed the expression of genes associated with quiescence, proliferation, DNA damage and apoptosis in HSC after SL-TBI. We found that LH suppression promoted higher levels of Hes1, Cdkn1a (p21), Bcl2 and Mcl1, genes previously identified to enforce HSC quiescence and survival (Fig. 1E). Consistent with these data, mice treated with the LHRH-Ant showed improved survival when exposed to successive doses of 5-fluorouracil, a well-established method to challenge HSC quiescence and self-renewal potential. It has been previously showing that promoting quiescence early after hematopoietic insults can mitigate HSC exhaustion and, ultimately, promote hematopoietic recovery and mouse survival. Mice receiving LHRH-Ant 24h after lethal TBI (840cGy) exposure showed a significant increase in survival compared to control animals. In fact, LH suppression spared LT-HSCs from radiation toxicity thus promoting hematopoietic recovery (Fig. 1F). Consistent with a negative role of LH on HSC recovery after radiation, mice deficient for LHR had a statistically significant increase in survival when exposed to L-TBI compared to control littermate.In conclusion our studies demonstrate that LH can directly regulate HSC quiescence, and that LH inhibition with a LHRH-Ant can be used as a radiation mitigation strategy. [Display omitted] DisclosuresJenq:Seres: Research Funding. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. van den Brink:Seres: Research Funding; Jazz Pharmaceuticals: Consultancy; Therakos Institute: Other: Speaking engagement; PureTech Health: Consultancy.

Thymic Reconstitution after Damage Is Influenced By Zinc Status

T cell reconstitution, which is dependent on thymus function, is significantly delayed after hematopoietic stem cell transplant; leading to morbidity and mortality due to infection and possibly even malignant relapse. Zinc, the second most abundant trace metal in the body, is crucial for thymic function: in fact, zinc deficiency of all causes lead to thymic atrophy with a consequent reduction in the number of circulating recent thymic emigrants (RTEs). However, the role of zinc in thymic reconstitution after chemotherapy and hematopoietic stem cell transplantation (HSCT) has not been investigated. Using a murine model of thymic function after acute damage, we found that mice showed reduced thymic cellularity after three weeks of a zinc deficient diet, and lower regenerative capacity after a sublethal dose of total body irradiation (SL-TBI) (Fig. a). Conversely, zinc supplementation in normal dietary condition caused increased thymic regeneration at time-points after a mouse model of allogeneic-HSCT (Fig. b). As a result, mice that received zinc supplementation demonstrated more naive T cells and more recent thymic emigrants in peripheral blood (Fig. c) after allo-HSCT. Mechanistically, we found that zinc supplementation could stimulates the proliferation of thymic epithelial cells (TECs), which are the main cell type responsible for supporting for T-cell development, likely via induction in the production of BMP4, a pro-regenerative cytokine produced by endothelial cells (EC) (Fig. d). In fact, treatment with the BMP-receptor antagonist dorsomorphin abrogated the effect of zinc supplementation (Fig. e), and ex vivo propagated ECs (exECs) were directly induced to produce BMP4 when stimulated in vitro for 24 hours with zinc sulfate (Fig. f). Following damage there is a switch in the availability of zinc from inside the cellular fraction to the extracellular fraction (Fig g), suggesting that zinc is normally used and stored in developing T-cells but after damage they release zinc in the extracellular space after cell death due to TBI. This release is enough to trigger the production of regenerating factors from radio resistant cells, such as EC. Consistent with this, we found higher levels of intracellular zinc in thymocytes from zinc-supplemented mice before TBI and higher levels of zinc in the extracellular fraction after damage (Fig h). Moreover, while there was no difference in BMP4 expression in cocultures of exEC in presence of SN from control and zinc-treated mice at day 0, BMP4 levels increased in the presence of SN harvested from mice that had previously received TBI (Fig i). In conclusion, we herein demonstrate that zinc is important for thymic regeneration, and zinc supplementation offers an innovative therapeutic strategy to improve T cell reconstitution in patients receiving allo-HSCT.

Latexin Inhibition Mitigates Irradiation Induced Hematopoietic Injury

Radiation-associated bone marrow (BM) injury is one of the most serious limiting factors of radiotherapy. Radiation-induced hematopoietic injury, no matter how transient or long lasting, can ultimately impair HSC function and decrease the HSC reserve, leading to increased risk for the development of BM failure or cancer. However, molecular mechanisms underlying radiation-induced HSC functional decline are largely unknown.We previously identified a stem cell regulatory gene, latexin (Lxn), as a novel negative regulator of HSCs in mice. HSCs in Lxn knockout mice (Lxn-/-) had increased self-renewal and survival. In our new findings, we surprisingly found that Lxn-/- mice had the significant survival advantages under lethal dose of total body irradiation (TBI). We further found that HSCs and hematopoietic progenitor cells (HPCs), measured by immunophenotypes and colony assay, recovered much faster in Lxn-/- mice than wild-type mice (WT) within one month after sub-lethal dose of TBI. The better preserved HSC/HPC pool was due to the decreased apoptosis in which the percentage of Annexin V + PI- apoptotic HSCs/HPCs cells was significantly lower in Lxn-/- mice than WT mice. These data suggest that Lxn inactivation protects HSCs and HPCs from radiation-induced cell death, thus mitigating acute hematopoietic suppression and conferring a survival advantage.To determine the long-term effect of TBI on Lxn-/- HSCs, we performed limiting dilution competitive repopulation unit assay (CRU), and found that Lxn-/- CRU was significantly higher than WT CRU. Moreover, we performed serial transplantation experiment, and found that Lxn-/- HSC continuously regenerated blood and bone marrow cells even at the 4th round of transplantation whereas WT HSCs were exhausted. These data provide robust evidence that Lxn inactivation protects functional long-term HSCs from radiation-induced injury. Radiation can increase the risk of hematological malignancy later in the life. We thus maintained a group of mice that were subject to either a single dose of 6.5Gy TBI or split low doses of TBI (2 Gy daily for 6 days), and monitored their gross condition and blood cell counts for 20 months. At 20 month post-radiation, we performed bone marrow analysis and histopathology analysis. We found that Lxn-/- mice did not spontaneously develop hematopoietic malignancies, their bone marrow HSCs/HPCs had normal population size, and bone marrow had normal histopathology. These data suggest that Lxn inactivation mitigates radiation-induced short-term myelosuppression and long-term HSC functional impairment without induction of hematologic malignancy.At the molecular level, we previously reported that Lxn sensitized leukemogenic cells to gamma-irradiation-induced cell-cycle arrest and cell death through Rps3 pathway, and Rps3 was a binding protein of Lxn. Rps3 has been shown to be involved in the NFkB pathway. We found that Rps3 bound Lxn in primary hematopoietic stem and progenitor cells (HSPCs) using Co-IP assay. Lxn-/- HSPCs had the increased expression of Rps3 and NFkB p65 before or post-irradiation. Knockdown of Rps3 in Lxn-/- HSPCs decreased NFkB p65 and increased radiation-induced apoptosis. Moreover, when Lxn-/- HSPCs were treated with NFkB p65 specific inhibitor, the similar phenotypes were also shown, suggesting that Lxn functions through Rps3-NFkB-mediated pro-survival pathway in primary HSPCs. We are currently proving this molecular pathway using the in vivo model by crossing p65 knockout mice with Lxn-/- mice.In conclusion, latexin inhibition mitigates irradiation induced hematopoietic injury via Rps3-NFkB-mediated pro-survival pathway. DisclosuresNo relevant conflicts of interest to declare.

Tissue Trace Element Change after Total Body Irradiation

Purpose: In this study, we examined the changes of tissue contents of trace elements and iron after total body irradiation (TBI) and their possible impact on late toxicities. Material and Methods: 20 female Wistar rats were randomly assigned to two groups – either radiation (n = 10) or control (n = 10). Rats in the radiation group received TBI of 5 Gy in a single fraction. Rats were sacrificed and tissue samples of heart, lung and kidney were taken 8 weeks after radiation. Tissue levels of zinc, copper, magnesium, manganese and iron analysis were performed with an atomic absorption spectrophotometer and suprapure grade standard solutions. One kidney of each animal was taken for electron microscopic analysis. Blood samples were collected from all animals and the blood chemistry related to kidney function was studied. Results: The kidney levels of Fe and Cu significantly increased 8 weeks after irradiation (p < 0.05). The Cu/Zn ratio did not reach statistical significance in any tissue, however in kidney, there was a tendency to rise (p = 0.08). Myocardium and lung content of trace elements and iron did not show any significant change 8 weeks after irradiation. Electron microscopic analysis showed significant injury in glomerular endothelial cells, renal tubules and thickening of basement membrane. Blood chemistry showed a significant rise in serum creatinine (p = 0.008) and calcium (p = 0.01) in the TBI group. Serum creatinine levels were 0.73 and 0.84 mg/dl, and serum calcium levels were 10.1 and 11.3 mg/dl in control and TBI groups, respectively. Conclusion: A sublethal dose of TBI causes deposition of Cu and Fe within the kidney after TBI. Deposition of these elements may have some additional role on the toxicity caused by direct radiation on the kidney.

Inhibition of Hematopoietic Recovery from Radiation-Induced Myelosuppression by Natural Killer Cells

We have examined the role of natural killer (NK) cells in situ in the recovery of marrow hematopoiesis in B6D2F1 mice receiving various doses of total-body irradiation (TBI) as a well-characterized model for treatment-induced myelosuppression. Applying an in situ cytotoxic approach for ablating NK 1.1 cells, we have demonstrated that NK 1.1 cells differentially inhibit the recovery of hematopoietic stem cells (CFU-S) and their progenitor cells committed to granulocyte-macrophage differentiation from a sublethal dose of TBI (9 Gy) while not affecting the recovery of progenitor cells committed to either erythroid or megakaryocyte differentiation from TBI. However, recoveries of CFU-S and progenitor cells were unaffected by the ablation of NK cells prior to a moderate dose of TBI (2 Gy). These findings provide in situ evidence that NK cells are potential inhibitors of hematopoietic recovery from treatment-induced myelosuppression.