Abstract
Abstract 4142
Background:
Injury to healthy (non-target) tissues still is a major limitation of radiation therapy, at least in part related to release of cytokines, including TNFα and IL-1β, which amplify tissue damage. Cytokine release is particularly prominent in patients who receive total body irradiation (TBI) before hematopoietic cell transplantation (HCT), as interactions of allogeneic donor cells with patient tissue contribute to the resulting “cytokine storm” and the development of graft versus-host-disease (GVHD). In murine models, administration of alpha-1 anti-trypsin (AAT) in the peri-transplant period decreased GVHD incidence and mortality. AAT, a member of the serine protease inhibitor (serpin) family, is a major protective protein in the circulation and has been used successfully in the clinic for other indications.
Methods:
We were interested in determining the potential benefits of AAT in preventing toxicity related to the transplant conditioning regimen, specifically TBI. AAT inhibits proteinase-3 (PR3) which, among other targets, cleaves IL-32 thereby leading to activation of TNFa and enhancing the cytokine storm. Since others have also suggested that AAT, via enhanced expression of heme oxygenase-1 (HMOX-1), leads to activation of Nrf2, thereby enhancing transcription of anti-inflammatory cytokines such as IL10 and the IL-1 receptor antagonist (IL1Ra), we determined the overall shift in the cytokine milieu and cell death. Thus, we irradiated male C3H/HeN mice (n = 5 mice per group; 6–8 weeks old) with sub-lethal doses (500, 600 and 700 cGy) of TBI from a 137Cs source. AAT (300 μg/animal) was administered intra-peritoneally 1 hour before and every 48 hours after TBI for a total of 6 doses. The effect of TBI/AAT on hematopoiesis was analyzed by growing granulocyte-macrophage colony forming units (GM-CFU) from bone marrow cells, on days 3, 7 and 14 after TBI.
Results:
Results were compared to those with cells from albumin-treated controls. Marrows (days 3 and 7) from AAT-treated mice generated higher GM-CFU counts than those from controls (mean = 25 vs. 5 colonies per 25 × 103 cells plated after 500cGy, and 15 vs. 3 colonies per 25 × 103 cells after 600 cGy). Peripheral blood and unsorted bone marrow from AAT-treated mice showed up-regulation of HMOX-1 and Nrf2 (30 and 50 log2 increase, respectively), and enhanced transcription of IL-10 and IL-1Ra (50 and 10 log2, respectively) compared to albumin treated donors. PR3 and TNFα, in contrast, were down-regulated (5 log2 and 7 log2 decrease in comparison to albumin treated controls).
The cytokine mitigating effects of AAT were accompanied by attenuation of ATM-p53-dependent DNA damage responses in bone marrow cells (determined on days 3, 7, and 14), showing a 3-fold decrease in p53 protein levels, and a 6-fold decrease in phospho-ATM in comparison to albumin treated controls (peak day 7). In addition, protein levels of caspase 3 and caspase 9 were decreased (2-fold and 5-fold, respectively; peak on day 7) in unsorted marrow cells and spleen lysates of AAT treated mice in comparison to albumin treated controls. Bone marrow histopathology revealed normo-cellularity and a decrease in cleaved caspase 3 staining in AAT-treated mice compared to albumin treated animals.
Summary and conclusions:
AAT treatment significantly mitigated the hematopoietic toxicity induced by sub-lethal TBI. The mechanism involves cytokine suppression, associated with attenuation of ATM-p53 mediated DNA damage response. Taken together, these data suggest that AAT treatment pre-TBI provides protection against radiation injury.
Disclosures:
No relevant conflicts of interest to declare.
Transplantation of Unirradiated Bone Marrow Cells after Total-Body Irradiation Prevents the Development of Thymic Lymphoma in Mice through Niche Competition
