C/EBPδ Deficiency Sensitizes Mice to Ionizing Radiation-Induced Hematopoietic and Intestinal Injury

<div><p>Knowledge of the mechanisms involved in the radiation response is critical for developing interventions to mitigate radiation-induced injury to normal tissues. Exposure to radiation leads to increased oxidative stress, DNA-damage, genomic instability and inflammation. The transcription factor CCAAT/enhancer binding protein delta (<i>Cebpd</i>; C/EBPδ is implicated in regulation of these same processes, but its role in radiation response is not known. We investigated the role of C/EBPδ in radiation-induced hematopoietic and intestinal injury using a <i>Cebpd</i> knockout mouse model. <i>Cebpd</i>−/− mice showed increased lethality at 7.4 and 8.5 Gy total-body irradiation (TBI), compared to <i>Cebpd</i>+/+ mice. Two weeks after a 6 Gy dose of TBI, <i>Cebpd</i>−/− mice showed decreased recovery of white blood cells, neutrophils, platelets, myeloid cells and bone marrow mononuclear cells, decreased colony-forming ability of bone marrow progenitor cells, and increased apoptosis of hematopoietic progenitor and stem cells compared to <i>Cebpd+/+</i> controls. <i>Cebpd</i>−/− mice exhibited a significant dose-dependent decrease in intestinal crypt survival and in plasma citrulline levels compared to <i>Cebpd+/+</i> mice after exposure to radiation. This was accompanied by significantly decreased expression of γ-H2AX in <i>Cebpd</i>−/− intestinal crypts and villi at 1 h post-TBI, increased mitotic index at 24 h post-TBI, and increase in apoptosis in intestinal crypts and stromal cells of <i>Cebpd</i>−/− compared to <i>Cebpd+/+</i> mice at 4 h post-irradiation. This study uncovers a novel biological function for C/EBPδ in promoting the response to radiation-induced DNA-damage and in protecting hematopoietic and intestinal tissues from radiation-induced injury.</p></div

<i>Cebpd−/−</i> mice showed increased radiosensitivity to TBI.

<p>Thirty-day survival of <i>Cebpd−/−</i> mice and <i>Cebpd+/+</i> control mice exposed to 7.4 Gy (n = 7 per genotype) or 8.5 Gy (n = 12 mice per genotype) of TBI. <i>P = 0.02</i> for 7.4 Gy; <i>P</i><0.0001 for 8.5 Gy, as calculated by Logrank (Mantel-Cox) test. The numbers in parentheses indicate the number of animals that survived.</p

<i>Cebpd−/−</i> mice had increased apoptosis and mitotic index and decreased levels of γ-H2AX in intestinal crypts, post-TBI.

<p>(A) Representative images of radiation-induced DNA fragmentation (TUNEL, green staining), DNA-damage marker γ -H2AX (red staining), and cellular nuclei (DAPI, blue staining) of proximal jejunums harvested from <i>Cebpd +/+</i> and <i>Cebpd</i>−/− mice at indicated times after exposure to 7.4 Gy TBI (magnification 10X). (B, C) Quantification of TUNEL-positive cells in intestinal crypts and stromal cells of the villi at indicated time-points after exposure to IR. (D, E) Quantification of γ-H2AX expression levels in intestinal crypts and villi at indicated time-points after exposure to IR. Values are presented as mean ± SEM, n = 4 per genotype per group. (F) Proximal jejunums of <i>Cebpd+/+</i> and <i>Cebpd</i>−/− mice were harvested at 0 h (No IR) and 24 h (IR) after exposure to 7.4 Gy TBI; representative images of immunohistochemical staining of phospho-histone H3 (Ser28) taken at 40× magnification. (G) Phospho-histone H3 (Ser28)-positive cells across approximately 100 intestinal crypts from unirradiated (No IR) and irradiated (IR) <i>Cebpd +/+</i> and <i>Cebpd</i>−/− mice were scored and represented as mean ± SEM, n = 4 per group.</p

<i>Cebpd−/−</i> deficiency enhanced radiation-induced myelosuppression.

<p>Peripheral blood B cells, T cells, and myeloid cells from unirradiated (No IR) and irradiated (IR) <i>Cebpd+/+</i> and <i>Cebpd</i>−/− mice (n = 3/genotype) were enumerated 14 days after exposure to 6 Gy TBI by phenotyping (A, C, E) and expressed as percent of total WBCs (B, D, F). All data are represented as mean ± SEM.</p

<i>Cebpd</i>-deficiency resulted in radiosensitization of bone marrow mononuclear cells and bone marrow progenitors.

<p>(A–B) BMCs and BM-MNCs were enumerated per pair of femurs and tibias per mouse from unirradiated (No IR) and irradiated (IR) <i>Cebpd+/+</i> and <i>Cebpd</i>−/− mice (n = 3/genotype) 14 days after 6 Gy TBI. (C–E) Effects of TBI are depicted for CFU-GEMM, CFU-GM, and BFU-E assays of BMC precursors from unirradiated and irradiated <i>Cebpd +/+</i> and <i>Cebpd</i>−/− mice 14 days after sublethal TBI (6 Gy). All data are represented as mean ± SEM.</p

<i>Cebpd−/−</i> mice showed impaired post-irradiation recovery of WBCs, neutrophils and platelets.

<p>(A) WBCs (n = 6), (B) neutrophils (n = 3), and (C) platelets (n = 6) from unirradiated (No IR) and irradiated (IR) <i>Cebpd+/+</i> and <i>Cebpd</i>−/− mice were counted 14 days after exposure to 6 Gy TBI. All data are represented as mean ± standard error mean (SEM).</p