Carbon Monoxide Therapy Reduces Reactive Oxygen Species Production and the Short-Term Hematopoietic Stem Cell Population In Heme-Oxygenase-1 Knockout Mice

Abstract 4767

Previous studies have demonstrated that heme-oxygenase-1 (HO-1), the rate limiting enzyme in the catabolism of heme, is a regulator of the balance between hematopoiesis and stem cell preservation under stress. HO-1 produces three by-products, carbon monoxide, biliverdin/bilirubin, and iron/ferritin, all of which have demonstrated antioxidant and anti-apoptotic properties. HO-1^-/- mice display oxidative stress, anemia, and leukocytosis with chronic inflammation. We hypothesize that therapy with inhaled carbon monoxide (CO) may reduce stress hematopoiesis in HO-1^-/- mice, restoring them to normal hematopoiesis. We have previously reported that treatment with 250 ppm CO for 1 h raises mouse carboxyhemoglobin to 14–20% which returns to baseline within 24 hours. Therefore, in order to test this hypothesis we treated 25 week old HO-1^-/- mice and HO-1^+/+ mice (n=7/strain) with 250 ppm inhaled CO for 1 h/day, 3 days/week for eight weeks and compared them to an equal number of untreated HO-1^-/- and HO-1^+/+ mice. We demonstrate that after four weeks, CO-treated HO-1^-/- mice have a total white blood cell count of 11.9 ± 3.2 K/μ L compared to 22.6 ± 3.2 K/μ L in untreated HO-1^-/- mice (p<0.05), with HO-1^+/+ mice having no treatment effect. After 8-weeks of treatment the mice were sacrificed and methylcellulose colony-forming unit assays and flow cytometry were performed on bone marrow to assess their hematopoietic potential. Total methylcellulose colony-forming units were similar in both HO-1^-/- and HO-1^+/+ mice with CO-treatments significantly (p<0.05) increasing the total number of colonies per animal. Of note, in both the CO-treated HO-1^-/- and HO-1^+/+ mice the CFU-GM were not significantly affected. Cell cycle analysis of the bone marrow using propidium iodide demonstrates that untreated HO-1^-/- mice have a significantly decreased percent of cells in S-phase compared to untreated HO-1^+/+ mice. Treatment with CO significantly (p<0.05) increases the percent of cells in S-phase in HO-1^-/- mice but not HO-1^+/+ mice. Similarly, untreated HO-1^-/- mice have a lower frequency of cells in sub-G₁ phase compared to untreated HO-1^+/+ mice, however CO-treatments significantly (p<0.01) increase this frequency in HO-1^-/- mice to levels comparable with untreated and CO-treated HO-1^+/+ mice. Reactive oxygen species (ROS) production in lineage^-, c-kit⁺, Sca-1⁺ (KLS) cell population was assessed using 5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate dye. HO-1^-/- mice have a significantly (p<0.001) increased proportion of ROS positive KLS cells compared with HO-1^+/+ mice. Interestingly, the CO-treated HO-1^-/- mice had a significantly higher proportion of KLS cells staining positive for ROS, but the mean fluorescent intensity of this population was significantly (p<0.05) decreased compared to untreated HO-1^-/- mice. This indicates that there is an overall decrease in ROS in the KLS cells of CO-treated HO-1^-/- mice compared to untreated HO-1^-/- mice. Analysis of long-term, short-term, and multipotent progenitor cell populations was conducted and reveals significant changes at the level of the short-term hematopoietic progenitor population (ST-HSC). Specifically, untreated HO-1^-/- mice have a significantly increased percent of ST-HSC cells compared to untreated HO-1^+/+ mice. Treatment with CO significantly (p<0.05) decreases the percent of ST-HSC cells in HO-1^-/- mice but not HO-1^+/+ mice. Combined this data indicates that CO therapy is able to modify the hematopoietic potential of HO-1^-/- mice. We propose a model in which CO-mediated signaling initiates a homeostatic conditioning program in stem cells to balance hematopoiesis and stem cell preservation.