Downregulation of Heme Oxygenase 1 (HO-1) Activity in Hematopoietic Cells Enhances Their Engraftment After Transplantation

Abstract

Heme oxygenase 1 (HO-1) is an inducible stress-response enzyme that not only catalyzes the degradation of heme (e.g., released from erythrocytes) but also has an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury. HO-1 has a well-documented anti-inflammatory potential, and HO-1 has been reported to have a negative effect on adhesion and migration of neutrophils in acute inflammation in a model of peritonitis. This finding is supported by our recent observation that hematopoietic stem progenitor cells (HSPCs) from HO-1 KO mice are easy mobilizers, since they respond better to peripheral blood chemotactic gradients than wild-type littermates. Based on these findings, we hypothesized that transient inhibition of HO-1 by nontoxic small-molecule inhibitors would enhance migration of HSPCs in response to bone marrow chemoattractants and thereby facilitate their homing. To directly address this issue, we generated several human hematopoietic cell lines in which HO-1 was upregulated or downregulated. We also exposed murine and human BM-derived cells to small-molecule activators and inhibitors of HO-1. Our results indicate that HO-1 is an inhibitor of hematopoietic cell migration in response to crucial BM homing chemoattractants such as stromal-derived factor 1 (SDF-1) and sphingosine-1-phosphate (S1P). Most importantly, our in vitro and in vivo animal experiments demonstrate for the first time that transiently inhibiting HO-1 activity in HSPCs by small-molecule inhibitors improves HSPC engraftment. We propose that this simple and inexpensive strategy could be employed in the clinical setting to improve engraftment of HSPCs, particularly in those situations in which the number of HSPCs available for transplant is limited (e.g., when transplanting umbilical cord blood).

Figure 1. Impact of HO-1 upregulation on chemotaxis and adhesion of human hematopoietic cell lines (K562, Raji and Jurkat)

(A, top). Western blot detection of HO-1 expression levels in hematopoietic cell lines engineered for HO-1 overexpression. The same membranes were reprobed with β-actin to confirm equal loading of total protein. Legend: C- control, UR – upregulation. (A, bottom). HO-1 expression was evaluated at the mRNA level by real-time PCR. Results from three independent experiments are pooled together. *p ≤ 0.005. (B) Fibronectin adhesion assay for HO-1 overexpressing cell lines. The number of adherent cells is indicated, and data from three separate experiments are pooled together. *p ≤ 0.01. (C) The chemotactic responsiveness of HO-1-overexpressing cells to medium alone (−) and to stromal-derived factor 1 (SDF-1) or sphingosine-1-phosphate (S1P) gradients compared with migration of control parental cells. Results are combined from three independent experiments. *p ≤ 0.05.

Figure 2. Impact of HO-1 downregulation on chemotaxis and adhesion of hematopoietic cell lines (Raji and Nalm6)

(A, top) Western blot detection of HO-1 expression levels in hematopoietic cell lines with downregulated HO-1. The membranes were reprobed with β-actin to confirm equal loading of total protein. Legend: C- control, DR – downregulation. (A, bottom) HO-1 expression at the mRNA level according to real-time PCR. Data from three independent experiments are pooled together. *p ≤ 0.005. (B) Adhesion to fibronectin of cell lines in which HO-1 had been downregulated. The number of adherent cells is indicated, and data from three separate experiments are pooled together. *p ≤ 0.01. (C) The chemotactic responsiveness of cells with downregulated HO-1 to medium alone (−) and to stromal-derived factor 1 (SDF-1) or sphingosine-1-phosphate (S1P) gradients in comparison with parental control cells. Results are combined from three independent experiments. *p ≤ 0.05

Figure 3. The influence of a small-molecule HO-1 inhibitor (SnPP) on chemotaxis and adhesion of murine BM-MNCs and on in vivo homing and engraftment of HSPCs

(A) The chemotactic responsiveness to SDF-1 and S1P gradients of murine BM-MNCs (evaluated by FACS) and clonogenic CFU-GM progenitors exposed to two different dosages of HO-1 inhibitor. Results are combined from three independent experiments.*p ≥ 0.05. (B) The effect of HO-1 inhibition by SnPP on adhesion of murine BM-MNCs to fibronectin. The results are shown as the percentage relative to control cells unexposed to SnPP (control). Data from four separate experiments are pooled together. *p ≤ 0.01. (C) Increase in homing of HO-1 inhibited HSPCs from GFP⁺ to the BM of normal wild type (WT) animals. Lethally irradiated WT mice (six animals per group) were transplanted with 3 × 10⁶ GFP⁺ BM-MNCs pre-incubated with 50 μM SnPP for 1 h. Twenty-four hours after transplantation, femoral BMMNCs were harvested, the number of GFP⁺ cells in murine BM was evaluated by FACS (left), and the number of clonogenic GFP⁺ CFU-GM progenitors was enumerated in an in vitro colony assay (right). Both results were compared with the numbers obtained for WT mice transplanted with GFP⁺ BMMNCs untreated with SnPP. No colonies were formed in lethally irradiated and non-transplanted mice (irradiation control). The data in both panels represent the combined results from two independent experiments (n = 12). *p ≤ 0.05. (D) Defect in short-term engraftment of HSPCs exposed to SnPP in BM. Lethally irradiated mice (six mice per group) were transplanted with 1.5 × 10⁵ BM-MNCs from WT mice incubated with medium alone (control) or SnPP (50 μM) for 1 h. Twelve days after transplantation, femoral BMMNCs were harvested and plated in in vitro cultures to enumerate the number of CFU-GM progenitors (left), and spleens were removed to count the number of CFU-S colonies (right). The data in both panels represent the combined results from two independent experiments (n = 12). *p ≤ 0.05.

Figure 4. Accelerated recovery of peripheral blood counts in mice transplanted with BM-MNCs exposed to SnPP

Lethally irradiated mice were transplanted with 2.5 × 10⁵ BM-MNCs incubated with 50 μM SnPP. White blood cells (A) and platelets (B) were counted at intervals (at 0, 5, 7, 11, 16, 21, 28 and 90 days after transplantation). Results are combined from two independent experiments (six mice per group, n = 12). *p ≤ 0.05.

Figure 5. SnPP, a small-molecule inhibitor of HO-1, enhances chemotaxis and decreases adhesion of human peripheral blood (PB)- and umbilical cord blood (UCB)-derived cells

(A and B, left) - effect of HO-1 inhibition by SnPP on the chemotactic responsiveness of UCB- (A) and PB-derived (B) CFU-GM progenitors to SDF-1 and S1P gradients compared with cells exposed to medium alone (control). Results are evaluated by FACS and are combined from two independent experiments.*p ≥ 0.05. (A and B, right) Adhesion of UCB- (A) and PB-derived (B) cells to fibronectin. The results are shown as the percentage relative to control cells unexposed to SnPP. Results from two separate experiments are pooled together. *p ≤ 0.01.