Pre-Transplantation Blockade of TNF-α-Mediated Oxygen Species Accumulation Protects Hematopoietic Stem Cells

Abstract

Hematopoietic stem cell (HSC) transplantation (HSCT) for malignancy requires toxic preconditioning to maximize anti-tumor effects and donor-HSC engraftment. While this induces bone marrow (BM)-localized inflammation, how this BM environmental change affects transplanted HSCs in vivo remains largely unknown. We here report that, depending on interval between irradiation and HSCT, residence within lethally irradiated recipient BM compromises donor-HSC reconstitution ability. Both in vivo and in vitro we demonstrate that, among inflammatory cytokines, TNF-a plays a role in HSC damage: TNF-a stimulation leads to accumulation of reactive oxygen species (ROS) in highly purified hematopoietic stem/progenitor cells (HSCs/HSPCs). Transplantation of flow-cytometry-sorted murine HSCs reveals damaging effects of accumulated ROS on HSCs. Shortterm incubation either with an specific inhibitor of tumor necrosis factor receptor 1 signaling or an antioxidant N-acetyl-L-cysteine (NAC) prevents TNF-a-mediated ROS accumulation in HSCs. Importantly, pre-transplantation exposure to NAC successfully demonstrats protective effects in inflammatory BM on graft-HSCs, exhibiting better reconstitution capability than that of nonprotected control grafts. We thus suggest that in vivo protection of graft-HSCs from BM inflammation is a feasible and attractive approach, which may lead to improved hematopoietic reconstitution kinetics in transplantation with myeloablative conditioning that inevitably causes inflammation in recipient BM. STEM CELLS 2017;35:989-1002 SIGNIFICANCE STATEMENTThis study shows the following: (a) depending on interval between TBI and HSCT, residence within lethally irradiated recipient BM compromises donor-HSC reconstitution ability; (b) elevated levels of TNF-a in inflamed BM is responsible for the effect; (c) TNF-a induces formation of ROS through an NADPH oxidase in donor HSCs, leading to impaired reconstitution ability; (d) Pre-incubation with an antioxidant NAC that suppresses TNF-a-stimulated ROS production successfully protects transplanted HSCs from BM inflammation. We eventually came up with the proposal of "in vivo stem cell protection" as a novel therapeutic concept in HSCT. These findings have implications for the basic HSC biology and for the improvement of an HSCT outcome for patients.

Rescuing Self: Transient Isolation and Autologous Transplantation of Bone Marrow Mitigates Radiation-Induced Hematopoietic Syndrome and Mortality in Mice

“…We supported this assumption by demonstrating that the isolation and cryopreservation of BM grafts more efficiently preserves different HSPCs than those of the BM of an irradiated animal, at least during the first 24 h after lethal TBI. This result is in concordance with a recent report by Ishida et al ( 32 ) who showed that elevated tumor necrosis factor alpha (TNF-α) levels in inflamed BM contribute to the compromised reconstitution of donor BM. However, such a direct comparison between in vivo and a vial containing BM frozen to −80°C is complex considering the number of influencing variables involved.…”

“…Notably, the reconstituted hematopoiesis post AL-BMT showed a shift toward the myeloid lineage, and the M-bias was strongly evident in the mice that received the BM collected 8 h after the induction of hypometabolism. A recent study suggested that the transfer of LT-HSCs pretreated with an antioxidant, N -acetyl cysteine, led to myeloid reconstitution ( 32 ). The M-bias observed in this study could partially be explained by considering that SJNP-1, which was used for inducing HMS, is an effective free radical scavenger and antioxidant.…”

“…The Lin − cell fraction was washed with PBS + 2% FBS, and the cell number was volumetrically determined through flow cytometry ( 30 ). For analyzing different HSPC populations, the Lin − fraction was preincubated with anti-CD16/32 antibody (0.5 μL/10 6 cells; Biolegend, CA, USA) to block the Fcγ receptors and then stained with anti-Sca1-PE (1 μL/10 6 cells; Biolegend), c-Kit-APC-Cy7 (0.5 μL/10 6 cells; Biolegend), and CD34-PerCP/Cyp5.5 (0.8 μL/10 6 cells; Biolegend) on ice, in dark for 1 h. The frequencies of hematopoietic progenitor cells (HPCs; Lin − Sca1 − c-kit + cells), KSL (Lin − Sca1 + c-kit + cells), short-term HSCs (ST-HSCs; Lin − Sca1 + c-kit + CD34 + cells) ( 31 ), and long-term HSCs (LT-HSCs; Lin − Sca1 + c-kit + CD34 − cells) ( 32 ) were analyzed using BD Accuri C6 software. Appropriate isotypes and single positive controls were also acquired whenever required for compensation, and at least 20,000 cells were acquired for each sample.…”

Granulocyte-derived TNFα promotes vascular and hematopoietic regeneration in the bone marrow

“…Some reports suggest that TNFα acts on HSPCs to promote their maintenance in the steady state and engraftment during regeneration 31 , 35 , 36 . Other reports suggest that TNFα acts on HSPCs to inhibit their function during homeostasis and that TNFα-receptor blockade prior to transplantation promotes HSPC survival 37 , 38 . Our results support the notion that TNFα has pro-regenerative effects on HSPCs.…”

Single cell transplantation—Golden standard of hematopoietic stem cell heterogeneity study

“…Evi1 [28] , Tie2 [24] 等标记. 这种利用特异基因标记的方法 [19] . 实验操作中可 根据实际照射源的类型(钴60或者X-ray)以及小鼠的耐 受程度和实验的可操作性等方面协调进行.…”